Bbm book 2013 final version with cover full bleed

!!!!!!!!!!!!!!!!!!!!!!!!!!!!On!the!cover:!!The!Bassler!Lab!studies!quorum!sensing—the!process!of!cell9cell!communication!in!bacteria.! Quorum! sensing! enables! populations! of! bacteria! to! regulate! gene! expression,! and!therefore! behavior,! on! a! community9wide! scale.! Using! quorum! sensing,! bacteria! can! switch!between! two! distinct! gene! expression! programs:! one! that! is! favored! at! low! cell! density! for!individual,! asocial! behaviors! and! another! that! is! favored! at! high! cell! density! for! social,! group!behaviors.! The! Bassler! Lab! was! instrumental! in! defining! the! quorum9sensing! system! of! Vibrio&harveyi.! Notably,! the! quorum9sensing! behaviors! of! this! bacterium! include! bioluminescence,! as!shown!in!the!photograph.!!!!!Funding! for!BBM2013!was!made!possible! in!part! by!1!R13!AI! 102383901! from! the!National! Institute!of!Allergy! and!Infectious! Diseases.! The! views! expressed! in! written! conference! materials! or! publications! and! by! speakers! and!moderators!do!not!necessarily!reflect!the!official!policies!of!the!Department!of!Health!and!Human!Services;!nor!does!mention!of!trade!names,!commercial!practices,!or!organizations!imply!endorsement!by!the!U.S.!Government.!

2013!Boston!Bacterial!Meeting!7!Schedule!and!Introduction!

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!

Friday,!June!14th!

Time! Event! Speakers! Session!Chair!!

12:00!pm! Registration! ! !

12:45!pm! Opening!Remarks! ! !

1:00!pm! Session!I!

EmilyKate!McDonough!Benjamin!Wolfe!Alexander!Elsholz!Tami!Lieberman!François!Lebreton!Sriram!Kosuri!

Andrew!Goodman!(Yale!University)!

3:00!pm! Coffee!Break! ! !

3:30!9!4:40!pm!!! Poster!Session!I! #!1970! !

4:40!pm! Session!II!

Dipti!Nayak!Linfeng!Huang!Diana!Morales!Michael!Wollenberg!Wolfram!Möbius!

Rachel!Dutton!(Harvard!University)!

6:30!9!8:30!pm! Reception!! ! !!

Saturday,!June!15th!

Time! Event! Speakers! Session!Chair!

9:00!am! Coffee! ! !

9:30!am!!! Session!III!

Kristina!Jonas!Matthew!Cabeen!Xavier!Meniche!Pushkar!Lele!

Jodi!Camberg!(University!of!Rhode!Island)!

10:50!am! Coffee!Break! ! !

11:20!am! Session!IV!

Marina!Besprozvannaya!Burak!Okumus!Lia!Cardarelli!!Naveen!Sinha!Antonio!Gomes!

Susan!He!(UMass9Lowell)!

1:00!pm!!! Lunch! ! !

2:00!pm!!! Session!V!

Elizabeth!Mearls!Matthew!Ramsey!Pete!Chandrangsu!Henry!Haiser!Patrick!Degnan!

Tim!van!Opijnen!(Boston!College)!

3:40!9!4:50!pm! Poster!Session!II! #719140! !

4:5595:00!pm! Poster!prize!award! ! !

5:00!pm! Keynote!Speaker! Bonnie!Bassler! !

6:00!9!8:00!pm! Reception! ! !

!

2013!Boston!Bacterial!Meeting!7!Schedule!and!Introduction!

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!

Welcome!!!Dear!Participants,!!On!behalf!of! the!entire!organizing!committee,!we!would! like! to!welcome!you! to! the!nineteenth!annual! Boston! Bacterial! Meeting! (BBM).! For! the! past! nineteen! years,! the! BBM! has! provided! a!unique!forum!for!bacteriologists!from!across!New!England!to!share!research!and!exchange!ideas!in!a! scientifically! enriching,! collegial! atmosphere.! This! year,! the! BBM! received! more! abstract!submissions!than!ever,!an!especially!impressive!feat!given!last!year's!record9breaking!turnout.!The!quality!of!and!enthusiasm!for!bacteriology!research!in!the!Boston!area!is!as!strong!as!ever.!!!The!BBM!would!not!be!possible!without!the!enthusiastic!participation!of!scientists! like!you.!This!year,!we!have!twenty9five!excellent!talks!touching!on!many!aspects!of!bacteriology!research!and!well!over!one!hundred!poster!presentations.!One!of!the!most!important!goals!of!the!meeting!is!to!promote!collaboration!in!science,!and!we!encourage!you!to!go!out!and!meet!your!colleagues.!!We!would!like!to!thank!our!faculty!sponsors:!Roberto!Kolter,!Richard!Losick,!John!Mekalanos,!and!Andrew!Wright.!We!are!also!indebted!to!the!hard!work!of!the!graduate!students!and!postdoctoral!fellows!who!volunteer!their!time!to!organize!the!meeting!and!ensure!that!it!runs!smoothly.!Thank!you!!!Our! keynote! speaker! this! year! is! Prof.! Bonnie!Bassler! from!Princeton!University.!We!have!been!trying!for!years!to!have!Prof.!Bassler! join!us,!and!we!are!delighted!to!have!her!this!year.!We!are!looking!forward!with!enthusiasm!to!her!Saturday!evening!talk.!!We!hope! that! you!enjoy! the!nineteenth!annual!Boston!Bacterial!Meeting!and! that! you! find! the!next!two!days!both!scientifically!and!socially!enriching.!!!!Thank!you!for!joining!us!!

Matt!Cabeen!and!Edel!Hyland!Co9chairs,!BBM2013!

!!!

Meeting!Announcements!!

• Please!silence!your!mobile!devices!before!entering!the!lecture!hall.!• Please!keep!your!name!badge!on!at!all!times!during!the!meeting.!Due!to!the!large!numbers!

of!attendees!this!year,!you!will!be!asked!to!present!your!badge!in!order!to!get!into!the!keynote!lecture.!

• Restrooms!are!located!in!the!basement.!• Room!103B!is!available!as!a!mothers’!room.!

2013!Boston!Bacterial!Meeting!7!Keynote!Speaker!

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KEYNOTE!SPEAKER!!

BONNIE!L.!BASSLER,!PH.D.!!

!!

!Bonnie! L.! Bassler! is! a! Howard! Hughes!Medical! Institute! Investigator! and! is! Squibb! Professor! of!Molecular! Biology! at! Princeton! University.! She! performed! her! undergraduate! studies! at! the!University!of!California9Davis!and!earned!her!PhD!at!Johns!Hopkins!University.!She!began!working!on! quorum! sensing! during! her! postdoctoral! fellowship! with!Michael! Silverman! at! the! Agouron!Institute! in! La! Jolla,! CA,!where! she!discovered! that!Vibrio& harveyi! uses!more! than!one! signaling!molecule!for!quorum!sensing.!!!!!! !!Dr.!Bassler's!research!at!Princeton!has!continued!to!uncover!the!surprising!mechanisms!by!which!bacteria!communicate.!She!discovered!that!bacteria!can!accomplish! interspecies!communication!using! quorum9sensing! molecules! as! a! common! molecular! language! that! she! calls! "bacterial!Esperanto".!The!molecules!used!for!such!interspecies!communication,!collectively!known!as!AI92,!held!more!surprises.!To!find!the!structure!of!V.&harveyi!AI92,!Dr.!Bassler!had!to!crystallize!the!entire!ligand9bound!receptor!of!AI92! in!collaboration!with!Princeton!crystallographer!Fred!Hughson.!To!their! surprise,! AI92! bound! the! element! boron,! which! has! few! biological! roles,! as! a! necessary!cofactor.! Moreover,! AI92! molecules! spontaneously! interconvert! among! different! structures,!providing!the!basis!for!interspecies!communication!using!AI92.! !!More! recent! work! from! the! Bassler! Lab! has! shown! how! small! regulatory! RNAs! control! Vibrio!quorum9sensing! cascades.! She! is! now! studying! how! the! behaviors! of! individual! cells! relate! to!population9level! behavior! during! quorum! sensing.! Her! lab! is! also! conducting! studies! aimed! at!developing! pro9! and! anti9quorum9sensing! molecules! that! have! potential! as! new! therapeutics.!!Dr.! Bassler! has! received! numerous! awards,! including! a!MacArthur! "Genius"! Fellowship! and! the!L'oréal9UNESCO! Award.! She! has! been! elected! to! the! National! Academy! of! Sciences! and! the!American!Academy!of!Arts!and!Sciences.!

2013!Boston!Bacterial!Meeting!7!New!Faculty!

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New!members!of!the!academic!community!!As! part! of! BBM’s! goal! to! foster! interactions! and! collaborations!between! various! individuals! and!their! institutions,!we!would! like! to!highlight! the!new!members!of!our! research! community!who!have! recently!begun! their! appointments.!!We!encourage!everyone! to!provide! these!new! faculty!members!with!a!warm!welcome!to!our!Boston9area!bacteriology!community!!!!Bree!Aldridge!Department!of!Molecular!Biology!and!Microbiology,!Department!of!Biomedical!Engineering,[email protected]!http://sackler.tufts.edu/Faculty9and9Research/Faculty9Profiles/Bree9Aldridge9Profile!!

We! are! a! multidisciplinary! research! team,! integrating! quantitative! single9cell!measurement!with!computational!modeling!and!analysis!to!create!intuitive!descriptions!of!complex!cell!biology.!Our!research!focuses!on!understanding!how!mycobacteria! tolerate!stress!and!perturb!host!cell!biology!to!evade!killing!by!antibiotic!treatment!and!the!host!immune!response.!We!integrate!single9cell!measurements!and!computational!modeling!to!quantitatively! describe! stress! tolerance! and! virulence!mechanisms! of!mycobacteria! and!the! fate!of! infected!macrophages.!Our!approach! is!motivated!by!our! recent! finding! that!the! asymmetric! growth! pattern! of! mycobacteria! creates! subpopulations! of! cells! with!different! growth! parameters! and! tolerance! to! antibiotic! treatment.!We! aim! to! use! this!knowledge! to! engineer! improved! therapeutics! for! treating! tuberculosis! and! other!mycobacterial!diseases.!

!!Jodi!Camberg!Cell!and!Molecular!Biology!Department,[email protected]!!http://cels.uri.edu/cmb/CMB_FacultyView.aspx?fname=Jodi&lname=Camberg!!

Research! in! our! lab! addresses! the! biochemical! mechanisms! and! physiological! roles! of!energy9dependent!molecular!machines! in!complex!cell!processes!such!as! the!cell!division!pathway! in! bacteria.! Energy9dependent! molecular! machines! are! proteins! that! harness!chemical! energy! released! upon! nucleotide! hydrolysis! and! translate! that! energy! into!mechanical!work.!The!cell!division!pathway! requires! the!contributing!activities!of! several!molecular! machines! whose! activities! must! be! precisely! coordinated! and! regulated! both!spatially! and! temporally! for! cells! to! divide.! Cell! division! entails!multiple! steps,! including!assembly! of! division! protein! complexes! at! midcell,! membrane! constriction,! insertion! of!new! cell! wall! components! and! separation! of! daughter! cells.! Our! laboratory! is! currently!focused! on! studying! the! biochemical!mechanisms! of! proteins! that!modulate! the! cellular!position!of!the!division!machinery,!referred!to!as!the!Min!system,!as!well!as!the!machinery!

that!promotes!constriction!(FtsZ!and!FtsZ9interacting!proteins).!!A!second!focus!of!our!group!is!regulated!proteolysis.!Protein!degradation!is!utilized!by!the!cell!to!maintain!the!

cellular!proteome!and!promote!homeostasis.!Regulated!proteolysis!can!be!used!to!control!cellular!responses!to!stimuli!and!modulate! cellular! processes.! The! ATP9dependent! chaperone! protease! complex! known! as! ClpXP! is! a!molecular!machine!that!unfolds!protein!substrates!bearing!specific!recognition!tags!and!degrades!them.!One!substrate!degraded!by!ClpXP!in!E.&coli!is!the!essential!protein!FtsZ.!Our!lab!is!working!on!how!degradation!of!cell!division!proteins!such!as!FtsZ!modulates!the!process!of!cell!division!in!E.&coli.!This!work!will!lead!to!a!better!understanding!of!the!fundamental!process! of! cell! division! and! the! biochemical! mechanisms! of! substrate! recognition! and! protein! unfolding! by! ATP9dependent!molecular!chaperones!and!degradation!by!cognate!proteases.!!


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Yunrong!(Win)!Chai!Department!of!Biology,[email protected]!http://www.northeastern.edu/biology/people/faculty/yunrong9chai/!!

Biofilms! are! the! predominant! life! form! of! microbes! in! nature,! and! are! medically! and!environmentally!significant.!My!lab!uses!Bacillus&subtilis!and!other!related!Bacilli&species!as!model!microorganisms! to! study! biofilm! formation! and! other!multicellular! behaviors.!We!hope! to! understand! how! biofilm! formation! in! B.& subtilis! is! governed! by! a! complex!regulatory! network! in! response! to! a! variety! of! environmental! signals,! and! how! these!signals! trigger! and! modulate! biofilm! formation! at! different! stages! during! multicellular!development.!We!also!hope!to!understand!the!role!of!beneficial!biofilms!in!bacteria9host!interactions! and! host! protection! by! using! both! B.& subtilis! and! Lactobacillus! as! model!microorganisms.!!

!!Jason!Crawford! !Departments!of!Chemistry!and!Microbial!Pathogenesis,!Chemical!Biology!Institute,[email protected]!http://www.chem.yale.edu/faculty/crawford.html!

!The! Crawford! laboratory! focuses! on! developing! and! systematically! applying! genome!sequence9guided!methods! for! the! discovery! of! novel! genetically! encoded! small!molecules!from!mutualistic!and!pathogenic!bacteria.!To!decode!the!chemical9signaling!events!between!microbes! and! their! animal! hosts,! our! research! combines! the! disciplines! of! natural! product!biosynthesis,! microbial! small! molecule! characterization,! molecular! mode! of! action,! and!microbiology.!!! With! the! explosion! of!microbial! genome! sequence! data! available,! it! is! now! evident!that!microbes!harbor!the!genetic!potential!to!contribute!several!orders!of!magnitude!more!pharmacologically! relevant!molecules! than! are! currently! known.!However,! tight! regulatory!control! of! the! majority! of! bioactive! small! molecule! metabolic! pathways! in! the! producing!

organism! blocks! their! functional! elucidation! in! the! lab.! To! overcome! this! challenge,! we! recapitulate! natural! host9pathogen!interactions!to!understand!and!stimulate!unknown!biosynthetic!pathways!that!may!not!normally!be!turned!on!under!typical!lab!conditions.!In!addition,!we!utilize!microbial!genomic!analyses,!protein!biochemistry,!and!genetic!manipulation!to!discover!the!novel!enzyme!systems!and!their!functional!roles!in!the!context!of!their!ecological!niche.!Many!of!the!highly!unusual!“orphan”!biosynthetic!gene!clusters!identified!in!our!analysis!are!suspected!of!synthesizing!novel,! structurally! diverse,! and! biologically! active! small! molecules.! These! types! of! “cryptic”! naturally! produced!molecules!often!regulate!complex!interactions!with!their!animal!hosts,!hold!a!rich!history!of!being!utilized!as!human!drugs,! and! serve!as! excellent!molecular!probes! for! identifying!new!drug! targets! for! a!wide! variety!of!diseases.!Our!group!is!particularly!interested!in!the!molecular!communications!between!a!family!of!prolific!bioactive!small!molecule9producing!Gamma9proteobacterial!pathogens!and!their!afflicted!animal!hosts!–!insects!and!humans.!!!!!!!!!!


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Kristen!DeAngelis!Department!of!Microbiology,!University!of!Massachusetts,[email protected]!http://www.micro.umass.edu/faculty9and9research/kristen9deangelis!!

My!work!focuses!on!understanding!microbes!both!from!individual!physiological!perspectives!as!well!as!broad!ecological!perspectives.!Specifically,!I!am!interested!in!effects!that!climate!change!has!on!soil!microbial!communities!and!applying!the!results!towards!improvement!of!next9generation! biofuels.! We! aim! to! examine! soil! carbon! dynamics,! with! a! focus! on!microbial! carbon!storage!and!greenhouse!gas!emissions! in! the! rhizosphere!and!within! the!context!of!plant9microbial! interactions.!The!microbial!role! in!ecosystem!function!is!difficult!to! define! partly! because! microbial! functions! feed! back! at! many! scales.! At! the! single9cell!scale,! they! sense! and! respond! to! their! environments! through! two9component! regulatory!systems.! At! the! population9level! scale,! there! are!myriad! chemical! inter9! and! intra9species!communications! that! control! group! behaviors.! At! the! community! scale,! composition!changes!with!environmental!factors,!altering!nutrient!pools!and!process!rates.!My!research!

program! is! intended! to! address! this! problem! by! exploring! the! following! questions:! How! are!microbial! community!structure! and! function! related?!What! interactions!with! the! environment! or!with! other! organisms! control!microbial!activity?!What!can!be!gained!or!lost!from!examining!natural!versus!controlled,!laboratory!systems?!!!Andrew!Goodman!Yale!Microbial!Diversity!Institute,[email protected]!http://bbs.yale.edu/microbiology/people/andrew_goodman.profile!!

!What!are!the!mechanisms!of!cooperation!and!competition!among!human!gut!microbes,!and!how!do! these! interactions!shape!community!composition?!What!are! the!consequences!of!interpersonal! variation! in! human! gut!microbiomes?!Our! lab! combines!microbial! genetics,!functional! genomics,! and! germfree! animal! studies! to! dissect! the! conversations! that! are!occurring!within!the!gut!microbiota!and!between!these!microbes!and!their!hosts.!!!!!

!!Guixin!(Susan)!He!Department!of!Clinical!Lab!and!Nutritional!Sciences,!University!of!Massachusetts,[email protected]!http://www.uml.edu/Biomed9Biotech/faculty/He9Guixin9Susan.aspx!

!I! have! been!working! on! antibiotic! resistance!mechanisms! and! cell!membrane! transporters! in!Pseudomonas&aeruginosa!and!Enterobacter&cloacae! for!eight!years.!!Currently!we!are!focusing!on! the! following! studies:! 1)! Investigating! the! presence! of! disinfectant9resistant! bacteria! in!clinical! settings;! 2)! Analyzing! resistance! genes! in! antibiotic9resistant! enteric! bacteria;! 3)!Examining!the!accumulation/efflux!of!antibiotics!in!the!bacterial!cells;!and!4)!Developing!nano9approaches!to!solve!the!issue!of!antibiotic!resistance.!!!


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Vanja!Klepac7Ceraj!Department!of!Biological!Sciences,[email protected]!http://new.wellesley.edu/biology/faculty/klepac9ceraj!!

Microbial! communities! comprise! a! complex,! often! stable,! milieu! of! many! different!organisms! modified! by! physical! and! chemical! perturbations! over! space! and! time.! It! is!becoming!increasingly!clear!that!microbial!communities!play!an!crucial!role!in!maintaining!the! health! of! an! environment,! whether! a! human! body! or! a! lake,! and! it! is! therefore!important!that!we!understand!which!variables!cause!community!shifts!and!how!microbial!interactions! contribute! to! the! stability! of! such! communities.! !My! research! interests! are!focused! on! understanding! how! microbial! communities! are! formed,! their! stability! and!resilience! in! relation! to!particular!environmental! variables,!microbe9microbe! interactions,!and!how!these!interactions!shape!evolution!in!these!populations.!To!this!end,!my!research!

fosters!collaboration!among!scientists! from!different!disciplines! including!biology,! chemistry,!geology!and!medicine,!and!is!pursued!in!collaboration!with!investigators!at!MIT,!the!Forsyth!Institute,!and!other!institutions.!!!Yasu!Morita! ! !Department!of!Microbiology,!University!of!Massachusetts,[email protected]!http://www.micro.umass.edu/faculty9and9research/yasu9morita!!

The!genus!Mycobacterium!includes!a!number!of!pathogenic!species!that!cause!various!diseases!such! as! tuberculosis! and! leprosy.! The! research! goal! of! our! laboratory! is! to! understand! the!pathogenesis!of!these!medically!important!bacteria,!especially!focusing!on!the!biogenesis!and!functions!of! the!plasma!membrane! (PM)!and!cell!wall! (CW).! In! fact,! the!multi9layered,!highly!impermeable! complex! of! PM/CW! represents! a! major! virulence! determinant! of! pathogenic!mycobacteria.!While!PM/CW!biogenesis!is!a!proven!drug!target,!the!synthesis!and!functions!of!PM/CW!components!and! the!assembly!of! the!PM/CW!complex! remain!poorly!defined!at! the!molecular! level.!Our!research! is!aimed!at!clarifying!several!key!aspects!of!PM/CW!biogenesis!and!functions!using!glycobiological,!biochemical,!and!cell!biological!approaches.!

!!Wai7Leung!Ng! !Department!of!Molecular!Biology!and!Microbiology,[email protected]!http://sackler.tufts.edu/Faculty9and9Research/Faculty9Profiles/Wai9Leung9Ng9Profile!!

Group! behaviors! that! are! critical! for! survival! in! harsh! environments,! such! as! biofilm!formation,! virulence! factor! production,! and! genetic! exchange,! are! seemingly! futile! if!performed!by!a!single!bacterium!acting!alone.!To!solve!this!problem,!bacteria!depend!on!a!cell9to9cell! communication! process! called! quorum! sensing! (QS).! Through! production! and!detection! of! extracellular! chemicals! called! autoinducers,! bacteria! use! QS! to! monitor! cell!population!density!and!complexity,!and!synchronize!the!gene!expression!of!the!group!to!act!in!unison.!Our!lab!uses!a!multi9disciplinary!approach!to!understand!how!pathogenic!Vibrio!species! communicate! with! each! other.! Through! these! studies,! our! goal! is! to! reveal! the!molecular! mechanisms! underpinning! QS,! and! to! understand! how! QS! controls! Vibrio!virulence!and!physiology.!In!turn,!we!hope!to!understand!how!group!behaviors!evolve.!We!will!also!use!these!findings!to!develop!novel!therapeutics!to!combat!infectious!diseases!by!

manipulating!cell9to9cell!communications.!!


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Alexandra!Purdy!Department!of!Biology,[email protected]!https://www.amherst.edu/people/facstaff/apurdy!!

Many!bacterial!species!use!carefully!regulated!attachment!and!virulence!mechanisms!to!colonize!and!sometimes!sicken!their!eukaryotic!hosts.!My!laboratory!aims!to!understand!the!complex!molecular!“conversations”!that!underlie!these!interactions!by!studying!Vibrio&cholerae,! the! Gram9negative! bacterium! that! causes! cholera,! an! acute! diarrheal! disease!that!affects!hundreds!of!thousands!of!people!each!year.!!Vibrio& cholerae! is! naturally! found! in! aquatic! environments,! often! attached! to!phytoplankton,! aquatic! arthropods! such! as! copepods,! and! larger! organisms.!V.& cholerae!also!can!be!carried!by! terrestrial!arthropods! such!as!houseflies.!Our! laboratory!explores!interactions! between! V.& cholerae! and! arthropods! using! Drosophila& melanogaster,! the!common! fruit! fly,! as! a! model! system.!In! the! course! of! our! work,! we! have! discovered!specific! V.& cholerae! genes! that! are! required! for! virulence! and! colonization! in!Drosophila.!These! include! genes! that! likely! initiate! signal! transduction! cascades! that!

regulate! expression! of! V.& cholerae! genes! in! the! fly.!Our! lab! is! interested! in! understanding! how! these! regulatory!cascades!work;!that!is,!what!genes!do!they!regulate?!What!environmental!signals!do!they!sense?!How!are!they!turned!on!and!off?!!Ultimately,!this!project!will!reveal!the!genetic!mechanisms!that!allow!V.&cholerae!to!associate!with!arthropods!in!order!to!survive!and!spread!in!the!rivers,!lakes,!ponds,!and!oceans!that!let!the!disease!persist!in!areas!where!it!is!endemic,!and!spread!to!distant!locales.!!Our!ultimate!goal! is!to!better!understand!the!ecology!and!evolution!of!a!pathogen!of!major!public!health!importance!by!integrating!genomic,!genetic,!molecular!and!environmental!approaches.!

!!Tim!van!Opijnen!Biology!Department,[email protected]!http://www.bc.edu/content/bc/schools/cas/biology/facadmin/vanopijnen.html!!

We!work! on!microbial! systems! biology! and! try! to! understand! a! bacterium! as! a! complete!system!by!applying!a!combination!of!high9throughput!robotics,!next!generation!sequencing!and! computational!biology.! The!goal!of! the! lab! is! to!develop!new!antibiotics! and!engineer!bacteria!with!new!properties!that!can!aid!in!curing!disease.!!To!make!our!research!go!lightning!fast,!we!are!the!proud!owner!of!a!unique!state9of9the9art!robotics!system,!which!we!use!extensively!to!focus!on!three!subjects:!1)& Antibiotics.! We! utilize! cutting! edge! genome9wide,! experimental! and! bioinformatical!systems! approaches,! several! of! which! we! ourselves! have! recently! developed,! in! order! to!construct!drug/gene!interaction!networks!that!mediate!the!bacterial!antibiotic!responses.!2)&GenomeBwide& strategies.& An! important! goal! in! modern! biology! is! to! understand! the!relationship!between!genotype!and!phenotype;!what!constitutes!a!phenotype,!which!genes!are! involved! and! how! they! interact! to! provide! an! efficient! yet! robust! response! to!

environmental! change.! We! recently! developed! the! now! widely! used! massively! parallel! sequencing! technique,! Tn9seq.!New!work!in!the!lab!focuses!on!developing!strategies!that!automate!the!discovery!of!genotype9phenotype!links!and!the!placement!of!genes!in!their!pathways.!3)&Engineering&bacteria.&Microbes!are!extremists,!being!found!on!the!most! inhospitable! places! on! earth.! In! the! lab!we! uncover! the! capabilities! of! different! bacterial! species! in! order! to!create!a!biological! toolbox!that! is! filled!with!components!that!can!be!used!to!engineer!bacteria!with!new!traits!and!novel!applicability.!!

2013!Boston!Bacterial!Meeting!7!Oral!Presentations!

9

Friday!|!Session!I,!1!pm!to!3!pm!!

1.! EmilyKate!McDonough!!

Use!of!a!high!throughput!genetic!selection!to!identify!regulators!of!the!Vibrio&cholerae&infection7induced!gene,!xds&&EmilyKate!McDonough,!David!Lazinski,!and!Andrew!Camilli!!!

2.! Benjamin!Wolfe!! A!global!view!of!the!taxonomic!and!functional!diversity!of!cheese!rind!microbial!communities!!Benjamin!E.!Wolfe,!Julie!E.!Button,!and!Rachel!J.!Dutton!!

3.! Alexander!Elsholz! A!post7translational!feed7back!loop!by!a!tyrosine!kinase!regulates!exopolysaccharide!production!in!Bacillus&subtilis!!Alexander!Elsholz1,!Richard!Losick1!!

4.! Tami!Lieberman!! Pathogen!genetic!variation!within!single!clinical!samples!provides!a!signature!of!selective!pressures!!Tami!D.!Lieberman,!Kelly!B.!Flett,!Idan!Yelin,!Thomas!R.!Martin,!Alexander!J.!McAdam,!Gregory!P.!Priebe,!&!Roy!Kishony!!!

5.! François!Lebreton! Emergence!of!epidemic!multi7drug!resistant!Enterococcus&faecium!from!animal!and!commensal!strains!!François!Lebreton,!Willem!van!Schaik,!Abigail!McGuire,!Jennifer!Wortman,!Bruce!Birren,!Ashlee!Earl,!Michael!S.!Gilmore

!

!6.! Sriram!Kosuri! Using!multiplex!DNA!synthesis!to!study!transcription!and!translation!in!E.&coli!!

Daniel!B.!Goodman,!Sriram!Kosuri,!and!George!M.!Church!!!!!Friday!|!Session!II,!4:40!pm!to!6:20!pm!!7.! Dipti!Nayak! A!molecular!mechanism!for!formaldehyde!detoxification!and!growth!in!

Methylobacterium!!Dipti!D.!Nayak,!Milya!Davlieva,!Yousif!Shamoo!and!Christopher!J.!Marx!!!

8.! Linfeng!Huang! Practical!and!functional!implications!of!siRNA7like!small!RNAs!in!bacteria!!Linfeng!Huang,!Padraig!Deighan,!Jingmin!Jin,!Elaine!Lee,!Larry!McReynolds,!Ann!Hochschild,!Judy!Lieberman!!!

9.! Diana!Morales! Invading!the!privacy!of!Enterococcal!invaders!!Morales!DK.,!Chai!L.,!Ramsey!M.,!Inoue,!T.,!Gilmore,!M.,!and!Kolter!R.!!

10.! Michael!Wollenberg!! Propionibacterium&acnes7produced!porphyrins!induce!Staphylococcus&aureus&aggregation!!Wollenberg,!M.S.,!Claesen,!J.,!Fernandez9Escapa,!I.,!Fischbach,!M.A.,!and!K.P.!Lemon!!!

11.!!!

Wolfram!Möbius! Range!expansions!of!microorganisms!in!structured!environments!Wolfram!Möbius,!Andrew!W.!Murray,!and!David!R.!Nelson!!

!!!!


10

Saturday!|!Session!III,!9:30!am!to!10:50!am!!12.! Kristina!Jonas! Proteotoxic!stress!induces!cell!cycle!arrest!by!triggering!the!degradation!of!the!

replication!initiator!DnaA!!Kristina!Jonas,!Jing!Liu,!Peter!Chien,!and!Michael!Laub!!!

13.! Matthew!!Cabeen! Cooperation!and!virulence!factor!production!by!a!quorum7sensing!mutant!of!a!pathogenic!bacterium!!Matthew!T!Cabeen!and!Richard!Losick!!!

14.! Xavier!Meniche!! The!Mycobacterial!growth!organelle!is!positioned!by!DivIVA!at!a!sub!polar!site!!Meniche!X,!Siegrist!MS,!Bertozzi!CR,!and!Sassetti!CM!!!

15.! Pushkar!Lele! Dynamics!of!mechanosensing!in!the!bacterial!flagellar!motor!!Pushkar!P.!Lele,!Basarab!G.!Hosu,!and!Howard!C.!Berg

!

!!!Saturday!|!Session!IV,!11:20!am!to!1!pm!!16.! Marina!

Besprozvannaya!SpoIIIE!achieves!directional!DNA!translocation!through!allosteric!regulation!of!ATPase!activity!by!an!accessory!domain!!Marina!Besprozvannaya!and!Briana!M.!Burton!!

17.! Burak!Okumus! MACS:!microfluidics7assisted!cell!screening!!Burak!Okumus,!Raul!Fernandez9Lopez,!Dirk!Landgraf

!,!Dann!Huh,!Sadik!Yildiz,!Erdal!Toprak!

and!Johan!Paulsson!!!

18.! Lia!Cardarelli! Self!recognition!in!Proteus&mirabilis!is!minimally!mediated!by!a!single!protein7protein!interaction!!Lia!Cardarelli,!Christina!Saak,!and!Karine!A.!Gibbs!!

19.! Naveen!Sinha! A!whole7colony!technique!to!study!spatiotemporal!patterns!of!cellular!differentiation!in!Bacillus&subtilis!biofilms!!Naveen!N!Sinha,!James!N!Wilking,!Agnese!Seminara,!David!A!Weitz,!and!Michael!P!Brenner!!!

20.! Antonio!Gomes! Optimal!time7scheduled!therapy!effectively!fights!antibiotic!resistance!by!exploiting!the!cost!of!resistance!!Antonio!LC!Gomes,!James!E!Galagan,!and!Daniel!Segre!!

!!!Saturday!|!Session!V,!2!pm!to!3:40!pm!!21.! Elizabeth!Mearls!! The!identification!of!several!histidine!kinases!involved!in!the!metabolic!shut7down!of!

Clostridium&thermocellum!!Elizabeth!B.!Mearls,!Javier!A.!Izquierdo,!and!Lee!R.!Lynd!!!

22.! Matthew!Ramsey! Novel!pheromone7mediated!sensitivity!accounts!for!inhibition!of!vancomycin7resistant!Enterococcus&faecalis!by!commensal!enterococci!!Matthew!M.!Ramsey,!Lynn!Hancock,!Marcus!Rauch,!Paul!Himes,!and!Michael!Gilmore!!


11

!!23.!

!Pete!Chandrangsu!

!Methylglyoxal!resistance!in!Bacillus&subtilis:!contributions!of!bacillithiol7dependent!and!7independent!pathways!!Pete!Chandrangsu!and!John!D.!Helmann!!!

24.!!

Henry!Haiser! Predicting!and!manipulating!drug!inactivation!by!the!human!gut!microbiome!!Henry!J.!Haiser,!David!B.!Gootenberg,!Kelly!Chatman,!Gopal!Sirasani,!Emily!P.!Balskus,!and!Peter!J.!Turnbaugh!!

25.! Patrick!H.!Degnan! Genetic!redundancy!and!specificity!in!the!human!gut!microbiome!!Patrick!H.!Degnan,!Natasha!Barry,!Kenny!Mok,!Michiko!E.!Taga!and!Andrew!L.!Goodman!

2013!Boston!Bacterial!Meeting!7!Oral!Presentation!Abstracts!

12

1! !Use!of!a!high!throughput!genetic!selection!to!identify!regulators!of!the!Vibrio&cholerae&infection7induced!gene,!xds&!!

EmilyKate!McDonough1,!David!Lazinski

1,!and!Andrew!Camilli

1!!

1Howard!Hughes!Medical!Institute!and!Department!of!Molecular!Biology!and!Microbiology,!Tufts!University!School!of!Medicine!!!As!a!waterborne!human!pathogen,!V.&cholerae!undergoes!adaptive!shifts!in!gene!expression!throughout!the!different!stages!of!its!life!cycle.!Recently,!our!laboratory!identified!57!V.&cholerae&genes!that!are!induced!near!the!end!of!the!infection!cycle.!Several!of!these! ‘late’! genes! were! shown! to! prepare! the! bacteria! for! transition! to! the! aquatic! environment,! rather! than! contributing! to!colonization!or!virulence!of!the!pathogen.!One! late!gene,!xds,! is!a!secreted!exonuclease!that!modulates!biofilm!growth.!To! learn!more! about! the! signals! that! trigger! xds! expression! during! infection,! we! implemented! a! genetic! selection! coupled! with! high!throughput!DNA! sequencing! to! identify! regulators!of! this! late! gene.!We! identified! the!phosphate9starvation! response! regulator,!PhoB,!as!an!activator!of!xds.!We!confirmed!PhoB!as!a!positive!regulator!of!xds!both!in&vitro!and!in&vivo,!i.e.,!during!infection.!We!are!currently!working!on!defining!the! interaction!between!PhoB!and!the!xds&promoter.!Our!work! indicates!that!xds! is! induced!under!phosphate9limiting!conditions,!such!as!during!the!late!stage!of!infection!of!the!small!intestine.!Additionally,!we!hypothesize,!based!on!this!and!other!work,!that!xds!may!play!an!important!role!in!the!utilization!of!DNA!as!a!phosphate!source.!!!

2! ! A!global!view!of!the!taxonomic!and!functional!diversity!of!cheese!rind!microbial!communities!

!

Benjamin!E.!Wolfe1,!Julie!E.!Button

1,!and!Rachel!J.!Dutton

1!!

1FAS!Center!for!Systems!Biology,!Harvard!University!!!Although!humans!have!been!using!microbes!to!make!cheese!for!thousands!of!years,!we!know!little!about!the!diversity!of!cheese!microbial!communities!and!the!factors!that!shape!this!diversity.!Microbes!colonize!the!surface!of!naturally!aged!cheeses!and!form!multi9species!biofilms!(rinds)!that!contribute!to!flavor!development.!We!used!amplicon!and!metagenomic!sequencing!to!assess!the!taxonomic!and!functional!diversity!of!rind!microbial!communities!from136!artisan!cheeses!made!across!the!Northern!Hemisphere.!We! sampled! rinds! that!were!washed! (washed),! inoculated!with!filamentous! fungi! (bloomy),!or! left!unperturbed! (natural)!during!aging.!!Taxonomic!diversity!of! cheese! rind!microbial! communities! is! low! (~10925! taxa!per! cheese)!and! is! largely! structured!by! rind! type!(washed,!bloomy!or!natural)!not!geography,!source!of!milk!(cow,!goat,!or!sheep),!or!treatment!of!milk!(raw!or!pasteurized).!Genes!associated!with!flavor!development!are!unevenly!distributed!across! the!three!rind!types,! indicating! functional!divergence!due!to!aging!techniques.!When!we!mimic!aging!techniques!in!the!lab,!we!can!experimentally!create!three!distinct!rind!communities!from!a!common!pool!of!species.!Cheese!rind!microbial!communities!are!a!simple!and!tractable!system!that!will!provide!new!opportunities!to!discover!the!fundamental!drivers!of!microbial!community!processes.!!!

3! ! A!post7translational!feed7back!loop!by!a!tyrosine!kinase!regulates!exopolysaccharide!production!in!Bacillus&subtilis!

!Alexander!Elsholz1,!Richard!Losick1!1Molecular!and!Cellular!Biology,!Harvard!University!!Exopolysaccharides! are! a! common! feature! of! the! self9produced! extracellular! matrix! that! holds! cells! together! in! structured!communities!called!biofilms.! In!many!bacteria,! tyrosine!kinases!are! involved! in!controlling! the!production!of!exopolysaccharides,!but!the!signal!that!regulates!this!family!of!kinases!remains!elusive.!We!found!that!in!Bacillus!subtilis!a!tyrosine!kinase!mediates!a!post9translational,!feed9back!loop!that!controls!the!production!of!exopolysaccharide.!Evidence!supports!the!following!positive!and!negative!feedback!model!in!which!the!signal!is!exopolysaccharide!itself.!The!activity!of!the!kinase!depends!on!a!membrane9bound!receptor! protein,!which! regulates! the! kinase! in! response! to! the! levels! of! exopolysaccharide!outside! of! the! cell.! At! low! levels! of!exopolysaccharide,! the! kinase! is! stimulated! and! phosphorylates! at! least! one! of! the! biosynthetic! enzymes! involved! in!exopolysaccharide!production,!enhancing!its!activity.!When!the!level!of!exopolysaccharide!reaches!a!threshold!level,!the!kinase!is!inactivated!by!autophosphorylation,!shutting!down!exopolysaccharide!production.!Thus!exopolysaccharide!production!is!controlled!homeostatically!by!a!regulatory!tyrosine!kinase!whose!activity!depends!on!exopolysaccharide!levels.!!!


13

4! ! Pathogen!genetic!variation!within!single!clinical!samples!provides!a!signature!of!selective!pressures!

!

Tami!D.!Lieberman1,!Kelly!B.!Flett

2,!Idan!Yelin

3,!Thomas!R.!Martin

4,!Alexander!J.!McAdam

5,Gregory!P.!Priebe

2,6,!&!Roy!Kishony

1,7!!1

Department!of!Systems!Biology,!Harvard!Medical!School,!2Division!of!Infectious!Diseases,!Boston!Children’s!Hospital!and!Harvard!

Medical! School,!3Faculty! of! Biology,! Technion9Israel! Institute! of! Technology,

! 4Division! of! Respiratory!Diseases,! Boston! Children’s!

Hospital!and!Harvard!Medical!School,!5Department!of!Laboratory!Medicine,!Boston!Children’s!Hospital!and!Harvard!Medical!School,!6

Division! of! Critical! Care!Medicine,! Boston! Children’s! Hospital! and! Harvard!Medical! School,!7School! of! Engineering! and! Applied!

Sciences,!Harvard!University.!!!Bacterial!pathogens!acquire!mutations!during! infection!as!they!adapt!to!challenges!posed!by!the!environment!within!the!human!host.! Recent! studies! tracking! bacterial! evolution! within! individual! patients! have! identified! mutations! responsible! for! antibiotic!resistance,!the!rate!at!which!mutations!accumulate,!and!specific!bacterial!genes!and!pathways!that!are!under!selective!pressure.!Such! studies! typically! use! sequencing! of! a! single! colony! isolate! from! each! clinical! sample,! giving! only! a! limited! picture! of! the!diversity!of!the!pathogen!population!at!the!time!of!sampling.!Here,!using!colony!re9sequencing!and!deep!population!sequencing!of!Burkholderia&dolosa& from!people!with! cystic!fibrosis,!we! identify!extensive! intrastrain!genomic!diversity!within! individual! clinical!samples.!We!find!that!mutations!rarely!fix!within!a!patient's!pathogen!population—instead,!diversifying!lineages!coexist!for!many!years.!When!strong!selection! is!acting!on!a!gene,!multiple!adaptive!mutations!arise!yet!coexist,!generating! lasting!allele!diversity!that! provides! a! signature! of! past! selection.! In! the! pathogen! populations! we! studied,! genes! involved! in! outer9membrane!components,! iron! scavenging!and!antibiotic! resistance!all! showed! signs!of!within9patient! selection.!These! results!offer!a!general!approach!for!diagnosing!selective!pressures!acting!on!a!pathogen!within!the!human!host!using!single!clinical!samples.!!!

5! ! Emergence!of!epidemic!multi7drug!resistant!Enterococcus&faecium!from!animal!and!commensal!strains!

!

François!Lebreton*1,2,!Willem!van!Schaik*

1,2,3,!Abigail!McGuire*

2,!Jennifer!Wortman

2,!Bruce!Birren

2,!Ashlee!Earl

2,!Michael!S.!

Gilmore1,2!!

*Equal!contributions.!1)!Departments!of!Ophthalmology,!Microbiology!and!Immunobiology,!Harvard!Medical!School,!Boston.!2)!The!Broad!Institute,!Cambridge.!3)!Department!of!Microbiology,!University!Medical!Center!Utrecht,!The!Netherlands.!!!The!enterococci!emerged!as!leading!causes!of!multidrug!resistant!(MDR)!nosocomial!infection!in!the!1980’s,!and!are!now!common!causes! of! outbreaks.!We! examined! the! genomes! of! a! collection! of! E.& faecium! strains! that! span! the! breadth! of! the! species! to!understand!the!scope!of!diversity!of!the!species,!as!well!as!the!relationship!between!epidemic/MDR!isolates,!isolates!from!animals!and!human!commensal!strains.!We!were!able!to!distinguish!the!lineage!responsible!for!MDR!human!infection,!and!to!measure!the!evolutionary!distances!from!commensal!human!and!animal!flora.!The!MDR!lineage!emerged!about!75!years!ago!concomitant!with!the! introduction! of! antibiotics.! Examining! gene! enrichment! patterns! and! other! evolutionary! drivers,! we! identified! factors! that!contributed!to!the!emergence!of!the!observed!lineages.!These!results!show!the!impact!of!antibiotic!use!on!the!outgrowth!of!MDR!E.&faecium!in!the!antibiotic!era.!!!

6! ! Using!multiplex!DNA!synthesis!to!study!transcription!and!translation!in!E.&coli!

!!Daniel!B.!Goodman

1,2,3,!Sriram!Kosuri

2,3,!and!George!M.!Church

2,3!!

1Program!in!Bioinformatics!and!Integrative!Genomics,!Harvard9MIT!Division!of!Health!Science!and!Technology!

2Wyss! Institute!for!

Biologically!Inspired!Engineering,!Harvard!University,!3Department!of!Genetics,!Harvard!Medical!School!!

!The!unpredictability! of! gene!expression! from!a! given!DNA! sequence! limits! our! understanding!of! bacterial! systems!and!makes! it!difficult!to!reliably!engineer!them.!The!small!number!of!natural!sequences!available!is!insufficient!for!a!complete!understanding!of!this!complex!process.!To!address!this!problem,!we!synthesized!27,000!combinations!of!common!promoters,!ribosome!binding!sites,!and!N9terminal!coding!sequences!in!E.!coli!(over!5!megabases!of!unique!nonrandom!sequence)!and!simultaneously!measured!DNA,!RNA,!and!protein! levels! from!the!entire! library.!Using!a! simple!additive!model,!we! found! that!RNA!and!protein!expression!were!within!2x!of!predicted!levels!80%!and!64%!of!the!time!respectively.!This!large!dataset!also!allowed!for!quantitation!of!global!effects,!including! how! GC! content! and! secondary! structure! affect! translation! efficiency! and! how! translation! rate! alters!mRNA! stability.!Perhaps!most! surprisingly,! we! found! that! the! use! of! rare! codons! at! the! N9terminus! strongly! increases! expression! compared! to!common!codons.! Finally,! this! approach! suggests! the!possibility!of! studying!natural! and! synthetic! gene! regulation!and! regulatory!networks!an!unprecedented!scale.!!


14

7! ! A!molecular!mechanism!for!formaldehyde!detoxification!and!growth!in!Methylobacterium!

!

Dipti!D.!Nayak1,!Milya!Davlieva

2,!Yousif!Shamoo

2,3!and!Christopher!J.!Marx

1,4!!

1Department! of! Organismic! and! Evolutionary! Biology,! Harvard! University,! Cambridge! MA,!

2Department! of! Biochemistry,! Rice!

University,!Houston!TX.!2Department!of!Ecology!and!Evolutionary!Biology,!Rice!University,!Houston!TX.

!4Faculty!of!Arts!and!Sciences!

Center!for!Systems!Biology,!Harvard!University,!Cambridge!MA.!!!Members!of!the!Methylobacterium&genus!are!facultative!methylotrophs;!they!grow!on!reduced!single!carbon!compounds!such!as!methanol.!During!methylotrophic!metabolism!carbon! is!oxidized! to! formaldehyde!prior! to!assimilation!and! further!dissimilation.!However,!paradoxically,!these!bacteria!can’t!grow!on!formaldehyde.!In!order!to!understand!the!molecular!basis!of!this!paradox!we!experimentally!evolved!populations!of!Methylobacterium&extorquens!PA1! to!grow!on!20!mM!formaldehyde.!Sequencing!evolved!isolates!revealed!mutations!in!two!genes!across!replicate!populations.!The!first!gene,!efgA,!encodes!a!protein!that!has!a!Domain!of!Unknown! Function! Type! 336.! Phylogenetic! analysis! of! EfgA! revealed! an! exclusive! association! with! methylotrophs! across! the!bacterial!domain.!Genetic!analysis!of!EfgA!revealed,!almost!counter!intuitively,!that!loss!of!function!mutations!in!this!protein!result!in!formaldehyde!growth.!The!crystal!structure!of!EfgA!revealed!that!it!plays!a!regulatory!role!and!is!potentially!involved!in!a!signal9transduction! cascade! responding! to! formaldehyde! concentrations! in! the! cell.! The! second! protein,! EfgB,! is! a! putative!adenylate/guanylate! cyclase! that,! based! on! preliminary! results,! regulates! a! novel! stress! response! mechanism! in! response! to!aldehydes.!By!using!experimental!evolution!as!a!genetic!tool!we!have!uncovered!a!molecular!mechanism!for!formaldehyde!growth!and!detoxification!in!Methylobacterium.!!!!

8! ! Practical!and!functional!implications!of!siRNA7like!small!RNAs!in!bacteria!

!Linfeng!Huang

1,!Padraig!Deighan

2,3,!Jingmin!Jin

4,!Elaine!Lee

1,!Larry!McReynolds

4,!Ann!Hochschild

2,!Judy!Lieberman

1!!

1Program! in!Cellular!and!Molecular!Medicine,!Boston!Children's!Hospital,!and!Department!of!Pediatrics,!Harvard!Medical!School,!Boston! MA

2!Department! of! Microbiology! and! Immunobiology,! Harvard! Medical! School,! Boston! MA

3!Department! of! Biology,!

Emmanuel!College,!Boston!MA4!New!England!Biolabs,!Ipswich!MA!USA!!

!RNA! interference!by!double9stranded! small! interfering!RNAs! (siRNA)! suppresses!gene!expression!by! inducing! the!degradation!of!mRNAs!bearing!complementarity.!Although!siRNAs!are!considered!to!be!specific!to!eukaryotes,!we!discovered!unexpectedly!that!ectopic! expression! of! p19,! a! plant! viral! siRNA9binding! protein,! stabilized! a! cryptic! siRNA9like! species! in! both! Gram9positive! and!negative!bacteria.!These!siRNAs,!which!we!named!pro9siRNA!for!“prokaryotic!siRNA”,!are!RNase!III9dependent!products!and!have!the! same! chemical! characteristics! as! eukaryotic! siRNAs.! Following! this! discovery,! we! developed! a! method! of! producing! and!purifying!recombinant!pro9siRNAs!from!E.&coli!cells!that!efficiently!and!specifically!knock!down!target!genes!in!mammalian!cells.!In!addition! to! this! practical! application! for! pro9siRNAs,! wefound! that! p19! expression! stabilized! endogenous! bacterial! pro9siRNAs.!These!were!generated!from!RNase!III9dependent!processing!of!overlapping!sense!and!antisense!transcripts.!Most!bacterial!genes!had!representative!pro9siRNAs,!indicating!that!most!bacterial!genes!have!at!least!partially!overlapping!antisense!transcripts.!Many!of!these!antisense!transcripts!were!previously!uncharacterized!because!they!are!short9lived!in!strains!with!active!RNase!III.!Our!data!raise!the!possibility!that!RNase!III9dependent!regulation!of!mRNAs!by!antisense!RNAs!is!widespread!across!the!bacterial!genome.!!!

9! ! Invading!the!privacy!of!Enterococcal!invaders!

!Morales!DK.1,!Chai!L.1,!Connery,!A.,2!Ramsey!M.3,!!Ausubel,!F.,2!Gilmore,!M.3,!and!Kolter!R.1!1Department! of! Microbiology! and! Immunobiology,! Harvard! Medical! School,! 2Department! of! Molecular! Microbiology,!Massachusetts! General! Hospital! and! Department! of! Genetics,! Harvard! Medical! School.! 3Department! of! Ophthalmology! and!Department!of!Microbiology!and!Immunobiology,!Harvard!Medical!School.!!!Enterococcus& faecalis& is! a! non9motile! opportunistic! pathogen! that! normally! resides! in! the! gastrointestinal! tract! of! humans.!Increasing! interest! in! enterococcal! virulence!has!been!prompted!by! concerns! that! the!majority! of! strains! can! resist!most!of! the!currently!available!antimicrobial!agents.!Improvement!in!treating!serious!enterococcal!infections!will!likely!depend!upon!a!clearer!understanding!of!virulence!traits!and!host9pathogen!interactions.!E.&faecalis&can!adhere!to!and!invade!human!tissues,!a!trait!that!is!important! for! colonizing! human! sites! different! that! the! gastrointestinal! tract.!We! have! found! that!E.& faecalis! is! also! capable! of!invading! agar! surfaces.! This! phenomenon! is! controlled! by! the! composition! of! its! growth! environment;! the! presence! of!carbohydrates,!such!as!glucose,!prevented!this!invasive!behavior.!!We!screened!an!E.&faecalis!transposon!mutant!library!for!strains!


15

that!were!incapable!of!invading!agar.!!By!analyzing!mutants!defective!in!agar!invasion,!we!found!that!invasion!and!cell!aggregation!were!promoted!by! the!production!of!an!extracellular!polysaccharide!derived! from!N9acetyl9glucosamine.! !Additionally,!we! found!that!invasion!could!be!controlled!by!an!“inversion!mediated”!phase!variation!system,!which!has!not!been!previously!described!in!this!organism.!Finally,!consistent!with!our!hypothesis!that!invasiveness!is!required!for!virulence,!mutants!defective!in!invasion!were!also!defective!in!killing!the!model!host!organism!Caenorhabditis&elegans.!Our!studies!indicate!that!this!novel!invasive!behavior!is!a!useful!tool!to!identify!new!virulence!traits!in!E.&faecalis.!!!

10! ! Propionibacterium&acnes7produced!porphyrins!induce!Staphylococcus&aureus&aggregation!

!

Wollenberg,!M.S.1,!Claesen,!J.

2,!Fernandez9Escapa,!I.

1,!Fischbach,!M.A.

2,!and!K.P.!Lemon

1,3!!

1Department!of!Microbiology,!Forsyth!Institute;!

2Department!of!Bioengineering!and!TherapeuticSciences,!University!of!California,!

San!Francisco;!3Division!of!Infectious!Diseases,!Boston!Children’s!Hospital!

!Propionibacterium!and!Staphylococcus!are!two!of!the!most!common!bacterial!genera!detected! in!the!microbiota!of!adult!human!nostrils.&Our!lab!has!observed!that!nostril!Propionibacterium&produce!diffusible!substances!affecting!the!growth!of!Staphylococcus&aureus.&We!have!identified!coproporphyrin!III!as!a!P.&acnes9produced!substance!that!affects!S.&aureus&aggregation.!!The!cell9free!supernatant!of!a!P.&acnes&culture!was! fractionated;!each!fraction!was!tested!for!S.&aureus&aggregation!activity.!One!inducing!fraction!was!visibly!orange/brown!and!strongly!absorbant!at!400!nanometers.!HPLC!and!LC/mass!spectrometry!analyses!confirmed!that!this!fraction!contained!the!porphyrin!derivatives!coproporphyin!I!(CI)!and!coproporphyrin!III!(CIII).!!In&vitro,!both!CI!and!CIII!caused!S.&aureus&aggregation,!however,!only!coproporphyrin!III!caused!S.&aureus&aggregation!under!iron9limited! culture! conditions.!Novel,! affinity9based!purification!of!S.& aureus&proteins! aggregating!with!CIII!was!used! to! identify!CIII9associated! proteins.! These! proteins! include! the! well9known,! iron9regulated,! heme9binding! protein! IsdA! and! an! unknown,!hypothetical!protein!that!is!highly!conserved!among!the!Staphyloccocus&genus.!!This!work!addresses!a!long9standing!mystery!–!Why&do&Propionibacterium&spp.&producerelatively&large&extracellular&amounts&of&the&

porphyrin&coproporphyrin& III,&which& is&considered&ametabolic&“deadBend”?!We!speculate!that!the!answer!might!be!an!example!of!either!interspecific!competition!for!iron!or!interspecific!cooperation!for!biofilm!formation!among!the!nostril!microbiota.!!!

11! ! Range!expansions!of!microorganisms!in!structured!environments!

!

Wolfram!Möbius1,!Andrew!W.!Murray

2,!and!David!R.!Nelson

1!!

1Department!of!Physics!and!FAS!Center!for!Systems!Biology,!Harvard!University,!

2FAS!Center!for!Systems!Biology!and!Department!

of!Molecular!and!Cellular!Biology,!Harvard!University!!!We!employ!both!experimental!and!theoretical!methods!to!understand!how!species!invade!structured!environments!and!how!such!range! expansions! affect! their! genetic! diversity.!Wethereby! focus! on! continuous! environments! with! finite9sized! regions! of! poor!growth!conditions.!In!particular,!we!study!an!experimental!model!system,!in!which!a!population!of!bacteriophage!T7!spreads!on!a!lawn!of! susceptible!E.&coli!bacteria!while!a! region!of! resistant!bacteria,! i.e.,!a! region!with!poor!growth!conditions! for! the!phage,!poses!an!obstacle! to!growth!of! the!plaque.!We!use!an! inkjet!printing!assay! to!create! single! regions!of! resistant!bacteria!around!which! the! plaque! grows! and! examine! the! plaque! dynamics! quantitatively.! Reaction9diffusion! modeling! and! a! coarse9grained!description! allow! us! to! understand! the! dynamics! in! such! simplified! geometries.! We! predict! that! during! the! encounter! of! an!obstacle,!alleles!present! in!the!expanding!population!are!driven!to!extinction,!are!unaffected,!or!even!sweep!through!part!of!the!population,!depending!on! their! spatial! distribution! relative! to! the! region!of!poor! growth!and!purely! for! geometric! reasons.! This!effect!of! ‘selection!by!geometry’! is! confirmed!using! stochastic! simulations!and!a! range!expansion!of!fluorescently! labeled!E.& coli!strains.!!!!!!!!!


16

12! ! Proteotoxic!stress!induces!a!cell!cycle!arrest!by!triggering!the!degradation!of!the!replication!initiator!DnaA!

!

Kristina!Jonas1,!Jing!Liu

3,!Peter!Chien

3,!and!Michael!Laub

1,2!!

1Department! of! Biology! and!

2Howard! Hughes! Medical! Institute,! Massachusetts! Institute! of! Technology,!

3Department! of!

Biochemistry!and!Molecular!Biology,!University!of!Massachusetts!Amherst!!!The!decision!to!initiate!DNA!replication!is!a!critical!step!in!the!cell!cycle!of!all!organisms.!Cells!often!delay!replication!in!the!face!of!stressful!conditions,!but!the!underlying!mechanisms!remain!incompletely!defined.!Here,!we!demonstrate!in!Caulobacter&crescentus!that!proteotoxic!stress!induces!a!cell!cycle!arrest!by!triggering!the!degradation!of!DnaA,!the!conserved!replication!initiator.!We!find!that!the!Hsp70/DnaK!chaperone,!a!key!player!in!protein!folding!and!the!regulation!of!the!heat!shock!response,!is!required!for!DNA!replication!and!the!accumulation!of!stable!DnaA.!A!depletion!of!available!DnaK,!either!through!genetic!manipulation!or!heat!shock,!induces!synthesis!of!the!Lon!protease,!which!can!directly!degrade!DnaA.!Moreover,!our!data!suggest!that!unfolded!proteins,!which!accumulate! following! a! loss! of! DnaK! or! under! various! stress! conditions,! allosterically! activate! Lon! to! degrade! DnaA,! thereby!ensuring! cell! cycle! arrest.! Our! work! reveals! a! new! cellular! mechanism! for! regulating! DNA! replication! under! adverse! growth!conditions.!Additionally,!our!data! indicate! that!unfolded!proteins! can!actively!and!directly! alter! substrate! recognition!by! cellular!proteases.!!!

13! ! Cooperation!and!virulence!factor!production!by!a!quorum7sensing!mutant!of!a!pathogenic!bacterium!

!Matthew!T!Cabeen!and!Richard!Losick!!Molecular!and!Cellular!Biology,!Harvard!University!!!Many!important!virulence!factors!of!the!human!pathogen!Pseudomonas&aeruginosa!are!under!quorum9sensing!control,!making!it!paradoxical! that!quorum9sensing9defective! lasR!mutants!commonly!emerge! in!chronic! infections!and!are!associated!with!disease!progression.!One!explanation! is! that! lasR&mutants!gain!a!growth!advantage!by!social!cheating.!Here,!we!show!that! lasR!mutants!have!a!delayed!but!hyperactive!quorum9sensing!response!that!causes!strong!virulence!factor!overproduction.!We!provide!evidence!that!the!absence!of!a!negative!regulator!in!lasR!mutant!cells!permits!a!subset!of!quorum9regulated!genes,!including!those!encoding!important!virulence!factors,!to!be!overexpressed.!In!mixed!cultures!of!lasR!and!wild9type!cells,!lasR!cells!retain!their!overproduction!phenotype!and!cooperate!with!wild9type!cells!to!yield!novel!hyper9rugose!biofilm!colony!morphology.!We!further!show!that! lasR!cells!eventually!cooperate!with!respect!to!virulence!factor!production!even!under!conditions!typically!used!to!enforce!cheating.!Our!results!imply!that!lasR!populations!are!hyper9virulent,!explaining!their!clinical!prevalence.!We!speculate!that!the!lasR!mutation!is!an!adaptive!mechanism!by!which!P.&aeruginosa!can!enact!altered!quorum!sensing!and!virulence!behavior.!!!

14! ! The!Mycobacterial!growth!organelle!is!positioned!by!DivIVA!at!a!sub!polar!site!

!Meniche!X

1,!Siegrist!MS

2,!Bertozzi!CR

2,!and!Sassetti!CM

1!!

1Department! of! Microbiology! and! Physiological! Systems,! UMASS! Medical! School,!

2Department! of! Chemistry,! University! of!

California,!Berkeley!!While!many!bacilli!elongate!via!lateral!cell!wall!deposition,!elongation!of!the!mycobacterial!cell!is!largely!restricted!to!the!old!pole.!The!mycobacterial!cell!wall!is!a!complex!multilayered!structure!composed!of!peptidoglycan,!arabinogalactan!and!mycolic!acids.!We!investigated!the!ultrastructure!and!organizing!principles!underlying!the!polar!growth!complex! in!Mycobacteria.!Terminal!cell!wall!synthetic!enzymes!were!found!at!the!cell!pole!and!in!dynamic!foci!along!thelateral!cell!wall!body.!However,!the!proteins!were!only!co9localized! at! the! pole,! consistent! with! acoordinated! function! in! cell! elongation! only! at! this! site.! CoIP! experiments! defined!interactions!between!the!ACC!complex!required!for!mycolic!acids!precursor!synthesis!and!the!intermediate!filament,!DivIVA.!Time9lapse!microscopy! demonstrated! that!DivIVA! is! enriched! at! the! growing! pole,! and!depletion! of! the! protein! formed! spherical! cell!shapes.!We! propose! that!mycobacterial! DivIVA! participates! in! organizing! the! growth! organelle! by! recruiting! cell! wall! synthetic!proteins!to!the!growth!site.!Further!investigation!of!the!growth!pole!by!colocalization!experiments!revealed!that!DivIVA!marks!the!tip! of! the! cell,! while! terminal! cell! wall! synthetic! enzymes! localize! to! a! distinct! sub9polar! space.! Bioorthogonal! labeling! of! PG!precursors!verified!that!nascent!cell!wall!is!deposited!at!this!sub9polar!site.!!!


17

15! ! Dynamics!of!mechanosensing!in!the!bacterial!flagellar!motor!

!

Pushkar!P.!Lele!1,!Basarab!G.!Hosu

!1,!and!Howard!C.!Berg

!!

1Department!of!Molecular!and!Cellular!Biology,!Harvard!University,!Cambridge!MA!02138.!!!Mechanosensing!by!flagella!is!thought!to!trigger!bacterial!swarmer9cell!differentiation,!an!important!step!in!pathogenesis.!How!flagellar!motors!sense!mechanical!stimuli!is!not!known.!To!study!this!problem,!we!suddenly!increased!the!viscous!drag!on!motors!by!a!large!factor,!from!very!low!loads!experienced!by!motors!driving!hooks!with!short!filament!stubs,!to!high!loads,!experienced!by!motors!driving!19μm!latex!beads.!From!the!initial!speed!(after!the!load!change),!we!inferred!that!motors!running!at!low!loads!are!driven!by!1!or!at!most!2!force9generating!units.!Following!the!load!change,!motors!gradually!adapted!by!increasing!their!speeds!in!a!stepwise!manner!(in!a!few!minutes).!Single9motor!TIRF!imaging!of!YFP9MotB!(part!of!a!stator!force9generating!unit)!confirmed!that!the!response!to!sudden!increments!in!load!occurred!by!the!addition!of!new!force9generating!units.!We!estimate!that!6911!force9generating!units!drive!motors!at!high!loads.!Wild9type!motors!and!motors!locked!in!the!clockwise!or!counter9clockwise!state!behaved!in!a!similar!manner,!as!did!motors!in!cells!deleted!for!flta!or!for!genes!in!the!chemotaxis!signaling!pathway.!Thus,!the!stators!themselves!probably!act!as!dynamic!mechanosensors. How!such!changes!might!impact!cell!development!remains!an!interesting!question.!!!

16! ! SpoIIIE!achieves!directional!DNA!translocation!through!allosteric!regulation!of!ATPase!activity!by!an!accessory!domain!!Marina!Besprozvannaya,!and!Briana!M.!Burton!

Molecular!and!Cellular!Biology,!Harvard!University!!!Bacterial! chromosome! segregation! utilizes! highly! conserved! directional! translocases! of! the! SpoIIIE/FtsK! family.! These! proteins!employ! an! accessory! DNA9binding! domain! (gamma)! to! dictate! directionality! of! DNA! transport.! It! remains! unclear! how! the!interaction!of!gamma!with!specific!recognition!sequences!coordinates!directional!DNA!translocation.!Here!we!demonstrate!that!the!gamma!domain!of!SpoIIIE!inhibits!ATPase!activity!in!the!absence!of!DNA,!but!stimulates!ATPase!activity!through!sequence9specific!DNA!recognition.!Furthermore,!maintaining!the!integrity!of!gamma9gamma!inter9subunit!interactions!is!necessary!for!appropriate!sequence! recognition! and! inhibition! of! ATP! turnover! in! the! absence! of! substrate.! Consistent! with! these! findings,! the! gamma!domain!is!necessary!for!maintaining!rapid!DNA!transport!along!the!length!of!the!chromosome! in&vivo.!Together!our!data!provide!the! molecular! mechanism! behind! the! critical! step! of! directional! activation! that! is! coordinated! by! allosteric! regulation! of! ATP!turnover! by! the! gamma! domain.! Thus,! we! propose! a! coordinated! stimulation! model! in! which! the! gamma! domain! translates!substrate!DNA!sequence!information!to!the!motor!domain.!!!

17! ! MACS:!microfluidics7assisted!cell!screening!

!

Burak!Okumus1,!Raul!Fernandez9Lopez

2,!Dirk!Landgraf

!3,!Dann!Huh

4,!Sadik!Yildiz

5,!Erdal!Toprak

6!and!Johan!Paulsson

7!!

1,2Department! of! Systems! Biology,! Harvard!Medical! School;!

3Whitehead! Institute! for! Biomedical! Research,!MIT;!

4FAS! Center! for!

Systems! Biology,! Harvard!University;!5,6Faculty! of! Engineering! and!Natural! Sciences,! Sabanci! University,! Turkey;!

7Department! of!

Systems!Biology,!Harvard!Medical!School!!!We! developed! a! microfluidic! technique! so9called! MicrofluidicsBAssisted& Cell& Screening! (MACS),! which! can! be! used! for:! 1)!Automated,! high7throughput! imaging.! By!means! of! a! growth! chamber! upstream! of!MACS,! we! could!monitor! the! density! of! a!growing!Escherichia&coli!culture!in!real9time!within!a!temperature9controlled!incubator!while!taking!images!of!(total!of!~!0.1!million)!single!cells!in!a!fully!hands9off!fashion!with!minimal!user!intervention.!Our!results!capture!the!progression!of!population!growth!at!the! single9cell! level! with! high! temporal/spatial! resolution,! and! ample! statistics! simultaneously! on! cell9size! and! chromosome9replication!activity!along!the!growth!curve.!2)!In>vivo&single7molecule!counting.!Under!certain!parameters,!MACS!can!greatly!slow9down!the!diffusion!of!cytoplasmic!molecules!in!E.&coli,!which!enables!digital!counting!of!low9abundance!proteins!via!standard!total9internal9reflection9fluorescence! (TIRF)! microscopy.! 3)! Capturing! rare! events.! Operating! MACS! in! a! mode! where! cells! flow!continuously!as!a!single!layer!through!the!field!of!view!enables!sifting!through!millions!of!cells!per!hour.!On9the9fly!image!analysis!then!allows! for!acquiring!a!detailed! snapshot!of! the! cell! of! interest.! Finally,!MACS!holds! the!potential! to! lend! itself! for! studying!other! cell! types!with! unprecedented! capabilities! such! as! automated! screening! of! libraries! and! cell! populations! for! rare! events,!protein!abundance!quantification!with!single9molecule!accuracy,!and!eventually!single9cell!imaging9based!sorting.!


18

18! ! Self!recognition!in!Proteus&mirabilis!is!minimally!mediated!by!a!single!protein7protein!interaction!!!Lia!Cardarelli,!Christina!Saak,!and!Karine!A.!Gibbs!Molecular!and!Cellular!Biology,!Harvard!University!!!Distinguishing!between!“self”!and!“nonself”! is!a!phenomenon! likely! involved! in!kin!selection.!We!previously! identified!a!six9gene!operon,!ids,!which!is!required!for!the!identification!of!self!and!the!territorial!exclusion!of!nonself!in!the!pathogen!Proteus&mirabilis.!Particularly!interesting!are!genes!idsD!and!idsE,!which!encode!the!genetic!determinants!of!self.!We!hypothesized!that!short,!strain9specific!sequences!within!IdsD!and!IdsE!are!together!sufficient!to!define!self!and!that!a!specific!interaction!between!these!regions!mediates! self! recognition,! similar! to! antibody9antigen! interactions.! Consistent!with! this! prediction,! co9immunoprecipitation! and!mass!spectrometry!analyses!indicate!that!IdsD!and!IdsE!have!overlapping!protein!interaction!profiles.!Furthermore,!IdsD!and!IdsE!from! the! same! strain! interact! in& vitro,& whereas! the! interaction! between! IdsD! and! IdsE! from! different! strains! is! abrogated.!Remarkably,!deletion!mapping!showed!that!the!domain!containing!strain9specific!amino!acids!of! IdsE!is!required!for! IdsD!binding!and!that!exchanging!the!IdsE!strain9specific!residues!to!a!non9self!sequence!concomitantly!changes!the!binding!specificity!of!IdsE.!Taken! together,! these!data! indicate! that! the! identification!of! self! in!P.mirabilis! is!minimally!mediated!by! two!proteins,! IdsD!and!IdsE,! providing! mechanistic! insights! into! self! recognition! and! establishing! P.mirabilis& as! an! excellent! model! for! kin! selection!behaviors.!!!

19! ! A!whole7colony!technique!to!study!spatiotemporal!patterns!of!cellular!differentiation!in!Bacillus&subtilis!biofilms!

!Naveen!N!Sinha

1,!James!N!Wilking

1,!Agnese!Seminara

2,!David!A!Weitz

1,!and!Michael!P!Brenner

1!!1

School! of! Engineering! and! Applied! Sciences,! Harvard! University,!2Institut! Non! Linéaire! de! Nice,! Le! Centre! national! de! la!

recherche!scientifique!!!Most!bacteria!live!in!surface9attached!colonies!known!as!biofilms,!which!contain!numerous!distinct!cell!types!embedded!in!a!self9produced!extracellular!polymer.!Cellular!differentiation! is!primarily!a! result!of!extracellular! signals,! including!nutrient!availability!and! chemical! signals! from! other! cells.! These! signals! change! over! time,! leading! to! spatial! and! temporal! patterns! in! the! relative!populations! of! cell! types.! Although! the! response! of! single! cells! to! extracellular! signalshas! been! investigated,! the! connection!between! external! cues! and! spatiotemporal! patterns! of! populations! of! different! cell! types! within! an! intact! biofilm! is! not! well!understood.!Here!we!demonstrate!a!method! for!measuring!macroscale,! spatiotemporal!patterns!of!gene!expression! in!bacterial!biofilms! and! quantitatively! linking! these! patterns! to! external! cues.! Bacillus& subtilis! is! our! model! system,! since! its! pathway! to!sporulation! has! been! well! studied.! We! combine! quantitative! analysis! of! fluorescence! microscopy! images! of! labeled! cells! and!measurements!of!nutrient! concentrations!with!a! simple!physical!model.!With! this!approach,! it!may!be!possible! to!elucidate! the!transition!rates!between!cell!types!as!a!function!of!nutrient!levels!and!other!signals!within!a!colony.&!!!

20! ! Optimal!time7scheduled!therapy!effectively!fights!antibiotic!resistance!by!exploiting!the!cost!of!resistance!

!Antonio!LC!Gomes

1,!James!E!Galagan

1,2,!and!Daniel!Segre

1,2,3!!

1Bioinformatics! Program,!Boston!University,!

2Department!of! Biomedical! Engineering,! Boston!University,!

1Department!of! Biology,!

Boston!University!!!Drug!resistance!is!a!common!problem!in!the!fight!against!infectious!diseases.!Recent!studies!have!shown!conditions!(which!we!call!antiR)!that!are!able!to!select!against!resistant!strains.!However,!no!specific!drug!administration!strategies!based!on!this!property!exist!yet.!Here,!we!compare!growth!of!resistant!versus!sensitive!strains!under!different!treatments!(no!drugs,!antibiotic,!and!antiR),!and!show!how!a!precisely!timed!combination!of!treatments!can!defeat!resistant!strains.!Our!analysis! is!based!on!a!mathematical!model!of!infection!and!immunity!in!which!antibiotic!resistance!is!conferred!by!a!costly!plasmid.!Based!on!this!model,!we!estimate!treatment!schedules!that!would!lead!to!a!complete!elimination!of!both!sensitive!and!resistant!strains.!In!particular,!we!obtained!an!expression!that!describes!the!time!necessary!to!turn!aresistant!infection!into!sensitive!(tclear).!This!time!depends!on!the!rates!of!pathogen!division,!growth!and!plasmid!loss.!Finally,!we!estimated!tclear!using!available!empirical!data!and!found!that!the!rate!of!resistance!loss!increases!up!to!15!times!under!antiR!treatment!compared!to!a!regime!of!no!treatment.!An!extension!of!our!findings!to!population!models!suggests!quantitative!optimization!to!therapeutic!plans!for!clearing!antibiotic9resistant! infections,! including!novel!drug9cycling!strategies.!


19

21! ! The!identification!of!several!histidine!kinases!involved!in!the!metabolic!shut7down!of!Clostridium&thermocellum!

!

Elizabeth!B.!Mearls1!Javier!A.!Izquierdo

2,!and!Lee!R.!Lynd

2!!

1Department!of!Biological!Sciences,!Dartmouth!College,!

2Thayer!School!of!Engineering,!Dartmouth!College!!

!Clostridium&thermocellum!is!a!Gram9positive!anaerobe!that!solubilizes!cellulosic!biomass!and!produces!ethanol!as!a!byproduct!of!its!metabolism.!When! faced! with! unfavorable! growth! conditions! or! changes! in! environment,! C.& thermocellum! can! enter! different!resting! states! that! render! it! metabolically! unproductive.!We! have! initiated! efforts! to! better! understand! both! the! physiological!triggers!and!molecular!mechanisms!that!lead!to!the!development!of!resting!cell!types,!which!include!spores!and!L9forms.!To!better!understand!what!environmental!conditions!lead!to!the!formation!of!resting!cell!forms,!C.&thermocellum!cultures!were!subjected!to!a!variety!of!stresses.!Spore!formation!was!induced!by!changes!in!carbon!source!and!oxygen!exposure,!both!resulting!in!~7%!spores,!whereas! L9forms! were! induced! by! depletion! of! soluble! substrate,! resulting! in! ~98%! L9forms.! Subsequently,! we! identified! four!histidine! kinases! involved! in! the! onset! of! spore! formation,! and! confirmed! their! roles! via! deletion,! complementation,! and! over9expression!analysis.!In!addition,!one!kinase!deletion!strain!had!an!L9form!phenotype,!suggesting!said!kinase!plays!a!role!in!L9form!formation.!Understanding!how!resting!cells!form!will!provide!insight!into!how!C.&thermocellum&copes!with!environmental!stressors!and!lead!to!the!development!of!more!robust!strains!of!C.&thermocellum.!!!

22! ! Novel!pheromone7mediated!sensitivity!accounts!for!inhibition!of!vancomycin!resistant!Enterococcus&faecalis!by!commensal!enterococci!!Matthew!M.!Ramsey

1,!Lynn!Hancock

2,!Marcus!Rauch

1,!Paul!Himes

1,!and!Michael!Gilmore

1!!

1Department!of!Ophthalmology,!and!Department!of!Microbiology!and!Immunobiology,!Harvard!Medical!School,!Massachusetts!Eye!and!Ear!Infirmary,!

2Division!of!Biology,!Kansas!State!University!

!Enterococci!are!commensal!bacteria!that!occur!in!the!gut!of!a!wide!range!of!animals!and!have!recently!emerged!as!leading!causes!of!multidrug9resistant!hospital!infection.!Compared!to!commensal!enterococci,!the!genomes!of!multidrug9resistant!clinical!isolates!possess!numerous!mobile!elements,!including!a!pathogenicity!island,!phage,!transposons,!and!plasmids!that!encode!virulence!and!antibiotic! resistance,! often! constituting! over! a! quarter! of! the! genome.! To! determine! the! extent! to! which! accretion! of! mobile!elements!in!clinical!strains!affects!their!ability!to!compete!in!a!GI!tract!consortium,!we!tested!the!ability!of!the!prototype!multidrug9resistant!E.& faecalis! strain!V583! to!proliferate! in! coculture!with!human! fecal! flora.!We! found! that!V583!growth!was! inhibited! in!mixed!culture,!under!conditions!where!commensal!enterococci!flourished.!We!found!that!this!V583!inhibition!was!dependent!on!pheromone!production!by!endogenous! fecal! commensal! enterococci,! and! that! this! inhibition! involved!a! specific! constellation!of!mobile!elements!found!in!V583!and!several!other!multidrug9resistant!enterococci.!These!data!show!that!the!enrichment!of!mobile!elements! found! in! pathogenic! multidrug9resistant! E.& faecalis! comes! with! a! fitness! cost! incompatible! with! commensal! strains,!highlighting!the!importance!of!maintaining!normal!flora!as!a!barrier!to!multidrug9resistant!infection.!!!

23! ! Methylglyoxal!resistance!in!Bacillus&subtilis:!contributions!of!bacillithiol7dependent!and!independent!pathways!

!

Pete!Chandrangsu1!and!John!D.!Helmann

1!!

1Department!of!Microbiology,!Cornell!University!!!Methylglyoxal! (MG),! an!α,β! unsaturated! aldehyde,! is! a! toxic,! electrophilic! byproduct! of! glycolysis.! Exposure! to!MG! leads! to! cell!death! as! a! result! of! DNA! damage! and! protein! inactivation.! In! Gram9negative! bacteria,! protection! from!MG! is! conferred! by! the!glutathione9dependent!glyoxalase!pathway.!Glutathione! is!absent! from! low9GC!Gram9positive!Firmicutes!such!as!Bacillus&subtilis.!The! identification!of!bacillithiol! (BSH)!as! the!major! low9molecular9weight! thiol! in! the!Firmicutes! raises! the!possibility! that!BSH! is!involved! in! MG! detoxification.! Our! genetic! studies! have! identified! BSH9dependent! and! BSH9independent! MG! detoxification!pathways.! The! BSH9dependent! pathway! utilizes! the! glyoxalase! I! (YwbC)! and! glyoxalase! II! (YurT)! enzymes! to! convert!MG! to! D9

lactate.!The!critical!step!in!this!pathway!is!theactivation!of!the!KhtSTU!K+/H

+!antiporter,!which!leads!to!cytoplasmic!acidification!and!

protection! from! the! harmful! effects! of! MG.! We! demonstrate! that! cytoplasmic! acidification! independent! of! BSH! or! KhtSTU! is!sufficient!for!protection!from!MG.!We!have!also!identified!two!BSH9independent!pathways.!The!first!involves!YdeA,!predicted!to!be!a!glyoxalase!III!that!converts!MG!to!D9lactate,!and!the!second!involves!YhdN,!previously!shown!to!be!a!broad!specificity!aldo9keto!reductase!that!converts!MG!to!acetol.!!


20

24! ! Predicting!and!manipulating!drug!inactivation!by!the!human!gut!microbiome!

!

Henry!J.!Haiser1,!David!B.!Gootenberg

1,!Kelly!Chatman

1,!Gopal!Sirasani

2,!Emily!P.!Balskus

2,and!Peter!J.!Turnbaugh

1!!

1FAS!Center!for!Systems!Biology,!Harvard!University;!

2Department!of!Chemistry!and!Chemical!Biology,!Harvard!University!

!The! trillions! of! microorganisms! in! the! human! gastrointestinal! tract! are! an! underexplored! aspect! of! pharmacology.! Despite!numerous!examples!of!microbial!effects!on!drug!efficacy!and!toxicity,!there!is!often!an!incomplete!understanding!of!the!underlying!mechanisms.! We! have! dissected! the! inactivation! of! the! commonly! prescribed! cardiac! glycoside,! digoxin,! by! the! gut!Actinobacterium,! Eggerthella& lenta.! Whole! genome! transcriptional! profiling,! comparative! genomics,! and! culture9based! assays!revealed!a!cytochrome9encoding!operon!up9regulated!by!digoxin,!absent!in!non9metabolizing!E.&lenta!strains,!and!predictive!of!the!efficiency!of!digoxin!inactivation!by!thehuman!gut!microbiome.!Digoxin!inactivation!was!further!enhanced!by!microbial!interactions!andinhibited!by!the!amino!acid!arginine.!Pharmacokinetic!studies!using!gnotobiotic!mice!revealed!that! increasing!dietary!protein!reduces!the!in!vivo!metabolism!of!digoxin!by!E.&lenta,!with!significant!changes!to!drug!concentration!in!the!urine!and!serum.!These!results!emphasize!the!importance!of!viewing!pharmacology!from!the!perspective!of!both!our!human!and!microbial!genomes.!!!

25! ! Genetic!redundancy!and!specificity!in!the!human!gut!microbiome!

!

Patrick!H.!Degnan1,!Natasha!Barry

1,!Kenny!Mok

2,!Michiko!E.!Taga

2!and!Andrew!L.!Goodman

1!!

1Department!of!Microbial!Pathogenesis!and!Microbial!Diversity!Institute,!Yale!University!

2Department!of!Plant!&!Microbial!Biology,!

University!of!California!Berkeley!!!Genome! sequencing! efforts! have! revealed! the! complete! nucleotide! sequences! of! hundreds! of! human! gut! symbionts,! but! the!mechanisms!required!for!microbial!competition!and!cooperation!inthe!gut!are!largely!undefined.!Human!gut!microbes!depend!on!proteins!with! distant! homology! to! vitamin! B12! transporters! to! colonize! the!mammalian! gut! in! gnotobiotic!mouse!models.! Our!analysis!of!over!300!sequenced!human!gut!microbial!genomes!identified!elaborate!systems!for!uptake!of!vitamin!B12!and!related!compounds! (corrinoids)!and!multiple!paralogous! transporters!within!many!of! these!genomes.!To!understand!how!these!systems!determine! the! fitness! of! human! gut! microbes! in& vivo,! we! combined! genetic,! gnotobiotic,! and! computational! approaches! to!characterize! corrinoid! transport! in! the! prominent! human! gut! symbiont!Bacteroides& thetaiotaomicron.!We!demonstrate! that! the!role!of!corrinoid!exchange!in!shaping!community!assembly!can!be!modulated!through!control!of!host!diet!in!gnotobiotic!mice.!Our!experiments!also!show!that!each!of!the!three!outer!membrane!corrinoid!transporters!encoded!in!the!B.&thetaiotaomicron&genome!can! facilitate! B129dependent! growth! in& vitro.! Competition! assays! further! reveal! patterns! of! corrinoid! specificity.! Our! study!underlines!the!critical!role!of!the!small!molecule!exchange!in!mediating!human!gut!microbial!community!dynamics.!!!

2013!Boston!Bacterial!Meeting!7!Poster!Presentations!

Posters!1!9!70:!!Friday!Posters!71!9!140:!!Saturday

21

Presenter! Title!!

Poster!#!

Andres!Abin9Fuenter!!

Mechanistic!understanding!of!microbial!desulfurization!!Andres!Abin9Fuentes,!Magdy!El9Said!Mohamed,!Kristala!LJ!Prather,!and!Daniel!IC!Wang

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

!

16!

Kelly!Aldridge! A!co7cultivation!assay!to!detect!small7molecule7mediated!interactions!between!common!members!of!the!nostril!microbiota!!Kelly!L.!Aldridge,!Michael!S.!Wollenberg,!and!Katherine!P.!Lemon!

134!

!Gilad!Antler!!

!Combined!S733!and!O718!Isotope!Tracing!of!Intracellular!Sulfur!Metabolism!during!Microbial!Sulfate!Reduction!Gilad!Antler,!Tanja!Bosak,!Shuhei!Ono,!Orit!Sivan,!and!Alexandra!V.!Turchyn!!

!127!!

Seblewongel!Asrat! Innate!immune!strategies!that!respond!to!Legionella&pneumophila!intracellular!replication!!Seblewongel!Asrat,!Aisling!Dugan!and!Ralph!Isberg!!

120!

Elham!Azizi! Predictive!Models!of!Gene!Regulation!for!Mycobacterium&tuberculosis!!Elham! Azizi,! Matthew! Peterson,! Antonio! Gomes,! Anna! Lyubetskaya,! and! James!Galagan!

!

22!

Christina!E.!Baer! Novel! cell! division! and! growth! genes! discovered! using! high7throughput!microscopy!!Christina!E.!Baer!and!Christopher!M.!Sassetti!!

74!

Alicia!E.!Ballok! Epoxide7Mediated!Changes!in!OMV!Packaging!Affect!Pseudomonas&aeruginosa!Virulence!Alicia!E.!Ballok!and!George!A.!O’Toole!!

10!

Matheus!Quintana!Barreto!

Use!of!a!waste!stream!for!bioplastic!production!by!Ralstonia&eutropha&Matheus!Quintana!Barreto,!Christopher!Brigham!!

119!

Owen!Bennion! Single!cell!characterization!of!drug!tolerance!in!mycobacteria!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Owen!Bennion,!Anh!Hoang,!Jemila!Kester,!Marta!Fernandez9Suarez,!Danielle!Heller,!Daniel!Irimia,!Mehmet!Toner,!Sarah!Fortune,!Bree!Aldridge!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

99!

Ryan!W.!Benson! DNA!damage!and!resulting!functional!interactions!between!DNA!Pol!III!holoenzyme!subunits!and!DNA!Pol!IV!in!Escherichia&coli!!Ryan!W.!Benson,!Thomas!J.!Rands,!and!Veronica!Godoy!

!

98!

Brittany!Berdy! Bacterial!slow!growth!–!a!common!survival!strategy?!Brittany!Berdy,!Sandra!Buerger,!Alexander!Sousa,!Ethan!Edson,!Kieran!Halligan,!Lucy!Ling,!Amy!Spoering,!and!Slava!Epstein!

!

57!

Mehmet!Berkmen! Expression!of!antibodies!in!SHuffle!!Michael9Paul!Robinson,!Julie!Lobstein,!Thomas!J.!Mansell,!Corinna!Tuckey,!Paul!Riggs,!Paul!Colussi,!Chris!Noren,!Chris!Taron,!Matthew!P.!DeLisa!and!Mehmet!Berkmen!!

137!

Katherine!E.!Berry! E.&coli!mgtA!leader!region!encodes!an!Hfq7dependent!sRNA!!Katherine!E.!Berry!and!Ann!Hochschild�!!

58!

Anthony!Bissell! Lassomycin,!a!novel!lasso!peptide!kills!Mycobacteria&tuberculosis!by!targeting!the!ATP7dependent!protease!ClpC1P1P2!!!Ekaterina!Gavrish,!Clarissa!Sit;!Olga!Kandror,!Amy!Spoering,!Aaron!Peoples,!Lucy!Ling,!Ashley!Fetterman,!Dallas!Hughes,!Shugeng!Cao,!Anthony!Bissell,!Heather!Torrey,!Tatos!Akopian,!Andreas!Mueller,!Alfred!Goldberg,!Jon!Clardy,!and!Kim!Lewis!

95!

! !!

!



22

Presenter! Title!!

Poster!#!

Adam!S.!Bitzer! Interaction7induced! Motility! of! Pseudomonas& fluorescens! Pf071! and! Pedobacter!V48!Adam.!S.!Bitzer,!Sarah.!C.!Seaton,!Stuart.!B.!Levy,!Mark.!W.!Silby!

!

73!

Kristina!Boguslawski! Plasmid! encoded! quorum! sensing! regulators! control! competence! development!andsporulation!in!Bacillus&subtilis&&Kristina!Boguslawski!and!Kevin!Griffith!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

27!

Lindsey!Bomar! Lipid7dependent! Inhibition! of! the! Pathobiont! Streptococcus& pneumoniae! by!Corynebacterium!Primary!Nostril!Isolates!Lindsey!Bomar,!Silvio!Brugger,!and!Katherine!P.!Lemon!!

136!

Bijou!Bose! Intragenic!and!extragenic!suppressors!improve!the!sporulation!efficiency!of!Bacillus&subtilis&spoIIIEΔγ!mutants!Bijou!Bose,!Marina!Besprozvannaya!and!Briana!M.!Burton!!

21!

Chelsea!Boyd! Structural!features!of!the!Pseudomonas&fluorescens!biofilm!adhesin!LapA!required!for!biofilm!formation!and!cell!surface!localization!!Chelsea!D.!Boyd,!Ivan!E.!Ivanov,!Mary!E.!Schwartz,!Debashree!Chatterjee,!Peter!D.!Newell,!Lynne!Turnbull,!Michael!S.!Johnson,!Cynthia!B.!Whitchurch,!Terri!Camesano,!Holger!Sondermann,!and!George!O’Toole�!!

118!

S.R.!Brinsmade!!

A!Spectrum!of!CodY!Activities!Prioritizes!Gene!Expression!and!Metabolism!in!Bacillus&subtilis&&S.!R.!Brinsmade,!E.!L.!Alexander,!J.!Livny,!Arion!I.!Stettner,!Daniel!Segrè,!K.!Y.!Rhee,!A.!L.!Sonenshein

!!

!

35!

Autumn!Brown! Toxin7Antitoxin!Interferases!in!Stress!Response!and!Persister!Formation!!Autumn!Brown,!Sarah!Rowe!and!Kim!Lewis!!

82!

Laura!Brown! Characterization!of!the!E.&coli!replisome7associated!putative!helicase!YoaA!Laura!T.!Brown,!Deani!Cooper,!and!Susan!T.!Lovett!!

3!!

Julie!E.!Button! An!experimental!model!system!for!probing!microbial!community!interactions!and!dynamics!!Julie!E.!Button,!Laura!M.!Sanchez,!Marcela!Santarelli,!Benjamin!E.!Wolfe,!Pieter!C.!Dorrestein,!and!Rachel!J.!Dutton!!

85!

Ewen!Cameron! Targeted!protein!degradation!platform!for!tunable!control!of!synthetic!and!endogenousbacterial!systems!!Ewen!Cameron!and!Jim!Collins

!

!

23!

Bo!Cao! Genome7wide!mapping!of!the!Bacterial!DNA!Phosphorothioatome!using!Single!Molecule,!Real7Time!Sequencing!!Bo!Cao,!Tyson!A!Clark,!Michael!S!DeMott,!Qiuxiang!Cheng,!Zixin!Deng,!Matthew!Boitano,!Khai!Luong,!Stephen!W!Turner,!Delin!You,!Peter!C!Dedon,!Jonas!Korlach!!!

138!

Rachel!N.!Carmody! Cooking!alters!gut!microbial!ecology!!Rachel!N.!Carmody!and!Peter!J.!Turnbaugh!

!

90!

Clair!Chatelle! Converting!a!thiol!reductase!(DsbG)!into!a!disulfide!bond!isomerase!(DsbC)!using!a!novel!in&vivo!genetic!screen!Claire!Chatelle,!Stéphanie!Kraemer,!Caroline!Roggemans,!Na!Ke,!Paul!Riggs,!Mehmet!Berkmen

!!!!!!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

29!

Scott!S.!Chilton! DEAD!DNA!Transport:!a!mutational!analysis!of!a!competence!helicase/translocase!Scott!S.!Chilton!and!Briana!M.!Burton!

53!



23

Presenter! Title!!

Poster!#!

Yok!Hian!Chionh! Dynamic!reprogramming!of!tRNA!modifications!is!linked!to!activation!of!the!Dos!regulon!in!hypoxia7induced!mycobacterial!dormancy!Yok!Hian!Chionh,!Fabian!Hia,!Sylvie!Alonso,!Peter!C.!Dedon!!

129!

Brandon!Chiou! Exploring!the!dynamics!within!photosynthetic!communities!that!degrade!cellulose!and!fix!nitrogen!Emily!Mamula,!Brandon!Chiou,!Jea!Young!Park,!and!Jean!Huang!

!

91!

Philicia!Chow! Biofilm!formation!by!the!metabolically!versatile!phototroph!Rhodopseudomonas&palustris&&Philicia!Chow,!Chase!Kernen,!Jean!Huang,!and!Rebecca!Christianson!

!

106!

Sean!Clarke! Hypermutation!in!Experimentally!Evolved!Vibrio&&Sean!Clarke,!Sonia!Timberlake,!Arne!Materna,!Eric!Alm!!

36!

Olga!Danilchanka! Role!of!Type!6!Secretion!System!of!Pseudomonas&aeruginosa!for!Interaction!with!a!Host!Olga!Danilchanka,!David!Skurnik,!Charlotte!Gaultier,!Gerald!Pier!and!John!Mekalanos!!

107!

Rita!de!Cassia!Pessotti! Endophytic!actinobacterial!interactions!lead!to!developmental!changes!and!bioactivity!against!a!gram7negative!phytopathogen!Rita!de!Cassia!Pessotti,!Matthew!Francis!Traxler,!Monica!Tallarico!Pupo,!Jon!Clardy,!Roberto!Guillermo!Kolter!

!

56!

Dennise!A.!de!Jesús9Díaz!

Subversion!of!the!host!cell!cycle!by!Legionella&pneumophila&Dennise!A.!de!Jesús9Díaz!and!Ralph!R.!Isberg!!

94!

Kaila!Deiorio9Haggar! Phylogenetic!Distribution!of!B.&subtilis!Ribosomal!Autogenous!Regulatory!Elements!Kaila!Deiorio9Haggar,!Arianne!M.!Babina,!Jon!Anthony,!and!Michelle!M.!Meyer!!

121!

Nicholas!A.!DeLateur! Computational!prediction!and!validation!of!putative!ketosteroid!isomerase!(KSI)!structuralgenomics!proteins!Nicholas!A.!DeLateur,!Srinivas!Somarowthu,!Colleen!Shea,!Mark!Naniong,!Mary!Jo!Ondrechen,!Penny!J.!Beuning

!!!!!!!!!!!!!!!!!!!!!!!!!!

!

5!

Kurt!M.!Dahlstrom! Pseudomonas&fluorescens!as!a!Model!System!for!Cyclic!Diguanylate!Signaling!Kurt!M.!Dahlstrom,!Peter!Newell,!Holger!Sondermann,!and!George!O’Toole!!

48!

Marcus!Dillon! The!influence!of!operon!organization!on!the!evolutionary!rate!of!bacterial!genomes!Marcus!Dillon!and!Vaughn!Cooper!

!

97!

Tobias!Dörr! Substrate! specificity! of! an! elongation7specific! peptidoglycan! endopeptidase! and!its!implications!for!cell!wall!architecture!and!growth!of!Vibrio&cholera!!Tobias!Dörr,!Felipe!Cava,!Hubert!Lam,!and!Matthew!K!Waldor!!

43!

Daniel!P.!Dowling! Structural!Elements!of!the!NRPS!Cyclization!Domain!and!its!Intermodule!Comunication!Domain!Daniel!P.!Dowling,!Yan!Kung,!Anna!K.!Croft,!Koli!Taghizadeh,!Christopher!T.!Walsh,!and!Catherine!L.!Drennan

!

!

13!

Laura!Filkins! PSEUDOMONAS>STAPHYLOCOCCUS&INTER7BACTERIAL!INTERACTIONS!IN!THE!RESPIRATORY!TRACT!OF!CYSTIC!FIBROSIS!INFANTS!Laura!Filkins,!Juliette!Madan,!and!George!O'Toole!

!

101!



24

Presenter! Title!!

Poster!#!

Kelly!Fimlaid! Global!analysis!of!&Clostridium&difficile&sporulation!sigma!factors!using!RNA7Sequencing!!Kelly!A.!Fimlaid,!Jeffrey!P.!Bond,!Kristin!C.!Schutz,!Emily!E.!Putnam,!Jacqueline!M.!Leung,!Trevor!D.!Lawley,!and!Aimee!Shen

!!!!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

8!

Kenny!Flynn! Biotic!Interactions!Drive!Contingent!Adaptation!of!Pseudomonas&aeruginosa!Biofilms!Kenny!Flynn!and!Vaughn!Cooper!

!

79!

Luciane!S.!Fonseca! DNA!damage!response!in!L.&interrogans:!the!role!of!two!LexA!repressors!!Luciane!S!Fonseca,!Josefa!B!da!Silva,!Alan!D!Grossman,!Renata!MA!da!Costa!and!Paulo!L!Ho!!

60!

Christopher!Ford! Sterile!granulomas!develop!during!active!tuberculosis:!local!disparities!despite!global!effects!Philana!Ling!Lin,!Christopher!B.!Ford,!M.!Teresa!Coleman,!Amy!J.!Myers,!Thomas!Ioerger,!James!Sacchettini,!Sarah!M.!Fortune,!JoAnne!L.!Flynn!!

113!

Lucy!Foulston! An!elegant!solution!for!the!production!of!biofilm!matrix!protein!by!Staphylococcus&aureus&Lucy!Foulston,!Alexander!Elsholz!and!Richard!Losick!!

139!

Yang!Fu! Validation!of!a!cis7regulatory!RNA!element!for!ribosomal!proteins!S6!and!S18!Yang!Fu,!Mark!Soo,!Michelle!M.!Meyer!!

114!

Michael!A.!Funk! Structural!insight!into!anaerobic!toluene!activation!by!benzylsuccinate!synthase!Michael!A.!Funk,!E.!Neil!G.!Marsh,!Catherine!L.!Drennan!!

30!

Ellen!Fynan! The!Effect!of!Bisphenol7A!on!Representative!Gut!Microbiota!!Kimberly!Mohareb,!Michelle!Wheeler,!and!Ellen!Fynan!

!

135!

Tantan!Gao! Studying!the!role!of!the!bacterial!tyrosine!kinase!YwqD!in!biofilm!formation!in!Bacillus&subtilis&Tantan!Gao,!Alexander!Elsholz,!Yunrong!Chai,!and!Richard!Losick!

!

76!

Elsa!Bou!Ghanem! Pneumolysin!targets!CD73,!a!host!immune7modulatory!5’7nucleotidase!that!promotes!resistance!to!invasive!Streptococcus&pneumoniae&infection!Elsa!Bou!Ghanem,!Stacie!Clark,!Sara!Roggensack,!Michael!Pereira,!Sally!McIver,!Philip!Haydon!and!John!Leong!!

59!

Sonali!Ghosh! The!Cause!of!Superdormancy!in!Bacillus&subtilis!Spore!Germination!Sonali!Ghosh!and!Peter!Setlow!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

15!

Marcus!Gibson! Structural!Insights!into!an!Oxalate!Oxidoreductase!Marcus!I.!Gibson,!Ed!J.!Brignole,!Elizabeth!Pierce,!Stephen!W.!Ragsdale,!and!Catherine!L.!Drennan!!

125!

Sarah!Gignoux9Wolfsohn!

Next7Generation!16s!Profiles!of!White!Band!Disease!in!the!Threatened!Coral!Acropora&Cervicornis!!Sarah!Gignoux9Wolfsohn!and!Steven!Vollmer!!

18!

Elizabeth!Halvorsen! The!role!of!InlF!in!Listeria&monocytogenes&infection!of!the!brain!!!Elizabeth!Moritz!Halvorsen!and!Darren!E.!Higgins�!

133!

!!!!

!!!!

!!!!



25

Presenter! Title!!

Poster!#!

Stavroula!K.!Hatzios! A!Chemoproteomic!Approach!to!Enzyme!Discovery!During!Vibrio&cholerae!Infection!!Stavroula!K.!Hatzios,!Julianne!Martell,!Diana!Munera,!Soren!Abel,!Eranthie!Weerapana,!and!Matthew!K.!Waldor!!

88!

Jennifer!M.!Hayashi! Protein!localization!to!membrane!subdomains!in!Mycobacterium&smegmatis!!Jennifer!M!Hayashi!and!Yasu!S!Morita!!

96!

Andrew!D.!Hempstead! Modulation!of!the!Host!Unfolded!Protein!Response!by!Legionella&pneumophila!Andrew!D.!Hempstead!and!Ralph!R.!Isberg!

!

69!

Gary!E.!Heussler! Beyond!Immunity:!New!Insights!into!the!Pseudomonas&aeruginosa!CRISPR/Cas!System!!Gary!E.!Heussler,!Kyle!C.!Cady,!and!George!A.!O’Toole�!!

122!

Jessica!K.!Jarett! Environmental!influences!and!biotic!interactions!shaping!the!coral!holobiont!!Jessica!K.!Jarett,!Cara!L.!Fiore,!Daniel!A.!Brazeau,!and!Michael!P.!Lesser

!!!!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

25!

Peng!Jiao! A!New!Method!for!Fermentation!Process!Optimization!of!a!Complex!System!!Peng! Jiao,!Ray!Li,!Nida!Sayed,!Wenglong!Lin,!Maureen!Hamilton,!Michelle!Chopra,!Seann!O’Connell,!Khursheed!Karim,!Sangram!Yadav,!Sohan!Patel,!Longkuan!Xiang!&!Steven!Harris!!!

34!

Christopher!M.!Johnson!

Composition!of!the!cell!membrane!affects!the!efficiency!of!bacterial!conjugation!Christopher!M.!Johnson!and!Alan!D.!Grossman�!!

67!

James!E.!Kath! Concentration7dependent!interactions!with!the!sliding!clamp!serve!as!a!switch!for!regulating!E.&coli!polymerase!exchange!James!E!Kath,!Seungwoo!Chang,!Slobodan!Jergic,!Deena!Jacob,!Justin!MH!Heltzel,!Mark!D!Sutton,!Graham!C!Walker,!Nicholas!E!Dixon,!and!Joseph!J!Loparo!!

75!

Meaghan!Keane! Analysis!of!the!roles!of!Shewanella&oneidensis!Hfq!in!cell!growth!and!oxidative!stress!response!!Christopher!Brennan,!Meaghan!Keane,!Taylor!Hunt,!Nick!Mazzucca,!Jessica!Leonard,!and!Brett!Pellock!!!

72!

Karen!J.!Kieser! PonA1!controls!cell!elongation!and!morphology!in!mycobacteria!!!Karen!J.!Kieser,!Jemila!C.!Kester,!Michael!C.!Chao,!Sarah!M.!Fortune,!Eric!J.!Rubin.!

62!

! ! !Natalia!V.!Kirienko! Pseudomonas!aeruginosa!Disrupts!Caenorhabditis!elegans!Iron!Homeostasis,!

Causing!a!Hypoxic!Response!and!Death!Natalia!V.!Kirienko,!Daniel!R.!Kirienko,!Gary!Ruvkun,!Frederick!M.!Ausubel!!

!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

17!

Annette!E.!LaBauve! Pseudomonas&aeruginosa&detection!of!host7derived!sphingosine!impacts!survival!during!lung!infection!Annette!E.!LaBauve!and!Matthew!J.!Wargo!!

51!

Cristina!Landeta! Inhibition!of!disulfide!bond!formation!in!gram7negative!bacteria!Cristina!Landeta,!Jessica!Blazyk,!Feras!Hatahet,!Dana!Boyd,!Holly!Arnold,!Rachel!Dutton,!Markus!Eser!and!Jon!Beckwith�!!

1!

Sara!Leiman! Revisiting!the!Role!of!D7amino!acids!in!Bacillus&subtilis&biofilm!formation!Sara!Leiman,!Roberto!Kolter,!and!Richard!Losick�!!

68!

Maxime!Leroux! An!insertion!mutation!in!priA!that!splits!its!phenotype!Maxime!Leroux,!Niketa!Jani!and!Steven!Sandler!

!

47!



26

Presenter! Title!!

Poster!#!

Vivian!Leung! Disruption!of!Septation!Results!in!Bipolar!Growth!in!Mycobacteria&Vivian!Leung,!Cara!Boutte,!and!Eric!J.!Rubin!!

104!

Kathryn!Levasseur! Determining!the!location!of!virulence7associated!transcription!factors!in!Francisella&tularensis&Kathryn!Levasseur,!Cathy!Su,!and!Simon!Dove!&

7!

Lorraine!Ling! ClpX,!a!bacterial!AAA+!unfoldase,!targets!the!Mu!transpososome!complex!via!a!multivalent!recognition!signal!Lorraine!Ling,!Aliaa!Abdelhakim,!Robert!T.!Sauer,!Sherwin!P.!Montano,!Phoebe!A.!Rice,!Tania!A.!Baker�!!

115!

Kristin!Little!! Expression!of!ids&genes!in!Proteus&mirabilis&is!stably!bimodal!and!likely!regulated!by!non7heritable!factors!Kristin!Little!and!Karine!Gibbs!!

78!

Songnian!Liu! Mercury!resistance!of!two!magnetotactic!bacteria!strains!and!their!potential!ability!to!reduce!mercury!by!biosynthetic!magnetite!Songnian!Liu!and!Heather!Wiatrowski!! !!

19!

Jarukit!Edward!Long! Exogenous!Carbohydrates!and!Glycerol!are!Required!for!Growth!During!Mtb&Infection!Jarukit!Edward!Long!and!Christopher!Sassetti!!

92!

Rustin!Lovewell! Bacterial!Motility!Facilitates!Phagocyte!Recognition!and!Uptake!of!P.&aeruginosa!and!other!Gram7Negative!Pathogens!Rustin!Lovewell,!Eyal!Amiel,!George!O’Toole,!and!Brent!Berwin!!

49!

Douglas!C.!Marshall! Transcriptome!Analysis!of!Adaptation!to!Water!Stress!by!Pseudomonas&fluorescens!Pf071!Growing!in!Soil!Douglas!C.!Marshall,!Stuart!B.!Levy,!and!Mark!W.!Silby�!!

131!

Brenley!McIntosh! CACAO:!Linking!evidence7based!gene!annotation!to!education!!!!!!!!Brenley!McIntosh

!and!Jim!Hu!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

28!

Jamie!Meadows! Regulation!of!carnitine!catabolism!in!Pseudomonas&aeruginosa&Jamie!Meadows!and!Matthew!Wargo!&

50!

Brian!Meehan! The!Pathways!for!Periplasmic!Disulfide!Bond!Formation!are!Essential!for!Growth!of!Escherichia&coli!and!Mycobacterium&smegmatis!!Brian!Meehan,!Rachel!Dutton,!Na!Ke,!and!Jon!Beckwith!

!

52!

Stephanie!L.!Mitchell! VieB!plays!an!inhibitory!role!in!the!VieSAB!three7component!signal!transduction!system!in!Vibrio&cholerae.!Stephanie!L.!Mitchell,!Ayman!Ismail,!and!Andrew!Camilli!! !!

42!

Georgeanna!M.!Morton!

Oligomeric!State!of!the!Mating!ATPase!ConE!!Georgeanna!M.!Morton,!Kyle!J.!Swerdlow,!and!!Melanie!B.!Berkmen!!!!!!!&

11!

Xiangyu!Mou! Contribution!of!vah1,&plp,&and&rtxA!to!the!cytotoxicity!of!Vibrio&anguillarum&against!epithelial!cells,!leukocytes!and!erythrocytes!Xiangyu!Mou,!Edward!J.!Spinard,!David!A.!Parra,!J.!Oriol!Sunyer!and!David!R.!Nelson!!

44!

Paul!Muller! Germinant7Free!Stochastic!Germination!of!Wild!Type!Bacillus&subtilis!Spores:!Observations!in!Favor!of!the!Scout!Hypothesis!Paul!Muller!and!Slava!Epstein!!

46!



27

Presenter! Title!!

Poster!#!

Pallavi!P.!Murugkar! Identifying!a!growth!factor!for!previously!uncultivated!bacteria!from!the!oral!cavity!Pallavi!P.!Murugkar,!Eric!J.!Dimise,!Eric!J.!Stewart,!Jon!Clardy,!and!Kim!Lewis!!

102!

Sidrotun!Naim! Quorum!Sensing!of!Vibrio&harveyi:&An!in&vivo&Study!in!Shrimp!Sidrotun!Naim,!Kevin!Fitzsimmons,!Donald!V.!Lightner!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

26!

Asha!Nayar! Proof!of!dual!targeting!and!novel!mode!of!action!for!non7quinolone!bacterial!topoisomerase!type!II!inhibitors!Asha!Nayar,!Mark!Cronin,!Folkert!Reck,!Gregory!Stone!and!Thomas!Dougherty!!

117!

Peter!Nguyen! Programmable!Biofilm7Based!Materials!from!Engineered!Curli!Nanofibers!Peter!Q.!Nguyen,!Zsofia!Botyanszki,!Pei!Kun!R.!Tay!!and!Neel!S.!Joshi!!

38!

Amy!Nwaobasi! The!Effects!of!DinD!on!RecA!structures!using!Fluorescent!Microscopy!!Amy!Nwaobasi!and!Steven!Sandler!!!!

31!

Maghnus!O’Seaghdha! Streptolysin!O!and!its!co7toxin!NAD7glycohydrolase!protect!group!A!Streptococcus!from!xenophagic!killing!!!Maghnus!O’Seaghdha!and!Michael!R.!Wessels�!!

110!

Rajat!Kumar!Pal! Regulation! of! the! iron! acquisition! and! utilization! response! by! extracellular! iron7scavenging!molecules!under!iron!replete!conditions!in!Bacillus&subtilis!!Rajat!Kumar!Pal!and!Kevin!Griffith!!

81!

Sarju!Patel! Functional!diversity!of!five!homologous!Cu+7ATPases!present!in!Sinorhizobium&meliloti!!Patel!S.,!Padilla9Benavides!T.,!Collins!J.!and!Argüello,!J.M.!!

63!

Shaaretha!Pelly! Characterization!of!the!Mcr11!small,!non7coding!RNA!of!Mycobacterium&tuberculosis!!Shaaretha!Pelly,!Cara!Smith,!William!R.!Bishai,!Gyanu!Lamichhane�!!

105!

Jon!Penterman! Molecular!control!of!the!Sinorhizobium&meliloti&cell!cycle!by!host!cationic!peptides!during!symbiosis!Jon!Penterman!and!Graham!Walker!!

108!

Kyle!J.!Perry! A!differential!fluorescence7based!genetic!screen!identifies!PepP!as!a!Listeria&monocytogenes&determinant!required!for!optimal!intracellular!replication!!Kyle!J.!Perry!and!Darren!E.!Higgins!&

20!

Jessica!Polka! DNA!segregation!on!sticky!treadmills:!a!plasmid!centromere!promotes!nucleation,!stability,!and!bundling!of!the!actin7like!filament!AlfA!!Jessica!K.!Polka,!R.!Dyche!Mullins

!

!

37!

Laurent!Potvin9Trottier! Stochasticity!of!gene!expression!of!a!synthetic!oscillator!under!constant!growth!conditions!Laurent!Potvin9Trottier,!Nate!Lord!and!Johan!Paulsson!

!

84!

Katherine!E.!Price! Polymicrobial!interactions!in!the!cystic!fibrosis!lung!Katherine!E.!Price,!Amanda!A.!Naimie,!Charles!Bay,!Edward!F.!Griffin,!and!George!O’Toole!!

100!

Oliver!Purcell! Towards!a!whole7cell!modeling!approach!for!synthetic!biology!Oliver!Purcell,!Bonny!Jain,!Jonathan!Karr,!Markus!Covert,!Timothy!Lu!!

66!

! ! !



28

Presenter! Title!!

Poster!#!

Ardeshir!Rineh! Photodynamic!inactivation!of!anaerobic!bacteria!in!the!absence!of!oxygen!Ardeshir!Rineh,!George!P.!Tegos,!Conor!L.!Evans,!Michael!J.!Kelso!and!Michael!R.!Hamblin

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

!

9!

Emily!S.C.!Rittershaus! cAMP!remodels!metabolic!flux!in!M.&tuberculosis!via!protein!lysine!acetylation!Emily!S.!C.!Rittershaus,!Seung9hun!Baek,!and!Christopher!Sassetti!!!

86!

Amy!Rohlfing! Interaction!between!PigR!and!the!MglA7SspA!complex!in!Francisella&tularensis!is!necessary!for!virulence!gene!expression!Amy!Rohlfing!and!Simon!Dove!!

83!

Chloe!Roussel9Rossin! Defining!genes!implicated!in!a!gene!addition!process!in!Escherichia&coli&Chloe!Roussel99Rossin,!Elisabeth!Raleigh!

!

40!

Jacob!Rubens! Analog!Genetic!Circuits!Enable!Complex!Computations!in!Living!Cells!Jacob!R.!Rubens,!Ramiz!Daniel,!Rahul!Sarpeshkar,!and!Timothy!K.!Lu!!!

2!

Brandon!S.!Russell! Development!and!Characterization!of!a!BSL72!Wistar!Rat!Model!of!Pulmonary!Mycobacterial!Infection!Brandon!S.!Russell,!Sasilada!Sirirungruang,!Nicola!M.!A.!Parry,!Megan!E.!McBee,!James!G.!Fox,!and!Peter!C.!Dedon

!

!

12!

Christina!C.!Saak! Variable! regions! in! self7identity! proteins! are! important! for! self7recognition! and!other!aspects!of!surface7based!growth!in!Proteus&mirabilis!!Christina!C.!Saak,!and!Karine!A.!Gibbs!!

111!

John!P.!Santa!Maria!Jr.! Mapping!the!Cell!Wall!Interactome!of!Staphylococcus&aureus&&John!P.!Santa!Maria!Jr.,!Ama!Sadaka,!Samir!H.!Moussa,!Stephanie!Brown,!Jason!Zhang,!Eric!Rubin,!Michael!Gilmore,!and!Suzanne!Walker

!!!!!!!!!!!!!!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

4!

Kara!B.!Schallies! Sinorhizobium&meliloti&Cell!Cycle!Regulation!of!DivK!and!CtrA!Karla!B.!Schallies,!and!Katherine!E.!Gibson�!!

126!

Ruth!Schmidt! The!role!of!volatiles!as!infochemicals!in!bacterial!inter7specific!interactions!Ruth!Schmidt,!Kees!Hordijk,!Saskia!Gerards,!Wietse!de!Boer!and!Paolina!Garbeva�!!

130!

Maia!Schoonmaker! The!Transpeptidase!Landscape!of!Mycobacterium&tuberculosis&&&&&&&Maia!Schoonmaker,!William!Bishai,!Gyanu!Lamichhane

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&!

32!

Sarah!C.!Seaton! Co7culturing!of!Pseudomonas&and!Pedobacter&induces!an!interspecies!social!trait!Sarah!C.!Seaton,!Adam!Bitzer,!Stuart!B.!Levy,!and!Mark!W.!Silby�!!

93!

Charlotter!A.!Seid! The!bacterial!replication!initiator!DnaA!and!its!regulator!YabA!associate!with!several!chromosomal!regions!bound!by!the!nucleoid7associated!protein!Rok!in!Bacillus&subtilis!!Charlotte!A.!Seid,!Tracy!A.!Washington,!Houra!Merrikh,!and!Alan!D.!Grossman�!!

64!

Benedicte!Sempe! Role!of!StreptolysinO!and!NAD+7glycohydrolase!during!interaction!of!GroupA!Streptococcus!with!macrophages!Benedicte!Sempe!and!Michael!Wessels!

!

65!

Alecia!N.!Septer! Novel!self7recognition!genes!contribute!to!territorial!behavior!in!Proteus&mirabilis&Alecia!N.!Septer!and!Karine!A.!Gibbs!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!&

14!

Bijaya!Sharma! Identification!of!a!Growth!Factor!for!Marine!Uncultured!Bacteria!Bijaya!Sharma,!Anthony!D’Onofrio,!Eric!Dimise,!Eric!J.!Stewart,!Jon!Clardy,!and!Kim!Lewis!

128!



29

Presenter! Title!!

Poster!#!

Onkar!Sharma! Anthrax!toxin7mediated!cellular!delivery!of!streptococcal!NAD!glycohydrolase!Onkar!Sharma,!M.!N.!O’!Seaghdha!and!M.!R.!Wessels!!

61!

Scarlet!S.!Shell! Widespread!mRNA!processing!shapes!the!Mycobacterium!tuberculosis!transcriptome!Scarlet!S.!Shell*,!Thomas!R.!Ioerger*,!Michael!R.!Chase*,!James!C.!Sacchettini,!and!Sarah!M.!Fortune!

!

80!

Betty!Slinger! Parallel!evolution!of!bacterial!RNA!cis7regulators!for!ribosomal!protein!S15!Betty!Slinger,!Molly!Gilligan,!and!Michelle!M.!Meyer!!!

71!

William!E.!Spears! Reprogramming!bacterial!secretion!systems!for!therapeutic!applications!Analise!Z!Reeves,!William!E!Spears,!Amy!J!Wagers,!Cammie!F!Lesser! !

!

45!

Rachel!Staples! Pleiotropy!positively!correlates!with!the!direct!effect!of!beneficial!mutations!Rachel!Staples,!David!Morejon,!and!Vaughn!Cooper�!!

140!

Kerry9Ann!V.!Stewart! Levels!of!Germinant!Receptors!in!the!Bacillus!subtilis!Spore!Inner!Membrane!and!Their!Relationship!to!Spore!Germination!rates!Kerry9Ann!V.!Stewart,!Xuan!Yi,!Sonali!Ghosh,!and!Peter!Setlow!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

24!

Philip!Strandwitz! Discovery!of!a!Novel!Bacterial!Growth!Factor!for!the!Human!Gut!Microbiota!Philip!Strandwitz,!Ki9Hyun!Kim,!Eric!Stewart,!Jon!Clardy,!and!Kim!Lewis�!!

124!

Kyle!J.!Swerdlow! Oligomerization!and!assembly!of!a!DNA!translocation!channel!Kyle!J.!Swerdlow,!Gianna!R.!Mancuso,!April!Culananay,!Stefanie!Baril,!Melanie!B.!Berkmen!!!!!!!!!!!!&

41!

Jessie!Thompson! The!high&light>inducible!genes!of!the!marine!cyanobacterium!Prochlorococcus:&&A!diverse!dynamic,!high!copy!number!gene!family!Jessie!Thompson*,!Greg!Kettler*,!Steve!Biller,!Julie!Miller,!Lisa!Moore,!and!Sallie!W.!Chisholm!!

132!

Amaris!Torres9Delgado! More!than!just!a!molecular!matchmaker:!ClpS!adaptor!protein!controls!ClpAP!substrate!selection!Amaris!Torres9Delgado,!Robert!T.!Sauer!and!Tania!A.!Baker!

&

54!

Michael!D.!Valentino! Genomics!of!the!Conjunctival!Pathogen!Streptococcus&pneumonia&Michael!D.!Valentino,!Jason!W.!Rosch,!Wolfgang!Haas,!Christine!M.!Sanfilippo,!Robert!A.!Carter,!Michael!E.!Zegans,!Bernard!Beall5,!Elaine!I.!Tuomanen,!Timothy!W.!Morris!and!Michael!S.!Gilmore!!

77!

Nicole!M.!Vega! Salmonella&typhimurium!intercepts!E.&coli!signaling!to!enhance!antibiotic!tolerance!Nicole!M.!Vega,Kyle!R.!Allison,!Amanda!N.!Samuels,!Mark!S.!Klempner,!and!James!J.!Collins�!!

70!

Marissa!G.!Viola! Conserved! residues! near! the! FtsZ! C7terminus! contribute! to! recognition! and!degradation!of!FtsZ!by!the!ATP7dependent!protease!ClpXP!Marissa!G.!Viola,!Sue!Wickner,!and!Jodi!L.!Camberg!!

112!

Ben!Waldman! How!is!cell!division!inhibited!in!a!strain!of!E.&coli&K>12&lacking!the!Rep!helicase?!Ben!Waldman,!Jaymes!Farrell,!and!Steven!Sandler!

!

116!

Yingjiao!Wang! Mercury!Challenge!to!Dissimilatory!Metal!Reducing!Bacteria!!Yingjiao!Wang,!Tyler!Robison!and!Heather!Wiatrowski!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

33!



30

Presenter! Title!!

Poster!#!

Larissa!M.!Wenren! Mapping!a!network!for!self!recognition!in!Proteus&mirabilis&Larissa!M.!Wenren,!Nora!L.!Sullivan,!Lia!Cardarelli,!Alecia!N.!Septer,!and!Karine!A.!Gibbs!!!!!!!!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

109!

Carl!N.!Wivagg! Wall!teichoic!acids!influence!mreB!motion!in!Bacillus!subtilis!Carl!N.!Wivagg,!Saman!Hussain,!Michael!F.!Dion,!and!Ethan!C.!Garner!!

102!

Laurel!D.!Wright! Library!assembly!of!orthogonal!genetic!memory!devices!Lei!Yang,!Timothy!Lu,!Christopher!Voigt!!

87!

Lei!Yang! Library!assembly!of!orthogonal!genetic!memory!devices!Lei!Yang,!Timothy!Lu,!Christopher!Voigt!!

55!

Grace!J.!Yuen! Enterococcus!infection!of!C.&elegans!as!a!model!of!innate!immunity!Grace!J.!Yuen!and!Frederick!M.!Ausubel!!

123!

Yanija!J.!Zhang! Genomic!Profiling!of!Bacterial!Strategies!for!Counteracting!Host!Defenses!in!Tuberculosis!Yanjia!J.!Zhang,!Thomas!R.!Ioerger,!Alissa!C.!Rothchild,!Curtis!Huttenhower,!Christopher!M.!Sassetti,!James!C.!Sacchettini,!Samuel!M.!Behar,!Eric!J.!Rubin!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

6!

Wenjing!Zhao! The!probiotic!bacterium,!Phaeobacter&gallaeciensis! S4,!down7regulates!virulence!factor!production!in!the!oyster!pathogen,!Vibrio&tubiashii&Wenjing!Zhao,!Christine!Dao,!David!C.!Rowley,!and!David!R.!Nelson!

39!

! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !

2013!Boston!Bacterial!Meeting!7!Poster!Presentation!Abstracts!

31

1! ! Inhibition!of!disulfide!bond!formation!in!Gram7negative!bacteria!

!Cristina!Landeta1,!Jessica!Blazyk1,!Feras!Hatahet1,!Dana!Boyd1,!Holly!Arnold2,!Rachel!Dutton3,!Markus!Eser4!and!Jon!Beckwith1!1Department!of!Microbiology! and! Immunobiology,!Harvard!Medical! School.! 2University! of!Oregon,! Eugene,!OR.! 3FAS,! Center! for!Systems!Biology,!Harvard!University.!4Affimed!therapeutics,!Heidelberg!Germany!!Disulfide! bonds! are! important! for! the! stability! and! activity! of! many! exported! proteins.! In! bacteria! the!in& vivo! disulfide! bond!formation!requires!enzymes!that!are!capable!of!catalyzing!this!process.!These!enzymes!are!members!of!oxidoreductases,!belonging!in!most!cases!to!the!thioredoxin!superfamily,!which!is!characterized!by!a!motif!that!has!two!cysteins!separated!by!two!amino!acids.!In!Escherichia& coli! these!enzymes!are!DsbA!and!DsbB!whereas! in!Mycobacterium& tuberculosis! the! system! includes!DsbA9like!and!VKOR!(Vitamin!K!epOxide!Reductase)!enzymes.!The!disulfide!bond!formation!enzymes!are!essential!for!E.&coli!and!M.&tuberculosis!survivals!as!well!as!they!are! involved! in!the!folding!of!many!virulence!factors! in!gram9negative!pathogens.!We!have!developed!a!sensitive!high9throughput!screening!for!the!identification!of!small!molecule!inhibitors!of!MtbVKOR!and!EcDsbB!enzymes.!We!have!identified!10!inhibitors!of!EcDsbB!and!through!looking!for!relationship!between!chemical!structure!and!activity!we!have!found!four!more!potent!inhibitors.!Furthermore!we!have!tested!those!inhibitors!in!an!E.&coli!strain!expressing!DsbB!proteins!from!other!gram9negative!bacteria!and!found!some!of!them!did!inhibit!these!as!well!as!the!E.&coli!enzyme.!We!are!further!characterizing!and!testing!the!inhibitors!in!pathogenic!strains.!!!

2! ! Analog!Genetic!Circuits!Enable!Complex!Computations!in!Living!Cells!

!Jacob!R.!Rubens3,4,5,!Ramiz!Daniel1,3,5,6,!Rahul!Sarpeshkar1,3,6,!and!Timothy!K.!Lu1,2,3,5!!!1!Department!of!Electrical!Engineering!and!Computer!Science,!MIT,!2!Department!of!Biological!Engineering,!MIT,!3Synthetic!Biology!Center,!MIT,!4MIT!Microbiology!Program,!MIT!,!5Synthetic!Biology!Group,!Research!Laboratory!of!Electronics,!MIT,!6Analog!Circuits!and!Biological!Systems!Group,!Research!Laboratory!of!Electronics,!MIT!!Digital!logic,!wherein!genes!are!turned!on!or!off,!is!the!predominant!paradigm!for!engineering!synthetic!gene!networks.!However,!digital! logic!has!proved!difficult! to! scale!and!other!approaches!may!be!needed! for!efficient! computation! in! living! cells! given! the!resource9limited!environment!and!the!small!number!of!well9characterized!genetic!parts.!Here,!we!utilize!graded!positive!feedback!to!engineer!synthetic!gene!circuits!that!respond! log9linearly!to!a!wide9dynamic!range!of! input! inducer!concentrations.!This!wide9dynamic!range!behavior!enables!us!to!construct!synthetic!gene!networks!using!an!analog!approach!and!we!demonstrate!how!the!circuits! can! be! composed! together! to! implement! addition! and! ratiometric! computations.! Furthermore,! the! circuits! can! be!architected! to! have! tunable! transfer! functions! controlled! by! front9end! digital! logic,! allowing! them! to! be! readily! interfaced!with!existing!digital!gene!networks.!Analog!gene!circuits!open!new!applications!for!synthetic!biology!that!require!complex!computations!and!wide9dynamic! range!bio9sensing,! and!a!hybrid! analog9digital! approach! to! gene!networks!may!be!essential! to!build! complex!synthetic!biological!systems.!!!!!

3! ! Characterization!of!the!E.&coli!replisome7associated!putative!helicase!YoaA!

!Laura!T.!Brown,!Deani!Cooper,!and!Susan!T.!Lovett!!!!!!!!!!!!!!!!! ! ! ! ! ! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Department!of!Biology!and!Rosenstiel!Center,!Brandeis!University,!Waltham,!MA!!

Azidothymidine,!(AZT)!a!replication!chain!terminating!nucleotide,!is!incorporated!at!the!replication!fork,!leading!to!DNA!damage!if!left!unrepaired.!Using!a!high!expression9suppressor!screen,!we!found!that!expression!of!DNA!polymerase!III!holoenzyme!subunit,!Chi!(HolC)!and!the!putative!helicase,!YoaA,!suppresses!sensitivity!to!AZT!in!a!variety!of!strains.!The!two!proteins!have!a!documented!physical!interaction.!Mutants!in!yoaA!and!holC!are!sensitive!to!AZT!damage.!Suppression!of!AZT!sensitivity!by!either!YoaA!or!Chi!is!dependent!on! the!other!protein.!YoaA! is!a!putative!Fe9S!cluster,!Rad3/XPD! family!helicase.!An!E.& coli!paralog! to!YoaA,!DinG,!has!documented! 5’! to! 3’! helicase! activity,! functional! Fe9S! and! ATPase! domains.! Suppression! of! AZT! sensitivity! by! YoaA! requires!functional!ATPase!and!Fe9S!cluster!domains.!Both!proteins!are! induced!by! the!SOS!DNA!damage! response,!which! involves!RecA,!LexA!and!other!proteins.!Promoter! induction!assays! indicate!dependence!of!yoaA! induction!on!LexA!suppressor!cleavage! from!a!putative!LexA!box.!Chi!is!an!accessory!protein!of!the!clamp!loader!complex!that!interacts!with!SSB.!Our!working!hypothesis!is!that!Chi’s!interaction!with!SSB!at!the!replication!fork!recruits!the!repair!helicase,!YoaA,!which!unwinds!the!nascent!strand!to!facilitate!repair.!


32

4! ! Mapping!the!Cell!Wall!Interactome!of!Staphylococcus&aureus&!

!

John!P.!Santa!Maria!Jr.1,!Ama!Sadaka

2,!Samir!H.!Moussa

1,!Stephanie!Brown

3,!Jason!Zhang

1,!Eric!Rubin

1,!Michael!Gilmore

4,!and!

Suzanne!Walker2!!

1Department!of!Microbiology!&!Immunobiology,!Harvard!University,!

2Department!of!Ophthalmology,!University!of!Cincinnati,!

3Department!of!Biological!Chemistry,!John!Innes!Centre,!

4Department!of!Ophthalmology,!Massachusetts!Eye!and!Ear!Infirmary!!

!The!bacterial!cell!envelope!has!been!the!subject!of!intense!study!for!decades,!but!how!its!substituents!interact!to!coordinate!cell!growth! and! division! remains! a!mystery.! Recent! evidencehas! demonstrated! that! wall! teichoic! acids,! anionic! glycopolymers! that!comprise!a!large!fraction!of!the!cell!wall,!play!important!roles!in!key!envelope!processes,!such!as!peptidoglycan!biosynthesis!and!cell!separation.!We!combined!TnSeq!with!a!small!molecule!that!inhibits!the!production!of!wall!teichoic!acids!in!S.&aureus&to!identify!previously! unknown! synthetic! lethal! interactions! between! cell! envelope! pathways.! Exploring! these! interactions! will! help!elucidatecell!envelope!biogenesis!and!may!provide!new!targets!for!combination!antibacterial!therapies.!

5! ! Computational!prediction!and!validation!of!putative!ketosteroid!isomerase!(KSI)!structuralgenomics!proteins!

!Nicholas!A.!DeLateur,!Srinivas!Somarowthu,!Colleen!Shea,!Mark!Naniong,!Mary!Jo!Ondrechen,!Penny!J.!Beuning

!!!!!!!!!!!!!!!!!!!!!!!!!

Department!of!Chemistry!and!Chemical!Biology,!Northeastern!University!!!Ketosteroid!Isomerase!(KSI)!performs!an!important!biological!function!in!the!metabolism!of!many!bacteria!by!degrading!steroids.!Pseudomonas&putida!KSI!(PpKSI)! is!strikingly!efficient!and!selective.!Three!putative!KSIs! identified!from!Structural!Genomics!were!analyzed!by!THEMATICS!(THEoretical!Microscopic!Anomalous!TItration!CurveS)!and!POOL!(Partial!OrderOptimum!Likelihood),!then!characterized!in&vitro!to!determine!their!KSI!activity.!A!putative!KSI!from!Mycobacterium&tuberculosis!(MtKSI)!was!predicted!to!have!ketosteroid!isomerase9like!activity!but!putative!KSIs!from!Mesorhizobium&loti!and!Pectobacterium&atrosepticum!were!predicted!by!these! methods! not! to! have! such! activity.! Biochemical! experiments! reveal! that! theputative! KSI! MtKSI! does! indeed! possess! KSI!activity,! although!with! reduced!efficiency! compared! to!PpKSI.! The! two!proteins!predicted! to!be!misannotated,!MlKSI! and!PaKSI,!exhibited!no!detectable!KSI!activity.!To! investigate! this! lower!efficiency! in! the!correctly!annotated!KSI,!we!engineered!the!MtKSI!active!site!to!resemble!more!closely!that!of!PpKSI,!and!found!that!most!of!these!mutations!alone!or!in!tandem!significantly!lowered!rather!than!increased!activity.!!!!

6! ! Genomic!Profiling!of!Bacterial!Strategies!for!Counteracting!Host!Defenses!in!Tuberculosis!

!Yanjia! J.! Zhang,! Thomas! R.! Ioerger,! Alissa! C.! Rothchild,! Curtis! Huttenhower,! Christopher! M.! Sassetti,! James! C.! Sacchettini,!Samuel!M.!Behar,!Eric!J.!Rubin!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Department!of!Immunology!and!Infectious!Disease,!Harvard!School!of!Public!Health,!Boston,!MA,!USA!!&

Mycobacterium&tuberculosis!(Mtb)!persists!as!a!key!global!health!pathogen!because!of!its!ability!to!counteract!host!immunity.!The!relative! importance!of!various! immune!compartments! to!Mtb!host! immunity! is!well!documented,!but! the!physical! stresses! they!impose!and!the!bacterial!determinants!of!surviving!these!stresses!have!not!been!extensively!characterized.!We!performed!a!series!of! genome9scale! screens! to! find! these! bacterial! determinants! of! counteracting! immunestresses,! sets! of! genes,! which! we! term!“counteractomes.”! The! CD4! counteractome—the! Mtb! genes! required! for! surviving! the! stresses! imposed! by! CD4! T! cells—was!particularly! interesting.! It!revealed!that!CD4!T!cells!attempt!to!starve!Mtb!of!an!essential!amino!acid,!tryptophan,!forcing!Mtb!to!make! its!own!tryptophan!to!survive! in&vivo.!We! leveraged!this!finding! to!design!a!novel! therapeutic!strategy,! showing! that!CD49mediated! tryptophan! starvation! can! synergize! with! small! molecule! inhibitors! of! bacterial! tryptophan! synthesis! to! kill! Mtb! in!infected!macrophages! and!mice.! Together,! our! findings! demonstrate! that! the!Mtb! determinants! for! surviving! host! immunity—Mtb’s! immune!counteractomes—serve!as!probes!of!host! immunity,!uncovering!immune9mediated!stresses!that!can!be!leveraged!for!therapeutic!discovery.!


33

7! ! Determining!the!location!of!virulence7associated!transcription!factors!in!Francisella&tularensis&

!Kathryn!Levasseur,!Cathy!Su,!and!Simon!Dove!Division!of!Infectious!Diseases,!Boston!Children’s!Hospital,!Boston!MA!02115!!The!regulation!of!virulence!factors! in!the!pathogen!Francisella&tularensis! is! incompletely!understood.!At!the!transcriptional! level,!the!proteins!MglA,!SspA,!and!PigR!are!thought!to!positively!control!a!common!set!of!genes!by!forming!a!complex!that!associates!with!RNA!polymerase.!However,!it!is!not!known!if!MglA,!SspA,!and!PigR!are!found!solely!at!promoters!of!regulated!genes.!It!is!also!unclear! whether! these! proteins! are! found! only! with! the! RNA! polymerase! holoenzyme! at! the! promoter,! if! they! associate! with!elongating!RNA!polymerase,!or!both.!To!address!these!questions,!we!performed!chromatin!immunoprecipitation!followed!by!next!generation! sequencing! (ChIP9Seq)! to! identify! the! genomic! DNA9binding! locations! of! the! MglA9SspA! complex,! PigR,! and! RNA!polymerase.! This! has! yielded! information! about! how! a! common! set! of! virulence9associated! genes! is! regulated.! Additionally,!we!have! performed! ChIP9Seq! on! other! putative! transcription! factors! implicated! in! virulence! gene! regulation.! These! studies! have!allowed!us!to!identify!promoters!across!the!genome,!as!well!as!given!us!a!more!in9depth!understanding!of!gene!regulation!at!the!level!of!transcription!in!F.&tularensis.!!!

8! ! Global!analysis!of!&Clostridium&difficile&sporulation!sigma!factors!using!RNA7Sequencing!

!

Kelly!A.!Fimlaid1,2,!Jeffrey!P.!Bond

1,!Kristin!C.!Schutz

1,!Emily!E.!Putnam

1,!Jacqueline!M.!Leung

1,2,!Trevor!D.!Lawley

3,!and!Aimee!Shen

1!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

1Department! of! Microbiology! and! Molecular! Genetics,! University! of! Vermont,! Burlington,! Vermont,! 05405,! USA;

2!Program! in!

Cellular,!Molecular!&!Biomedical!Sciences,!University!of!Vermont,!05405,!USA;!3Microbial!Pathogenesis!Laboratory,!The!Wellcome!

Trust!Sanger!Institute,!Wellcome!Trust!Genome!Campus,!Hinxton,!CB10!1SA,!United!Kingdom.!! The!Gram9positive!pathogen!Clostridium&difficile! is!a!leading!cause!of!health9care!associated!diarrhea!due!to!its!ability!to!produce!highly!transmissible!spores.!Since!C.&difficile! spores!are!resistant!to!most!antimicrobials,! they!persist! in!the!environment!and!can!cause!life!threatening,!recurrent!infections.!Despite!their!importance,!little!is!known!about!the!basic!biology!of!C.&difficile!spores.!In!particular,! the! transcriptional! program! activated! during! sporulation! has! notbeen! described! in!C.& difficile! or! any! other! clostridial!

species.! Based! on! studies! of! other! spore9forming! bacteria,! the! sigma! factors! σF,! σ

E,! σ

G,! and! σ

K! are! predicted! to! regulate! the!

transcriptional! program! of! C.& difficile.! To! identify! genes! induced! by! these! sigma! factors! during! sporulation,! we! generated!sporulation!sigma!factor!mutants!and!used!RNA9Seq!to!determine!their!transcriptomes,respectively.!This!analysis!revealed!that!C.&difficile!sporulation!is!regulated!differently!compared!to!the!model!spore9former!Bacillus&subtilis!and!other!Clostridium!spp.,!since!

σG! activity! requires! σ

F! for! post9translational! activation! but! does! not! require! σ

E! activity.! Overall,! σ

F,! σ

E,! σ

G,! and! σ

K,activated! 185!

genes,!including!many!that!are!unique!to!C.&difficile.!Further!characterization!of!these!spore!proteins!may!lead!to!the!development!of!novel!therapeutics!and!vaccines!that!can!control!C.&difficile&infections.!!!!!

9! ! Photodynamic!inactivation!of!anaerobic!bacteria!in!the!absence!of!oxygen!

!

Ardeshir!Rineh1,!George!P.!Tegos

2,!Conor!L.!Evans

3,4,!Michael!J.!Kelso

1!and!Michael!R.!Hamblin

3,4!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

1School!of!Chemistry,!University!of!Wollongong,!Wollongong,!Australia;!

2Department!of!Pathology,!School!of!Medicine,!University!

of!New!Mexico,!Albuquerque,!USA;!3Wellman!Center!for!Photomedicine,!Massachusetts!General!Hospital,!Boston,!Massachusetts,!

USA;!4Department!of!Dermatology,!Harvard!Medical!School,!Boston,!Massachusetts,!USA!!

Non9antibiotic! approach,! photodynamic! therapy! (PDT),! combines! a! nontoxic! photoactivatable! photosensitizer! (PS),!with! specific!wavelength! of! visible! light! to! inactivate! the!microbial! cells.! The! PDT!mechanism! is! based!on! the! ability! of! the! PS! to! absorb! the!energy!of!light!and!exited!to!triplet!state.!This!long9term!exited!PS!has!a!good!chance!to!interact!with!other!substrates!or!oxygen!molecule!to!produce!Reactive!oxygen!species!(ROS)!such!as!singlet!oxygen!and!free!radical!that!are!toxic!to!the!bacteria!cells.!The!exited! PS! can! return! to! the! ground! state! by! reaction!with! oxygen,! generating! oxygen! superoxide! radical! and! other! oxygenated!products!during!type!I!mechanism.!Type!II!mechanism,!however,!involves!an!energy!transfer!process!from!the!tripletstate!of!PS!to!ground9state!oxygen!giving!a!highly!reactive!singlet!state!of!oxygen.!Measuring!the!amount!of!fluorescence!released!from!proper!probes!during!interaction!with!PSs!is!one!of!theexperiments!to!understanding!the!photoreaction!pathway!mechanism.!Methylene!blue! for! instance! has! been! indicated! that! produces! radicals! through! both! type! I! and! type! II! mechanisms.! However,! some! new!derivatives!of!methylene!blue! like!EtNBS!has!been!revealed!produce!free!radicals! just!based!on!type!I!mechanism!in! low!level!of!


34

oxygen.! In!this!study,!we!used!EtNBS!asan!oxygen!independent!photosensitizer!for!photodynamic! inactivation!of!some!anaerobic!bacteria!like!S.&aureus!and!E.&coli!in!the!absence!of!oxygen!in!anaerobic!chamber.!!!

10! ! Epoxide7Mediated!Changes!in!OMV!Packaging!Affect!Pseudomonas&aeruginosa!Virulence!

!Alicia!E.!Ballok

!and!George!A.!O’Toole!

Department!of!Microbiology!and!Immunology,!Geisel!School!of!Medicine!at!Dartmouth!!&

Pseudomonas& aeruginosa! produces!membrane! blebs! or! outer!membrane! vesicles! (OMVs)! thatcontain! virulence! factors! that! are!important! for! pathogenesis! including!phospholipases,! alkaline!phosphatases! and! the!CFTR! inhibitory! factor! (Cif).! Cif,! an! epoxide!hydrolase,! is! transcriptionally! regulated!by!epoxides,!which! serve!as! ligands!of! the! repressor,!CifR.!We! tested!whether!epoxides!have!an!effect!on!Cif!level!in!OMVs.!We!identified!specific!epoxides!that!increased!Cif!packaging!into!OMVs!in!a!CifR9independent!fashion.!The!outer!membrane!protein,!OprF,!was!also!increased!in!OMVs!under!these!conditions.!Conversely,!proteins!such!as!the!phospholipases!were!reduced,!while!alkaline!phosphatase!activity!was!unaltered.!Additionally,!we!demonstrated!that!OMV!shape!and! density! were! also! affected! by! epoxide! treatment,! with! two! distinct! vesicle! fractions! present!when! cells! were! treated!with!epoxides.! Vesicles! isolated! from! the! two! density! fractions! exhibited! different! protein! profiles.! Furthermore,! we! identified!transposon!mutants!that!are!altered!in!OMV!packaging.!Finally,!epithelial!cell!cytotoxicity!was!reduced!in!the!vesicles!formed!in!the!presence!of!EBH.!Overall,!our!data!demonstrate!that!epoxides!have!profound!effects!on!vesicle!packaging!in!P.&aeruginosa,!and!that!there! may! be! differential! packaging! of! virulence! factors! in! OMVs,! which! has! functional! consequences! on! host9pathogen!interactions.! !!

11! ! Oligomeric!State!of!the!Mating!ATPase!ConE!!!&

!Georgeanna!M.!Morton1,!Kyle!J.!Swerdlow1,!and!!Melanie!B.!Berkmen1!!!!!! ! ! ! ! ! ! !!!!!!!!1Chemistry!and!Biochemistry,!Suffolk!University;!Boston!MA!!During!conjugation,!mating!pores!allow!bacteria&to!transfer!DNA!to!recipient!cells.!ConE!is!one!of!several!proteins!that!make!up!the!mating! pore! of!Bacillus& subtilis.! ConE,! and! its9characterized! homologs! VirB4! and! TrwK,! belongs! to! the! FtsK9HerA! superfamily! of!ATPases!which! contain! a! conserved! arginine! finger! that! is! a! good! predictor! for! ring! formation! and! inter9protomer! interactions.!Several! superfamily! members! have! been! purified! and! characterized! in& vitro;! they! form! monomers,! dimers,! and! higher! order!oligomers! in! solution! and! generally! form!hexamers!when! crystallized.!We! tested! if! ConE!oligomerizes! and!how! the!mutation!of!ConE’s! arginine! finger! affects! its! oligomerization.! His69ConE! and! mutant! His69ConE! (R726A)! were! purified! via! nickel! affinity!chromatography.! Purification!was!monitored! via! SDS9PAGE!and!multimeric! states!were!determined!by!BN9PAGE.!We! found! that!both!wild!type!and!mutant!His69ConE!form!monomers,!dimers,!and!higher!oligomers.!Addition!of!ATP,!single9stranded!DNA,!and!double9stranded!DNA!did!not!dramatically!alter!the!oligomeric!states.!!!!!

12! ! Development!and!Characterization!of!a!BSL72!Wistar!Rat!Model!of!Pulmonary!Mycobacterial!Infection!

!

Brandon! S.! Russell1,! Sasilada! Sirirungruang

2,! Nicola!M.! A.! Parry

3,! Megan! E.! McBee

1,4,! James! G.! Fox

1,3,5,! and! Peter! C.! Dedon

1,4,5!

1Department!of!Biological!Engineering,!MIT,!

2Department!of!Chemistry,!MIT,!

3Division!of!Comparative!Medicine,!MIT,!

4Infectious!

Disease!IRG,!Singapore9MIT!Alliance!for!Research!and!Technology,!5Center!for!Environmental!Health!Sciences,!MIT!!

!The!increasing!occurrence!of!multidrug9resistant!strains!of!Mycobacterium!tuberculosis!(Mtb)!poses!a!substantial!threat!to!global!public!health.!With!the!goal!of!accelerating!translational!research,!we!sought!to!develop!a!biosafety!level!2!(BSL92)!animal!model!of!pulmonary!Mycobacterium!infection.!Mycobacterium!bovis!bacille!Calmette9Guérin!(BCG)!was!chosen!as!aBSL92!surrogate!for!Mtb,!and!a! rat!model!was! chosen!due! to! low! cost,! availability!of! reagents,! and! larger! volume!of!bodily!fluids.! Thirty9four!69week!old!

outbred! female!Wistar! rats!were!dosed!enodotracheally!with!1x108!CFU!BCG.!Cohorts! (n=6)!were!necropsied!at!1,!2,!3,!4!and!8!

weekspost! infection.! The! success! rate! of! infection! was! 87.5%! (21/24),! and! bacillary! load! peaked! atweek! 1! with! an! average! of!

3.4x103! CFU! /! mg! lung! tissue.! Flow! cytometry! revealed! peak! innate! immune! activation! at! weeks! 394,! with! significant! (p<0.05)!

increases! in! granulocytes,!CD3+,MHCII

+,! and!CD11bc

+MHCII

+! cells.! Lung!histopathology! showed!extensive! inflammation!with!well!

organized,! coalescing! granulomas.! Forthcoming! serum! and! tissue! cytokine! profiles! willcomplete! our! analysis.! This! model! is!affordable,!easy!to!use,!and!displays!persistent!infection,!significant!immune!infiltration,!and!robust!pathology.!We!believe!it!will!be!


35

useful!for!drug!development!and!metabolomics!studies.!!!!

13! ! Structural!Elements!of!the!NRPS!Cyclization!Domain!and!its!Intermodule!Communication!Domain!

!

Daniel! P.! Dowling1,! Yan! Kung

2,! Anna! K.! Croft

3,! Koli! Taghizadeh

4,! Christopher! T.! Walsh

5,! and! Catherine! L.! Drennan

1,4,6!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

1Howard! Hughes! Medical! Institute,! Massachusetts! Institute! of! Technology,!

2Department! of! Chemistry,! Bryn! Mawr! College,!

3Department! of! Chemical! and! Environmental! Engineering,! University! of! Nottingham,!

4Center! for! Environmental! and! Health!

Sciences,! Massachusetts! Institute! of! Technology,!5Departments! of! Biological! Chemistry! and! Molecular! Pharmacology,! Harvard!

University,!6Departments!of!Chemistry!and!Biology,!Massachusetts!Institute!of!Technology!!

!Manipulation! of! known! natural! product! biosynthetic! gene! clusters! provides! an! attractive! method! for! generating! novel! “non9natural”!natural!products!for!drug!discovery,!and!an!increase!in!structural!information!of!how!these!systems!function!is!vital!for!the!design!of!efficient!systems.!Polyketide!synthases!(PKSs)!and!nonribosomal!peptide!synthetases!(NRPSs)!enable!theincorporation!of!acyl! and! amino! acid! groups! into! a! growing! chemical! chain.!Many! NRPS! products! contain! heterocycles,! which! confer! increased!compound! stability! in! addition! to! constraining! the! molecular! structure! to! a! bioactive! conformation.! One! such! system,!theepothilone! biosynthetic! cluster,! is! a! hybrid! PKS/NRPS! responsible! for! the! generation! of! theantitumor! epothilones.! Here! we!report! the! first! crystal! structure! of! an! NRPS! cyclization! domain,! that! of! the! EpoB! module! (EpoBcy),! which! catalyzes! both! the!formation!and!cyclization!of!Nacetyl9L9cysteine!to!a!thiazole.!This!structure!has!enabled!the!identification!of!active!sister!residues,!which!were!mutated!and!probed!for!product!formation!by!LC9MS.!Additionally,!thecrystal!structure!of!EpoBcy!represents!the!first!structural!depiction!of!an!intermodule!communication!domain!naturally!tethered!to!its!neighboring!protein!module,!and!molecular!dynamics!simulations!highlight!the!flexibility!of!the!communication!domain!with!respect!to!the!cyclization!domain.!!!!

14! ! Novel!self7recognition!genes!contribute!to!territorial!behavior!in!Proteus&mirabilis&

!Alecia!N.!Septer!and!Karine!A.!Gibbs!Molecular!and!Cellular!Biology,!Harvard!University!!!The! ability! to! distinguish! self! from!nonself! is! a! behavior! found! across! biology! and! is! required! for! territoriality.! In! the! bacterium!Proteus&mirabilis,!self!recognition!can!be!observed!when!kin!populations!merge!but!non9kin!populations!separate.!Previously,!the!idr!gene!cluster!wasidentified!in!a!screen!for!mutants!unable!to!recognize!their!parent!as!self,!however!the!specific!role!of!idr!in!self!recognition!is!unknown.!Given!that!IdrD!and!IdrE!are!related!to!proteins!that!function!as!toxin/anti9toxin!systems!in!bacteria!as!well!as!synaptic!partner!matching!and!trans9cell!signaling!in!neurons,!we!predict!that!the!idr!gene!products!may!have!similar!functions!in!P.mirabilis! self! recognition.!Consistent!with! idrD/idrE! encoding!a! toxin/anti9toxin! system,!expression!of! idrE& in& trans! in!an! idrDE9deficient!strain!is!sufficient!to!restore!a!merge!phenotype!with!the!parent.!Moreover,!while!the!parent!strain!territorially!excludes!non9kin!in!co9swarm!assays,!the!idrDE9deficient!strain!is!impaired!in!this!behavior,!and!heterologous!expression!of!idrE!in!a!non9kin!strain! is! sufficient! to! reverse! the! outcome! of! territorial! exclusion! with! the! idrDE+! parent.! Taken! together,! these! data! support!models! for! either! a! toxin/anti9toxin! or! partner9matching! function! for! IdrDE,! and! suggest! that! idr! genes! play! a! central! role! in!terriroriality.!&!!!

15! ! The!Cause!of!Superdormancy!in!Bacillus&subtilis!Spore!Germination!

!Sonali!Ghosh!and!Peter!Setlow!Department!of!Molecular,!Microbial!and!Structural!Biology,!University!of!Connecticut!Health!Center,!Farmington,!CT!!!Members!of!Bacillus&and&Clostridium!species!sporulate!under!adverse!conditions!and!the!resultant!spores!can!remain!metabolically!dormant! for! years.! However,! when! conditions! are! favorable,! spores! can! return! to! active! growth! through! the! process! of!germination.! Specific! nutrient! germinants! that! trigger! spore! germination! are! recognized! by! specific! germinant! receptors! (GRs).!There! is! tremendous!heterogeneity! in!the!germination!of! individual!spores! in!populations,and!the!reasons! for! this!heterogeneity!have! been! unclear.! In! this! study,! Bacillus& subtilis! spores! that! germinated! extremely! poorly! with! saturating! levels! of! nutrient!germinants,! termed! superdormant! (SD)! spores,!were! isolated! from! spore! populations.! The! germination! of! the! dormant! and! SD!spores! with! various! germinants! was! measured,! as! were! the! levels! of! a! number! of! germination! proteins! including! germinant!receptor!(GR)!subunits,!SpoVAD!and!GerD.!Levels!of!the!germination!proteins!GerD!and!SpoVAD!were!identical!in!dormant!and!SD!


36

spores.!However,!levels!of!subunits!of!a!GR!or!GRs!activated!in!SD!spore!isolation!were!69!to!109fold!lower!than!in!dormant!spores,!while! levels!of! subunits!of!GRs!not!activated! in!SD! spore! isolation!were!≤!2fold! lower.!These! results! indicate! that! low! level!of!a!specific!GR!or!GRs!is!a!major!cause!of!spore!superdormancy.!!!!

16! ! Mechanistic!understanding!of!microbial!desulferization&

!Andres!Abin9Fuentes

1,!Magdy!El9Said!Mohamed

2,!Kristala!LJ!Prather

1,!and!Daniel!IC!Wang

1!

1Department!of!Chemical!Engineering,!Massachusetts!Institute!of!Technology,!

2R&DC,!Saudi!Aramco!!

!Microbial! desulfurization! (BDS)! has! gained! interest! due! to! the! ability! of! certain! biocatalysts! to! desulfurize! compounds! that! are!recalcitrant!to!the!currently!employed!hydrodesulfurization!(HDS)!technology.!Two!of!the!major!obstacles!to!commercialization!of!BDS! are!mass! transport! limitations! and! the! inability! to!maintain! biocatalyst! activity! over! time.!Mass! transport! limitations!were!studied! in! a! small9scale! model! system! consisting! of! a! resting! cell! suspension! of! Rhodococcus& erythropolis! IGTS8! mixed! with!hexadecane! containing! dibenzothiophene! (DBT).! The! effective! diffusivity! of! the! substrate!within! the! biocatalyst! aggregates!was!

estimated!to!be600!times!smaller!than! its!diffusivity! in!water.! Intense!agitation!(over!10,000!W/m3)! is! required!to!overcome!the!

mass!transport!limitation!due!to!biocatalyst!aggregation,!which!may!besignificant!when!scaling9up.!Retention!of! the!product,!29hydroxybiphenyl! (HBP),!by!the!biocatalyst!was!correlated!with!reduction! in!biocatalyst!activity.!

Addition!of!an!HBP9selective!adsorbent!to!the!BDS!system!was!successful!in!reducing!the!HBP!retention!by!the!biocatalyst!109fold,!with!cytoplasmic!HBP!concentrations!reduced!to!approximately!260!μM.!Despite!reduced!HBPretention!within!the!biocatalyst,!the!biocatalyst! activity! could!not!be! improved! in! the!presence!of! the! resin!because!HBP! levels!under!100!μM!were! found! to! inhibit!three!of!the!four!pathway!enzymes.!!!

17! ! Pseudomonas! aeruginosa! Disrupts! Caenorhabditis! elegans! Iron! Homeostasis,! Causing! a! Hypoxic! Response! and!Death!!Natalia!V.!Kirienko1,2,!Daniel!R.!Kirienko1,2,!Gary!Ruvkun1,2,!Frederick!M.!Ausubel1,!1!Department!of!Molecular!Biology,!Massachusetts!General!Hospital,!2!Department!of!Genetics,!Harvard!Medical!School! The! opportunistic! pathogen! Pseudomonas! aeruginosa& causes! serious! human! infections,! but! effective! treatments! and! the!mechanisms!mediating!pathogenesis!remain!elusive.!Caenorhabditis&elegans&shares!innate!immune!pathways!with!humans,!making!it!invaluable!to!investigate!infection.!In!addition,!multiple!bacterial!virulence!factors!necessary!for!human!infection!play!roles!in!C.&elegans!pathogenesis.!To!determine!how!P.&aeruginosa!disrupts!host!biology,!we!studied!how!P.&aeruginosa! kills!C.&elegans! in!a!liquid9based!pathogenesis!model.!We!found!that!P.&aeruginosa9mediated!killing!does!not!require!quorum9sensing!pathways!or!host!colonization,!but!relies!on!two9component!sensor!KinB,!which!is!necessary!for!virulence!in!murine!and!zebrafish!infection!models.!We! used! the! liquid! C.& elegans–PA14! assay! to! carry! out! high9throughput! screening! of! chemical! libraries.! In! addition! to! known!antimicrobials!and!chemotherapeutics,!we!identified!that!iron!chelators!alleviate!P.&aeruginosa9mediated!killing.!Consistent!with!a!role! for! iron! in!P.&aeruginosa!pathogenesis,! the!bacterial! siderophore!pyoverdin!was!required! for!virulence!and!was!sufficient! to!induce! a! hypoxic! response! and! death! in! the! absence! of! bacteria.! Loss! of! the! C.& elegans! hypoxia9inducing! factorHIF91,! which!regulates! iron! homeostasis,! exacerbated! P.& aeruginosa! pathogenesis,! further! linking! hypoxia! and! killing.! As! pyoverdin! is!indispensable!for!virulence!in!mice,!pyoverdin9mediated!hypoxia!is!likely!to!be!relevant!in!mammalian!pathogenesis!as!well.!!!

18! ! Next7Generation!16s!Profiles!of!White!Band!Disease!in!the!Threatened!Coral!Acropora&Cervicornis!

!Sarah!Gignoux9Wolfsohn!and!Steven!Vollmer!Department!of!Marine!and!Environmental!Sciences,!Northeastern!University!!White!Band!Disease!(WBD)!has!lead!to!the!destruction!of!up!to!95%!of!the!Caribbean!corals!Acropora&cervicornis&and&A.&palmata&

since!its!discovery!in!1979,!spreading!rapidly!throughout!the!Caribbean!by!unknown!means.!We!have!confirmed!that!WBD!is!caused!by!a!bacterium,!but!the!specific!pathogen!is!still!unknown.!!Here,!we!examine!16s!next9generation!profiles!of!diseased!and!healthy!A.&cervicornis&collected!in!Bocas!Del!Toro,!Panama!from!2009!and!2010.!!The!V6!region!of!the!16srRNA!gene!of!54!samples!were!Illumina!sequenced.!!These!samples!were!collected!in!the!field!from!paired!healthy!and!diseased!corals!at!three!sites.!!Comparing!healthy! and!diseased! samples! across! sites! and! years,!we! see! significant!differences! in! the!bacterial! communities!of!healthy! and!diseased! corals! as! well! as! differences! between! sites! and! between! years.& Taxa! that! have! been! identified! as! significantly! more!


37

abundant!in!diseased!vs.!healthy!corals!are!considered!putative!pathogens.!!The!creation!of!multi9year!multi9site!16s!metagenomic!profiles! of! diseased! and!healthy!A.& cervicornis&greatly! inform!our! search! for! a!WBD!pathogen!and!understanding!of! the! shifting!bacterial!communities!of!WBD.!!!!

19! ! Mercury! resistance! of! two! magnetotactic! bacteria! strains! and! their! potential! ability! to! reduce! mercury! by!biosynthetic!magnetite!!Songnian!Liu!and!Heather!Wiatrowski!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!Biology!Department,!Clark!University!!Mercury!is!a!toxic!element!that!is!in!many!aquatic!environments!due!to!atmospheric!deposition.!Some!aquatic!bacteria!are!capable!of!reducing!ionic!mercury![Hg(II)]!to!the!less!toxic,!volatile!elemental!mercury![Hg(0)]!through!the!use!of!the!mercuric!reductase,!and! others! are! capable! of! reducing! Hg(II)! to! Hg(0)! through! the! production! of!minerals! containing! reduced! iron.!Magnetotactic!bacteria,!in!the!phylum!!Alphaproteobacteria,!are!capable!of!producing!magnetite!(Fe3O4)!in!chains!of!elongated,!iron9rich!crystals!called! magnetosomes.! Previous! research! demonstrated! synthetic! magnetite! can! reduce! Hg(II)! to! Hg(0).! In! this! study,!Magnetospirillum&gryphiswaldense&MSR91!and!Magnetospirillum&magnetotacticum&MS91!were!cultivated!with!2.5µM,!5µM,!10µM,!25µM,!and!50µM!Hg(II).!Relatively!high!mercury!resistance!was!demonstrated,!with!minimum!inhibitory!concentrations!of!10µM!and!25µM,!respectively.!Synthetic!magnetite!(0.067g/L!and!0.2g/L)!was!confirmed!to!reduce!40%!or!50%!Hg(II)!to!Hg(0)!in!30mM!PIPES! buffer! solution! (pH=6.7).! Although! both! strains! failed! to! reduce! Hg(II)! to! Hg(0)! in! culture,! we! will! determine! if! isolated!magnetosomes!can!reduce!Hg(II)!to!Hg(0).!!!

20! ! A!differential!fluorescence7based!genetic!screen!identifies!PepP!as!a!Listeria&monocytogenes&determinant!required!for!optimal!intracellular!replication!!Kyle!J.!Perry!and!Darren!E.!Higgins!Department!of!Microbiology!and!Immunobiology,!Harvard!Medical!School,!Boston,!MA!!&

Listeria&monocytogenes! is! a!Gram9positive! intracellular! pathogen! capable! of! causing! severe! invasive! disease!with! high!mortality!rates! in! humans.!While! previous! studies! have! largely! elucidated! the! bacterial! and! host! cell!mechanisms! necessary! for! invasion,!vacuolar! escape,! and! subsequent! cell9to9cell! spread,! the! L.& monocytogenes! factors! required! for! rapid! replication! within! the!restrictive!environment!of!the!host!cell!cytosol!are!poorly!understood.!Here!we!describe!adifferential!fluorescence9based!genetic!screen!utilizing!fluorescence9activated!cell! sorting! (FACS)!and!high9throughput!microscopy! to! identify!L.&monocytogenes&mutants!defective!for!optimal!intracellular!replication.!Bacteria!harboring!a!deletion!of!one!of!the!identified!genes,pepP,!were!defective!for!growth!in!primary!murine!macrophages,!plaque!formation!in!monolayers!of!L2!fibroblasts,!and!attenuated!during!in&vivo!infection!of! mice;! thus! validating! theability! of! the! screening! method! to! identify! intracellular! replication9defective! mutants.! Genetic!complementation!of! the!pepP! deletion! strain! rescued! the! in& vitro& intracellular! infection!and! in& vivo&virulence!defects.!Additional!characterization!of!pepP!deletion!and!catalytic!null!mutant!strains!suggest!PepP!functions!during!vacuolar!escape!and!cell9to9cell!spread.!Western!blotanalysis!of!listeriolysin!O!(LLO)!levels,!a!L.&monocytogenes&determinant!required!for!efficient!vacuolar!escape,!indicated! that!PepP! is! required! for!optimal! LLO!production.! These!findings!demonstrate! that! the!differential! fluorescence9based!screening! approach! provides! a! powerful! toolfor! the! identification! of! intracellular! replication! determinants! in! multiple! bacterial!systems!and!identifies!PepP!as!an!additional!regulator!of!LLO,!a!key!L.&monocytogenes&virulence!factor.!!!!

21! ! Intragenic!and!extragenic!suppressors!improve!the!sporulation!efficiency!of!Bacillus&subtilis&spoIIIEΔγ!mutants!

!Bijou!Bose,!Marina!Besprozvannaya,!and!Briana!M.!Burton!Department!of!Molecular!and!Cellular!Biology,!Harvard!University!!In!response!to!nutrient!starvation,!Bacillus&subtilis!undergoes!a!developmental!process!leading!to!the!formation!of!stress9resistant!spores.! Early! in! this! process,! asymmetric! septation! divides! the! cell! into! two! compartments.! The! smaller! compartment! or!“forespore”! eventually! becomes! the! spore,! and! the! larger! compartment! or! “mother! cell”! eventually! lyses.! The! asymmetric!septation! traps!~30%!of!one!chromosome! inside! the! forespore,!and! the! remaining!~70%!must!be!actively! transported! in!by! the!ATPase!SpoIIIE.!SpoIIIE!possesses!a!γ!domain!which!helps!orient!translocation!by!recognizing!specific!chromosomal!sequences.!Cells!producing!SpoIIIE!lacking!its!γ!domain!(SpoIIIEΔγ)!sporulate!significantly!less!well!than!wild!type.!We!found!that!when!spoIIIEΔγ!cells!were!subjected!to!successive!rounds!of!sporulation,!selection!for!spores,!and!outgrowth,!spontaneous!suppressor!mutations!arose!that!greatly!improved!sporulation!efficiencies.!We!isolated!some!suppressor!mutations!within!the!spoIIIEΔγ!!gene!and!others!that!


38

were! extragenic.! We! identified! mutations! in! the! extragenic! suppressor! strains! by! whole9genome! sequencing.! Several! of! these!mutations!appear!to!affect!levels!or!activity!of!the!mother9cell9specific!σ!factor,!σE.!Models!for!how!σE!might!influence!chromosome!translocation!during!sporulation!in!spoIIIEΔγ!cells!will!be!presented.!!!

22! ! Predictive!Models!of!Gene!Regulation!for!Mycobacterium&tuberculosis!

!Elham!Azizi

1,!Matthew!Peterson

2,!Antonio!Gomes

1,!Anna!Lyubetskaya

1,!and!James!Galagan

1,2,3,4!

1Bioinformatics!Program,!

2Biomedical!Engineering!and!

3Microbiology!Departments,!Boston!University,!

4Broad!Institute!of!Harvard!

and!MIT!!!We!have!generated!the!first!genome!scale!model!of!the!M.!tuberculosis!regulatory!network!and!combined!this!network!with!the!first! comprehensive! profiling! of! mRNA,! proteins,! metabolitesand! lipids! in! MTB! during! hypoxia! and! re9aeration.! Adaptations! to!hypoxia! and!metabolic! alterations! such! as! switching! to! catabolism! of! host! lipids! are! thought! to! play! a! prominent! role! in!MTB!pathogenesis.!To!date,!we!have!mapped!regulatory!bindings!of!more!than!100!Transcription!Factors!(TFs)!in!MTB!through!a!high9throughput!system!based!on!ChIP9Seq.!We!have!developedcomputational!predictive!models!of!gene!expression!by!integrating!this!network!with!transcriptomics!data!obtained!from!the!induction!of!the!same!factors.!Our!regulatory!models!areable!to!predict!gene!expression! in!hypoxic!conditions!as!well!as!expressions! in!knock9outs!of!particular!regulators.! In!order!to! identify!the!key!factors!contributing!to!metabolic!alterations,!we!have!also!coupled!regulatory!and!metabolic!models,!to!translate!the!predicted!impacts!of!TF!perturbations!to!the!level!of!metabolite!productions.!!

23! ! Targeted!protein!degradation!platform!for!tunable!control!of!synthetic!and!endogenous!bacterial!systems!

!Ewen!Cameron

1,2!and!Jim!Collins

1,2,3!

1Department! of! Biomedical! Engineering,! Boston,!

2Howard! Hughes! Medical! Institute,!

3Wyss! Institute! for! Biologically! Inspired!

Engineering,!Harvard!University!!!Tunable!control!of!protein!degradation!in!bacteria!would!expand!the!genetic!tool!set!available!to!probe!natural!cellular!systems!and!develop!synthetic!gene!circuits.!Here!we!use!components!of!the!Mesoplasma&florum!tmRNA!system,!identified!by!Gur!and!Sauer,!to!create!an!orthogonal!degradation!system!in!E.&coli.!Variants!with!mutations!in!non9overlapping!regions!in!the!protein!degradation!tag! (PDT)! provide! independent! control! of! both! the! initial! level! and! the! inducible! degradation! rate! of! the! targeted! protein.!We!demonstrate! the! system’s! use! in! synthetic! circuitdevelopment! and! in! exogenous! control! of! core! bacterial! processes,! including!peptidoglycan!biosynthesis,!cell!division!and!chemotactic!motility.!The!synthetic!degradation!system!is!facile!and!modular,!requiring!a! small! peptide! tag! and! a! single! protease! gene.! It! does! not! require! disruption! of! host! systems! to! function! and! can! likely! be!transferred!to!other!bacteria!with!minimal!modification.!!!

24! ! Levels! of! Germinant! Receptors! in! the! Bacillus! subtilis! Spore! Inner! Membrane! and! Their! Relationship! to! Spore!Germination!rates!!Kerry9Ann!V.!Stewart,!Xuan!Yi,!Sonali!Ghosh,!and!Peter!Setlow!Department!of!Molecular,!Microbial,!and!Structural!Biology,!University!of!Connecticut!Health!Center!!!Bacillus!spores!are!dormant!and!resistant!to!harsh!environmental!conditions,!but!can!revert!to!growing!cells!through!the!process!of!germination.!Bacillus!subtilis!spores!have!three!major!germinant!receptors! (GRs),!GerA,!GerB,!and!GerK,!each!composed!of!A,!B,!and!C!subunits,!that!trigger!germination!via!specific!and!different!nutrient!germinants!by!a!largely!unknown!mechanism.!GerA!and!GerB/GerK!are! located! in!the!spore! inner!membrane!(IM).!We!hypothesize!that!these!GRs!can! interact!with!each!other!and!thus!affect!rates!of!germination!when!one!or!more!GRs!are!overexpressed.!We!germinated!wild9type!and!various!GR!mutant!spores!with!a! range! of! nutrient! germinant! concentrations! and! determined! levels! of! GR! A! and! C! subunitsin! these! spores.! Additionally,! we!determined!the!number!of!these!molecules!in!wild9type!sporest!o!facilitate!establishment!of!mathematical!models!of!germination!kinetics!and! to!understand!GR!assembly!within! the! IM.!Our! results! indicate! that!a!GR’s! level!does!not! change!when!one!or! two!other!GRs!are!overexpressed,!although!there!can!be!dramatic!affects!on!rates!of!germination.!!!!


39

25! ! Environmental!influences!and!biotic!interactions!shaping!the!coral!holobiont!

!

Jessica!K.!Jarett1,!Cara!L.!Fiore

1,!Daniel!A.!Brazeau

2,!and!Michael!P.!Lesser

1!

1Department!of!Molecular,!Cellular,!and!Biomedical!Sciences,!University!of!New!Hampshire,!

2School!of!Pharmacy,!University!of!New!

England!!!Corals! reefs! are! one! of! the! most! diverse! ecosystems! on! earth.! Reef9building! corals! contain! photosynthetic! dinoflagellate!endosymbionts!(Symbiodinium!sp.),!and!diverse!communities!of!prokaryotes!that!are!believed!to!play!multiple!roles.!The!Caribbean!coral!Montastraea&cavernosa&hosts!intracellular!nitrogen9fixing!cyanobacterial!symbionts!in!some!colonies,!apparent!as!an!orange!colored!morph,!while! conspecifics! are! brown.!We! compared! the! genetic! diversity! of! coralhosts,! Symbiodinium,! and! prokaryotic!communities!in!the!same!coral!colonies!and!in!water!samples,!at!multiple!locations!across!the!Caribbean.!Prokaryotic!communities!were!profiled!using!16S! rRNA!454!pyrosequencing,!Symbiodinium!were! typed!using!cloning! libraries!and!sequencing!of! the! ITS!2!region! of! rRNA,! and! coral! hosts! were! genotyped! using! AFLP,! a! genetic! fingerprinting! technique.! Colony! color! did! not! affect!prokaryotic!or!Symbiodinium! communities,!but!differences!between! sampling! locations!were!highly! significant! for!Symbiodinium!and! prokaryotic! communities! from! corals! and! water.! Symbiodinium& and! prokaryotic! community! patterns! were! not! significantly!correlated,!and!coral!hosts!of!different! colors!and! from!different! locations! formed!genetically!distinct!populations.!We!conclude!that!environmental! factors!are!an! important! influence!on!coral9associated!prokaryotic! communities,!while! the!fine9scale!genetic!differences!in!Symbiodinium!and!coral!hosts!observed!in!this!study!appear!to!be!less!influential.!!!

26! ! Quorum!Sensing!of!Vibrio&harveyi:&An!in&vivo&Study!in!Shrimp!

!

Sidrotun!Naim1,3*

,!Kevin!Fitzsimmons1,!Donald!V.!Lightner

2!

1Department! of! Soil,! Water! &! Environmental! Science,! University! of! Arizona,! Tucson,! AZ,2! Department! of! Veterinary! Science! &!Microbiology,! University! of! Arizona,! Tucson,! AZ,! 3Center! for! Sustainable! Aquaculture! &! Pathology! Studies,! Surya! University,!Serpong,!Indonesia!&

Vibrio&harveyi! is!the!most!important!bacterial!pathogen!in!shrimp!(Penaeus&vannamei).!A!series!of!challenge!studies!using!shrimp!was! conducted! to! confirm! the! in& vitro! findings! from! the! last! two! decades,! that! Quorum! Sensing! (QS)! regulates! virulence! of!V.&harveyi.! The! mortality! rates! of! the! wild9type! and! QS! mutants! suggest! that! QS! defined,! when! specific! virulence! genes! were!expressed!or!repressed.!The!final!mortality!rates!of!the!wild9type!(75%),!LCD!(80%),!and!HCD(90%),!show!that!V.&harveyi!mutants!that!are!unable!to!appropriately!control!the!timing!of!QSgene!expression!are!nonetheless,!virulent.!As!QS!in!V.&harveyi!consists!of!three!different!circuits,!further!experiments!deployed!six!mutants!lacking!either!a!synthase!or!a!receptor!for!each!circuit.!Mortality!rates!in!different!groups!indicated!that!the!CAI91!circuit!is!the!most!crucial!for!virulence,!followed!by!AI92!and!HAI91.!Microbiology,!histopathology,! and! PCR! analysisconfirmed! the! presence! of! V.& harveyi& in! the! hemolymph! of! dead! shrimp.! To! the! best! of! the!authors’! knowledge,! this! is! the! first! report! that! the! CqsS! gene! in! the! CAI91! cascade! is! the!most! important! determinant! for! the!virulence!of!V.&harveyi&in!shrimp.!!!

27! ! Plasmid! encoded! quorum! sensing! regulators! control! competence! development! and! sporulation! in!Bacillus&subtilis&!!Kristina!Boguslawski!and!Kevin!Griffith!Microbiology!Department,!University!of!Massachusetts9Amherst!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Roughly!half!of!the!bacterial!diversity!in!the!human!intestinal!tract!is!unable!to!be!cultured!in!the!laboratory.!Due!to!the!role!of!the!human!microbiome!in!health!and!disease,!there!is!substantial!interest!in!its!characterization;!however,!to!study!these!species!they!must! be! cultivated! in& vitro.! It! is! known! that! bacterial! species! rely! on! host! organisms! for! growth! factors! absent! from! laboratory!media.! Similarly,! many! processes! in! bacteria! are! regulated! by! autoinduction! –! a! phenomenon! governed! by! concentrations! of!secreted!signaling!molecules.!Here!we!hypothesize!that!human!hormones!and!bacterial!quorum!sensing!factors!may!be!essential!growth!factors!for!this!missing!diversity.!We!tested!this!hypothesis!by!comparing!total!colony!forming!units!of!diluted!fecal!samples!grown!with!and!without!the!proposed!growth!factors,!and!significantly!more!colonies!grew!on!plates!containing!autoinducer92!(AI92),! cyclic9adenosine!monophosphate! (cAMP),! and! norepinephrine.! Colonies! grown!with! added! AI92,! cAMP,! and! norepinephrine!were! identified! by! 16S! rRNA! sequence,! revealing! the! presence! of! several! novel! species,! including! representatives! seen! on! the!Human! Microbiome! Project’s! “Most! Wanted! Genome”! list.! These! results! demonstrate! that! the! addition! of! bacterial! and! host!signaling!factors!to!laboratory!medium!may!increase!the!recovery!of!previously!uncultured!bacteria!from!the!human!gut.!!


40

28! ! CACAO:!Linking!evidence7based!gene!annotation!to!education!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

!

Brenley!McIntosh1!and!Jim!Hu

1!!

1Department!of!Biochemistry!&!Biophysics,!Texas!A&M!University!!!The!Gene!Ontology!(GO)!project!provides!a!set!of!terms!describing!the!attributes!of!genes!and!their!products!that!greatly!simplifies!their!annotation,!but! information!available!about!protein!function!greatly!exceeds!the!current!rate!of!manual!curation.!Here,!we!describe! the! CACAO(Community! Assessment! of! Community! Annotation! with! Ontologies),! which! links! functional! annotation! to!education.!Teams!of!students!compete!over!multiple!rounds!to!add,!review!and!refine!functional!annotations!that!comply!with!the!standards! of! the! Gene! Ontology! (GO)! Consortium.! During! each! round,! teams! have! one! week! to! find! scientific! papers,! analyze!experiments!and!add!functional!annotations!to!protein!pages!on!GONUTS!(http://!gowiki.tamu.edu)!and!are!given!points!for!each!complete!annotation.!During!the!second!week!ofa!round,!teams!challenge!other!teams’!annotations,!performing!peer!review!of!the!understanding!of!experiments!cited!as!well!as!an!opportunity!to!steal!points!for!identifying!and!correcting!errors.!So!far,!CACAO!has!been! incorporated! into! genomics,! genetics,! cell! biology! and! other! courses! at! more! than! twenty! campuses! in! the! US! and! UK.!Assessment! of! the! quality! and! quantity! of! curation! by! students! by! experienced! curators! will! be! described! and! has! shown! that!thousands!of!annotations!are!suitable!for!deposition!with!the!GO!Consortium!and!other!major!database!resources.!!!

29! ! Converting!a!thiol!reductase!(DsbG)!into!a!disulfide!bond!isomerase!(DsbC)!using!a!novel!in&vivo!genetic!screen!

!

Claire!Chatelle1,!Stéphanie!Kraemer

1,2,!Caroline!Roggemans

1,3,!Na!Ke

1,!Paul!Riggs

1,!Mehmet!Berkmen

1!

1New!England!Biolabs,!Ipswich,!MA;!

2Firalis,!Huningue,!France;!

3Novartis,!Basel,!Switzerland!!

!The! post9translational! formation! of! disulfide! bonds! is! essential! for! the! folding! of!many! non9cytoplasmic! proteins.! However,! the!correct!pairing!of!cysteines!in!a!protein!with!multipledisulfide!bonds!is!inherently!fraught!with!error.!In!the!periplasm!of!E.&coli,!a!protein!containing!multiple!cysteines!can!be!mis9oxidized!by!DsbA,!resulting!in!misfolding,!low!yields!and!diseasein!organisms.!Mis9oxidized!proteins!require!the!corrective!action!of!a!disulfide!bond! isomerase,!DsbC.!Studying!the!mechanistic!details!of!oxidative!protein!folding!in&vivo!is!a!challenging!process.!Indeed,!although!disulfide!bond!isomerization!has!been!demonstrated!in&vitro!since!the!1960’s,!the!in&vivo!mechanism!is!still!not!fully!understood.!!

We! have! developed! a! genetic! approach! to! elucidate! the!mechanistic! details! of! disulfide! bond! isomerization! in!E& coli.! The!result!is!the!first!genetic!blue/white!screen!using!mutant!PhoA*,!which!permits!the!detection!of!disulfide!bond!isomerase!activity!in&vivo.!Using!this!screen,!wehave!isolated!mutants!of!DsbG!(a!paralogue!of!DsbC)!with!new!isomerase!activity.!!

Our!results!indicate!that!single!amino!acids!substitutions!in!DsbG!are!sufficient!to!convertthis!disulfide!bond!reductase!into!an!isomerase.!Using!biochemical!characterization,!we!have!separated!the!disulfide!bond!isomerization!and!reduction!activities!of!these!enzymes!with!single!point!mutations.!Our!results!demonstrate!the!effectiveness!of!this!screen!and!open!the!pathway!toisolating!novel!redox!enzymes!and!elucidate!mechanisms.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

30! ! Structural!insight!into!anaerobic!toluene!activation!by!benzylsuccinate!synthase!

!

Michael!A.!Funk1,!E.!Neil!G.!Marsh

2,!Catherine!L.!Drennan

1,3,4!

Departments! of!1Chemistry! and!

3Biology,! Massachusetts! Institute! of! Technology;!

2Department! of! Chemistry! and! Biological!

Chemistry,!University!of!Michigan;!4Howard!Hughes!Medical!Institute!Investigator!!

!Toluene! is! an! abundant! pollutant! that! is! consumed! by! certain! microorganisms! as! a! source! of! energy! and! carbon.! Anaerobic!degradation!of!toluene!by!Thauera&aromatica!is!initiated!by!benzylsuccinate!synthase!(BSS),!a!member!of!the!glycyl!radical!enzyme!(GRE)! family.! BSScatalyzes! the! addition!of! toluene! to! fumarate! through! several! radical! intermediates! to! form!benzylsuccinate,! a!product! that! can! be! further! degraded! through! anaerobic! respiration.! To! better! understand! the! mechanistic! details! of! the! BSS!reaction,!we!used!X9ray! crystallography! to!determine! the! structure!of! the!BSS! catalytic! subunit,! thus! illuminating! the!active! site!architecture.!Substrate!soaking!experiments!reveal!a!tight!binding!site!for!fumarate!at!the!core!of!the!enzyme,!with!a!distinct!set!of!residues! conserved! within! this! class! of! GREs.! A! binding! site! for! toluene! was! identified! based! on! soaking! experiments! and!computational! docking.! These! snapshots! of! BSS! reveal! how! the! enzyme! senses! substrates! and! controls! the! reactive! radical!intermediates! required! for! this! amazing! transformation.! BSS! is! the! first! structurally! characterized! member! of! the! Xsuccinate!synthase!subclass!of!GREs,!and!structural!modeling!of!related!enzymes!reveals!howthese!enzymes!selectively!perform!chemistry!on!a!range!of!substrates,!opening!the!door!for!engineering!on!this!powerful!class!of!enzymes.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!


41

31! ! The!Effects!of!DinD!on!RecA!structures!using!Fluorescent!Microscopy!!!

!Amy!Nwaobasi1,!Steven!Sandler!2!! ! ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! ! !!!!!!!!!!!1Biochemistry!and!Molecular!Biology!Department,!University!of!Massachusetts!Amherst,!2Microbiology!Department,!University!of!Massachusetts!Amherst!!Homologous!Recombination!is!important!for!creating!genetic!diversity,!repairing!breaks!in!the!DNA!and!broken!replication!forks.!In!Escherichia& coli,! RecA! plays! an! essential! role! in! recombination! and! regulation! of! the! SOS! Response! (a! major! response! to! DNA!damage).!SOS!proteins,!DinD!and!DinI,!modulate!the!activity!and!stability!of!RecA!structure!in!vitro.!DinD!is!thought!to!disassemble!RecA! filaments! in! the! process! of! recombination! while! DinI! can! either! stabilize! (low! ratio)! or! de9stabilize! (high! ratio)! of! RecA!filaments!depending!on! its! concentration.!Proteolysis! in!bacteria! is! important! in! regulating!processes!by!degrading!key!proteins.!ClpXP!and!Lon!are!proteases!that!have!been!shown!to!degrade!DinID!and!RecA!respectively!during!the!SOS!Response.!This!work!explores!the!relationship!between!DinI,!DinD,!ClpP,!Lon!and!RecA!in&vivo!in!log!phase!cells!and!lexA!defective!cells!(SOS!constitutive!expression)!using!quantitative!microscopy!of!structures!made!by!RecA9GFP.!!!!

32! ! The!Transpeptidase!Landscape!of!Mycobacterium&tuberculosis&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&!

!

Maia!Schoonmaker1,!William!Bishai

1,2,!Gyanu!Lamichhane

1!

1Center!for!Tuberculosis!Research,!Johns!Hopkins!University!School!of!Medicine,!1550!OrleansSt,!Baltimore,!MD!21231.!

2Kwazulu9

Natal!Research!Institute!for!Tuberculosis!and!HIV,!Durban!4001,!South!Africa!!The!peptidoglycan!layer!is!a!vital!structure!of!the!bacterial!cell!wall!and!is!essential!for!both!growth!and!survival.!Targeted!by!one!of!the!most!commonly!used!antibiotics,!namely!the!βlactams,!peptidoglycan!synthesis!is!blocked!at!the!final!step!of!the!biosynthesis!pathway.Classically,!4,39transpeptidases!catalyze!this!final!cross9linking!step!that! is! targeted.!However,! the!existence!of!a!second!class! of! transpeptidase,! the! 3,39transpeptidase,! was! recently! discovered.!Mycobacterium& tuberculosis,! the! etiological! agent! of!Tuberculosis,! is! known! to! possessredundant! 3,39transpeptidases.! Here,! we! demonstrate! that! loss! of! LdtMt1! and! LdtMt2,! two!known!3,39transpeptidases,! leads! to!alterations! in! the!cell! surface!morphology,! the! intracellular!morphology,!cellular!physiology,!growth!kinetics!and!virulence!of!Mycobacterium&tuberculosis.!Furthermore,!we!show!that!a!combined!loss!of!LdtMt1!and!LdtMt2!leads!to!susceptibility!to!both!βlactam!and!glycopeptide!antibiotics,!neither!of!which!have!been!shown!to!have!activity!against!wild9type!Mycobacterium& tuberculosis.! These! findings! suggest! that! a! combination! of! 3,3transpeptidase! inhibitors! and! peptidoglycan!targeting!antibiotics!could!effectively!treat!tuberculosis.!!!!

33! ! Mercury!Challenge!to!Dissimilatory!Metal!Reducing!Bacteria!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

!Yingjiao!Wang,!Tyler!Robison!and!Heather!Wiatrowski! ! !!!!!!!!!!!!!!!!!!!!Biology!department,!Clark!University!!!Despite!detailed!knowledge!about!how!Hg!damages!mammalian!cells,!we!know!nearly!nothing!about!the!mechanisms!of!Hg!toxicity!in!bacteria.!This! is!critical!because!bacteria!are!responsiblefor!transforming!Hg(II)! to!the! less!toxic,!volatile!Hg(0),!and!the!potent!neurotoxic!substance,!MeHg.!We!explored!the!effects!of!Hg(II)!on!glutathione!and!thioredoxin!in!two!dissimilatory!metal!reducing!bacteria,!Shewanella&oneidensis&MR91!and!Geobacter&sulfurreducens!PCA.!MR91!responded!differently!to!Hg(II)!in!fumarate!reducing!conditions!and!aerobic!conditions.!The!minimum!inhibitory!concentrations!were!0.25µM!under!aerobic!conditions!and!2µM!under!fumarate!reducing!conditions.!Without!Hg(II)!exposure,!MR91!produced!more!total!glutathioneunder!aerobic!conditions!than!under!fumarate!reducing!conditions!(21.83±1.04!and!16.47±0.23!nmol!GSH/µg!protein,!respectively).!After!a!one!hour!exposure!to!0.5µM!Hg(II),!totalglutathione!dropped!to!8.66±0.97!nmol!GSH/µg!protein!under!fumarate!reducing!conditions!with73.38±14.83%!survival!cells,! but! it! remained! unchanged! under! aerobic! conditions!with! 27.68±7.92%! survival.! In! addition,!more! intracellular! Hg(II)!was!detected!under! fumaratereducing! conditions! (19.03±2.85%)! than!under!aerobic! conditions! (13.55±0.98%).!When! [Hg(II)]>0.5µM,!there!was!no!glutathione!under!aerobic!conditions!and!<1%!cells!survived.In!PCA,!Hg(II)!treatment!with!Hg(II)!caused!the!oxidation!of!thioredoxin.!However,!in!MR91,!thioredoxin!remained!unchanged!by!Hg(II)!treatment.!!!!!!!


42

34! ! A!New!Method!for!Fermentation!Process!Optimization!of!a!Complex!System!

!Peng! Jiao,! Ray! Li,!Nida! Sayed,!Wenglong! Lin,!Maureen!Hamilton,!Michelle! Chopra,! Seann!O’Connell,! Khursheed!Karim,! Sangram!Yadav,!Sohan!Patel,!Longkuan!Xiang!&!Steven!Harris! ! ! ! ! ! ! !!!!!! ! ! ! ! ! !!!!!!Lonza!Biologics!Inc.,!Fermentation!R&D,!Hopkinton,!MA!!!Design!of!Experiments!(DoE)!has!been!widely!used!for!fermentation!development!and!optimization.!However,!traditional!statistical!DoE!methods!can!be! time9consuming!and!cost!prohibitive! for!complex!systems!with!over!20! factors!due! to! the! large!number!of!experimentalruns.!A!novel!methodology!based!on!system!theories!and!principles,!rather!than!traditional!statistics9based!DOE,!was!used! to! optimize! a! complex! fermentation! process!with! 66! parameters! (including! base!media,! feed! solutions,! and! fermentation!operation!parameters!with!a! Lonza!proprietary!E.! coli! XS!expression! strain).!A!49phase! study! including14! fermentation! runs!was!executed.!The!optimized!conditions!resulted!in!a!39fold!increase!in!the!target!protein!titer!(from!4.2! g/L! at! initial! baseline! conditions! to! 13.2! g/L! As! a! comparison,! an! independent! study! using! statistical! DoE!methodology!was!executed!to!optimize!the!target!protein!productivity!by!evaluating!base!media!and!process!conditions.!This!DoE!approach,!which!included!only!6! factors!and!a! total!of!20!runs,! increased!the!target!protein!yield! to!10!g/L! from!the!baseline!conditions.!Overall,!even!with!relatively!fewer!runs,!the!novel!methodology!enabled!optimization!of!acomplex!system!with!66!parameters!and!achieved!higher!titer!compared!to!the!optimization!performed!using!the!traditional!statistics!DoE!method.!!!!

35! ! A!Spectrum!of!CodY!Activities!Prioritizes!Gene!Expression!and!Metabolism!in!Bacillus&subtilis&!

!S.!R.!Brinsmade

1,!E.!L.!Alexander

2,!J.!Livny

3,!Arion!I.!Stettner

4,!Daniel!Segrè

4,!5,!K.!Y.!Rhee

2,!A.!!

L.!Sonenshein1!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!!!!!!!!!!!!!!!!!!!!!!

1Tufts!Univ.! Sch.! of!Med.,! Boston,!MA,!

2Div.! of! Infectious!Diseases,!Dept.! of!Med.,!Weill! Cornell!Med.!Coll.,!New!York,!NY,!

3The!

Broad!Inst.!of!MIT!and!Harvard,!Cambridge,!MA,!4Departments!of!Biomedical!Engineering!and!

5Biology,!Boston!University,!Boston,!

MA!!!Global! regulators! that! bind! key! metabolites! allow! bacteria! to! rapidly! adapt! to! dynamic! environments! by! coordinating! the!expression! of! many! genes.! In! Bacillus& subtilis,& CodY! controls! dozens! of! genes,! but! the! threshold! activities! of! CodY! required! to!regulate!each!gene!are!unknown.!We!hypothesized!that!targets!of!CodY!are!regulated!based!on!affinity!for!each!binding!site.!Using!RNA9Seq,!we!profiled!the!transcriptomes!of!a!panel!of!B.&subtilis&mutant!strains!expressing!CodY!proteins!with!different! levels!of!residual! activity.! In! parallel,!we!used!masss!pectrometry9based!metabolomics! to!quantify! intracellular!metabolites! connected! to!central!metabolism.!196!genes!were!overexpressed!and!27!genes!were!underexpressed!>39fold! in! thecodY&null!mutant!strain.! In!strains!producing!CodY!variants!F71Y,!R61K!and!R61H,!with!varying! residual!activity,! the!patterns!of! transcript!abundances!were!consistent! with! a! hierarchical! organization! of! gene! expression.! Using! LC9MS,! we! detected! significant! increases! in! valine! and!intermediates! in!arginine,!proline!and!glutamate!metabolism,!as!well! as!decreases! in!pyruvate,!malate!and!cis9aconitate! in!codY!mutant! strains.!We! conclude! that! variations! in! CodY! activity! during! changes! in! nutrient! availability! in! the! environment! lead! to!sequential!expression!of!groups!of!genes!and!to!an!apparent!re9routing!of!key!metabolic!pathways.!!!!

36! ! Hypermutation!in!Experimentally!Evolved!Vibrio&!

!

Sean!Clarke1,!Sonia!Timberlake

1,!Arne!Materna

2,!Eric!Alm

1!! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

1Biological!Engineering,!MIT,!

2CLC!Bio!!

!Mutator! bacteria! are! known! to! arise! in! wild,! clinical,! and! experimentally! evolved! populations,! but! the! extent! of! their! adaptive!contribution!is!unknown.!In!the!work!here,!we!evolved!a!wild!Vibrio&splendidus&isolate!and!found!unprecedented!levels!of!mutation!in! three!of!nine! lineages.! These!mutator! lines! arose! independently,! and!each!of! them!accumulated!more! than!1500! single9base!mutations.!By!comparison,!there!are!only!302!single9base!differences!between!the!ancestor!strain!and!another!strain!isolated!in!the!wild.! Hypermutation! was! associated! with! a! deletion! resulting! from! improper! prophage! excision.! Members! of! this! family! of!prophages!are!found!in!other!proteobacteria,!including!well9studied!human!pathogens,!from!very!different!environments.!We!have!preliminary!evidence!that!this!mechanism!of!evolution!could!be!relevant!in!the!environment,!where!horizontal!gene!transfer!and!mobile!elements!play!known,!significant!roles!in!bacterial!evolution.!!!!


43

37! ! DNA! segregation! on! sticky! treadmills:! a! plasmid! centromere! promotes! nucleation,! stability,! and! bundling! of! the!actin7like!filament!AlfA&!

Jessica!K.!Polka1,!R.!Dyche!Mullins

2!!! ! ! ! ! ! ! ! ! ! ! ! ! ! !!!!!!!!!!!!!!!!

1Department!of!Systems!Biology,!Harvard!Medical!School,!

2Department!of!Cellular!and!Molecular!Pharmacology,!UCSF!&

!In! bacteria,! some!plasmids! are! partitioned! to! daughter! cells! by! assembly! of! actin9like! proteins(ALPs).! The! best! understood!ALP,!ParM,! relies!on!a!core!set!of!properties! for! its! function,! including:!dynamic! instability,! spontaneous!nucleation,!and!bidirectional!elongation.!AlfA,!an!ALP!that!pushes!plasmids!apart!in!Bacillus,!relies!on!a!different!set!of!underlying!properties!to!segregate!DNA.!AlfA!elongates!unidirectionally!and!is!not!dynamically!unstable;!its!assembly!and!disassembly!are!regulated!by!a!cofactor,!AlfB.!Free!AlfB! breaks! up! AlfA! bundles! and! promotes! filament! turnover! but,! when! bound! to! the! centromeric! DNA! sequence,! parN,! AlfB!nucleates! and! stabilizes!AlfA!filaments.!When! reconstituted! in& vitro,! this! system!creates!polarized,!motile! comet! tails!which! can!associate!by!antiparallel!filament!bundling!to!formbipolar,!DNA9segregating!spindles.!!!

38! ! Programmable!Biofilm7Based!Materials!from!Engineered!Curli!Nanofibers!

!Peter!Q.!Nguyen1,2,!Zsofia!Botyanszki2,3,!Pei!Kun!R.!Tay2,4!!and!Neel!S.!Joshi1,2!1Harvard! University! School! of! Engineering! and! Applied! Science,! 2Wyss! Institute! for! Biologically! Inspired! Engineering,! 3Harvard!University!Department!of!Chemistry!and!Chemical!Biology!4Harvard9MIT!Health!Sciences!and!Technology!!The! vast! majority! of! research! on! bacterial! biofilms! has! focused! on! preventing! their! formation! and! promoting! their! dispersal.!However,!this!has!resulted!in!an!overlooked!opportunity!to!develop!biofilms!as!functional!materials.!Here!we!demonstrate!a!new!technology! platform,! Biofilm9Integrated! Nanofiber! Display! (BIND),! in! which! we! genetically! engineer! the! display! of! functional!peptides! on! the! curli! system! of! E.! coli.! !In! our! platform,! the! bacterium! acts! as! a! molecular! foundry! for! the! generation! of! a!programmable!extracellular!nanofiber!scaffold!in!biofilms!that!can!be!designed!with!various!physicochemical!properties.!We!utilize!a!novel!fusion!site!on!the!structural!curli!gene,!csgA,!to!create!a!library!of!functional!CsgA9peptide!chimeras!with!a!range!of!peptide!sizes! and! secondary! structures.! Furthermore,! we! establish! the! kinetics! and! stability! of! the! BIND! nanofibers! and! show! that! the!peptides!are!fully!displayed,!as!designed.!Finally,!we!demonstrate!peptide!domain!functionality!in!BIND!biofilms!was!retained!after!assembly!into!biofilm!matrices.!We!envision!BIND!will!have!broad!applications!in!nanoelectronics,!industrial!biocatalysis,!optically!active!biological!coatings,!biosensors,!microbial!fuel!cells,!and!bioremediation.!!!

39! ! The! probiotic! bacterium,!Phaeobacter& gallaeciensis! S4,! down7regulates! virulence! factor! production! in! the! oyster!pathogen,!Vibrio&tubiashii!!Wenjing!Zhao1,!Christine!Dao2,!David!C.!Rowley2,!and!David!R.!Nelson1!!! ! ! ! ! ! ! !!!!!!!!!!!!!1Department!of!Cell!and!Molecular!Biology,!2Biomedical!and!Pharmaceutical!Sciences,!University!of!Rhode!Island,!Kingston,!RI!!&

Phaeobacter!gallaeciensis!S4!is!regarded!as!a!potential!probiotic!against!oyster!pathogen,!Vibrio&tubiashii.!S4!is!an!excellent!biofilm!former! and! able! to! produce! an! antibiotic,! tropodithetic! acid! (TDA).! These! two! factors! play! important! roles! in! probiotic! activity.!Recently,!we!found!that!culture!supernatant!of!S4!down9regulated!protease!activity!in!V.&tubiashii!cultures.!We!examined!effects!of!S4!supernatant!on!the!transcription!of!several!genes!that!are!involved!in!protease!activity!including!vtpA,!vtpB,!and!vtpR!(encoding!metalloprotease!A!and!B!and!transcriptional!regulator!of!metalloproteases,!respectively)!by!qRT9PCR.!Expression!of!vtpB!and!vtpR!were!reduced!by!40.6%!and!29.1%,!respectively,!compared!to!untreated!control.!In!contrast,!expression!of!vtpA!was!not!affected.!Additionally,!we! examined! effects! of! S4! supernatant! upon!other! genes! including! vthBA! (V.& tubiashii! hemolysin! genes)! and!vanT!(vtpR!homolog!in!V.&anguillarum).!The!culture!supernatant!did!not!affect!transcription!of!these!genes.!S4!supernatant!was!extracted!with!non9acidified!ethyl!acetate!and!protease!inhibitory!activity!showing!in!organic!phase.!These!results!suggest!that!S4!excretes!a!small!nonpolar!molecule,!not!TDA,!that!specifically!inhibits!protease!a!activity!of!V.&tubiashii.!This!compound!may!act!by!disrupting!the!quorum!sensing!pathway!that!activates!protease!transcription!of!V.&tubiashii.!!!!!!!!


44

40! ! Defining!genes!implicated!in!a!gene!addition!process!in!Escherichia&coli!

!Chloe!Roussel99Rossin1,!Elisabeth!Raleigh1!! ! ! ! ! ! ! ! ! ! ! ! !!!!!!!!!!!!!!!!!!1Research,!New!England!Biolabs!!Non9homologous!gene!exchange!mechanisms!allow!bacteria!to!acquire!new!genes,!providing!them!niche9adaptative!functions!like!pathogenesis.!Studying!the!mechanisms!of!horizontal!gene!transfer!(HGT)!contributes!to!discovery!of!new!gene!functions.!To!study!gene!addition,!we!focused!on!the!Immigration!Control!Region!(ICR),!a!region!coding!for!endonucleases!in!E.&coli.!Genetic!analyses!indicated! an! important! allelic! diversity! inside! the! ICR,! but! highly! conserved! genes! adjacent! to! it.! These! conserved! genes! could!promote! HGT.! We! are! investigating! the! potential! role! of! two! of! these! genes:! yjiA,! just! next! to! the! ICR;! and! yjiP,! a! candidate!transposase!nearby.!

To!ask! if! these! influence!gene!addition!during!HGT,!a!rhamnose9regulated!overexpression!system!was!used!to!produce!the!protein!of!interest!only!in!presence!of!rhamnose.!!We!determined!how!long!it!takes!for!a!LacZ!reporter!to!be!maximally!expressed!once!induced!and!whether!YjiA/YjiP!overexpression!is!toxic.!

We! arranged! a! conjugal! cross,! introducing! a! donor! ICR! with! a! gene! coding! for! tetracycline! (TetR)! into! a! nalidixic! acid!(NalR)!!recipient.!Recombinants!are!TetR,!NalR.!

Mating!experiments!were!carried!out!when!YjiA/YjiP!expression!in!the!recipient!was!maximal.!YjiP!overexpression!increased!the!frequency!of!recombinants.!Genetic!classification!of!recombinants!is!underway!!!

41! ! Oligomerization!and!assembly!of!a!DNA!translocation!channel&

!Kyle!J.!Swerdlow1,!Gianna!R.!Mancuso1,!April!Culananay1,!Stefanie!Baril1,!Melanie!B.!Berkmen1!!!!!!!!!!!!!!!! !!!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!! ! !!!!!!!!!!!!!!!!!!!!!!!

1Department!of!Chemistry!and!Biochemistry,!Suffolk!University,!Boston,!MA!!!Mating,! or! conjugation,! is! the! process! by! which! a! cell! horizontally! passes! genetic!information!to! another! cell! through! a! multi9protein! DNA! translocation! channel.!ConE!is! a! key! ATPase! of! the! mating! machinery! of! ICEBs1,! the! Integrative! and! Conjugative!element!of!Bacillus&subtilis.!Characterized!homologs!form!a!variety!of!oligomers!in!solution!and!crystallize!as!hexamers.!Previously,!we!showed!that!purified!His69ConE!is!a!mixture!of!dimers,!trimers,!and!higher9order!oligomers!via!Blue!Native!Polyacrylamide!Gel!Electrophoresis! (BN9PAGE).! To! more!thoroughly!understand! the! interactions! between! ConE! and! other! players! in! the! mating!machinery,!we!have!employed!a!bacterial!two9hybrid!system.!We!investigated!the!interaction!between!ConE!fused!to!the!T18!and!T25!domains!of!Bordetella&pertussis!adenylate!cyclase.!Using!β9galactosidase!assay!to!indirectly!report!cyclic9AMP!levels,!we!found!that! T259ConE!and!T189ConE! interact.!We!hope! to!use! this! technique! to!measure!possible! interactions!between!ConE! and!other!ICEBs1!proteins!that!make!up!the!DNA!translocation!channel.!!!!

42! ! VieB!plays!an!inhibitory!role!in!the!VieSAB!three7component!signal!transduction!system!in!Vibrio&cholerae.!

!Stephanie!L.!Mitchell,!Ayman!Ismail,!and!Andrew!Camilli!! ! ! ! ! !!!!!!!!!!!!!!!! ! ! ! ! ! !!!!!Molecular!Biology!and!Microbiology,!Tufts!University!&

Vibrio&cholerae! is!a! facultative!pathogen!that! lives! in! the!aquatic!environment!and!the!human!host.!The!ability!of!V.&cholerae! to!monitor!environmental!changes!as!it!transitions!between!these!diverse!environments!is!vital!to!its!pathogenic!lifestyle.!V.&cholerae!alters! gene! expression! by! sensing! external! stimuli! through! two9component! systems! (TCSs)! and! three9component! systems.! V.&cholerae!utilizes!VieSAB,!a!three9component!system,!which!harbors!the!sensor!kinase!(VieS)!and!response!regulator!(VieA),!but!also!contains! a! novel! auxiliary! protein! (VieB).! The!VieSAB! system! is! important! in! the! induction! of! virulence! genes! by! controlling! the!concentration!of!the!secondary!messenger!molecule,!cyclic9di9GMP.!While!VieSA!behave!similar!to!typical!TCSs,!the!role!of!VieB!is!unclear.! Using! purified! proteins! and! phosphotransfer! assays! we! show! that! VieB! causes! a! dramatic! decrease! in! the! amount! of!phosphorylated!VieA.!This!inhibition!appears!to!be!due!to!a!direct!physical!interaction!between!VieB!and!VieS.!These!data!point!to!an! inhibitory! role! of! VieB,! allowing! for! additional! control! over! signal! output.! Understanding! the!mechanism! of! how! the! VieSAB!system! functions!may!provide! further! insight! into! how!V.& cholerae! controls! virulence! gene! expression! as! it! transitions! from! the!aquatic!environment!into!the!host.!!!!!


45

43! ! Substrate! specificity! of! an! elongation7specific! peptidoglycan! endopeptidase! and! its! implications! for! cell! wall!architecture!and!growth!of!Vibrio&cholera!!Tobias!Dörr1,!Felipe!Cava2,!Hubert!Lam3,!and!Matthew!K!Waldor1!! ! ! ! ! ! ! ! ! ! !!!!1Division!of!Infectious!Diseases,!Brigham!and!Women’s!Hospital!and!Department!of!Microbiology!and!Immunobiology,!Harvard!!Medical! School! and!HHMI,! 2Centro!de!Biología!Molecular! Severo!Ochoa,!Universidad!Autónoma!de!Madrid9Consejo! Superior! de!Investigaciones!Científicas,!Madrid,!Spain,!3!current!address:!!Discovery!Research,!Sanofi!Pasteur,!Cambridge,!MA !!The! bacterial! cell!wall! consists! of! peptidoglycan! (PG),! a! sturdy!mesh! of! glycan! strands! cross9linked! by! short! peptides.! This! rigid!structure!constrains!cell!shape!and!size,!yet!is!dynamic!enough!to!accommodate!insertion!of!newly!synthesized!PG,!which!was!long!hypothesized,! and! recently! demonstrated,! to! require! cleavage!of! covalent! bonds!within! the! stem!peptides! that! link! PG’s! glycan!strands.!Here,!we!identify!several!genes!in!Vibrio&cholerae!that!collectively!are!required!for!growth!–!particularly!elongation!9!of!this!pathogen.! !V.& cholerae& encodes! three! putative! periplasmic! proteins! that! contain! both! PG9binding! domains! and! M23! family!peptidase!domains,!here!denoted!ShyA,!ShyB,!and!ShyC.!!While!none!is!essential!individually,!the!absence!of!both!ShyA!and!ShyC!results! in! synthetic! lethality,! while! the! absence! of! ShyA! and! ShyB! causes! a! significant! growth! deficiency.!! ShyA! is! a! D,D!endopeptidase! able! to! cleave! most! peptide! chain! crosslinks! in! V.& cholerae’s& PG.! !However,! ShyA! has! little! activity! against!muropeptides!containing!pentapeptides,!which!typically!characterize!newly!synthesized!material.!The!presence!of!ShyA!enhances!removal! of! short! PG! strands,! which! accumulate! due! to! PG!maturation.! Together,! our! findings! reveal! unexpected! regulation! of!endopeptidase!activity!by!intrinsic!substrate!cues!with!important!implications!for!the!mechanism!of!bacterial!growth.!!!

44! ! Contribution!of!vah1,&plp,&and&rtxA!to!the!cytotoxicity!of!Vibrio&anguillarum&against!epithelial!cells,!leukocytes!and!erythrocytes!!Xiangyu!Mou1,!Edward!J.!Spinard1,!David!A.!Parra2,!J.!Oriol!Sunyer2!and!David!R.!Nelson1!1Department!of!Cell!and!Molecular!Biology,!University!of!Rhode!Island,!Kingston,!RI!02881,!2Department!of!Pathobiology,!University!of!Pennsylvania,!Philadelphia,!PA!19104!!We! have! been! investigating! the! regulation! and! roles! of! the! hemolysin/cytotoxin! genes! in! pathogenesis! by!V.& anguillarum,! the!causative! agent! of! warm! water! vibriosis! in! fish.! We! have! shown! that! there! are! three! hemolysin! genes! (plp,! vah1,! and! rtxA)!organized! in! two!gene!clusters.! In! this! study,!we! investigated! the!contributions!of! the! three!hemolysin!genes! to! the!cytotoxicity!against! various! types! of! fish! cells.! !Our! results! showed! that! RtxA,! Vah1! and! Plp! in!V.& anguillarum! LB20! culture! supernatant! are!together!responsible!for!100%!of!the!hemolytic!activity!against!rainbow!trout!erythrocytes,!100%!of!the!cytotoxicity!against!Atlantic!salmon!kidney!(ASK)!cells,!and!609100%!of!the!cytotoxicity!against! leukocytes!from!spleen!(SPL)!and!head!kidney!(HK)!of!rainbow!trout.!Among!the!four!types!of!cells,!HK!cells!are!the!most!sensitive!to!RtxA.!ASK!cells!are!the!most!sensitive!to!Vah1,!followed!by!SPL!cells,!while!erythrocytes!are!the!most!sensitive!to!Plp.!These!data!suggest!that!the!three!hemolysins!have!different!rolls!during!V.&anguillarum!infections!of!fish.!Data!also!suggest!that!expression!of!the!three!hemolysin!genes!is!regulated,!in!part,!by!a!negative!feedback!mechanism.!!!

45! ! Reprogramming!bacterial!secretion!systems!for!therapeutic!applications!

!Analise!Z!Reeves1,2,3,!William!E!Spears1,!Amy!J!Wagers3,4,5,!Cammie!F!Lesser1,2,4!! ! ! ! ! ! !!!!!!!!!!!!!!!!!!!!!!!!

1Department! of! Medicine,! Division! of! Infectious! Diseases,! Massachusetts! General! Hospital,! 2Department! of! Microbiology! and!Immunobiology,!Harvard!Medical!School,! 3Department!of!Stem!Cell!and!Regenerative!Biology,!Harvard!University,! 4Harvard!Stem!Cell!Institute,!5Howard!Hughes!Medical!Institute!!Many! gram9negative! bacteria! utilize! type! 3! secretion! systems! (T3SSs)! to! deliver! effector! proteins! directly! into! host! cells.! The!addition! of! an!N9terminal! type! 3! secretion! signal! to! heterologous! proteins! allows! for! recognition! and! secretion! of! proteins! not!typically!recognized!as!T3SS!substrates.!We!aim!to!utilize!this!property!of!T3SSs!as!a!biological!tool!for!applications!requiring!the!directed!delivery!of!proteins! into!mammalian!cells.!We!have!engineered!non9pathogenic&E.coli! strains! that!encode!the!structural!components!of!the!Shigella&flexneri!type!3!secretion!machinery!on!a!low9copy!plasmid.!The!addition!of!the!first!50!amino!acids!of!an!effector!to!mammalian!proteins!is!sufficient!for!secretion!and!delivery!of!heterologous!proteins!into!mammalian!cells.!Experiments!utilizing! an! E& coli! strain! capable! of! secreting! and! delivering! the! myogenic! transcription! factor,! MyoD,! into! mouse! embryonic!fibroblasts! (MEFs)! demonstrate! that! delivery! of! MyoD! by! the! E.& coli! results! in! the! induction! of! muscle9cell! specific! genes! and!ultimately!drives!their!transdifferentiation!into!myosin!heavy!chain9expressing!muscle!cells,!demonstrating!that!this!approach!can!be!used!to!alter!the!developmental!fate!of!a!cell.!Ultimately,!we!seek!to!engineer!similar!E.coli!strains!that!utilize!T3SSs!to!deliver!defined!sets!of!proteins!for!therapeutic!purposes.!!


46

46! ! Germinant7Free! Stochastic!Germination! of!Wild! Type!Bacillus& subtilis! Spores:!Observations! in! Favor! of! the! Scout!Hypothesis!!Paul!Muller!and!Slava!Epstein!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!Biology!Department,!Northeastern!University!!Recently,! a! model! of! bacterial! survival! was! proposed! in! which! vegetative! non! spore9forming! cells! become! dormant! upon!experiencing!adverse!environmental!conditions!and!subsequently!singular!members!“awaken”!in!an!apparent!stochastic!manner!to!serve!as!a!population!level!environmental!sensor.!This!model,!termed!the!Scout!Hypothesis1,!has!since!received!empirical!support!via!very9long9term!incubation!studies!where!colony!formation!of!environmentally!sampled!prokaryotes!were!tracked,!on!the!same!plates,! for! nearly! two! years.! The! initial! formulation! of! the! Scout! Hypothesis! and! subsequent! empirical! support! were! made!specifically!with! non! spore9forming! organisms! in!mind.!Here,!we! ask!why! should! not! spore! formers! also! take! advantage! of! this!strategy?! In! the! literature,!and! through!personal! communication,!we! found!anecdotal!evidence! in! favor!of! the!Scout!Hypothesis!being!valid! for! spore9forming!organisms2.!Using!a!very! simple!method,!we! tracked!Bacillus& subtilis! spore!germination! in!96!well!plates,! for!over!5!weeks,! in!a!minimally!defined!broth!media! lacking!all!known!germination! factors.!Here,!we!report!evidence! in!support!of!the!Scout!Hypothesis!as!a!survival!strategy!in!the!spore!forming!organism!B.&subtilis.!!!

47! ! An!insertion!mutation!in!priA!that!splits!its!phenotype!

!Maxime!Leroux1,!Niketa!Jani1!and!Steven!Sandler1!! ! ! ! ! ! ! ! ! ! ! !!!!!!!!!!!!!!1Department!of!Microbiology,!University!of!Massachusetts,!Amherst!!In!E.&coli!there!are!three!pathways!for!replication!restart:!PriA9PriB,!PriA9PriC!and!PriC9Rep.!The!priA!gene!encodes!a!protein!with!an!N9terminal!DNA!binding!domain!and!a!C9terminal!domain!with!ATPase/helicase!activity.!PriB!and!priC!encode!redundant!functions!and!have!no!phenotype!by!themselves.!A!priA2::kan!insertion!mutant!grows!poorly,!has!high!SOS!expression,!partitioning!defects,!increased! sensitivity! to! UV! and! is! recombination! deficient.! It! is! synthetically! lethal! with! priC! and! rep! mutations.! We! have!characterized! a! new! priA! mutation,! priA316::cat.! It! is! an! insertion!mutation! at! the! same! position! as!priA2::kan.! Surprisingly,! it!behaves!essentially!as!wild!type!for!growth,!recombination!and!UV!resistance.!It!does!however!show!slightly!increased!levels!of!SOS!expression!and!longer!cells,!but!not!to!the!same!levels!of!priA2::kan.!Also,!priA316::cat!does!show!some!synthetic!phenotypes!with!priB!and!priC!mutations,!with!the!priB!mutation!showing!a!much!greater!effect!than!the&priC!mutation.! !But!there!is!no!synthetic!lethality!in!the!absence!of!priC!or!rep.!Analysis!of!priA316::cat!suggests!that!this!activity!derives!from!expression!of!both!N9terminal!and!C9terminal!parts!of!PriA!on!two!different!peptides.!!!

48! ! Pseudomonas&fluorescens!as!a!Model!System!for!Cyclic!Diguanylate!Signaling!

!Kurt!M.!Dahlstrom1,!Peter!Newell1,!Holger!Sondermann2,!and!George!O’Toole1!1Department!of!Microbiology!&!Immunology,!Geisel!School!of!Medicine!at!Dartmouth,!2Department!of!Molecular!Medicine,!College!of!Veterinary!Medicine,!Cornell!University!!Cyclic!diguanylate!(c9di9GMP)!is!a!signaling!molecule!ubiquitous!throughout!the!bacterial!domain.!Although!c9di9GMP!governs!many!diverse!processes,!it!is!unclear!how!it!specifies!its!targets!inside!an!unpartitioned!bacterial!cell!that!may!have!dozens!of!predicted!diguanylate!cyclases!(DGCs).!Here!we!use!biofilm!formation!in!the!gram9negative!bacterium!Pseudomonas&fluorescens&as!a!model!system! to! study! how! a! large,! dynamic! network! of! c9di9GMP! synthesizing,! degrading,! and! effector! proteins! can! produce! specific!outputs. !!!!!!!!!!!!!!

We! have! previously! demonstrated! that! only! four! DGCs! in!P.& fluorescens! impact! biofilm! formation.!We! selected! one!DGC,!GcbC,!to!ask!why!this!enzyme!plays!a!specific!role!in!biofilm!formation.!We!demonstrated!through!in&vivo!and!in&vitro!studies!that!GcbC!physically! interacts!with!LapD,!an!effector!protein! that!binds!c9di9GMP!and!promotes!biofilm! formation.!We! identified!and!mapped!point!mutations!that!disrupt!this!interaction!onto!the!LapD!and!GcbC!structures,!finding!a!ridge!of!amino!acids!near!the!c9di9GMP!binding! site! in! LapD!and!a! ring!of! amino!acids! surrounding! the! catalytic! site!of!GcbC.!Our! findings! suggest! that!physical!interactions!between!DGCs!and!effector!proteins!may!create!local!pockets!of!targeted!c9di9GMP!signaling,!providing!one!possible!mechanism!of!DGC!specificity.!!!!!


47

49! ! Bacterial! Motility! Facilitates! Phagocyte! Recognition! and! Uptake! of! P.& aeruginosa!and! other! Gram7negative!Pathogens!!Rustin!Lovewell1,!Eyal!Amiel2,!George!O’Toole1,!and!Brent!Berwin1!1Department!of!Microbiology!and!Immunology,!Dartmouth!Medical!Center;!2The!Trudeau!Institute!!The! manner! in! which! the! loss! of! P.& aeruginosa! flagellar! motility! impacts! innate! immunity! is! poorly! understood.! !The! current!paradigm! suggests! that! loss! of! flagellar! expression! enables! immune! evasion! due! to! loss! of! flagellar! receptor! engagement.!!We!demonstrate! that! loss! of! flagellar! function,! not! structure,! is! the! critical! factor! in! the! development! of! phagocytic!resistance.!! Specifically,! isogenic! mutants! deficient! in! flagellar! motility,! but! retaining! intact! flagella,! are! highly! resistant! to!phagocytosis! at! levels! comparable! to! those! of! flagellum9deficient!mutants.! !In!P.& aeruginosa,! two! flagellar! stator! complexes! are!partially9redundant! in! providing! rotational! torque.!! Mutations! in! these! complexes! reveal! that! phagocytic! susceptibility! is!proportional! to! bacterial! swimming! capability.! !Importantly,! even! minor! changes! in! motility! result! in! measurable! changes! in!phagocytic! evasion.! The!degree!of! phagocytic! response! is! predicated! largely! on! the! ability! to! respond! to!bacterial!motility! after!host9cell! contact,! since! non9swimming! mutants! exhibit! resistance! to! engulfment! even! after! phagocyte! contact! has! been!established.!! Finally,! we! show! that! the! critical! host! PI3K9AKT! signaling! pathway! is! selectively! activated! by! bacterial! motility.! In!accordance!with! clinical!observations,! these! findings! fit! a!model! for!a! selective!pressure!within! the!host!environment! for!down9regulated!motility!and!conversion!from!a!planktonic!to!increasingly!sessile!lifestyle.!!!

50! ! Regulation!of!carnitine!catabolism!in!Pseudomonas&aeruginosa&

!Jamie!Meadows1!and!Matthew!Wargo1!1Microbiology!and!Molecular!Genetics,!University!of!Vermont!!Background:!!Carnitine!is!important!for!β9oxidation!in!mammals!and!abundant!in!host!tissues.!!A!carnitine!catabolism!operon!(CCO)!had!previously!been!identified!in!Pseudomonas&aeruginosa!and!the!regulator,!CdhR,!is!a!member!of!the!AraC!transcription!factor!family.!!Our!goal!was!to!determine!how!CdhR!regulates!transcription!of!the!CCO.!Methods:!!Growth!assays!were!performed!with!P.&aerugniosa!PA14!wild!type!(WT)!and!an!unmarked!deletion!of!cdhR,!in!minimal!media! supplemented! with! 20mM! of! carnitine! or! gylcine! betaine.!! β9galactosidase! assays! were! conducted! with! PA14WT! or!Ã¢Ë†â€&cdhR&& carrying! a! caiX::lacZYA! promoter!! reporter! fusion! in! response! to! pyruvate! or! carnitine.!! Promoter! mapping! was!performed! by! β9galactosidase! assays! with! varying! lengths! of! the! PcaiX::lacZYA.! !CdhR! binding! to! the! caiX! promoter! was!demonstrated!by!EMSA!and!DNaseI!footprinting.!Results:!!Deletion!of!cdhR!abolished!growth!and!induction!the!CCO!in!the!presence!of!carnitine.!!The!growth!phenotype!on!carnitine!was!rescued!by!complementation!for!cdhR.!!CdhR!was!shown!to!bind!the!caiX!promoter!by!EMSA!and!DNaseI!footprinting.!Conclusion:!!CdhR!is!required!for!growth!on!carnitine!and!induction!of!CCO.! !CdhR!binds!to!the!caiX&promoter! in!a!concentration!dependent!manner!and!the!DNA!sequence!has!been!identified!through!DNaseI!footprinting.!!!

51! ! Pseudomonas&aeruginosa&detection!of!host7derived!sphingosine!impacts!survival!during!lung!infection!

!Annette!E.!LaBauve1!and!Matthew!J.!Wargo1!!1Microbiology!and!Molecular!Genetics,!University!of!Vermont!!Background:! Our! goal! is! to! understand! how!Pseudomonas& aeruginosa& responds! to! the!mammalian! lung,! using! the! response! to!pulmonary!surfactant!to! identify!transcripts! likely! induced!early!during! lung!colonization.!The!most!highly! induced!transcript!was!divergently!transcribed!from!an!uncharacterized!AraC9family!transcription!factor!SphR.!We!determined!the!inducing!component!of!surfactant,!the!role!of!SphR,!and!the!impact!of!this!response!on!P.&aeruginosa&survival.!Methods:! Reporter! fusions! to! lacZ! were! used! to! measure! transcriptional! induction.! Binding! of! SphR! to! promoters! was!demonstrated!by!EMSA.!The!SphR!regulon!was!determined!using!microarray.!Survival!during!infection!was!measured!using!a!mouse!model!of!acute!pneumonia.!Results:!The!highly!induced!transcript,!named!sphA,!was!induced!by!the!sphingomyelin!component!of!lung!surfactant,!specifically!its!metabolite!sphingosine,!and!induction!was!dependent!on!SphR.!!The!SphR!regulon!consists!of!three!promoters:!sphA,!sphBCD,!and!cerN.!!Deletion!of!sphR!results!in!reduced!bacterial!survival!in!the!presence!of!sphingosine!and!during!mouse!lung!infection.!Conclusion:! This! is! the! first! identification! of! a! sphingosine9sensitive! transcription! factor! in! bacteria.! SphR! regulation! may! be!important! in! many! eukaryotic! infection! sites.! We! are! currently! determining! the! role! of! each! SphR! regulon! member! in! the! P.&aeruginosa&response!to!sphingosine.!!


48

52! ! The! Pathways! for! Periplasmic! Disulfide! Bond! Formation! are! Essential! for! Growth! of! Escherichia& coli! and!Mycobacterium&smegmatis!!Brian!Meehan1,!Rachel!Dutton2,!Na!Ke3,!and!Jon!Beckwith1!1Department!of!Microbiology!and! Immunobiology,!Harvard!Medical!School,! 2FAS!Center! for!Systems!Biology,!Harvard!University,!3New!England!Biolabs!!Numerous! proteins! in! the! bacterial! periplasm! require! disulfide! bonds! for! stability! and! activity.!!While! these! disulfides! can! form!spontaneously,! the!rate!at!which!this!occurs! is!exceedingly!slow,!so!bacteria!have!evolved!enzymes!to!catalyze!the!process.!!The!most!thoroughly!studied!pathway!for!disulfide!bond!formation!is!the!Dsb!system!of!E.&coli,!in!which!the!periplasmic!DsbA!oxidizes!substrate!proteins! and! is! then! recycled!by! the!membrane9bound!DsbB.!! Such!a! system! is!present! in!many!bacterial! species,! but!Mycobacteria! appear! to! catalyze!disulfide!bond! formation!despite! the! lack!of! a!DsbB!homolog.!!We!have!previously! shown! that!Mycobacterial! VKOR! (vitamin! K! epoxide! reductase)! can! complement! a! DdsbB! strain! of! E.& coli,! suggesting! that! the! two! are!functionally! homologous.!! Here! we! demonstrate! that! VKOR! plays! a! key! role! in! disulfide! bond! formation! in! M.&

smegmatis.!! Intriguingly,! while! VKOR! is! known! to! be! essential! for! growth,! DsbB! has! always! been! understood! to! be!dispensable.!!However,!we! show! that!dsb!mutants! of!E.& coli! display! severe! growth!defects! under! anaerobic! conditions! or!when!plated!aerobically!on!solid!media!containing!agarose!rather!than!agar.!!Such!observations!suggest!that!both!enzymes!are!essential!and!might!therefore!provide!vulnerable!targets!for!new!classes!of!antibiotics.!!!

53! ! DEAD!DNA!Transport:!a!mutational!analysis!of!a!competence!helicase/translocase!

!Scott!S.!Chilton1!and!Briana!M.!Burton1!1Department!of!Molecular!and!Cellular!Biology,!Harvard!University!!In!bacteria,!horizontal!gene!transfer!(HGT)!is!a!major!mechanism!for!generating!genetic!diversity.!!The!predominant!HGT!processes!all!involve!the!transfer!of!large!macromolecules!across!cell!membranes.!!Genetic!competence!is!one!such!process!in!which!cells!are!able!to!take!up!DNA!from!their!environment.!!These!cells!become!transformed!when!the!acquired!genes!are!maintained!as!part!of!the!organism’s!genome!or!as!an!extra9chromosomal!element.!!Many!bacteria!carry!homologs!of!the!central!DNA!uptake!machinery!that! has! been!well! characterized! in!Bacillus& subtilis.!! The! core!machinery! is! comprised! of! a! DNA! binding! receptor! (ComEA),! an!aqueous! pore! (ComEC),! and! a! helicase/translocase! (ComFA).!! It! is! thought! that! ComFA! belongs! to! a! family! of! low! processivity!helicases/translocases,! the! DEAD9box! family.! Thus,! it! is! unclear! how! ComFA! contributes! to! this! highly! processive! DNA!transport.!! Here! we! conduct! a! mutational! analysis! of! ComFA! based! upon! sequence! homology! to! conserved! DEAD9box!helicase/translocase!motifs.!!Mutating!the!conserved!residues!disrupts!DNA!uptake!and!transformation,!suggesting!that!ComFA!is!in!fact! a!member!of! the!DEAD9box! family.!!Understanding!how! the!uptake!machinery! functions! expands!our! understanding!of! the!diversity!of!cellular!functions!that!employ!DEAD9box!helicases,!and!possibly!the!diversity!of!behaviors!of!the!protein!family.!!!!

54! ! More!than!just!a!molecular!matchmaker:!ClpS!adaptor!protein!controls!ClpAP!substrate!selection&

!Amaris!Torres9Delgado1,!Robert!T.!Sauer1!and!Tania!A.!Baker1,2!1Department!of!Biology,!Massachusetts!Institute!of!Technology,!2Howard!Hughes!Medical!Institute!!Energy9dependent! AAA+! proteases! carry! out! regulated! proteolysis! to! ensure! protein! quality! control! and! post9translational!regulation!of!many!cellular!processes.!Control!of!proteolysis!occurs!primarily!at! the! level!of! substrate! recognition,!which!can!be!modulated! by! adaptor! proteins.! The! E.& coli! ClpS! adaptor! protein! delivers! N9end! rule! substrates! to! the! ClpAP! protease! for!degradation,!while!inhibiting!degradation!of!ssrA!substrates.!To!understand!this!specificity!switch,!we!are!elucidating!the!steps!of!the!degradation!cycle!subject!to!ClpS9mediated!inhibition.!We!observed!that!ClpS!does!not!prevent!ssrA!substrate!binding!to!ClpA.!ClpS!must!then!inhibit!a!later!step!in!the!degradation!cycle,!as!the!enzyme!fails!to!degrade!ssrA!substrates!in!the!presence!of!ClpS.!We!probed!the!structural! features!of!ClpS!and!ClpA!that!are! important! for! inhibition.!We!found!that!engagement!of! the!ClpS!N9terminal!extension!(NTE)!by!ClpA!is!a!critical!step!in!the!mechanism!of!inhibition.!Furthermore,!experiments!manipulating!the!NTE!suggest! that! the! NTE! is! responsible! for! most! of! the! ClpS! inhibitory! activity.! We! favor! a! model! in! which! formation! of! the!ssrA•ClpA•ClpS! complex! involves! engagement! of! the! ClpS! NTE! by! the! ClpA! pore,!which! prevents! substrate! engagement! by! the!degradation!machinery!for!further!processing.!!!!


49

55! ! Library!assembly!of!orthogonal!genetic!memory!devices!

!Lei!Yang,!Timothy!Lu,!Christopher!Voigt!Synthetic!Biology!Center,!Massachusetts!Institute!of!Technology,!Cambridge,!MA!!Synthetic! memory! devices! have! been! used! to! accomplish! sophisticated! functionalities! such! as! genetic! multiplexers! and! digital!counters.! Stable!DNA9encoded!memory! can! be! achieved! using! large! serine! type! phage! integrases,!which! catalyze! unidirectional!DNA! recombination!between! the! cognate!attP!and!attB! sites.!Although! this! type!of! integrase! is! very!abundant! in!nature,!only!a!small! number! of! the! cognate! attP! and! attB! sites! have! been! identified,! which! limits! our! capability! to! design! large! and! complex!synthetic! networks! using!memory! devices.! Here! we! use! bioinformatic! approaches! to! explore! novel! large! serine! integrases! and!predict!their!attP/attB!sites.!The!predicted!library!was!then!evaluated!for!efficiency!and!orthogonality!using!a!fluorescence9based!reporter! system.! To! date,!we! have! successfully! assembled! a! library! of! 10! novel! integrase9based!memory! devices,!which!will! be!further!expanded!and!used!to!build!orthogonal!genetic!circuits!having!memory!functions.!!!

56! ! Endophytic! actinobacterial! interactions! lead! to! developmental! changes! and! bioactivity! against! a! Gram7negative!phytopathogen!!Rita!de!Cassia!Pessotti1,!Matthew!Francis!Traxler1,!Monica!Tallarico!Pupo2,!Jon!Clardy3,!Roberto!Guillermo!Kolter1!!1Department! of!Microbiology! and! Immunobiology,! Harvard!Medical! School,! 2Department! of! Pharmaceutical! Sciences,! School! of!Pharmaceutical! Sciences! of! Ribeirao! Preto! 9! University! of! Sao! Paulo,! 3Department! of! Biological! Chemistry! and! Molecular!Pharmacology,!Harvard!Medical!School!!Interspecies!interactions!can!influence!development!and!secondary!metabolism!in!microorganisms,!and!these!interactions!can!be!studied! by! co9culturing! different! bacteria! strains.! The! present! work! shows! interactions! between! 20! endophytic! actinobacteria!isolated!from!Tithonia&diversifolia! (Mexican!Sunflower)! roots.!These!strains!were!cultivated! in!pairs!on!R2YE!solid!medium!in!129well!plates.!After!seven!days!of!incubation,!phenotypes!were!analyzed!and!the!colonies!were!overlaid!with!soft9LB9agar!inoculated!with!Pseudomonas&syringae!and!incubated!for!24!hours.!Bioactivity!was!analyzed!by!visualization!of!inhibition!halos.!Many!different!interactions!were!detected.! Besides! developmental! changes! such! as! induced/inhibited! actinobacterial! sporulation,! and! inhibited!actinobacterial! growth,! several! interactions! also! induced! or! decreased! bioactivity! against! P.& syringae,! a! gram9negative!phytopathogen.!These!results!highlight!the!relevance!of!co9cultures!as!a!strategy!for!studying!chemical!ecology!and!for!antibiotics!discovery.! Furthermore,! these! experiments! suggest! that! endophytes! may! interact! with! each! other! inside! their! host,! possibly!conveying!protection!against!phytopathogen!infection.!!!

57! ! Bacterial!slow!growth!–!a!common!survival!strategy?!

!Brittany!Berdy1,!Sandra!Buerger2,!Alexander!Sousa1,!Ethan!Edson1,!Kieran!Halligan1,!Lucy!Ling3,!Amy!Spoering3,!and!Slava!Epstein1,3!1Department! of! Biology,! Northeastern! University,! Boston,! MA! 2Boston! University,! Boston,! MA! 3Novobiotic! Pharmaceuticals,!Cambridge!MA!!Microbial! slow!growth!has!been!proposed!as! a! strategy! to! combat! environmental! challenges.!We!previously!demonstrated! that!most! environmental! isolates! grew! slowly! upon! initial! incubation,! but! grew! quickly! upon! subcultivation.! A! minority! of! isolates!required! over! 180! days! to! grow! upon! subculturing.! We! examined! these! isolates! to! determine! if! they! represented! true! slow!growers.!!Alternatively,!slow!growth!may!have!been!due!to!nutritional!factors!absent!from!the!media.!We!tested!marine!and!soil!isolates! that! required! 6! or! 192!months! to! form! growth,! respectively,! using! the! original! isolation!medium! and! optimized!media.!Isolates! grew!more! quickly! upon! subcultivation! and!with! optimized!media! compared! to! the! initial! isolation.! For! one! strain,! we!determined!magnesium!and!sodium!chloride!were!sufficient!for!fast!growth.!These!data!support!the!idea!that!slow!growth!is!not!an!inherent!property!of!the!majority!of!environmental!isolates.!Initial!slow!growth!may!be!due!to!suboptimal!growth!conditions,!but!not! the! innate! inability! to! proliferate! quickly.!! This! conclusion! is! general! since! the!microorganisms! tested!were! spore9! and!non9spore! forming,! and! from! soil! and!marine! habitats.! These! findings!may! relate! to! uncultivability! since! slow! growth! during! initial!incubation!may!be!difficult!to!detect,!resulting!in!potential!misclassification!as!“unculturable”.!!!!!


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58! ! E.&coli!mgtA!leader!region!encodes!an!Hfq7dependent!sRNA!

!Katherine!E.!Berry!and!Ann!Hochschild!Department!of!Microbiology!and!Immunobiology,!Harvard!Medical!School!!Transcription! of! the! E.& coli& mgtA& gene,! which! encodes! an! inducible! magnesium! transporter,! is! subject! to! complex! regulation.!Transcription!initiation!is!controlled!by!PhoP/Q,!and!post9initiation!control!is!mediated!by!a!magnesium9dependent!riboswitch!and!by!a! regulatory!upstream!ORF.! In! the!course!of! investigating! transcription!pausing! in! the!5’!UTR!of!mgtA,!we!discovered!a!weak!intrinsic! terminator,! conserved! in! E.& coli& and! Shigella& strains,! that! is! located! between! the!mgtA! promoter! and! riboswitch.! An!attractive!possibility! is! that!this!terminator!could!serve!as!an!attenuator,!adding!to!the!regulatory!complexity!of!the&E.&coli!mgtA!locus.!However,!we!have!no!evidence! that!magnesium! influences! termination!efficiency! in& vivo.!Although! this! element! supports!only!~5910%!termination!in&vitro,!it!results!in!the!accumulation!of!a!very!prominent!~52!nt!RNA!product!in&vivo!that!we!have!named!MgtZ.!Under!certain!conditions,!MgtZ!steady9state!levels!are!dependent!on!the!RNA!chaperone!Hfq,!which!mediates!base9pairing!interactions!between!many!sRNAs!and!their!target!mRNAs,!suggesting!that!MgtZ!may!function!similarly.!Studies!are!underway!to!uncover!potential!targets!of!the!MgtZ!sRNA!and!investigate!a!potential!physiological!role!of!this!sRNA.!!!

59! ! Pneumolysin! targets! CD73,! a! host! immune7modulatory! 5’7nucleotidase! that! promotes! resistance!to!invasive!Streptococcus&pneumoniae&infection!!Elsa!Bou!Ghanem1,!Stacie!Clark1,!Sara!Roggensack1,!Michael!Pereira1,!Sally!McIver2,!Philip!Haydon2!and!John!Leong1!1Department!of!Molecular!Biology!and!Microbiology,!2!Department!of!Neuroscience,!Tufts!University!School!of!Medicine,!Boston!!Pulmonary! inflammation! upon! Streptococcus& pneumoniae! (pneumococcus)! infection! is! associated! with! poor! control! of! the!infection.!A!crucial!regulator!of!inflammation!is!extracellular!adenosine!that!is!produced!from!extracellular!ATP!by!the!cell!surface!5'9nucleotidase! CD73.! We! show! here! that! pharmacological! inhibition! or! genetic! ablation! of! CD73! dramatically! increased! the!susceptibility!of!mice!to!invasive!pneumococcal!infection.!Intriguingly,!S.&pneumoniae!targets!this!immune9modulatory!pathway!as!pulmonary! infection!triggered!a!decrease! in!surface9associated!CD73!on!mouse! lung!epithelial!cells.! Importantly,!this!decrease! in!surface! CD73! upon! pneumococcal& infection! was! neither! due! to! host! cell! death! nor! a! decrease! in! CD73! production! within! the!epithelial!cells.!Rather,!S.&pneumoniae!targeted!CD73!cell!surface!localization.!Using!pneumococcal!infection!of!a!lung!epithelial!cell!line,! we! modeled! CD73! decrease! in& vitro! to! identify! bacterial! factors! that! contribute! to! this! response.! We! found! that! the!cholesterol9dependent! cytolysin!pneumolysin!was!both! required!and! sufficient! to!decrease!CD73!expression!on! the! cell! surface.!Further,!the!pore9forming!activity!of!pneumolysin!was!crucial!for!this!reduction.!Our!data!suggest!that!pneumolysin!plays!a!novel!role! in! the! pathogenesis! of! S.& pneumoniae& infection! by! targeting! the! expression! of! a! cell! surface! nucleotidase! required! for!modulating!a!damaging!host!inflammatory!response.!!!

60! ! DNA!damage!response!in!L.&interrogans:!the!role!of!two!LexA!repressors!

!Luciane!S!Fonseca1,!Josefa!B!da!Silva1,!Alan!D!Grossman2,!Renata!MA!da!Costa1!and!Paulo!L!Ho1!1Centro!de!Biotecnologia,!Instituto!Butantan,!2Department!of!Biology,!Massachusetts!Institute!of!Technology!!Leptospira& interrogans! is! a! pathogen! capable! of! surviving! in! the! environment! for! weeks! before! infecting! its! next! host.! This!characteristic! results! in! exposure! to! a! wide! range! of! stress! agents.! Among! the! possible! stresses! leptospires! can! face! inside! or!outside!the!host!is!DNA!damage.!DNA!lesions!can!trigger!the!SOS!response,!which!is!regulated!by!the!RecA!recombinase!and!by!the!transcriptional!repressor!LexA.!This!study!investigates!the!behavior!of!L.&interrogans!in!response!to!DNA!damage.!We!show!that!L.&interrogans!serovar!Copenhageni!genome!contains!two!genes!encoding!putative!LexA!proteins!(lexA1!and!lexA2),!one!of!which!was!potentially! acquired! by! lateral! gene! transfer.!Both! repressors! are! upregulated! after! DNA! damage.! LexA1! ChIP9seq! experiments!revealed!a!regulon!of!up!to!30!genes,!including!lexA1,!lexA2,!and!the!SOS9core!genes!recA,!recN!and!dinB.!On!the!other!hand,!LexA2!probably!controls!a!smaller!number!of!genes,! including!its!own!promoter.!Therefore,!the!L.! interrogans!serovar!Copenhageni!SOS!regulon!may!be!even!more!complex!than!that!of!most!other!bacteria,!as!a!result!of!LexA1!and!LexA2!binding!to!divergent!motifs.!These!novel!aspects!of!the!DNA!damage!response!in!Leptospira!may!reflect!the!importance!of!this!pathway!in!virulence.!!!!!


51

61! ! Anthrax!toxin7mediated!cellular!delivery!of!streptococcal!NAD!glycohydrolase!

!Onkar!Sharma,!M.!N.!O’!Seaghdha!and!M.!R.!Wessels!Division!of!Infectious!Diseases,!Boston!Children's!Hospital,!Boston,!MA!!Group! A! Streptococcus! (GAS)! produces! several! virulence! factors! that! modulate! its! interactions! with! human! oropharyngeal!keratinocytes!and!thus!promote!persistence!in!the!host.!The!co9toxins!Streptolysin!O!(SLO)!and!NAD!glycohydrolase!(NADase)!are!two! such! important! factors.! NADase! translocation! into! cells! is! SLO9mediated.! To! study! specific! intracellular! effects! of! NAD!glycohydrolase!on!the!host!cell,!an!SLO9independent!NADase!delivery!system!was!needed.!Anthrax!toxin,!an!A9B!type!toxin!secreted!by!Bacillus&anthracis!was!evaluated!for!its!ability!to!translocate!NADase!into!cells.!Anthrax!toxin!comprises!protective!antigen! (PA)!and! the!catalytic!moieties!Lethal! factor! (LF)!and!Edema!Factor! (EF).!PA!has! the! receptor!binding!and!pore9forming!properties!to!translocate!the!catalytic!moieties!LF!and!EF!into!the!host!cell!cytosol.!LFn,!the!N9terminal!263!aa!translocation!domain!of!LF,!is!sufficient!to!deliver!fused!heterologous!protein!cargo!across!the!PA!pore.!We!generated!LFn!fusion! constructs! of!NADase,! enzymatically! inactive!NADase! (NADase(G330D)),! the! activity! domain! of!NADase! (190NADase)! and!190NADase(G330D)!and!studied! their!ability! to! translocate! into!human!oropharyngeal!keratinocytes! in!a!PA9dependent!manner.!The! results! demonstrate! that! the! anthrax! toxin! can! substitute! for! SLO! in! delivery! of! NADase! into! eukaryotic! cells,! allowing!functional!analysis!of!its!specific!intracellular!effects.!!!

62! ! PonA1!controls!cell!elongation!and!morphology!in!mycobacteria!!

!Karen!J.!Kieser,!Jemila!C.!Kester,!Michael!C.!Chao,!Sarah!M.!Fortune,!Eric!J.!Rubin.!Department!of!Immunology!and!Infectious!Diseases,!Harvard!School!of!Public!Health,!Boston,!MA!02115!!The! cell! wall! of! mycobacteria! provides! a! wealth! of! drug! targets! that! can! be! identified! through! a! better! understanding! of! the!mechanisms! of! cell! wall! assembly.!Many! enzymes! responsible! for! cell! wall!metabolism! are! essential! for! bacterial! survival;! how!these!enzymes!are!regulated!to!maintain!the!critical!balance!between!cell!elongation!and!septation!is,!however,!poorly!understood.!Here,!we!focus!on!PonA1,!which!unlike!most!penicillin!binding!proteins!is!essential!for!in&vitro&growth!of!M.&smegmatis&and!in&vivo&growth!of!M.&tuberculosis.!Using!genetic!allelic!exchange!and!timelapse!microscopy,!we!find!PonA1!is!a!central!determinant!of!cell!morphology,!regulating!this!process!through! its!essential!transglycosylase!(TG)!activity!and!transpeptidase!(TP)!activity.!We!show!that!moenomycin,!a!small!molecule!that!targets!TG!domains,! inhibits!mycobacterial!growth.!Timelapse!microscopy!demonstrates!that!PonA1’s!novel!phosphorylation!regulates!cell!elongation!rate.!Overexpression!suggests!that!PonA1!influences!pole!formation!by!altering!local!peptidoglycan!structure;!protein!truncation!shows!that!PonA1’s!control!over!cell!shape!and!length!is!modulated!by!its!regulation!of!protein!complexes!in!the!periplasmic!space.!These!data!reveal!novel!mechanisms!by!which!mycobacteria!control!cell!growth,!and!that!PonA1!and!its!associated!pathways!constitute!potential!drug!targets!to!inhibit!mycobacterial!growth.!!!!

63! ! Functional!diversity!of!five!homologous!Cu+7ATPases!present!in!Sinorhizobium&meliloti!!

!Patel!S.,!Padilla9Benavides!T.,!Collins!J.!and!Argüello,!J.M.!Department!of!Chemistry!and!Biochemistry,!Worcester!Polytechnic!Institute,!Worcester,!MA,!01609,!USA!!!Copper! is!an!essential!enzyme!cofactor,!but!at!high!concentrations! it!participates! in!Fenton!reactions!producing!cytotoxicity.!The!dual!nature!of!Cu+!is!important!for!host9microbe!interactions.!Cu+9ATPases!drives!cytosolic!metal!efflux,!protecting!bacteria!against!Cu+! toxicity.! Many! pathogenic/symbiotic! bacteria! express! several! independently! regulated! Cu+9ATPases! genes! with! different!putative! roles.! Testing! this! hypothesis,! we! characterized! the! functional! diversity! of! five! homologous! Cu+9ATPases! using!Sinorhizobium9alfalfa!symbiotic!model.!Mutation!of!each!gene!leads!to!distinct!phenotypes,!both,!in!free9living!organisms!or!during!plant! nodulation.!actP! is! induced!by! Cu+! and! its!mutation! leads! to! Cu+! accumulation.! cut4! is! induced!under! anaerobiosis! and! is!required! for! bacteroid!differentiation! in! nodule.! fixI1! and! fixI2! are! located!downstream!of! cytochrome! c! oxidase! (Cox)! operons.!Microaerobiosis!dependent!fixI1!expression!suggests!its!role!in!Cox!metallation!during!infection.!The!lack!of!induction!of!fixI2!by!any!stressor!and!the!failure!in!nodules!formation!by!mutant!strain!indicate!a!constitutive!participation!in!Cox!maturation.!cut5!present!a!novel! genetic! arrangement! that! is! not! regulated! by! Cu+! and! appears! to! be! required! for! nodulation! and!metallation! of! secreted!cuproproteins.!The!success!of!host9pathogen!interactions!relies!on!complex!Cu+!homeostasis,!where!Cu+9ATPases!control!cytosolic!Cu+!levels!and!metallation!of!cuproproteins.!!!


52

64! ! The!bacterial!replication!initiator!DnaA!and!its!regulator!YabA!associate!with!several!chromosomal!regions!bound!by!the!nucleoid7associated!protein!Rok!in!Bacillus&subtilis!!Charlotte!A.!Seid1,!Tracy!A.!Washington1,!Houra!Merrikh2,!and!Alan!D.!Grossman1!1Department!of!Biology,!Massachusetts!Institute!of!Technology,!2Department!of!Microbiology,!University!of!Washington!!We!found!an!unexpected!link!between!a!negative!regulator!of!DNA!replication!(YabA)!and!the!nucleoid9associated!protein!Rok!of!Bacillus&subtilis.!!YabA!interacts!with!the!replication!initiator!DnaA!and!the!sliding!clamp!of!the!replication!machinery,!DnaN.!!YabA!is! found! associated! with! the! replisome! and! with! chromosomal! regions! bound! by! DnaA,! including! the! origin! of! replication!oriC.!! Using! ChIP9seq,!we! found! that! YabA! and!DnaA! also! associate!with! chromosomal! regions! that! are! bound! by! the! nucleoid9associated!protein!Rok,!an!analogue!of!H9NS! from!gamma9proteobacteria.!!Association!of!YabA!and!DnaA!with! these!Rok9bound!regions!was! largely!dependent!on!Rok.!! In!contrast! to! the!DnaA9dependent!association!of!YabA!with!previously!described!DnaA9bound!regions,!YabA!remained!associated!with!the!Rok9bound!regions! in!the!absence!of!DnaA.!!Loss!of!YabA!did!not!abolish!the!binding!of!Rok!or!DnaA!to!these!regions.!!We!investigated!the!possible!consequences!of!this!co9association!on!replication!and!on!the!expression!of!genes!regulated!by!DnaA!or!Rok.!!Our!results!indicate!that!YabA!may!be!involved!in!cellular!physiology!beyond!its!known!role!as!a!negative!regulator!of!replication!initiation.!!!

65! ! Role!of!StreptolysinO!and!NAD+7glycohydrolase!during!interaction!of!Group!A!Streptococcus!with!macrophages!

!Benedicte!Sempe1!and!Michael!Wessels1!1Division!of!Infectious!Diseases,!Children’s!Hospital,!Boston,!MA!!!GroupA!Streptococcus!(GAS,!Streptococcus!pyogenes)!is!a!human9restricted!pathogen!responsible!for!diverse!diseases!ranging!from!non9invasive! infections! of! the! throat! (pharyngitis)! to! invasive! life9threatening! infections! (necrotizing! fasciitis,! bacteremia).! For!successful!establishment!of!infection,!GAS!must!persist!within!the!host!and!face!its!immune!system.!

As!the!front!line!of!innate!immune!response,!macrophages!are!one!of!the!earliest!immune!cells!encountered!by!GAS!during!invasive!infections.!Moreover,!macrophages!are!thought!to!be!reservoirs!of!GAS!for!dissemination!of!infection!and!continued!tissue!injury.!However,!little!is!known!about!the!mechanism!allowing!GAS!to!avoid!killing!by!macrophages.!

Among!the!extracellular!proteins!secreted!by!GAS!are!StreptolysinO!(Slo)!and!NAD9glycohydrolase!(NADase).!These!toxins!are!known!to!reduce!internalization,!promote!colonization!and!survival!of!GAS!in!host!cells.!The!purpose!of!this!work!is!to!investigate!the!role!of!these!toxins!during! interaction!of!GAS!with!macrophages!to!better!understand!the!underlying!mechanisms! leading!to!the!transition!from!non9invasive!to!invasive!diseases.!

We!report!that!Slo!and!NADase!are!required!for!cytotoxicity!and!survival!of!GAS!during!interaction!with!macrophages.!Then,!preliminary! results! show! that! Slo! and! NADase! do! not! inhibit! the! endo9lysosomal! fusion.! Further! studies! are! in! progress! to!determine!the!involvement!of!these!toxins!in!the!inhibition!of!the!acidification!of!the!phagolysosome.!!!

66! ! Towards!a!whole7cell!modeling!approach!for!synthetic!biology!

!Oliver!Purcell1,2,a),!Bonny!Jain1,a),!Jonathan!Karr3,!Markus!Covert4,!Timothy!Lu1,2!a)!These!authors!contributed!equally!to!this!work.!1Department& of& Electrical& Engineering& && Computer& Science,& Massachusetts& Institute& of& Technology,& 77& Massachusetts& Avenue,&

Cambridge,& MA& 02139,& USA,! 2Department& of& Biological& Engineering,& Massachusetts& Institute& of& Technology,& 77& Massachusetts&

Avenue,&Cambridge,&MA&02139,&USA,!3Graduate!Program&in&Biophysics,&Stanford&University,&318&Campus&Drive&West,&Stanford,&CA&

94305,&USA,!4Department&of&Bioengineering,&Stanford&University,&318&Campus&Drive,&Stanford,&CA&94305,&USA&&&

!Despite!rapid!advances!over!the!last!decade,!synthetic!biology!lacks!the!predictive!tools!needed!to!enable!rational!design.!Unlike!established!engineering!disciplines,!the!engineering!of!synthetic!gene!circuits!still!relies!heavily!on!experimental!trial9and9error,!a!time9consuming!and!inefficient!process!that!slows!down!the!biological!design!cycle.!This!reliance!on!experimental!tuning!is!because!current!modeling! approaches! are! unable! to!make! reliable! predictions! about! the! in& vivo! behavior! of! synthetic! circuits.! A!major!reason! for! this! lack! of! predictability! is! because! current!models! view! circuits! in! isolation,! ignoring! the! vast! number! of! complex!cellular! processes! that! impinge! on! the! dynamics! of! the! synthetic! circuit! and! vice! versa.! To! address! this! problem,!we! present! a!modeling!approach!for!the!design!of!synthetic!circuits!in!the!context!of!cellular!networks.!Using!the!recently!published!whole9cell!model!of!Mycoplasma&genitalium,&we!examined!the!effect!of!adding!genes!into!the!host!genome.!We!also!investigated!how!codon!usage!correlates!with!gene!expression!and!find!agreement!with!existing!experimental!results.!Finally,!we!successfully!implemented!a!synthetic!Goodwin!oscillator!in!the!whole9cell!model.!We!provide!an!updated!software!framework!for!the!whole9cell!model!that!


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lays!the!foundation!for!the!integration!of!whole9cell!models!with!synthetic!gene!circuit!models.!This!software!framework!is!made!freely!available!to!the!community!to!enable!future!extensions.!We!envision!that!this!approach!will!be!critical!to!transforming!the!field!of!synthetic!biology!into!a!rational!and!predictive!engineering!discipline.!!!

67! ! Composition!of!the!cell!membrane!affects!the!efficiency!of!bacterial!conjugation!

!Christopher!M.!Johnson!and!Alan!D.!Grossman!Department!of!Biology,!Massachusetts!Institute!of!Technology!!The!Integrative!and!Conjugative!Element!ICEBs1!of!Bacillus&subtilis!is!a!model!for!the!study!of!conjugation!in!Gram9positive!bacteria.!Using! a! transposon! mutagenesis! screen,! we! identified! genes! that! affect! the! ability! of! B.& subtilis! to! act! as! a! recipient! during!conjugation.! The! screen! identified! several! phospholipid! synthetases! that! consume! phosphatidylglycerol! to! modify! the! cell!membrane.! The! deletion! of! one! of! these,!mprF,! was! found! to! be! epistatic! to! deletions! of! each! of! the! others,! indicating! that!lysylphosphatidylglycerol,! the! phospholipid! synthesized! by! the!mprF! gene! product,! likely! plays! a! key! role! in! ICEBs19mediated!conjugation.!We!found!that!deleting!mprF!impairs!the!ability!of!ICEBs1!to!transfer!DNA!across!cell!membranes.!This!defect!occurs!if!the!mutation! is! in!either!the!donor!or!the!recipient,!and! is!additive! if!both!donors!and!recipients!carry!the!mutation.!The! loss!of!mprF!does!not!affect!heritability!of! ICEBs1,!suggesting!that!cytoplasmic!functions!are!not!affected.!The!mutation!does! impair!the!ability!of!ICEBs1!to!mobilize!a!co9resident!plasmid,!supporting!the!notion!that!it!is!the!transfer!of!DNA!across!the!membrane!that!is!affected.!We!are!currently!investigating!the!mechanism!by!which!mprF!acts.!!!

68! ! Revisiting!the!Role!of!D7amino!acids!in!Bacillus&subtilis&biofilm!formation!

!Sara!Leiman1,!Roberto!Kolter2,!and!Richard!Losick1!1Department! of! Molecular! and! Cellular! Biology,! Harvard! University,! 2Department! of! Microbiology! and! Immunology,! Harvard!Medical!School!!Our! laboratories!previously! reported! that!a!mixture!of!D9leucine,!D9methionine,!D9tryptophan,!and!D9tyrosine! (D9LMWY)! inhibits!Bacillus&subtilis&biofilms!through!a!mechanism! in!which!the!D9amino!acids!are! incorporated! into!peptidoglycan.!!To!delve!deeper!into! this! phenomenon,! we! isolated! B.& subtilis&mutants! that! survive! in! the! presence! of! growth9inhibitory! concentrations! of! D9LMWY.!!Whole!genome!sequencing!revealed!that!these!strains!were!mutant!in!the!pathways!for!charging!tyrosyl!tRNA!or!regulating!protein!quality.!!Such!mutations!compensate! for! the!absence!of!D9tyrosyl! tRNA!deacylase! in!B.&subtilis,!an!enzyme!that!prevents!misincorporation!of!D9tyrosine!into!nascent!proteins.!!Growth!curves!in!biofilm9promoting!medium!showed!that!the!concentrations!of!D9LMWY!previously!reported!to!inhibit!biofilm!formation!caused!a!growth!defect.!!Biofilm!assays!further!demonstrated!that!the!D9LMWY!resistant!mutants!formed!biofilm!in!the!presence!of!D9tyrosine!and!that!wild9type!cells!treated!with!equimolar!mixtures!of!D9!and!L9amino!acids!formed!biofilm!comparable!to!that!of!untreated!B.&subtilis.!!Moreover,!an!excess!of!D9alanine!did!not!reverse!the!biofilm9inhibitory!action!of!D9amino!acids!as!was!previously!reported.!!Together,!these!data!indicate!that!D9amino!acids!affect!biofilms!through!their!effects!on!growth.!!!!

69! ! Modulation!of!the!Host!Unfolded!Protein!Response!by!Legionella&pneumophila!

!Andrew!D.!Hempstead1!and!Ralph!R.!Isberg1,2!1Department!of!Molecular!Biology!and!Microbiology,!Tufts!University!School!of!Medicine,!2Howard!Hughes!Medical!Institute!!General! cell! stress! pathways! have! been! implicated! in! the! activation! of! immunomodulatory! factors,! pointing! to! a! role! of! these!pathways! in! the! innate! immune! response.!! Host! Toll9like! receptor! (TLR)! mediated! detection! of! Legionella& pneumophila,! the!causative! agent! of! Legionnaires’! disease,! activates! one! such! pathway,! the! IRE1α! branch! of! the! unfolded! protein! response!(UPR).!!Legionella!derived!pathogen!associated!molecular!patterns!(PAMPs),!in!conjunction!with!an!induced!UPR,!greatly!enhances!proinflammatory! cytokine! expression.!! Surprisingly,! L.& pneumophila! is! able! to! replicate! intracellularly! under! the! restrictive!environment!of!an!induced!UPR.!!This!may!in!part!be!due!to!the!ability!of!Legionella!to!inhibit!XBP1,!a!potent!transcription!factor,!which! acts! downstream! of! IRE1α.!! L.& pneumophila! limits& XBP1! transcript! processing,! protein! expression,! and! subsequent!transcriptional! activation!of! target! genes,! including! those!encoding!proinflammatory! cytokines.!! The!ability!of!L.& pneumophila! to!accomplish! this! is! dependent! on! the! Icm/Dot! type! IV! secretion! system! (T4SS)! and! is! partially! dependent! on! five! translocated!effectors! which! inhibit! host! translation.!! These! findings! provide! evidence! that! L.& pneumophila! may! be! able! to! limit! the! innate!immune!response!through!the!inhibition!of!a!host!cell!stress!pathway.!!


54

70! ! Salmonella&typhimurium!intercepts!E.&coli!signaling!to!enhance!antibiotic!tolerance!

!Nicole!M.!Vega,1,5!Kyle!R.!Allison,1!Amanda!N.!Samuels,1!Mark!S.!Klempner,2!and!James!J.!Collins1,3,4!1Howard! Hughes!Medical! Institute,! Department! of! Biomedical! Engineering,! and! Center! of! Synthetic! Biology,! Boston! University,!2MassBiologics,!University!of!Massachusetts!Medical!School,3School!of!Medicine,!Boston!University,!4Wyss!Institute!for!Biologically!!Inspired!Engineering,!Harvard!University,!5Department!of!Physics,!Massachusetts!Institute!of!Technology!!Bacterial! communication! plays! an! important! role! in!many! population9based! phenotypes! and! interspecies! interactions,! including!those!in!host!environments.!!Here,!we!show!that!the!intestinal!pathogen!Salmonella&typhimurium!increases!its!antibiotic!tolerance!in! response! to! indole,! even! though!S.& typhimurium! does! not! natively! produce! this! signal.!! Increased! antibiotic! tolerance! can! be!induced!in!S.&typhimurium!by!both!exogenous!indole!added!to!clonal!S.&typhimurium!populations!and!indole!produced!by!E.&coli!in!mixed!microbial! communities.!! Further,!we! find! that! indole! signaling! by!E.& coli! induces! S.& typhimurium! antibiotic! tolerance! in! a!Caenorhabditis&elegans!model!for!gastrointestinal!infection.!These!results!suggest!that!the!intestinal!pathogen!S.&typhimurium!can!intercept!indole!signaling!from!the!commensal!bacterium!E.&coli!in!order!to!enhance!its!antibiotic!tolerance!in!the!host!intestine.!!!!

71! ! Parallel!evolution!of!bacterial!RNA!cis7regulators!for!ribosomal!protein!S15!

!Betty!Slinger1,!Molly!Gilligan1,!and!Michelle!M.!Meyer1!!1Biology!Department,!Boston!College!!Of!the!RNA!regulators!characterized!in!bacteria,!from!riboswitches!to!sRNAs,!autogenous!regulatory!elements!controlling!ribosomal!protein!synthesis!were!among!the!first!to!be!discovered!over!40!years!ago.!!These!structured!RNA!cis9regulators!are!typically!in!the!5’!UTR!of!an!operon!and!interact!with!excess!levels!of!a!specific!ribosomal!protein!to!prevent!translation!of!the!entire!operon,!thus!balancing!levels!of!ribosomal!proteins!across!different!transcriptional!units.!!The!regulator!of!ribosomal!protein!S15!in!this!manner!represents!an!especially!interesting!example!of!parallel!evolution.!!Four!distinct!RNA!structures,!that!share!no!obvious!similarity!but!have!a!common!biological!function,!have!been!discovered!in!different!bacterial!phyla.!!We!are! interested!in!how!these!dissimilar!RNAs!interact!with!homologous!proteins!to!regulate!gene!expression.!!In&vivo!and! in&vitro&assessment!of!RNA9protein!binding!and!regulatory! interactions! between! partners! from! different! species! reveal! that! RNA! structures! from! distinct! species! often! do! not!respond! to! all! S15! homologs.!! This! suggests! that! each! RNA9protein! pairing!may! have! distinct! binding! determinants,! despite! the!overall! similarity! between! the! protein! partners.! This! example! of! parallel! evolution! highlights! the! differences! in! gene9regulatory!mechanisms!in!different!bacterial!phyla!and!the!flexibility!of!RNA!as!a!regulator.!!!!

72! ! Analysis!of!the!roles!of!Shewanella&oneidensis!Hfq!in!cell!growth!and!oxidative!stress!response!

Christopher!Brennan,!Meaghan!Keane,!Taylor!Hunt,!Nick!Mazzucca,!Jessica!Leonard,!and!Brett!Pellock!!Department!of!Biology,!Providence!College!!Hfq!is!an!RNA!chaperone!protein!broadly!implicated!in!sRNA!function!in!bacteria.!We!have!constructed!a!null!allele!of!the!gene!that!encodes!the!RNA!chaperone!Hfq!in!Shewanella&oneidensis!strain!MR91,!a!dissimilatory!metal!reducing!bacterium.!Loss!of!hfq! in!S.&oneidensis!results!in!a!variety!of!mutant!phenotypes,!all!of!which!are!fully!complemented!by!addition!of!a!plasmid9borne!copy!of!the!wild!type!hfq!gene.!Aerobic!cultures!of!the!hfq&mutant!grow!more!slowly!through!exponential!phase!than!wild!type!cultures.!In!addition,!hfq&mutant&cultures!reach!a!terminal!cell!density!in!stationary!phase!that!is!~2/3!of!that!observed!in!wild!type!cultures.!Loss!of!hfq!also!results! in!defects! in!anaerobic!growth!with!fumarate!as!the!terminal!electron!acceptor!and!reduced!efficiency!of!Cr(VI)!reduction.!Finally,!the!hfq&mutant!exhibits!a!striking!loss!of!colony!forming!units!in!extended!stationary!phase!and!is!highly!sensitive! to! oxidative! stress! induced! by! H2O2! or!methyl! viologen! (paraquat).!We! are! currently! investigating! the!mechanisms! by!which!Hfq!promotes!cell!growth!and!oxidative!stress!survival!in!S.&oneidensis.!!!!!!!!


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73! ! Interaction7induced!Motility!of!Pseudomonas&fluorescens!Pf071!and!Pedobacter!V48!

!Adam.!S.!Bitzer1,!Sarah.!C.!Seaton2,!Stuart.!B.!Levy2,!Mark.!W.!Silby1;!1Biology!Dept.,!Univ.!of!Massachusetts!Dartmouth,!North!Dartmouth,!MA,!2Tufts!Univ.!Sch.!of!Med.,!Boston,!MA!!Close! interactions! among! bacteria! are!widespread! in! nature,!where!multi9species! communities! prevail.! Here!we! an! interaction9induced!social!trait,!motility!across!an!agar!surface.!When!strains!P.!fluorescens!Pf091!and!Pedobacter!V48!grow!alone!2%!Tryptic!Soy! agar,! they! form! non9motile! colonies.!When! they! physically! interact,! a!motile!mixed! species! colony! spreads! across! the! agar!surface.!Microbiological!methods!have!been!used!to!physically!characterize!the!motile!colony,!and!mutagenesis!and!transcriptome!analysis!have!been!carried!out!to!dissect!the!mechanisms!governing!this!social!motility!trait.!!Transcriptome!analysis!of!Pf091!during!the!interaction!showed!228!genes!increase,!ranging!in!fold!change!of!1.37!to!43.36,!and!179!decrease,!ranging!from!91.45!to!917.06!(P<.05).!!Notable! among! these! is! the!up9regulation!of! two!gene! clusters! predicted! to! specify! benzoate! and! catechol! catabolism.!Changes! in! flagella!or! pili! genes!were!noted,! suggesting! that! increased!expression! is! unnecessary! for! social!motility,! but! altered!expression!accelerates!motility!as!seen!in!transposon!mutants.!These!data!are!beginning!to!reveal!the!mechanistic!basis!of!surface!locomotion!of!two!species!where!neither!could!move!alone.!!These!are!of!interest!in!understanding!microbial!community!dynamics!and!for!application!in!biocontrol,!where!enhanced!motility!on!root!surfaces!might!improve!plant!protection.!!!

74! ! Novel!cell!division!and!growth!genes!discovered!using!high7throughput!microscopy!

!Christina!E.!Baer1,2!and!Christopher!M.!Sassetti1,2!1Department! of!Microbiology! and! Physiological! Systems,! University! of!Massachusetts!Medical! School,! 2Howard! Hughes!Medical!Institute,!University!of!Massachusetts!Medical!School!!The! polar! growth! of! Mycobacterium& tuberculosis& (Mtb)! contrasts! strikingly! with! the! lateral! growth! found! in! E.coli! and! the!alternating!polar!and!lateral!growth!observed!in!B.subtilis.!!This!growth!pattern!necessitates!continual!coordinated!trafficking!of!cell!growth! and! division! machinery.!! In! comparison! to! the! two! canonical! models! for! bacterial! growth! and! division,! the! molecular!mechanisms!driving!the!spatial!specialization!of!the!mycobacterial!cell!are!not!well!understood.!!

To! identify! the! novel! genes! that! coordinate! cell! growth! and! division! in!mycobacteria,! we! constructed! a! library! of! 16,000!transposon!mutants! in! a!Mycobacterium& smegmatis& (Msmeg)! strain! carrying! fluorescent! protein! markers! at! the! poles! and! the!septa.!We!developed!a!high9throughput!microscopy!pipeline!capable!of!imaging!each!mutant!strain!at!high9resolution.!!Using!this!pipeline,!we!have!begun!to!identify!genes!responsible!for!growth!site!selection,!cell!length,!and!cell!diameter.!!

We! observe! that! mutants! with! defects! in! growth,! internal! functional! organization,! and! division! have! widely! variable!morphologies.!!!Data!analysis!reveals!candidates!for!functional!cell!division!and!cytoskeletal!proteins!in!many!pathways,! including!lipid! metabolism.!! Our! approach! for! screening! mutant! libraries! microscopically! provides! a! new! experimental! pipeline! for! the!identification!of!candidate!genes!responsible!for!cell!growth!and!division!in!other!organisms!and!under!diverse!conditions.!!!

75! ! Concentration7dependent! interactions!with! the! sliding! clamp! serve! as! a! switch! for! regulating! E.& coli! polymerase!exchange!!James!E!Kath1,!Seungwoo!Chang1,!Slobodan!Jergic2,!Deena!Jacob3,!Justin!MH!Heltzel4,!Mark!D!Sutton4,!Graham!C!Walker3,!Nicholas!E!Dixon2,!and!Joseph!J!Loparo1!1Department! of! Biological! Chemistry! and!Molecular! Pharmacology,! Harvard!Medical! School,! 2School! of! Chemistry,! University! of!Wollongong,!3Department!of!Biology,!Massachusetts! Institute!of!Technology,!4Department!of!Biochemistry,!University!of!Buffalo,!The!State!University!of!New!York!!Translesion! polymerases! confer! damage! tolerance! by! their! ability! to! synthesize! across! DNA! lesions.! To! better! elucidate! the!molecular! origins! of! translesion! polymerase! exchange,! we! have! developed! a! single9molecule! assay! that! can! measure! primer!extension! activity! on! individual!DNA! substrates.! The!dramatically! different! rates! of!DNA! synthesis! by! replicative! and! translesion!polymerases!allow!for!the!assignment!of!the!active!polymerase!at!the!primer!terminus,!providing!us!with!a!kinetic!description!of!polymerase! exchange! inaccessible! in! ensemble! biochemical! experiments.! We! have! utilized! this! assay! to! study! switching! of!Polymerase! (Pol)! III,! the! E.& coli! replicative! polymerase,! with! the! highly! conserved! translesion! polymerase,! Pol! IV! at! the! primer!terminus.!Polymerase!exchange! is!mediated!through! interactions!with!the!processivity! factor!Beta,!which! is!proposed!to!act!as!a!recruiting! platform! for! DNA! processing! enzymes.! We! find! that! both! Pol! IV! and! Pol! III! are! able! to! bind! Beta! in! an! inactive!conformation!while! the! other! is! carrying! out! synthesis.! At! SOS9uninduced! concentrations! of! Pol! IV,! the! processivity! of! Pol! III! is!


56

largely!unaffected!by! the!presence!of!Pol! IV!bound! to! the! sliding!clamp.!High!concentrations!of!Pol! IV,! consistent!with! the!SOS9induced!state,!lead!to!a!Beta9mediated!decrease!in!Pol!III!processivity.!!!

76! ! Studying!the!role!of!the!bacterial!tyrosine!kinase!YwqD!in!biofilm!formation!in!Bacillus&subtilis!

!Tantan!Gao1,!2,!3,!Alexander!Elsholz1,!Yunrong!Chai3,!and!Richard!Losick1!1Department!of!Molecular!and!Cellular!Biology,!Harvard!University,!2Department!of!Plant!Pathology,!China!Agricultural!University,!3Department!of!Biology,!Northeastern!University!!Undomesticated!Bacillus&subtilis!strains!form!structured,!multicellular!communities!known!as!biofilms.!Previous!work!showed!that!deletion! of! the! ywqCBF! operon! resulted! in! a! notable! defect! in! biofilm! formation.!Within! the! operon,! ywqD! encodes! a! bacterial!tyrosine!kinase!(BYK).!In!this!study,!we!carried!out!a!genetic!screen!searching!for!suppressors!of!ΔywqCBF!mutation!and!identified!one!such!suppressor.!Results! from!genetic!mapping!and!strain! reconstitution!confirmed!that! the!suppressor!mutation! is!a! single!nucleotide!change!in!the!5!prime!non9coding!region!of!the!sinR!mRNA.!SinR&is!a!repressor!for!the!matrix!operons!epsABO!and!tapABsipWByqxM.!By!using! lacZ!reporters,!we!showed!that!the!expression!of!sinR!was!lower!in!the!suppressor!mutant!whereas!epsABO!was! highly! up9regulated.! epsB! in! the! epsABO! operon! encodes! a! second! BYK! in! B.& subtilis,! which! strongly! resembles! YwqD.!We!therefore! hypothesize! that! in! the! suppressor! mutant,! epsB! up9regulation! compensates! the! loss! of! YwqD! and! thereby! rescues!biofilm!formation.!We!expressed!epsB!from!a!xylose!inducible!promoter!and!found!that!it!similarly!rescued!the!biofilm!phenotype!caused!ΔywqCBF.!Our!results!suggest!that!there!is!“crosstalk”!between!the!two!BYKs!in!B.&subtilis,&providing&a!possible!link!between!biofilm!formation!and!cell!physiology.!!!

77! ! Genomics!of!the!Conjunctival!Pathogen!Streptococcus&pneumoniae!

!Michael!D.!Valentino1,2,!Jason!W.!Rosch3,!Wolfgang!Haas6,!Christine!M.!Sanfilippo6,!Robert!A.!Carter3,!Michael!E.!Zegans4,!Bernard!Beall5,!Elaine!I.!Tuomanen3,!Timothy!W.!Morris6!and!Michael!S.!Gilmore1,2!1Department!of!Ophthalmology,!Massachusetts!Eye!and!Ear!Infirmary,!2Department!of!Microbiology!and!Immunobiology,!Harvard!Medical! School,! 3Department! of! Infectious! Diseases,! St.! Jude! Children’s! Research! Hospital,! 4Department! of! Microbiology! and!Immunology,! Geisel! School! of!Medicine! at! Dartmouth,! 5Streptococcus! Laboratory,! Centers! for! Disease! Control! and! Prevention,!6Bausch!and!Lomb,!Inc.!!Streptococcus&pneumoniae& is!a!prevalent!cause!of!bacterial!conjunctivitis,!especially! in!children.!One! lineage! in!particular,!ST448,!has!caused!multiple!outbreaks!of!conjunctivitis!and!was!recently!shown!by!our!group!to!be!a!major!contributor!to!non9outbreak!related!ocular!infections!throughout!the!US.!These!findings!suggest!that!some!lineages!of!S.&pneumoniae!possess!a!unique!ocular!tropism!and!are!capable!of!spawning!epidemics.!We!set!out!to!sequence!representative!strains!isolated!from!three!major!outbreaks!to!begin!to!identify!candidate!functions!these!outbreak!strains!possess!that!potentially!contribute!to!the!ability!to!cause!epidemic!conjunctivitis,!and!account!for!their!unusual!tropism.!Comparative!analysis!shows!that!the!genome!landscape!content!of!an!ST448!strain!is!substantially!different!from!even!the!nearest!strain,!and!possesses!a!number!of!genes!with!the!potential!to!contribute!to!its!prevalence!in!outbreak!and!non9outbreak!infections!and!tropism!for!the!ocular!surface!of!sequence!type!448.!!!

78! ! Expression!of!ids&genes!in!Proteus&mirabilis&is!stably!bimodal!and!likely!regulated!by!non7heritable!factors!

!Kristin!Little1!and!Karine!Gibbs1!1Department!of!Molecular!and!Cellular!Biology,!Harvard!University!!Swarming! populations! of! the! bacterium! Proteus& mirabilis& are! capable! of! self! recognition:! they! selectively! merge! with! isogenic!swarms! but! form! a!macroscopic! boundary! against! non9isogenic! swarms.! Expression! of! the! ids&genes! is! necessary! for! this! group!behavior.! Interestingly,!previous!studies!have!shown!that!a!subset!of!cells! in!a!clonal!population!does!not!express! ids&genes.!We!have! further! quantified! and! confirmed! ids& gene! bimodality.! Studies! utilizing! wild9type! P.& mirabilis! carrying! a! fluorescent! ids&expression! reporter! suggest! maximal! population9wide! ids& expression! at! the! transition! to! stationary! phase! in! liquid! culture.! To!examine!whether!ids&expression!state!is!determined!by!heritable!factors,!we!employed!Fluorescence!Activated!Cell!Sorting!(FACS)!to!separate!ids9expressing!from!non9ids9expressing!cells.!Individual!cells!of!each!expression!state!were!used!as!the!progenitor!cell!to!inoculate!subsequent!cultures.!After!approximately!30!generations!cells!were!resorted,!and!the!distribution!of!idsBexpressing!cells!was!determined.!We!found!after!60!generations! (two!passages),! the!percentage!of! idsBexpressing! (~80%)!and! idsBnon9expressing!


57

(~20%)!cells!was!maintained!across!all!samples.!This!result!suggests!that!ids&expression!state&is!bistable!and!non9heritable.!Cell9to9cell!heterogeneity!of! ids!expression!has!interesting!implications!for!self!recognition!and!boundary!formation,!and!its!role!in!these!group!behaviors!remains!unknown.!!!!

79! ! Biotic!Interactions!Drive!Contingent!Adaptation!of!Pseudomonas&aeruginosa!Biofilms!

!Kenny!Flynn1!and!Vaughn!Cooper1!1Department!of!Molecular,!Cellular!and!Biomedical!Sciences,!University!of!New!Hampshire,!Durham,!NH!03861!!Biofilms! and! associated! chronic! infections! are! known! to! harbor! great! biodiversity! but! the! consequences! of! this! diversity! on!community!function!are!not!well!understood.!!To!understand!the!forces!favoring!diversification,!we!experimentally!evolved!three!populations!from!a!clone!of!Pseudomonas&aeruginosa!PA14!for!540!generations!under!conditions!favoring!a!regular!cycle!of!biofilm!formation! and! dispersal.!! A! unique! set! of! colony! types! evolved! in! each! community,! and! the! lack! of! parallelism! was!puzzling.!! Sequencing! of! clones! and! the! complete! biofilm! community! revealed! that! co9existing! strains! shared! a! small! subset! of!mutations! that! fixed! in!conjunction!with!a!defect! in!DNA!mismatch!repair.!!More!evident!were! the!changing! targets!of! selection!with!prolonged!adaptation!initially!favoring!mutations!related!to!DNA!repair!followed!by!regulatory!mutations.!!This!ecological!and!genetic!succession!evidently!led!to!strongly!co9adapted!communities!with!distinct!phenotypes!related!to!variation!in!cyclic9di9GMP!metabolism.!!Manipulating!cyclic9di9GMP! levels!within! individual!mutants! in! the! focal!community!not!only!reduced!the! fitness!of!the!targeted!mutant!but!also!drastically!altered!community!composition!and!destabilized!the!biofilm.!!In!summary,!these!findings!suggest! that! adaptation! in! biofilm! environments! is! largely! driven! by! strong! biotic! interactions! between! residents! following!stochastic!patterns!of!colonization!and!adaptive!variation.!!!

80! ! Widespread!mRNA!processing!shapes!the!Mycobacterium!tuberculosis!transcriptome!

!Scarlet!S.!Shell1*,!Thomas!R.!Ioerger2*,!Michael!R.!Chase1*,!James!C.!Sacchettini3,!and!Sarah!M.!Fortune1!1Department!of! Immunology!and! Infectious!Diseases,!Harvard!School!of!Public!Health,! 2Department!of!Computer! Science,! Texas!A&M!University,!3Department!of!Biochemistry!and!Biophysics,!Texas!A&M!University!*These!authors!contributed!equally!!Prokaryotic! mRNAs! are! subject! to! processing! by! endonucleolytic! cleavage,! which! is! generally! thought! to! initiate! transcript!degradation;!however,!in!a!few!cases!this!type!of!processing!leads!to!stable!cleavage!products.!Additionally,!stable!structural!RNAs!such! as! rRNAs! and! tRNAs! require! processing! for! correct!maturation.! Here!we! report! unexpectedly! widespread! RNA! processing!across!the!M.&tuberculosis!transcriptome.!Using!an!Illumina!sequencing!platform!to!globally!map!stable,!processed!mRNA!5’!ends,!we!detected!approximately!3,000!high9confidence!processing!sites.!Nearly!80%!of!the!processing!sites!are!located!within!annotated!coding! sequences.! If! translated,!many! processed!mRNA! fragments!would! be! expected! to! produce! alternative! protein! isoforms.!Consistent! with! this! hypothesis,! mass! spectrometry! revealed! protein! N9termini! mapping! to! internal! start! codons! in! several!processed! genes.! In! other! cases!mRNA!processing! divides! polycistronic! transcripts! into! fragments! that! have! different! stabilities,!resulting! in!differential! transcript! abundance!among!genes!within! individual! operons.!Overall,! RNA!processing! appears! to!play! a!major!and!previously!under9appreciated!role!in!shaping!the!M.&tuberculosis!transcriptome!and!regulating!gene!expression.!To!our!knowledge,!this!is!the!first!report!of!transcriptome9wide!RNA!processing!site!identification!in!any!bacterium.!!!

81! ! Regulation!of! the! iron! acquisition! and!utilization! response! by! extracellular! iron7scavenging!molecules! under! iron!replete!conditions!in!Bacillus&subtilis!!Rajat!Kumar!Pal!and!Kevin!Griffith!Microbiology!Department,!University!of!Massachusetts9Amherst!!Iron! is! an! essential! nutrient! required! for! the! viability! of!microorganisms.!! Under! aerobic! conditions! at! neutral! pH,! iron! is! highly!insoluble!(10918!M),!yet!bacteria!normally!require!~1096!M!iron!for!survival.!!To!overcome!this!disparity,!one!strategy!bacteria!have!evolved!is!to!produce!iron!scavenging!molecules,!known!as!siderophores,!which!are!secreted!into!the!environment!and!have!a!high!affinity!for!ferric! iron.!!Specific!receptors! import!the!iron9siderophore!complexes!into!the!cell!to!satisfy!the!cell’s!requirement!for!iron.!! In! this! work,! we! identified! a! novel! iron! scavenging! activity! from! cultures! of! Bacillus& subtilis& grown! under! iron! replete!conditions!(5!μM!FeCl3).&&The!iron!scavenging!activity!is!regulated!in!a!density9dependent!manner!with!maximal!activity!present!in!cultures!grown!to!high!population!density,!while!little!activity!is!observed!at!low!density.!!We!determined!that!the!first!gene!of!the!


58

ywbLMNO!operon! is! required! for! the!uptake!of! iron!under! these!conditions.!!Work! is! currently!underway! to!determine!whether!other!gene!products!of!this!operon!are!required!for!iron!uptake!and!to!identify!the!corresponding!siderophore(s).!!This!work!is!likely!to!aid!in!our!identification!of!novel!siderophores!in!B.&subtilis!and!also!contribute!to!our!general!understanding!of!iron!acquisition!by!bacteria.!!!!!!!

82! ! Toxin7Antitoxin!Interferases!in!Stress!Response!and!Persister!Formation!

!Autumn!Brown,!Sarah!Rowe,!and!Kim!Lewis!Antimicrobial!Discovery!Center,!Department!of!Biology,!Northeastern!University!!Toxin9antitoxin! (TA)! systems! have! been! gaining!much! attention! in! recent! years.! !They! have! been! implicated! in! a!wide! range! of!cellular! functions! including! stress! response,!persister! formation,! and!biofilm! formation.! The! chromosome!of!Escherichia& coli! K12!harbors! 11! seemingly! redundant! TA! interferases.! In! each! of! these! systems! the! toxin! acts! as! an! mRNA! endonuclease! when! its!cognate! antitoxin! is! degraded! by! a! stress9induced! protease.! Previous! studies! have! shown! that! ectopic! overexpression! of! toxin!interferases! induces! the! formation! of! persisters.! However,! deletion! of! at! least! 6! TAs! is! required! to! see! a! 109fold! reduction! in!persister!formation.!We!hypothesized!that!these!interferases!may!be!induced!under!different!stress!conditions!and!have!a!role!in!adapting! to!stress.!We!have!generated!GFP9promoter! fusions! for! the!11!TA! interferases! in!order! to!monitor! their! induction! to!a!range!of!exogenous!and!native! stresses.!Our! results! show! that!between!one!and! three! interferases!are!upregulated!under!each!stress!condition.!This!finding!indicates!that!individual!interferases!play!a!role!during!some!but!not!all!stresses!and!may!provide!an!insight!into!the!redundancy!of!the!TA!interferases!in!E.&coli!as!well!as!other!bacteria.!Deletion!mutants!are!currently!being!tested!to!determine!if!TA!interferases!that!are!upregulated!during!a!stress!contribute!to!adaptation!or!survival!under!the!same!stress.!!!

83! ! Interaction! between! PigR! and! the! MglA7SspA! complex! in! Francisella& tularensis! is! necessary! for! virulence! gene!expression!!Amy!Rohlfing!and!Simon!Dove!Division!of!Infectious!Diseases,!Boston!Children’s!Hospital,!Harvard!Medical!School!!In! the!Gram9negative! intracellular!pathogen!Francisella& tularensis,! the!expression!of!many! virulence!genes! is! regulated!by! three!proteins,!MglA,!SspA,!and!PigR.!!Evidence!suggests!that!MglA!and!SspA!form!a!heterodimeric!complex!that!directly! interacts!with!PigR,! a! putative! DNA! binding! protein.!! To! investigate! the! role! of! this! interaction,! a! genetic! screen!was! performed! in!which!we!isolated! three! mutants! of! MglA! that! were! specifically! defective! for! interaction! with! PigR.! The! corresponding! residues! in! MglA!appear! to! lie! at! the!edges!of! a! pocket! formed!by! the!predicted! interface!between!MglA! and! SspA.! Three! additional! amino!acid!substitutions! were! made! within! this! pocket.!! The! corresponding! mutants! were! also! specifically! defective! for! interaction! with!PigR.!!To!determine!if!the!interaction!with!PigR!is!necessary!for!virulence!gene!expression,!the!six!MglA!mutants!were!expressed!in!an!ΔmglA&strain!of!F.&tularensis.!!The!mutants!were!unable!to!restore!expression!of!two!MglA!regulated!genes.!!This!indicates!that!the!interaction!between!PigR!and!the!MglA9SspA!complex!is!necessary!for!virulence!gene!expression!in!F.&tularensis!and!identifies!a!putative!pocket!within!MglA!that!appears!to!be!important!for!interaction!with!PigR.!!!

84! ! Stochasticity!of!gene!expression!of!a!synthetic!oscillator!under!constant!growth!conditions!

!Laurent!Potvin9Trottier1,2,!Nate!Lord2!and!Johan!Paulsson2!1Biophysics!program,!Harvard!University,!2Department!of!Systems!Biology,!Harvard!Medical!School!!Cellular!processes!are!inherently!stochastic:!random!birth!and!deaths!of!individual!molecules!create!fluctuations!in!concentration!that! propagate! through! reaction!networks.! Cells!must! thus! control,! suppress,! exploit! or! tolerate! fluctuations.!Many! such! ‘noisy’!systems!have!now!been!analyzed!both!experimentally!and!mathematically,!mostly!emphasizing!fluctuations!around!a!given!average!while!much!less!is!known!about!the!regulation!of!systems!with!dynamically!changing!averages.!We!therefore!studied!the!sources!of!fluctuations!in!a!classic!synthetic!oscillator,!the!so9called!repressilator.!By!using!a!microfluidic!chip!developed!in!the!lab,!we!were!able! to!measure! oscillations! in! the! level! of! fluorescent! proteins! in! bacteria! growing! in! a! constant! environment! for! hundreds! of!consecutive! generations,! across! thousands! of! different! cells.! The! results! showed! that! the! repressilator!works!much! better! than!previously! thought,! with! essentially! all! bacteria! displaying! clear! oscillations! over! long! time! periods.! By! further! analyzing! the!underlying!mechanisms!we! suggest! that! this! is! due! to!noise9induced! transitions,!where!a!noise9free! system!would!be!unable! to!display!sustained!oscillations,!but!fluctuations!constantly!trigger!new!oscillations.!A!better!understanding!of!this!system!could!be!


59

helpful!when!designing!new!synthetic!devices,!but!the!regularity!of!the!oscillations!also!allow!us!to!use!the!system!as!a!tool!to!finely!quantify!many!properties!of!fluorescent!reporter!genes!such!as!maturation!delays.!!!

85! ! An!experimental!model!system!for!probing!microbial!community!interactions!and!dynamics!

!Julie!E.!Button1,!Laura!M.!Sanchez2,!Marcela!Santarelli3,!Benjamin!E.!Wolfe1,!Pieter!C.!Dorrestein2,!and!Rachel!J.!Dutton1!1FAS! Center! for! Systems! Biology,! Harvard! University,! 2Skaggs! School! of! Pharmacy! and! Pharmaceutical! Sciences,! University! of!California,!San!Diego,!3Department!of!Genetics,!Biology!of!Microorganisms,!Anthropology,!Evolution,!University!of!Parma!!Microbes!typically!live!in!complex,!multi9species!communities,!but!little!is!known!about!interspecies!interactions!that!shape!these!communities.!!Mechanistic!analyses!of!microbial!behavior!in!multi9species!communities!require!an!experimentally!tractable!model!system! that! is! culturable,! simple,! and! reproducible,! and!work! done! in! our! lab! demonstrates! that! the!microbial! communities! of!cheese!rinds!exhibit!these!characteristics.!!Here,!we!use!the!cheese!rind!model!system!to!track!succession!in!a!microbial!community!and!define!microbial!relationships!that!affect!succession.!!By!sequencing!time!course!samples!taken!from!a!natural!rind!cheese,!we!determined! that! the! rind! community! is! initially! dominated! by! Staphylococcus! sp.! As! the! cheese! ages,! Staphylococcus! sp.! are!succeeded!by!Brevibacterium&sp.,!which!come!to!dominate!the!rind!community.&&We!reproduced!this!major!pattern!on!a!cheese9like! medium! using! a! simplified,! in& vitro! community! consisting! of! Staphylococcus& succinus& and! Brevibacterium& linens.!! We!determined! that! S.& succinus& stimulates! growth! of! B.& linens,! and! eventually,! co9culture! with! B.& linens! results! in! death! of! S.&succinus.&&We!have! also! found! that! the! two! species! specifically! respond! to! each! other’s! proximity!with! the! production! of! small!molecules.!!Work!defining!the!mechanisms!and!genetic!pathways!involved!is!ongoing.!!!

86! ! cAMP!remodels!metabolic!flux!in!M.&tuberculosis!via!protein!lysine!acetylation!!

!Emily!S.!C.!Rittershaus1,!Seung9hun!Baek1,!and!Christopher!Sassetti1,2!1Microbiology!and!Physiological!Systems,!University!of!Massachusetts!Medical!School,!2Howard!Hughes!Medical!Institute!!During! infection,!Mycobacterium& tuberculosis! can! enter! into! a! stable! nonreplicating! state! in! which! the! bacterium! is! relatively!insensitive!to!both!immune!insults!and!antibiotic!treatment.!!This!quiescent!state!requires!a!number!of!metabolic!alterations,!which!are!only!beginning!to!be!understood.!We!subjected!a!M.&tuberculosis!transposon!mutant!library!to!oxygen!or!carbon!limitation!in&vitro! and! identified!mutants! that! could! not! survive.! In! tandem,!we! utilized!mass! spectrometry! to! assess! changes! in!metabolite!concentration!during!the!adaptation!to!stasis.!Finally,!we!have!employed!13C!tracing!to!verify!the!differential!activity!of!metabolic!pathways.!!

Using! this! coordinated! approach! we! are! beginning! to! define! a!multistep! cascade! that! is! important! for! the! adaptation! to!hypoxic! stasis.!First,! redox!stress! triggers!a! rapid!shift!of!carbon! flux! into!storage!pathways,!which! transiently! inhibits!growth!by!depleting!the!acetyl9CoA!pool.!This!initial!response!induces!the!accumulation!of!cyclic!AMP!(cAMP).!!cAMP!appears!to!act,!at!least!in!part,! by! activating! the! protein! lysine! acetyl! transferase,!Mt9PAT! (Rv0998).!! Mutation! of! either! an! adenylate! cyclase! or!Mt9PAT!causes!significant!changes!in!carbon!flux!and!perturbs!the!redox!state!of!the!cytosol.!!Mutants!lacking!component!of!this!cascade!are! defective! for! survival! in! hypoxia,! indicating! that! these! alterations! in! carbon! metabolism! are! critical! for! adapting! to! the!nonreplicating!state.!!!

87! ! Characterization!of!a!single7strand!origin!of!replication!in!an!integrative!and!conjugative!element!

!Laurel!D.!Wright,!Christopher!M.!Johnson,!and!Alan!D.!Grossman!Department!of!Biology,!Massachusetts!Institute!of!Technology!!!!Integrative!and!conjugative!elements!(ICEs,!also!known!as!conjugative!transposons)!are!the!largest!family!of!bacterial!conjugative!elements.!!Bacillus&subtilis&ICEBs1&replicates!by!a!rolling!circle!mechanism.!!During!rolling!circle!plasmid!replication,!a!single!strand!origin! (sso)!enhances!the!conversion!of!template!single9stranded!DNA!to!double9stranded!DNA!by!forming!a!stem9loop!structure!that! recruits! host! factors! to! initiate! second! strand! DNA! synthesis.! We! identified! a! putative! sso& in! ICEBs1! based! on! sequence!similarity! to! an! sso& in! a! B.& subtilis! rolling! circle! replicating! plasmid.!!! Using! Southern! blotting! and! segregational! analysis,! we!demonstrated! that! the! ICEBs1& sso& was! functional.!! Furthermore,! we! visualized! sso& function! in! live,! individual! cells! using! a!fluorescently9tagged!single9strand!DNA!binding!protein!(SSB).!!!Current!experiments!are!aimed!at!looking!at!the!effects!of!the!sso&on!ICEBs1&biology.!!We!suspect!many!other!ICEs!contain!a!single!strand!origin!of!replication.!!


60

88! ! A!Chemoproteomic!Approach!to!Enzyme!Discovery!During!Vibrio&cholerae!Infection!

!Stavroula!K.!Hatzios193,!Julianne!Martell4,!Diana!Munera193,!Soren!Abel193,!Eranthie!Weerapana4,!and!Matthew!K.!Waldor193!1Division! of! Infectious! Diseases,! Brigham! and! Women’s! Hospital,! 2Department! of! Microbiology! and! Immunobiology,! Harvard!Medical!School,!3Howard!Hughes!Medical!Institute,!4Department!of!Chemistry,!Boston!College!!Transcriptional! profiling! and!proteomics! are! powerful! tools! for! analyzing! gene! expression! in! biological! systems.!!However,! post9transcriptional!and!post9translational!modifications!can!lead!to!an!imperfect!correlation!between!protein!abundance!and!enzyme!activity.!! Activity9based! protein! profiling! (ABPP)! is! a! chemoproteomic! technique! that! enables! the! direct! evaluation! of! enzyme!activity!within!complex!proteomes.!!Activity9based!probes!selectively! label!the!active!site!of!a!particular!enzyme!class,!facilitating!detection,!enrichment,!and!identification!of!labeled!enzymes!by!mass!spectrometry.!!We!used!ABPP!to!identify!bacterial!and!host!serine!hydrolases!that!are!active!during!infection!with!Vibrio&cholerae,!the!causative!agent!of!cholera.!!By!profiling!serine!hydrolase!activity! in! the! infant! rabbit! model! of! infection,! we! identified! several! proteases! and! lipases! that! may! mediate! host9pathogen!interactions.!!!

89! ! Analysis!of!Microbial!Communities!from!the!Massachusetts!Alternative!Septic!System!Testing!Center!

!Alexander!Vickers!and!Heather!Wiatrowski!!Biology!Department,!Clark!University!!!Despite! the! key! role! of! microbial! activity! in! septic! leach! fields,! we! know! little! about! the! structure! of! these! communities.!! We!generated! a! clone! library! of! 16s! ribosomal! RNA! (rRNA)! genes! from! sediments! collected! at! the!Massachusetts!Alternative! Septic!System! Test! Center! (MASSTC).!! The! sequences! for! the! 16s! rRNA! genes! were! processed! using! the! Greengenes! pipeline,! then!classified!by!the!Ribosomal!Database!Project!(RDP),!the!National!Center!for!Biotechnology!Information!with!their!Basic!Alignment!Search!Tool!(BLAST).!!Microbial!community!structures!were!similar!in!a!leach!field!that!was!properly!loaded!with!sewage!and!one!that!was!overloaded!with!sewage.!!Betaproteobacteria!is!the!most!common!taxon!in!both!overloaded!and!properly!loaded!septic!leach! fields! (39%! and! 46%,! respectively! and! 44%!overall).! !Gammaproteobacteria! and!Alphaproteobacteria!were! also! identified,!composing!18%!and!15%!of!the!classifications!made.!!Several!sequences!may!have!originated!from!the!human!gut,!4%!of!sequences!were! from!the!phylum!Firmicutes,! including!one!member!of! the!Ruminococcaceae! family,!and!11%!of! sequences!were! from!the!phylum!Bacteroidetes.!!!

90! ! Cooking!alters!gut!microbial!ecology!

!Rachel!N.!Carmody1!and!Peter!J.!Turnbaugh1!1FAS!Center!for!Systems!Biology,!Harvard!University!!Human! energy! gain! is! shaped! by! the! gut! microbiota,! which! plays! critical! roles! in! nutrient! digestion! and! synthesis.! While!contributions!of!the!gut!microbiota!to!energy!gain!from!diets!differing!in!quantity!or!composition!have!been!studied,!the!impacts!of!processing!a!given!diet!by!common!methods! like!cooking!remain!unknown.!Here,!we!test! the!hypothesis! that!cooking!alters!gut!microbial!ecology!by!interrogating!the!gut!microbiotas!of!conventional!BALB/c!mice!fed!standardized!diets!of!meat!or!sweet!potato!served! raw! or! cooked.! 16S! rRNA! gene! sequencing! and! microbial! RNAseq! conducted! on! distal! gut! samples! revealed! dramatic!impacts!of!cooking!on!microbial!community!structure!and!gene!expression,!particularly!on!the!sweet!potato!diet.!Shifts!associated!with! cooking,! which! occurred! within! a! single! day,! favored! microbial! taxonomic! groups! previously! linked! to! host! adiposity,!suggesting! that! cooking! may! promote! energy! gain! in! part! through! microbial! mechanisms.! Notably,! the! microbial! community!structures!of!mice! fed! raw! sweet!potato!were!more! similar! to!mice! fed!meat! than! to!mice! fed! cooked! sweet!potato,! providing!striking!support!for!a!view!beyond!caloric!content!and!macronutrient!profile! in!the!assessment!of!dietary!factors!shaping!the!gut!microbiota.!!!!!!!!


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91! ! Exploring!the!dynamics!within!photosynthetic!communities!that!degrade!cellulose!and!fix!nitrogen!

!Emily!Mamula1,!Brandon!Chiou1,!Jea!Young!Park1,!and!Jean!Huang1!1Huang!Lab,!Franklin!W.!Olin!College!of!Engineering!!Nutrient!cycling! is! carried!out!by!communities!of!bacteria! in!nature.! !Cellulose! is! the!most!abundant!polymer! in!nature,!but! it! is!nitrogen!poor.! !In!this!research!we!cultivate!and!investigate!a!variety!of!marine!and!freshwater!photosynthetic!communities!that!degrade!cellulose!and!fix!nitrogen.!!The!diversity!of!the!communities!decreases!over!time!with!culture!transfer.!!We!are!analyzing!the!communities!using!genome!sequencing!and!biochemistry.!!From!one!community,!we!cultured!a!nitrogen!fixing!photosynthetic!bacterium!that! is!a!Rhodopseudomonas!palustris.! !We!study!the!growth!and!metabolic!capabilities!of!the!R.!palustris! isolate!and!examine!its!nitrogen!fixing!contribution!to!the!community.!!We!wish!to!build!a!model!that!incorporates!this!data!to!predict!factors!for!successful!nutrient!cycling!by!the!community.!!!!!

92! ! Exogenous!Carbohydrates!and!Glycerol!are!Required!for!Growth!During!Mtb&Infection!

!Jarukit!Edward!Long1!and!Christopher!Sassetti1!1Microbiology!and!Physiological!Systems,!University!of!Massachusetts!Medical!School!!The!preferred!niche!for!Mycobacterium!tuberculosis!(Mtb)!throughout!most!infection!is!a!phagosome9like!compartment!of!the!host!macrophage.!How!Mtb!obtains!nutrients!in!this!compartment!is!unclear.!!Infection!with!Mtb!can!be!divided!into!two!distinct!stages:!the!early!acute!phase!and!the!late!chronic!phase.!!Previously!we!used!a!global!genetic!interaction!mapping!strategy!to!characterize!a! cholesterol! uptake! system,! called! “Mce4”.! This! transporter! is! only! required! for! survival! during! the! chronic! stage! of! infection,!suggesting!that!the!nutrients!available!to!the!bacterium!change!as!disease!progresses.!!The!carbon!source(s)!used!during!the!acute!phase! remain! unknown.! !To! determine! what! nutrients! are! acquired! during! early! infection! we! undertook! a! high9throughput!transposon! screen! for!mutants! that! are! defective! for! growth! during! acute! infection.!We! found! a! putative! carbohydrate! uptake!system!(rv2038cBrv2040c)&that!is!critical!for!growth!during!this!stage.!!Using!a!transposon!sequencing!(TnSeq)!approach!to!measure!global!genetic!interactions,!we!verified!that!this!system!supplies!hexose!to!the!bacterium.!!Furthermore,!the!loss!of!hexose!uptake!revealed!a!new!requirement!for!glycerol!acquisition.!!These!results!suggest!that!Mtb&acquires!exogenous!carbohydrates!as!well!as!glycerol!for!biosynthetic!purposes!during!the!early!stages!of!infection.!!!

93! ! Co7culturing!of!Pseudomonas&and!Pedobacter&induces!an!interspecies!social!trait!

!Sarah!C.!Seaton1,!Adam!Bitzer2,!Stuart!B.!Levy1,!and!Mark!W.!Silby2!1Department! of!Molecular! Biology! and!Microbiology,! Tufts!University! School! of!Medicine,! 2Department! of! Biology,!University! of!Massachusetts!Dartmouth!!Bacteria! often! exist! as! multi9species! communities,! wherein! physical! and! chemical! interactions! influence! the! physiology! of! the!organisms.! We! recently! reported! increased! antibiotic! production! when! the! soil! bacteria! Pseudomonas& fluorescens& Pf091! and!Pedobacter&V48! interact.!Here!we!describe!another! interaction9induced!trait—social!motility!across!a!hard!surface—and!work!to!define!the!mechanism!underlying!this!response.!A!genetic!screen!identified!Pseudomonas&mutants!with!a!hyper9motile!phenotype!in!the!presence!of!Pedobacter,&and!mutations&map!to!genes!required!for!flagellar!biosynthesis.!Such!mutants!show!enhanced!social!motility!despite!being!deficient!in!flagellar9mediated!swimming!motility.!Additional!Pseudomonas&mutants!deficient!in!type9IV!pili!biogenesis!indicate!that!social!motility!is!enhanced!in!the!absence!of!surface!pili.!Transcriptome!analysis!indicates!a!global!response!of!Pseudomonas!to!Pedobacter,!with!over!400!genes!showing!altered!expression!during!interaction.!Pseudomonas!flagella!and!pili!genes! show! reduced! expression,! while! genes! involved! in! benzoate! catabolism! exhibit! elevated! expression.! The! importance! of!aromatic! compound! catabolism! during! interaction! is! unclear,! but! could! involve!metabolic! exchange! or! co9metabolism.! Imaging!mass! spectrometry! allowed! spatial! analysis! of! metabolite! exchange! during! Pseudomonas9Pedobacter! interaction,! and! revealed!small!molecules!that!may!drive!the!coordinated!motility.!Together,!our!data!begin!to!reveal!the!mechanism!of!an!interspecies!social!trait.!!!!!!


62

94! ! Subversion!of!the!host!cell!cycle!by!Legionella&pneumophila!

!Dennise!A.!de!Jesús9Díaz1!and!Ralph!R.!Isberg,!Ph.D.1,2!1Department! of!Molecular! Biology! and!Microbiology,! Tufts! University! Sackler! School! of! Graduate! Biomedical! Sciences,! 2Howard!Hughes!Medical!Institute!!The! ubiquitous! Gram9negative! pathogen! Legionella& pneumophila! modulates! endocytic! and! immune! host! processes! to! ensure!formation! of! the! vacuole! compartment! where! it! replicates! intracellularly.! However,! we! hypothesize! that,! as! an! intracellular!pathogen,!L.&pneumophila!would!exploit!additional!host!networks!to!replicate.!Analyses!of!host!cell!cycle!dynamics!upon!Legionella!infection!revealed!that!L.&pneumophila!infected!cells!couldn’t!progress!through!the!cell!cycle!independently!of!the!cell!cycle!stage!the!host!cell!is!at!the!time!of!infection.!In!order!to!prevent!host!cells!from!cycling,!Legionella!requires!the!presence!of!a!functional!Type!4!Secretion!System,!as! cells! infected!with!a!L.&pneumophila!mutant!defective! for! translocation!have! the!ability! to!progress!through!the!cell!cycle.!!Interesting,!Legionella! intracellular!growth!is!enhanced!in!host!cells!present!at!either!G1!or!G2!of!the!host!cell! cycle,! but! reduced! in! cells! present! in! S9phase.! !Our! results! demonstrate! that! L.pneumophila! preferentially! infects! growth9arrested!hosts!because!S9phase!of!the!host!cell!cycle!is!an!unstable!environment!for!bacteria!replication;!the!Legionella!containing!vacuole!is!permeable!at!this!stage!allowing!the!detection!of!bacteria!from!the!cytosol.!!Thus,!by!stopping!cell!cycle!progression!L.&pneumophila!prevents!detrimental!consequences!and!preserves!its!ability!to!replicate.!!!!

95! ! Lassomycin,! a! novel!lasso! peptide!kills!Mycobacteria& tuberculosis!by! targeting! the! ATP7dependent! protease!ClpC1P1P2!!!Ekaterina! Gavrish1,! Clarissa! Sit2;! Olga! Kandror3,! Amy! Spoering4,! Aaron! Peoples4,! Lucy! Ling4,! Ashley! Fetterman4,! Dallas! Hughes4,!Shugeng!Cao2,!Anthony!Bissell1,!Heather!Torrey1,!Tatos!Akopian3,!Andreas!Mueller3,!Alfred!Goldberg3,!Jon!Clardy2,!and!Kim!Lewis1!1Antimicrobial!Discovery!Center,!Department!of!Biology,!Northeastern!University.!2Department!of!Biological!Chemistry!&!Molecular!Pharmacology,!Harvard!Medical!School!3Department!of!Cell!Biology,!Harvard!Medical!School.!4NovoBiotic!Pharmaceuticals,!LLC.!!Most! antibiotics!were! discovered! from! soil!microorganisms,! but! this! limited! resource!was! overmined! in! the! 60s.! !We! turned! to!uncultured!bacteria!as!an!untapped!source!of!novel!compounds,!using!in&situ!cultivation!and!other!methods!to!grow!them.!Extracts!from!a!collection!of!uncultured!strains!were!screened!in!whole9cell!assays!for!specific!activity!against&M.&tuberculosis.!We!identified!lassomycin,! a! novel! peptide! produced! by! an! actinomycetes! related! to! Lentzea& kentuckyensis.! Time9dependent! killing! showed!lassomycin! has! potent! bactericidal! activity! against! exponential! and! stationary!M.& tuberculosis.! The! compound! is! active! against!multi9drug!and!extensively9drug!resistant!strains!of!M.&tuberculosis.&Lassomycin! is!highly!selective!for!Mycobacteria.!Studies!with!mammalian! cell! lines! showed! no! cytotoxicity.! To! identify! the! target! of! lassomycin,! resistant! mutants! were! isolated! and! whole!genomes! sequenced.! Analysis! of! SNPs! from! six! independent! resistant! mutants! identified! the! target! as! ClpC1,! a! subunit! of! the!essential! mycobacterial! protease! ClpP1P2.! Lassomycin! activated! the! ATPase! activity! of! ClpC1! in& vitro.! !A! fluorescent! protein9degradation!assay!in!M.&smegmatis!suggests!that!lassomycin!might!also!activate!proteolytic!activity.!Uniform!labeling!of!the!peptide!and!NMR!studies!revealed!the!compound!consists!of!189amino!acids!where!the!N9terminal!residue!forms!an!89residue!ring.!A!novel!antibiotic!target!and!potent!killing!activity!makes!lassomycin!a!prime!candidate!for!antitubercular!drug!development.!!!!

96! ! Protein!localization!to!membrane!subdomains!in!Mycobacterium&smegmatis!!

!Jennifer!M!Hayashi!and!Yasu!S!Morita!Department!of!Microbiology,!University!of!Massachusetts!Amherst!&

Mycobacterium& tuberculosis! is! the! etiological! agent! of! tuberculosis,! and! like! other! mycobacteria! species,! it! has! a! complex!multilayered!cell!wall.!!This!structure!resembles!that!of!a!Gram9negative!bacterium!due!to!its!waxy!outer!membrane!composed!of!mycolic! acids! that! are! covalently! linked! to! a! peptidoglycan/arabinogalactan! layer.!! Previously,!we! reported! the! isolation! of! two!distinct!membrane!fractions,! the!PMf!and!the!PM9CW,!by!sucrose!gradient! fractionation!of!mechanically9lysed!cells.!!!We!further!demonstrated!that!!certain!biosynthetic!proteins!specifically!localize!to!either!of!the!two!membrane!fractions.!!A!canonical!marker!for!the!PMf!!is!PimB’,!a!mannosyltransferase!involved!in!phosphatidylinositol!mannoside!biosynthesis.!Despite!having!no!predicted!membrane9spanning!domains,!PimB’!consistently!localizes!to!the!PMf!when!fractionated.!However,!it!remains!unknown!how!PimB’!associates!with!the!PMf.!This!study!aims!to!determine!the!molecular!mechanism!of!PimB’!localization!to!the!PMf!in!Mycobacterium&

smegmatis,!a!model!organism!for!tuberculosis!research.!!We!have!created!several!truncation!and!point!mutants!of!PimB’!and!have!examined!their!localizations.!To!date,!our!data!suggest!that!there!may!be!two!distinct!domains!in!PimB’!that!are!necessary!for!its!membrane!association,!and!further!studies!are!underway!to!decipher!its!precise!localization!mechanism.!


63

97! ! The!influence!of!operon!organization!on!the!evolutionary!rate!of!bacterial!genomes!

!Marcus!Dillon1and!Vaughn!Cooper1!1Deparment!of!Molecular,!Cellular,!and!Biomedical!Sciences,!University!of!New!Hampshire!!Particularly! in! bacterial! genomes,! genes! are! often! organized! into! multi9gene! operons,! which! are! controlled! under! the! same!promoter!and!display! similar!expression!patterns.!!Moreover,!evidence! is! increasing! that!predictable!variation! in!expression!also!occurs!within!individual!operons,!where!genes!closer!to!the!site!of!transcription!initiation!are!expressed!more!than!genes!near!the!distal!end.!!Based!on!this!evidence,!purifying!selection!should!be!stronger!on!genes!closer!to!the!site!of!transcription!initiation!and!their!sequences!should!be!more!conserved.!!To!test!this!model,!we!used!orthologs!of!three!species!from!the!Burkholderia&cepacia!complex!to!calculate!evolutionary!rates!(dN!and!dS),!and!compared!these!rates!with!the!genes!predicted!operon!positions.!!Genes!found!within!operons!evolved!more! slowly! and!genes!within! the! same!operon! tended! to!evolve! as! a!unit.!! Furthermore,!within!operons,!both!evolutionary!rate!parameters!were! increased! in!genes!at! the!distal!end!of!operons!relative!to!those!at! the!site!of!transcription! initiation.!!These! findings! indicate!that!multiple!aspects!of!operons! influence!the!rate!at!which!genes!evolve,!which!has!important!implications!for!evolutionary!phenomenon!like!speciation!and!aging.!!!!

98! ! DNA! damage! and! resulting! functional! interactions! between! DNA! Pol! III! holoenzyme! subunits! and! DNA! Pol! IV!in!Escherichia&coli!!Ryan!W.!Benson,!Thomas!J.!Rands,!and!Veronica!Godoy!Department!of!Biological!Sciences,!Northeastern!University!!Polymerase! switching! and! protein9protein! interactions! between! DNA! polymerases! are! still! poorly! understood! processes.! Pol! IV!(DinB)! in!E.& coli!has!been! shown! to!play! a! role! separate! from! their! translesion! synthesis! (TLS)!or!polymerase!activities.!DinB! can!affect! replication! checkpoints,! although! this! has! only! been! observed! during!DinB! overexpression.! By! using! different!dnaE!alleles!that! encode! variants! of! the! Pol! IIIα9subunit! of! the! replicative! polymerase,! we! have! been! able! to! study! the!in& vivo!interaction!between! this! protein! and! DinB! variants.! Synthetic! sickness! or! lethality! is! observed! with! combinations! of! different! alleles!of!dnaE!and!dinB.! Phenotypic! variation! and! the! subsequent! reversion! to! wild! type! allow! identification! of! various! mutations!in!dinB&that!suppress!growth!defects.&Our!investigation!allows!greater!understanding!of!the!mechanisms!regulating!the!TLS!activity!and!alternative!roles!that!these!specialized!DNA!polymerases!play!in!cells!experiencing!DNA!damage!or!environmental!stress,!such!as!replication!checkpoints.!!!!

99! ! Single!cell!characterization!of!drug!tolerance!in!mycobacteria!

!Owen! Bennion1,! Anh! Hoang2,3,! Jemila! Kester2,!Marta! Fernandez9Suarez4,! Danielle! Heller2,! Daniel! Irimia4,!Mehmet! Toner4,! Sarah!Fortune2,!Bree!Aldridge1!1Department!of!Molecular!Biology!and!Microbiology!&!Department!of!Biomedical!Engineering,!Tufts!University,!USA,!2Department!of!Immunology!and!Infectious!Diseases,!Harvard!School!of!Public!Health,!USA,!3Center!for!Engineering!in!Medicine,!Massachusetts!General!Hospital,!USA,!4Daktari!Diagnostics,!USA!!Mycobacterium& tuberculosis! infects! billions! of! people!worldwide! and! kills!more! than! 1.5!million! per! year.! Tuberculosis! remains!extremely!difficult!to!treat,!requiring!months!to!years!of!antibiotic!therapy!to!cure.!The!variable!course!of!disease!and!treatment!response!suggests!that!functionally!heterogeneous!populations!of!mycobacteria!respond!differently!to!stress.!Using!a!quantitative!single9cell! approach,! we! show! that!mycobacteria! deterministically! generate! diversity! in! their! growth! characteristics! through! an!asymmetric!growth!pattern.!Coupled!with!a! cell! cycle! regulated!by! time!and!not! size,! this!asymmetry! creates! subpopulations!of!cells!with!distinct!growth!rates!and!cell!sizes!that!are!differentially!susceptible!to!clinically!relevant!classes!of!antibiotics.!Thus,!the!unusual! growth! pattern! intrinsic! to! mycobacteria! deterministically! creates! a! diverse! population! structure! that! may! underlie!phenotypes!previously! thought! to!be! controlled!by! external! stressors.!We!have! also!observed! variation! among!microcolonies! in!antibiotic!susceptibility!that!cannot!be!explained!by!growth!asymmetry.!We!have!established!markers!of!cell!cycle!state!to!test!the!hypothesis! that!cell!cycle!state! is!another!determinant!of!drug!susceptibility.!By! integrating! live9cell! imaging,! image!analysis,!and!mycobacterial! reporter! strains,!we! are! defining! the! characteristics! of! drug! tolerant! subpopulations!with! the! aim! of! engineering!improved!drug!therapies.!!!


64

100!! Polymicrobial!interactions!in!the!cystic!fibrosis!lung!

!Katherine!E.!Price1,!Amanda!A.!Naimie1,!Charles!Bay2,!Edward!F.!Griffin1,!and!George!O’Toole1!!1Department!of!Microbiology!and!Immunology,!Geisel!School!of!Medicine!at!Dartmouth!2The!Dartmouth!Institute,!Geisel!School!of!Medicine!at!Dartmouth!!Cystic!Fibrosis!(CF)! is!a!human!genetic!disorder,!which!results! in!a! lung!environment!that! is!highly!conducive!to!chronic!microbial!infection.! Historically,! CF! bacterial! infections! were! attributed! to! very! few! species,! predominately! Pseudomonas! aeruginosa.!However,! over! the! past! decade,! deep9sequencing! studies! have! demonstrated! that! the! CF! lung! can! harbor! a! highly! diverse,!polymicrobial!community.!We!have!expanded!our!in!vitro!co9culture!model!of!P.&aeruginosa!biofilm!formation!on!CF9derived!airway!cells,! to! include!this!broader!set!of!CF!airway!colonizers!to! investigate!their!contributions!to!CF! lung!disease,!particularly!as!they!relate! to! antibiotic! sensitivity! and/or! tolerance!of! the!population.!Mixed! co9culture!with!S.& salivarius& sensitizes!P.& aeruginosa! to!Cayston! (aztreonam),! a! commonly! prescribed! CF! maintenance! therapy! antibiotic.! This! sensitization! appears! to! be! S.& salivarius&specific& and! requires! the! presence! of! host! cells.! P.& aeruginosa! and! S.& salivarius! co9localize! on! CF! airway! cells! by! fluorescence!microscopy,! recapitulating! our! finding! that! P.& aeruginosa! and! Streptococci! co9localize! ex! vivo! in! fixed! sputum! samples! by!fluorescent!in!situ!hybridization.!This!co9localization!suggests!that!these!organisms!occupy!the!same!microenvironment!in!the!lung,!potentially!allowing!for!interactions!in!vivo.!Identification!of!this!sensitization!mechanism!is!ongoing.!!!

101!! Pseudomonas>Staphylococcus&inter7bacterial!interactions!in!the!respiratory!tract!of!cystic!fibrosis!infants!

!Laura!Filkins1,!Juliette!Madan2,!and!George!O'Toole2!1Department!of!Microbiology,!Geisel!Medical!School!at!Dartmouth,!2Division!of!Neonatology,!Dartmouth!Hitchcock!Medical!Center!!The!respiratory!tract!of!infants!with!cystic!fibrosis!is!colonized!by!diverse!bacterial!genera!within!the!first!month!of!life.!!In!a!cohort!of!13!infants!with!cystic!fibrosis,!Pseudomonas!was!detected!in!oropharyngeal!swabs,!by!deep!sequencing,!within!the!first!year!of!life! for! all! 13!patients.!!While!highly!prevalent,! the! relative! abundance!of!Pseudomonas! is! very! low!at! this! early! age.!! Further,! a!mixed! effects! linear! model! displayed! a! significant,! positive! correlation! between! increased! Pseudomonas! and! increased!Staphylococcus! relative! abundance! in! patient! samples! collected! longitudinally! over! the! first! 3! years! of! life.!! Importantly,! this!correlation!is!independent!of!patient!and!the!time!point!of!collection.!!In!an!effort!to!better!understand!the!interactions!between!Pseudomonas!and!Staphylococcus!as!cystic!fibrosis!patients!age!and!develop!chronic!respiratory!infections,!we!have!adapted!an!in&vitro! co9culture! model.!! Our! in& vitro! Pseudomonas9Staphylococcus! co9culture! analyses! reveal! dynamic! bacterial9bacterial!interactions!over! time.!!During! initial!co9culture!Staphylococcus!growth! is!enhanced!by!the!presence!of!Pseudomonas.!!However,!during! late! co9culture! biofilm! development,! Pseudomonas! outcompetes! and! kills! Staphylococcus.!! The! mechanism! of! these!interactions!is!driven!by!Pseudomonas!quorum!sensing!and!iron!acquisition!pathways!and!is!currently!being!elucidated.!!!!!!

102!! Wall!teichoic!acids!influence!MreB!motion!in!Bacillus&subtilis!

!Carl!N.!Wivagg1,!Saman!Hussain1,!Michael!F.!Dion1,!and!Ethan!C.!Garner1!1Department!of!Molecular!and!Cellular!Biology,!Harvard!University!!Wall! teichoic! acids! (WTAs)! are! polyanionic! sugar9phosphate! chains! that! firmicutes! covalently! attach! to! their! cell! walls.! !Loss! of!WTAs!under!standard!growth!conditions!leads!to!aberrant!septum!formation,!slowed!growth,!and,!in!rod9shaped!bacteria,!loss!of!the!rod!shape.!!We!use!fluorescent!protein!particle!tracking!to!show!that!during!loss!of!the!rod!shape,!the!characteristic!motion!of!Mbl,! a! protein! of! the! MreB! family! of! rod9associated! proteins,! becomes! heterogeneous.! !Though! it! remains! processive! and!unidirectional! for! individual!MreB!particles,! the!particles!cease! to!move!circumferentially!around!the! long!axis!of! the!cell.! !Some!individual!particles!move!across!the!cell!surface! in! intersecting!paths.! !This!observation!may!provide! insights! into!the!mechanism!that!underlies!MreB’s!association!with!the!rod!shape:!for!example,!the!observation!is!consistent!with!the!model!wherein!MreB!acts!as!a!curvature!sensor!whose!direction!of!motion!determines!the!short!axis!of!the!cell.!!!!!!


65

103!! Identifying!a!growth!factor!for!previously!uncultivated!bacteria!from!the!oral!cavity!

!Pallavi!P.!Murugkar1,!Eric!J.!Dimise2,!Eric!J.!Stewart1,!Jon!Clardy2,!and!Kim!Lewis1!1Department!of!Biology,!Northeastern!University,!Boston,!MA,!2Department!of!Biological!Chemistry!and!Molecular!Pharmacology,!Harvard!Medical!School,!Boston,!MA!!A!considerable!part!of!the!oral!microbiome!remains!uncultured,!and!this!presents!a!significant!barrier!to!understanding!its!role!in!health!and!disease.! Indirect!approaches!such!as!metagenomics!provide!valuable! information!on!the!microbiome,!but!we!need!to!culture!microorganisms!in!order!to!properly!study!them.!We!have!previously!discovered!that!many!natural!bacterial!isolates!from!environments!outside!of!the!human!body!were!uncultured!due!to!their!dependence!on!growth!factors!that!are!normally!provided!by!other!organisms!in!the!environment.!!Using!co9culture!technique,!we!similarly!find!that!many!bacteria!from!the!oral!cavity!will!only!grow!in!the!presence!of!a!cultivable!microorganism.!Consequently,!we!isolated!several!dependent!bacteria!from!the!oral!cavity!and!developed!a!screen!using!mutants!of!Escherichia&coli&to!identify!their!growth!factors.!Menaquinone!4!(MK4)!was!identified!as!the! growth! factor! for! inducing! growth! of! one! previously! uncultured! bacterium,! KLE1280.! KLE1280! is! closely! related! to!Porphyromonas& catoniae.! KLE1280! requires! both!MK4! and! heme! for! its! growth.!Whole! genome! sequencing! confirms! that! this!isolate!is!indeed!missing!the!menaquinone!biosynthesis!genes.!Two!other!species!of!Porphyromonas&were!also!dependent!on!MK4.!Other!uncultured!bacteria!might!be!deficient! in! the! same!or! similar!growth! factors.!Using! this!approach!may!allow!us! to! isolate!many!more!uncultured!organisms.!!!

104!! Disruption!of!Septation!Results!in!Bipolar!Growth!in!Mycobacteria!

!Vivian!Leung1,!Cara!Boutte2,!and!Eric!J.!Rubin2!1Faculty!of!Arts!and!Sciences,!Harvard!University,! 2Department!of! Immunology!and! Infectious!Diseases,!Harvard!School!of!Public!Health!!Most!bacteria!develop!their!cell!wall!in!two!different!modes:!during!elongation!peptidoglycan!is!inserted!along!the!lateral!walls,!and!during!septation!a!cross9wall!is!built!to!separate!daughter!cells.!Mycobacterial!cell!growth!is!fundamentally!different!from!that!of!other!bacteria.!Cells! grow!asymmetrically! from!one!pole,! and! lack!many!genes! that! are! critical! in!model!organisms.!Despite! the!global!relevance!of!pathogenic!M.&tuberculosis,!little!is!known!about!the!composition!and!function!of!machinery!that!govern!these!essential!processes.!

In! this! study,! we! sought! to! elucidate! how! septation! and! elongation! are! coordinated.! We! constructed! Mycobacterium&

smegmatis&strains!for!depletion!of!essential!cell!division!proteins!ftsZ,!penicillin9binding!protein!3!(pbp3)!and!MSMEG_4234.!Upon!depletion! of! these! three! factors,! cells! filament! and! branch! extensively,! demonstrating! the! role! of! PBP3! and! MSMEG_4234! in!division.!Abortive!septa!occur!in!depleted!filaments,!showing!that!cells!initiate!but!fail!to!complete!septation.!Using!a!pulse9chase!method!to!track!cell!growth,!we!show!that!while!wild9type!cells!elongate!unipolarly,!depleted!cells!grow!from!both!poles.!These!findings!suggest!that!these!proteins!are!involved!in!both!septation!and!the!maintenance!of!asymmetrical!growth,!which!indicates!that!these!processes!are!tightly!coordinated!and!regulated!by!common!factors.!!!

105!! Characterization!of!the!Mcr11!small,!non7coding!RNA!of!Mycobacterium&tuberculosis!

!Shaaretha!Pelly,!Cara!Smith,!William!R.!Bishai,!Gyanu!Lamichhane!Department!of!Medicine,!Johns!Hopkins!University!School!of!Medicine!!The!success!of!Mycobacterium&tuberculosis&(Mtb),!the!pathogen!that!causes!tuberculosis!(TB),!is!largely!due!to!its!ability!to!thrive!in!the!hostile!conditions!of!the!macrophage.!Although!the!response!of!Mtb&to!stress!has!been!widely!investigated,!studies!have!largely!focused!on!the!bacterium’s!regulatory!proteins.!The!existence!of!small,!non9coding!RNA!(sRNA)!in!Mtb&has!recently!come!to!light!and!shows!great!potential!in!revealing!new!mechanisms!of!adapting!to!stress.!We!began!our!study!of!Mtb!sRNAs!by!identifying!a!sequence!(Mcr11)!between!MT1302&and!MT1303&through!screening!a!cDNA!library.&We!found!this!sRNA!to!be!highly!expressed!and!regulated!by!both!cAMP!and!a! low!pH.!We!then!generated!an!overexpressing!recombinant!strain!of!Mcr11!and! investigated!the!effects! of! overexpression! on!Mtb& physiology! and! disease! progression! in! the! murine! model.! In! addition,! we! undertook! high9throughput! RNA! sequencing! to! determine! any! differential! gene! expression! as! a! result! of!Mcr11! overexpression! and! to! further!explore!the!non9coding!transcriptional!landscape!of!Mtb.!Based!on!these!studies,&we!can!conclude!that!sRNAs!and!the!non9coding!transcriptome!of!Mtb!are!of!immense!significance!in!furthering!our!understanding!of!the!genetic!regulation!of!the!TB!bacillus.!! !


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106!! Biofilm!formation!by!the!metabolically!versatile!phototroph!Rhodopseudomonas&palustris&!

!Philicia!Chow!1,!Chase!Kernen!1,!Jean!Huang!1,!and!Rebecca!Christianson!1!1Olin!College!!Biofilms,! which! consist! of! bacteria! living! cooperatively! within! a! polymer! matrix,! have! been! a! subject! of! study! within! both! the!biology! and! physics! communities.! This! paper! examines! the! biofilms! formed! by! the! metabolically! versatile! photosynthetic!bacterium,! !Rhodopseudomonas& palustris,! growing! via! different!metabolic!modes.! !R.& palustris!was! grown! in! flow! cell! chambers!using! identical!medium!conditions! in!either!the!presence!or!absence!of! light!and!oxygen.! !Under!these!conditions,!the!bacterium!grew!via!photoheterotrophic!or!heterotrophic!metabolisms.! !We!used!confocal! laser!scanning!microscopy!and!the! image!analysis!program! COMSTAT! to! quantitatively! analyze! and! compare! image! data! of! R.& palustris! biofilm! formation! arising! in! these! two!metabolic! modes.! !The! comparison! shows! significant! quantifiable! differences! in! structure! between! the! biofilms! formed! by! the!bacterium!growing!heterotrophically!and!those!growing!photoheterotrophically.!!A!basic!model!is!described!which!suggests!ways!in!which!the!metabolic!activities!of!the!cell!could!drive!biofilm!architecture!based!on!interaction!with!abiotic!parameters.!!!!!

107!! Role!of!Type!6!Secretion!System!of!Pseudomonas&aeruginosa!for!Interaction!with!a!Host!

!Olga!Danilchanka1,!David!Skurnik2,!Charlotte!Gaultier2,!Gerald!Pier2!and!John!Mekalanos1!1Departent!of!Microbiology!and!Immunobiology,!HMS,!2Division!of!Infectious!Disease,!Brigham!and!Women’s!Hospital,!HMS!!Protein! secretion!machineries!of! gram9negative!bacteria!are! complex!multiprotein! structures!dedicated! to! secretion!of!effectors!required!for!bacterial!pathogenesis.!Type!6!secretion!system!(T6SS)!has!been!described!to!be!important!for!interaction!of!bacteria!with!both!eukaryotic!and!bacterial!cells.!Recently,!we!showed!that!T6SS!cluster!1!of!P.&aeruginosa!is!required!for!T6SS!dueling!and!killing!of!heterologous!bacterial!species.!In!this!work!we!examined!a!role!of!P.&aeruginosa!T6SS!for!interaction!with!eukaryotic!cells.!In!particular,!we!showed!that!deletion!of!any!out!of!three!T6SS!clusters!of!P.&aeruginosa!results!in!reduced!cytotoxicity!toward!J774!and! RAW! 264.7! macrophage! cell! lines.! To! reveal! a! role! of! T6SS! during! infection,! we! used! a! murine! model! of! chronic! gut!colonization.!We! showed! that! the! level! of! colonization! by! T6SS! cluster! 1!mutant! is! similar! to! that! of! wild9type! bacteria! in! the!presence! of! antibiotics! that! are! required! to! prevent! growth! of! normal! microflora.! After! removal! of! antibiotics,! wild9type! P.&aeruginosa! is! outcompeted! by! microflora! and! cleared.! By! contrast,! T6SS! cluster! 1! mutant! was! able! to! persist! suggesting! an!interaction! of! T6SS!with! the! host,! either! through! a! direction! interaction!with! its!microbiota,! or! through! an! interaction!with! its!immune!system.!!!

108!! Molecular!control!of!the!Sinorhizobium&meliloti&cell!cycle!by!host!cationic!peptides!during!symbiosis!

!Jon!Penterman!and!Graham!Walker!Department!of!Biology,!Massachusetts!Institute!of!Biology,!Cambridge,!MA!!

!While!forming!a!symbiosis!with!plant!legume!hosts,!the!intracellular!bacterium!Sinorhizobium&meliloti!undergoes!a!dramatic!cellular!differentiation!that! is! required! for!nitrogen!fixation.! !One!aspect!of! this!differentiation! involves!endoreduplication! (replication!of!DNA! in! the! absence! of! cell! division),!which! increases! the! cell’s!metabolic! capacity! and! likely! the! efficiency! of! nitrogen! fixation.!!Cellular!differentiation!of!S.&meliloti& is!driven!by!a!cocktail!of!nodule9specific!cysteine!rich!cationic!peptides!produced!by!the!host!that! share! similarity!with!mammalian! and!plant! defensins! and!begins! soon! after! it! is! internalized!by!host! cells.! !How!S.&meliloti&

senses!host!cationic!peptides!and!alters! its!cell!cycle! is!not!understood.! !Here!we! leveraged!a!synchronization!protocol! to!probe!how! the! S.& meliloti! cell! cycle! is! affected! by! treatment! with! a! synthesized! host! cationic! peptide.! ! Microarray! experiments! on!synchronized! populations! treated! with! the! peptide! revealed! that! nearly! 15%! of! the! transcriptome! was! significantly! altered!compared! to! untreated! controls.! !Many!of! the! transcriptional! changes! reflected! the! activation!of! general! stress! and!membrane!stress! responses.! ! Interestingly,! several! well9defined! signaling! pathways! that! are! required! for! symbiosis! by! S.& meliloti,& and! for!infection! by! closely! related! pathogens,! are! strongly! induced! by! peptide! treatment,! suggesting! these! pathways! mediate! crucial!physiological!responses!to!cationic!peptides.!!Unlike!untreated!populations,!genes!that!specify!cell!polarity!(podJ),!regulate!cell!cycle!progression!(divK,&divJ,!pleD,&sciP,&ccrM),&and!mediate!cell!division!(ftsK,! ftsI,!xerC,!septum!formation! initiator)!were!not!properly!upregulated!in!a!cell!cycle9dependent!manner.!!Fluorescence!microscopy!on!peptide9treated!cells!showed!that!most!cells!lacked!Z9rings! even! though! ftsZ! gene! expression!was! normal.! ! Promoter! analysis! of! the! cell! cycle! and! division! genes! altered! by! peptide!treatment!suggests!that!CtrA,!a!master!cell!cycle!regulator,!regulates!them,!and!efforts!are!underway!to!assay!levels!of!CtrA!protein!and! phosphorylation! in! peptide9treated! cells.! ! Our! observations! indicate! that! host9derived! cationic! peptides! steer! S.&meliloti! to!endoreduplicate!by!primarily!blocking!cell!division!at!the!transcriptional!and!cellular!level.!!


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109!! Mapping!a!network!for!self!recognition!in!Proteus&mirabilis!

!

Larissa!M.!Wenren1,!Nora!L.!Sullivan

2,!Lia!Cardarelli

1,!Alecia!N.!Septer

1,!and!Karine!A.!Gibbs

1!

1Department!of!Molecular!and!Cellular!Biology,!Harvard!University,!

2W.M.!Keck!Science!Department,!Claremont!McKenna,!Pitzer!

and!Scripps!Colleges!! The! gram9negative! bacterium! Proteus& mirabilis! exhibits! territorial! behavior! that! can! be! macroscopically! observed.! On! nutrient!surfaces,! different! isogenic! populations! can! recognize! adjacent! populations! as! non9self! and! form! a! physical! boundary.! Previous!transposon!mutagenesis!screens!have! identified!three!gene!clusters!that!are! involved! in!this!self! recognition!capability.!We!have!now!begun!to!elucidate!how!these!components!contribute!to!territoriality!and!how!they!are!interconnected.!Our!results! indicate!that!the!ability!to!recognize!self!provides!an!advantage!for!obtaining!territory.!In!addition,!our!findings!suggest!that!a!putative!type!VI!secretion!system!(awidely!conserved!bacterial!secretion!apparatus)!serves!as!the!functional!intersection!point!for!export!of!self9determinant!proteins!encoded!by!two!gene!clusters!(ids!and!idr).!Moreover,!secretion!of!Ids!and!Idr!proteins!is!independent!of!one!another,! and! differing! phenotypes! suggest! that! these! two! sets! of! components! have! distinct! roles! in! both! self! recognition! and!territoriality.!This!work!provides!a!foundation!for!further!mapping!of!the!self!recognition!system!in!P.&mirabilis!and!other!bacteria.!!!!

110!! Streptolysin!O!and!its!co7toxin!NAD7glycohydrolase!protect!group!A!Streptococcus!from!xenophagic!killing!

!Maghnus!O’Seaghdha!and!Michael!R.!Wessels!Division!of!Infectious!Diseases,!Boston!Children’s!Hospital!and!Department!of!Pediatrics,!Harvard!Medical!School!!Group!A!Streptococcus!(GAS)!causes!pharyngitis!and!in!some!cases,!severe!invasive!infections.!GAS!can!be!internalized!and!killed!by!epithelial!cells! in&vitro,!a!process!that!may!contribute!to!local!innate!defense!against!pharyngeal!infection.!!Secretion!of!the!pore9forming!toxin!streptolysin!O!(SLO)!by!GAS!has!been!reported!to!stimulate!targeted!autophagy!(xenophagy)!upon!internalization!of!the! bacteria! by! epithelial! cells.! We! now! report! in9depth! investigation! of! xenophagy! in! response! to! GAS! infection! of! human!oropharyngeal! keratinocytes,! the! predominant! cell! type! of! the! pharyngeal! epithelium.!! We! found! that! SLO! expression! was!associated! with! prolonged! intracellular! survival;! unexpectedly,! expression! of! the! co9toxin! NADase! was! required! for! this! effect.!Shortly!after!internalization!of!GAS,!SLO9mediated!damage!to!the!bacteria9containing!vacuole!resulted!in!exposure!to!the!cytosol,!ubiquitination!of!GAS!and/or!associated!vacuolar!membrane!remnants,!and!engulfment!of!GAS!in!autophagosomes.!!We!also!found!that! production! of! streptolysin! S! could! mediate! targeting! of! GAS! to! autophagosomes! in! the! absence! of! SLO,! independent! of!ubiquitination.! Maturation! of! autophagosomes! was! prevented! by! SLO! pore9formation! and! by! SLO9mediated! translocation! of!NADase.!This!novel!activity!of!NADase!to!block!autophagic!killing!of!GAS!in!pharyngeal!cells!may!contribute!to!pharyngitis!treatment!failure,!relapse,!and!chronic!carriage.!!!

111!! Variable! regions! in! self7identity! proteins! are! important! for! self7recognition! and! other! aspects! of! surface7based!growth!in!Proteus&mirabilis!!Christina!C.!Saak1,!and!Karine!A.!Gibbs1!1!Molecular!and!Cellular!Biology,!Harvard!University!!Populations!of!the!gram9negative!bacterium!Proteus&mirabilis&display!the!social!behavior!of!self9!versus!non9self!recognition!when!moving! in! swarms!across!a! surface!and!macroscopic!boundaries! form!between!approaching! swarms!of!different! strains.! The! ids!operon!(idsABCDEF)!is!necessary!for!this!behavior,!as!a!Δids!strain!no!longer!merges!with!its!parent.!IdsD!and!IdsE!are!self9identity!encoding! proteins! that! likely! convey! uniqueness! through! their! variable! regions! (VRs).! Their! function! and! structure! are! currently!unknown,!however! they!are!predicted! to!each!have! two! transmembrane!domains.! The!work!presented!here!demonstrates! that!swapping!any!VR!of! idsD!or! idsE!between!disparate!strains!alters!self9recognition!patterns,!motility,!and!other!aspects!of!surface9based!growth!(developmental!cycle!and!colony!morphology).!Swapping!both!VRs!results!in!a!mutant!strain!that!no!longer!merges!with! its!parent!but! instead!with!a!strain!expressing!the!donor’s! ids! locus.!!These!results!strongly!suggest!the! involvement!of! IdsD!and!IdsE!VRs!in!the!signaling!cascade!for!self9recognition.!It!further!suggests!that!the!Ids!self9recognition!system!regulates!various!aspects!of!surface9based!growth!and!that!this!regulation!depends!on!the!presence!of!cognate!IdsD/IdsE!VRs!within!a!population.!!!!!!


68

112!! Conserved! residues! near! the! FtsZ! C7terminus! contribute! to! recognition! and! degradation! of! FtsZ! by! the! ATP7dependent!protease!ClpXP!!Marissa!G.!Viola1,!Sue!Wickner2,!and!Jodi!L.!Camberg1!1University! of! Rhode! Island,! Department! of! Cell! and!Molecular! Biology,! Kingston,! RI,! 02881! 2National! Cancer! Institute,! National!Institutes!of!Health,!Laboratory!of!Molecular!Biology,!Bethesda,!MD,!20892!!The!highly!conserved!protein!FtsZ!is!essential!for!cell!division!in!Escherichia&coli!and!forms!the!septal!ring,!or!Z9ring,!during!bacterial!cytokinesis.!The!Z9ring!is!a!large!dynamic!protein!complex!that!assembles!at!the!site!of!constriction.!Z9ring!assembly!is!modulated!by! several! cell! division!proteins! that! interact!with! FtsZ.! FtsZ! shares! structural! homology!with! the!eukaryotic!protein! tubulin! and!assembles! into! linear!filaments! in!vitro! in!the!presence!of!GTP.!FtsZ!monomers!contain!an!N9terminal!polymerization!domain,!an!unstructured! linker! region,! and! a! conserved! C9terminal! core! domain,! which! is! important! for! interactions! with! several! proteins!including!ClpXP.!The!ATP9dependent!protease!ClpXP!recognizes!protein!substrates!bearing!specific!recognition!signals,!unfolds!and!degrades!them.!We!identified!specific!residues!near!the!FtsZ!C9terminus!that!are!important!for!degradation!by!ClpXP!and!for!FtsZ!function.!To!elucidate!the!role!of!ClpXP!during!division!and!determine!if!degradation!of!FtsZ!by!ClpXP!modulates!Z9ring!dynamics,!we!performed!fluorescence!recovery!after!photobleaching!(FRAP)!of!Z9rings!in!live!cells!expressing!FtsZ!fused!to!green!fluorescent!protein! (GFP).! Our! results! suggest! that! ClpXP!modulates! Z9ring! dynamics! in! vivo! and! identify! specific! residues! in! FtsZ! that! are!important!for!recognition!and!degradation!by!ClpXP.!!!

113!! Sterile!granulomas!develop!during!active!tuberculosis:!local!disparities!despite!global!effects!

!Philana! Ling! Lin1*,! Christopher! B.! Ford2,3*,! M.! Teresa! Coleman4,! Amy! J.! Myers5,! Thomas! Ioerger6,! James! Sacchettini7,! Sarah! M.!Fortune2,3,8,!JoAnne!L.!Flynn5!1Department! of! Pediatrics,! Children's! Hospital! of! Pittsburgh! of! the! University! of! Pittsburgh! Medical! Center,! Pittsburgh,!Pennsylvania,!USA,!2!Department!of!Immunology!and!Infectious!Diseases,!Harvard!School!of!Public!Health,!Boston,!Massachusetts,!USA,!3!Broad!Institute!of!MIT!and!Harvard,!Cambridge,!Massachusetts,!USA,!4!Department!of!Radiology,!PET!Center,!University!of!Pittsburgh!School!of!Medicine,!Pittsburgh!PA!USA,!5!Department!of!Microbiology!and!Molecular!Genetics,!University!of!Pittsburgh!School! of!Medicine,! Pittsburgh,! Pennsylvania,! USA,! 6! Department! of! Computer! Science! and! Engineering,! Texas! A&M!University,!College! Station,! TX!USA,! 7! Department! of! Biochemistry! and! Biophysics,! Texas! A&M!University,! College! Station,! TX!USA,! 8! Ragon!Institute!of!MGH,!MIT,!and!Harvard,!Boston,!Massachusetts,!USA.!*!These!authors!contributed!equally!to!this!work!!Over! 30%! of! the! world’s! population! is! infected! with!Mycobacterium& tuberculosis! (Mtb),! yet! only! ~5910%! will! develop! clinical!disease.!Despite! significant! effort,!we!understand! little! about!what! distinguishes! individuals!who!progress! to! active! tuberculosis!(TB)!from!those!who!remain!latent!for!decades.!The!variable!course!of!disease!is!recapitulated!in!cynomolgus!macaques!infected!with!Mtb.!Active!disease! in!macaques! is!defined!by!clinical,!microbiologic!and! immunologic!signs!and!occurs! in!~45%!of!animals,!while!the!remaining!are!clinically!asymptomatic.!Here,!we!use!barcoded!Mtb!strains!and!quantitative!measures!of!culturable!and!cumulative!bacterial! burden! to! show! that!most! lesions! are! founded!by! a! single!bacterium!and! reach! similar!maximum!burdens.!Despite!common!origins,!the!fate!of!individual!lesions!varies!significantly!within!the!same!host.!Strikingly,!in!active!disease,!the!host!sterilizes!some!lesions!even!while!others!progress.!Our!data!suggest!that!lesional!heterogeneity!arises,!in!part,!through!differential!killing! of! bacteria! after! the! onset! of! adaptive! immunity.! Thus,! individual! lesions! follow! diverse! and! overlapping! trajectories,!suggesting! critical! responses! occur! at! a! local! level! to! ultimately! determine! the! clinical! outcome! of! infection.! Defining! the! local!factors!that!dictate!outcome!will!be!important!in!developing!effective!interventions!to!prevent!active!TB.!!!

114!! Validation!of!a!cis7regulatory!RNA!element!for!ribosomal!proteins!S6!and!S18!

!Yang!Fu,!Mark!Soo,!Michelle!M.!Meyer!Department!of!Biology,!Boston!College,!Chestnut!Hill!MA!02467!!Eubacteria!adopt!autogenous!regulation!for!ribosomal!protein!biosynthesis!to!maintain!a!balance!between!the!levels!of!ribosomal!RNAs! and! proteins! during! ribosome! assembly.! In! bacteria,! genes! coding! for! ribosomal! proteins! are! arranged! in! operons.! The!expression!of! an!operon! is! inhibited!when! a! cis9regulatory!RNA!element!within! the! 5’9UTR!or! intergenic! region!binds! ribosomal!protein(s)!translated!from!the!same!operon.!In!E.&coli,!around!2/3!ribosomal!proteins!are!controlled!by!12!such!RNA!elements.!The!regulation!of!the!remainder!is!unknown.!

Genes! coding! for! ribosomal! proteins! S6! and! S18! have! similar! genomic! organizations! in! many! bacteria! species! and! are!


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predicted!to!be! in! the!same!operon.!Our!studies!have!discovered,! for! the! first! time,! the!RNA!element!responsible! for!S6!operon!regulation.!

We!demonstrate!first!that!in!Bacillus&subtilis!the!3!genes!of!the!S6!operon!are!co9transcribed!and!identified!the!transcription!initiation!site.!Further!gel9shift!assays!and!nuclease!protection!assays!show!that!S6!RNA!element!prefers! interaction!with!S6/S18!dimer!protein,!and!the!Shine9Dalgarno!sequence!of!S6!operon!is!directly!involved!in!protein!binding.!Based!on!our!findings,!we!speculate!that!this!RNA!element!regulates!the!expression!of!S6!operon!by!binding!S6!and!S18!as!a!dimer.!!!

115!! ClpX,!a!bacterial!AAA+!unfoldase,!targets!the!Mu!transpososome!complex!via!a!multivalent!recognition!signal!

!Lorraine!Ling1,!Aliaa!Abdelhakim1!,!Robert!T.!Sauer1,!Sherwin!P.!Montano2,!Phoebe!A.!Rice2,!Tania!A.!Baker1,3!1Biology,!Massachusetts!Institute!of!Technology,!2Biochemistry!and!Molecular!Biology,!University!of!Chicago,!3HHMI!!The! AAA+! unfoldase! ClpX! targets! proteins! for! remodeling! and! degradation.! Although! some! peptide! signals! (tags)! have! been!discovered,!clear!recognition!rules!and!consensus!motifs!are!still!undefined.!!To!elucidate!these!motifs,!we!examined!an!E.coli!ClpX!substrate,!phage!MuA!transposase.!Initially!monomeric,!subunits!assemble!into!the!transpososome!(Mu9t),!a!stable!tetramer!bound!to! phage! DNA.! ClpX! remodels! transpososomes,! triggering! the! DNA! repair/replication! phases! of! viral! integration.! Previous!work!identified!a!peptide!tag!located!at!MuA’s!C9terminus!required!for!ClpX’s!recognition!of!both!oligomeric!states.!Furthermore,!ClpX!recognizes!Mu9t!109fold!more!strongly! than!monomers.!We!sought! to! identify! sequences! in!MuA!specifically! responsible! for! the!enhanced!recognition!of!Mu9t.!Through!mutagenesis!of!MuA’s!C9terminal!domain,!we!found!a!short!sequence!region!~40!residues!upstream!from!the!tag!that!is!critical!for!discrimination!of!the!complex.!This!tethering!motif!binds!directly!to!ClpX’s!N9domain.!To!dissect!how!the! tethering!motif!and! tag!work! together,!we!assayed!Mu9t!with!mixed!mutant!subunits.!We!observed!an!additive!effect! of!mutations! in! the! tethering!motif,!with! as!much! as! a! 109fold!weaker! recognition! of! complexes!when! all! subunits! carry!mutations.!Our!model! proposes! that!MuA!oligomerization! generates! a!multivalent! recognition! signal! specifying! transpososomes!over!monomers.!!!

116!! How!is!cell!division!inhibited!in!a!strain!of!E.&coli&K>12&lacking!the!Rep!helicase?!

!Ben! Waldman1,! Jaymes! Farrell2,! and! Steven! Sandler1! 1Department! of! Microbiology,! University! of! Massachusetts9Amherst,!2Department!of!Biochemistry!and!Molecular!Biology,!University!of!Massachusetts9Amherst!!Resolution!of! collisions! between! transcriptional! complexes! and!DNA! replication! forks! is! essential! for! survival.! In!E.& coli,! the!Rep!helicase!plays!an!important!role!in!this!process.!Microscopic!analysis!of!Δrep!mutants!shows!they!are!on!average!39fold!longer!than!wildtype.! The! longer! Δrep! cell! length! is! suppressed! by! rpoC*,! a! mutated! RNA! polymerase! that! can! suppress! defects! due! to!transcription/replication! conflicts.! Deletion! of! the! nucleoid! occlusion! factor! slmA& prevents! filamentation,! as! do! removal! of! the!nucleoid! associated! protein! genes! fis,! ihfA& or! ybaB.! Using! a! zipABgfp& fusion! and! time9lapse! photography,! it! is! seen! that! Δrep!mutants!fail!to!resolve!FtsZ9rings!to!successful!division!57.7%!of!the!time!(failure!rate! is!6.6%!in!wildtype).!Z9rings!formed!on!the!nucleoid!decay!at!a!higher!frequency,!consistent!with!SlmA9mediated!depolymerization.!Many!FtsZ9rings,!though!originally!formed!between!partitioned!nucleoids,!are!ultimately!decayed!on!nucleoids!following!a!“departioning”!event,!suggesting!rearrangement!of!the! nucleoid! to! inhibit! cell! division.! It! is! hypothesized! that!when!Δrep!mutants! fail! to! resolve! transcription/replication! conflicts,!origin!proximal!regions!of!the!chromosome!with!a!higher!concentration!of!SlmA!binding!sites!are!spatially!redistributed!in!the!cell!through!a!Fis,!IhfA,!and!YbaB!dependent!manner,!and!this!rearrangement!inhibits!cell!division.!!!

117!! Proof!of!dual!targeting!and!novel!mode!of!action!for!non7quinolone!bacterial!topoisomerase!type!II!inhibitors!

!Asha!Nayar,!Mark!Cronin,!Folkert!Reck,!Gregory!Stone!and!Thomas!Dougherty!Infection!Bioscience,!Infection!iMed,!AstraZeneca!R&D,!Waltham,!MA.!!The!goal!of!this!study!was!to!confirm!the!target!of!non9quinolone!topoisomerase!type!II!!inhibitors!in!Pseudomonas&aeruginosa!and!Escherichia&coli,!through!mutant!generation!followed!by!targeted!sequencing.!First!step!mutants!of!P.&aeruginosa!had!mutations!in!the! efflux! pump! regulator& nfxB.! Second! step!mutants! had! additional! mutations! in! Asp82! of! subunit! A! of! gyrase& (gyrA).&& These!second! step!mutants!were! not! cross! resistant! to! fluoroquinolones.! !However,! in!E.coli,! resistant!mutants! could! not! be! isolated.!Based!on! the! results!obtained!with!P.aeruginosa,!Asp82!gyrA!mutations!were! recombineered! into!E.coli.&Resulting! strains&had&a!marginal!two9fold!increase!in!MIC.!Subsequently,!these!constructs!were!used!to!isolate!second!step!mutants!which!had!additional!mutations! in!Topoisomerase! IV!genes,!parC&or&parE.!These!mutants!were!not!cross!resistant!to!fluoroquinolones.!!Since! in!E.&coli&


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resistant! colonies! could! only! be! isolated! by! introducing! mutation! in! gyrA,! this! non9quinolone! topoisomerase! type! II! inhibitor!appears!to!act!through!dual!target!inhibition!in!this!species,!by!targeting!gyrase!(GyrA!)!and!topoisomerase!IV!(ParC).!!!Target!based!mutations! did! not! alter! susceptibility! to! fluoroquinolones,! further! supporting! a! distinct! mode! of! action! for! this! non9quinolone!compound!series!compared!to!fluoroquinolones,!despite!targeting!the!same!proteins.!!!

118!! Structural! features!of! the!Pseudomonas& fluorescens! biofilm!adhesin! LapA! required! for!biofilm! formation!and! cell!surface!localization!!Chelsea!D.!Boyd1,!Ivan!E.!Ivanov2,!Mary!E.!Schwartz2,!Debashree!Chatterjee3,!Peter!D.!Newell1,!Lynne!Turnbull4,!Michael!S.!Johnson4,!Cynthia!B.!Whitchurch4,!Terri!Camesano2,!Holger!Sondermann3,!and!George!O’Toole1!1Department!of!Microbiology!and! Immunology,!Geisel!School!of!Medicine!at!Dartmouth,!Hanover,!NH,!2Department!of!Chemical!Engineering,!Worcester!Polytechnic!Institute,!Worcester,!MA,!3Department!of!Molecular!Medicine,!College!of!Veterinary!Medicine,!Cornell!University,!Ithaca,!NY,!4Microbial!Imagine!Facility,!the!ithree!institute,!University!of!Technology!Sydney,!Sydney,!New!Wales,!Australia!!The! localization!of! the!adhesin!protein!LapA!to! the!cell9surface! is!a!key! regulatory!step! required!by!Pseudomonas& fluorescens! to!irreversibility!attach!to!a!surface!and!form!a!biofilm.!LapA!is!a!member!of!a!diverse!family!of!predicted!bacterial!adhesins,!with!low!sequence! similarity.! However,! these! proteins! share! predicted! domains.! Through! mutagenesis! of! these! predicted! features,! we!determine! the! significance!of! each! feature! in! relation! to! LapA!export! and! localization! to! the! cell9surface!and! function! in!biofilm!formation.!Our!results!suggest!that!the!Type!1!secretion!signal!is!required!for!export!and!cell9surface!localization.!Deletion!of!the!central!repetitive!region!within!LapA!results!in!an!inability!to!form!a!biofilm,!suggesting!that!these!repeats!are!required!for!biofilm!formation.!

Previously! our!work!has! shown! that! the!N9terminus!of! LapA! is! required! for! cleavage!by! the!periplasmic! cysteine!protease!LapG! and! release! of! the! protein! from! the! cell! surface! in! conditions! unfavorable! for! biofilm! formation.! Together! with! our!mutagenesis!analysis!of!LapA,!outer!membrane!topology!mapping!methods!suggest!that!the!N9terminus!of!LapA!is!a!cell!surface9associated! domain.! Ongoing! work! is! focused! on! deciphering! the! mechanism! of! LapA! release! from! the! outer! membrane! upon!cleavage!by!LapG.!!!

119!! Use!of!a!waste!stream!for!bioplastic!production!by!Ralstonia&eutropha!

!Matheus!Quintana!Barreto1,!Christopher!Brigham1!1Department!of!Bioengineering,!University!of!Massachusetts!Dartmouth!&

Ralstonia&eutropha&is!a!gram9negative!b9proteobacterium!that!produces!polyhydroxyalkanoates!(PHAs),!alternatives!for!petroleum9based!polymers,!when!grown!in!a!nutrient9limiting!environment!with!plentiful!carbon.!The!purpose!of!our!project!is!to!engineer!a!R.& eutropha& strain! that! can! degrade! chitin! from! shrimp! and! lobster! shells! and! produce! poly939hydroxybutyrate! (PHB)! from! N9acetylglucosamine! (NAG).! We! are! evaluating! the! growth! of! R.& eutropha& under! different! conditions! in! order! to! find! the! best!conditions!for!growth!and!PHB!production.!Our!results!show!that!R.&eutropha&grows!better!and!faster!on!fructose!than!on!NAG!as!the!sole!carbon!source!and!that!its!growth&on!NAG!decreases!proportionally!as!carbon!source!concentration!increases.!The!addition!of!small!amounts!of!fructose!to!NAG!cultures!reduces!the!extended!lag!phase!in!all!growth!media!tested.!Our!results!indicate!that!high!concentrations!of!NAG!decrease!growth!of!R.&eutropha,&whereas!the!presence!of!fructose!assists!in!the!metabolism!of!NAG!by!result! of! some! heretofore! unknown! mechanism.! Our! goals! are! to! evaluate! PHB! production! in! the! different! conditions! tested,!examine! the! gene! expression! patterns! of! NAG9grown! cells! in! the! presence! and! absence! of! fructose,! engineer! R.& eutropha& to!degrade!chitin!and!evaluate!the!resulting!growth!and!PHB!production.!!!

120!! Innate!immune!strategies!that!respond!to!Legionella&pneumophila!intracellular!replication!

!Seblewongel!Asrat1,!Aisling!Dugan1!and!Ralph!Isberg1!1Molecular!Biology!and!Microbiology,!Tufts!University!School!of!Medicine!!Many! pathogens,! particularly! those! that! require! their! host! for! survival,! have! devised!mechanisms! to! subvert! the! host! immune!response! in! order! to! survive! and! replicate! intracellularly.! Legionella& pneumophila,! the! causative! agent! of! Legionnaires’! disease,!promotes!intracellular!growth!by!translocating!proteins! into!its!host!cytosol!through!its!type!IV!protein!secretion!machinery.!This!allows!construction!of!a!replication!compartment!composed!of!membrane!derived!from!the!host!cell!early!secretory!apparatus.!At!


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least!5!of!the!bacterial! translocated!proteins! inhibit!host!protein!synthesis!at!the!elongation!step.!! Interestingly,! infection!with!L.&pneumophila! leads! to! the!production!of! few! stress! response! genes,! such! as! pro9inflammatory! cytokines,!which! are! produced! in!spite!of!the!fact!that!host!cell!protein!synthesis!is!blocked!by!the!bacterium.!Selective!synthesis!of!these!proteins!allows!the!host!to!control! L.& pneumophila! infection,! but! the! mechanism! by! which! the! proteins! bypass! the! translational! block! exerted! by! L.&pneumophila!is!not!understood.!The!study!presented!here!elucidates!how!innate!immune!molecules!are!produced!under!conditions!in!which!protein!synthesis! is! inhibited!by!pathogens.! In!addition,!our!results!give!insight! into!the!mechanism!by!which!the!innate!immune!system!distinguishes!between!virulent!pathogens!from!harmless!microbes!and!mount!a!specific!response.!!!

121!! Phylogenetic!Distribution!of!B.&subtilis!Ribosomal!Autogenous!Regulatory!Elements!

!Kaila!Deiorio9Haggar,!Arianne!M.!Babina,!Jon!Anthony,!and!Michelle!M.!Meyer!Department!of!Biology,!Boston!College,!Chestnut!Hill!MA!02467!!Ribosomal!protein!operons!in!bacteria!are!regulated!by!RNA!secondary!structures!within!their!mRNA!transcripts.!These!structures!interact!with!specific!r9proteins!to!control!expression,!thus!balancing!the!synthesis!of!ribosomal!proteins!and!RNA,!thereby!ensuring!proper!ribosome!synthesis!and!assembly.!In!Bacilli,!there!are!several!such!ribosomal9protein!autogenous!regulatory!RNAs!that!have!been! experimentally! confirmed,! and! several! additional! putative! RNA! structures! that! have! been! identified! through! comparative!genomics.!We! aim! to! characterize! the! evolutionary! distribution! of! these! regulatory! RNA! structures! and! validate! their! biological!functionality!in!gram9positive!organisms.!To!assess!this,!we!created,!or!modified!existing,!structural!alignments!for!these!RNAs!and!used!them!to!perform!homology!searches.!We!have!determined!that!most!structures!exhibit!a!narrow!phylogenetic!distribution,!predominately!relegated!to!the!Firmicute!class!Bacilli.!In!addition,!we!demonstrated!that!several!of!the!putative!RNA!structures!are!actively! transcribed! in! B.! subtilis! and! confirmed! their! operon! structures.! This! finding! suggests! that! there! are! likely! many! non9homologous!RNA!regulatory!elements! regulating! ribosomal!protein!biosynthesis! that! still!await!discovery!and!characterization! in!other!bacterial!species.!!!

122!! Beyond!Immunity:!New!Insights!into!the!Pseudomonas&aeruginosa!CRISPR/Cas!System!

!Gary!E.!Heussler,!Kyle!C.!Cady,!and!George!A.!O’Toole!Department!of!Microbiology!and!Immunology,!Geisel!School!of!Medicine!at!Dartmouth,!Hanover,!NH,!03755!!Studies!of!clustered!regularly!interspaced!short!palindromic!repeats!(CRISPRs)!and!CRISPR9associated!(Cas)!genes!have!resulted!in!a!model!whereby!CRISPRs!function!as!a!defense!system!against!bacteriophage!infection!and!conjugative!plasmid!transfer.!Previously!we! have! shown! an! alternate! function! in!which! the! interaction! between! the! type! I9F! CRISPR! region! of!Pseudomonas& aeruginosa!strain!UCBPP9PA14!and!bacteriophage!DMS3!resulted!in!an!inhibition!of!biofilm!formation!by!P.&aeruginosa!lysogenized!with!DMS3.!Here!we!demonstrate!the!type!I!CRISPR!region!of!P.&aeruginosa!is!also!capable!of!functioning!as!a!defense!system!against!infection!of!various!bacteriophage! including!DMS3,!and! that! this!drastic!change! in!phenotypic!output! is!dependent!on!a!single!nucleotide!mutation! in! the! target! of! CRISPR2! Spacer! 1! encoded! small! RNA! (crRNAC2S1).! Additionally,! we! demonstrate! the! presence! of! the!crRNAC2S1!targeted!protospacer!on!the!bacterial!chromosome!is!sufficient!for!CRISPR!dependent!biofilm!inhibition.!These!data!have!important!implications!for!understanding!the!role!for!CRISPR/Cas!systems!in!bacterial!biology!and!evolution.!!!

123!! Enterococcus!infection!of!C.&elegans!as!a!model!of!innate!immunity!

!Grace!J.!Yuen1,3!and!Frederick!M.!Ausubel2,3!1Program!in!Immunology,!2Department!of!Genetics,!Harvard!Medical!School,!and!3Department!of!Molecular!Biology,!Massachusetts!General!Hospital,!Boston,!MA.!&

Enterococcus!is!a!Gram9positive!commensal!that!is!also!an!important!opportunistic!pathogen.!Most!human!enterococcal!infections!are!caused!by!either!E.&faecalis!or!E.&faecium.!We!have!previously!shown!that!infection!with!either!species!leads!to!gut!distention,!but!only!E.& faecalis! is!able!to!establish!a!persistent!and! lethal! infection! in!the!nematode.!We!now!provide!evidence!that!at! least!three! canonical! C.& elegans! immune! signaling! pathways! are! important! for! survival! during! infection! with! either! E.& faecalis! or! E.&faecium,!suggesting!that!an!active!host!response!is!required!to!keep!E.&faecium!infection!“in!check”!in!the!worm!intestine.!

To! further! characterize! the! C.& elegans! host! response! to! Enterococcus! infections,! we! used! genome9wide! transcriptional!profiling!of!nematodes!feeding!on!E.&faecalis!and!E.&faecium,!as!well!as!two!controls,!heat9killed!E.&coli!and!live!Bacillus&subtilis.!We!found!that!relative!to!the!B.&subtilis&control,!E.&faecalis!and!E.&faecium!caused!the!upregulation!of!249!and!166!genes,!respectively,!


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of!which!105! genes!were! common! to!both,! comprising! the!Enterococcus! gene! signature.! Shared!by!both!Enterococcus! infection!signatures!were! genes! relating! to! oxidation/reduction,! fatty! acid!metabolism,! and! C9type! lectins.! Additionally,! the!Enterococcus!infection! gene! signature! is! fairly! distinct! from! the! P.& aeruginosa,! S.& aureus,! and! C.& albicans! infection! signatures.! Studies! are!underway! in! understanding! the! biology! of! Enterococcus! infection! in! C.& elegans! and! identifying! novel! Enterococcus9activated!pathways.!!!

124!! Discovery!of!a!Novel!Bacterial!Growth!Factor!for!the!Human!Gut!Microbiota!

!Philip!Strandwitz1,!Ki9Hyun!Kim2,!Eric!Stewart1,!Jon!Clardy2,!and!Kim!Lewis1!1Department! of! Biology,! Northeastern! University,! 2Department! of! Biological! Chemistry! and! Molecular! Pharmacology,! Harvard!Medical!School!!Roughly!half!the!bacterial!species!in!the!human!gut!have!not!been!cultured.!Due!to!the!role!of!the!human!microbiome!in!health!and!disease,! there! is! interest! in! its! characterization;! however,! to! properly! study! these! species! they! must! be! cultivated! in& vitro.!Previously,! our! group! found! that! “uncultured”! bacteria! depend! on! neighboring! “helper”! bacteria! for! growth! factors.! For! these!helper9uncultured! pairs,! Escherichia& coli! could! also! act! as! a! helper.! Here,! we! hypothesize! that! if! helper9uncultured! pairs! were!isolated! in!which!E.&coli&could!not!act!as!a!helper,! those!pairs!would!be!sources!of!novel!growth!factors.!We!isolated!such!a!pair!from!human!feces,!where!Dorea&longicatena!induced!growth!of!KLE1738.!KLE1738!is!found!on!the!NIH’s!“Most!Wanted”!list,!and!is!distantly! related!to! its!nearest! relative!by!16S!rRNA!sequence.&Bacteroides& fragilis!also! induces!growth!of!KLE1738,!and!bioassay!driven! purification! is! being! performed! to! isolate! the! inducing! factor! in! its! supernatant.! Concurrently,! a! transposon! library! of!B.&fragilis! is!being!screened!to! identify!the!gene(s)!responsible!for!production!of!the! inducing!factor.!These!results!indicate!that!gut!commensals!continue!to!be!a!valuable!source!for!bacteria!growth!factors,!which!allow!access!to!missing!bacterial!diversity.!!!

125!! Structural!Insights!into!an!Oxalate!Oxidoreductase!

!Marcus!I.!Gibson1,!Ed!J.!Brignole1,4,!Elizabeth!Pierce2,!Stephen!W.!Ragsdale2,!and!Catherine!L.!Drennan1,3,4!Departments!of!1Chemistry!and!3Biology,!Massachusetts!Institute!of!Technology,!2Department!of!Biological!Chemistry,!University!of!Michigan,!4Howard!Hughes!Medical!Institute!!Thiamine!pyrophosphate! (TPP)! is!a!cofactor! that!enables!enzymatic!catalysis!across!a!broad!spectrum!of!metabolic!chemistry.! In!Moorella&thermoacetica,!the!TPP9dependent!enzyme!oxalate!oxidoreductase!(OOR)!allows!growth!on!oxalate!and!carbon!dioxide,!via! the!Wood9Ljungdahl!pathway,!even! in! the!presence!of!other!electron!acceptors.!Unlike!other!oxalate9transforming!enzymes,!OOR!performs!the!oxidation!of!oxalate!in!the!absence!of!molecular!oxygen,!directing!the!electrons!through!a!ferredoxin!domain!to!the!enzymes!involved!in!acetogensis,!while!releasing!two!molecules!of!CO2.!We!present!the!first!high9resolution!structure!of!OOR!which,! in! conjunction!with! structures!of! a! related!pyruvate:ferredoxin!oxidoreductase,!may! tell! us!more!about!how!nature,! and!TPP9enzymes!in!particular,!can!perform!one9carbon!chemistry.!!!

126!! Sinorhizobium&meliloti&Cell!Cycle!Regulation!of!DivK!and!CtrA!

!Karla!B.!Schallies1,!and!Katherine!E.!Gibson2!1Biology!Department,!University!of!Massachusetts!Boston!&

Sinorhizobium&meliloti&is!an!alphaproteobacteria!that!grows!as!a!free9living!organism!in!soil!or!as!a!symbiont!within!legumes.&As!a!free9living! organism,! S.& meliloti& undergoes! asymmetrical! cell! division! with! once9and9only9once! DNA! replication! per! cell! cycle;!however,! inside!the!plant,! the!bacterium!differentiates! into!a!bacteroid! through!a!novel!cell! cycle!pathway!of!endoreduplication!without!cell!division.!My!goal!is!to!understand!the!two9component!signaling!pathway!(TCS)!that!regulates!these!distinct!cell!cycle!outcomes.!Based!on!the!Caulobacter&crescentus!model,!I!hypothesize!this!TCS!pathway!requires!PleC,!DivJ,!and!two!novel!histidine!kinases,!CbrA!and!CbrB,!to!regulate!phosphorylation!of!the!response!regulator!DivK.!I!further!predict!these!interactions!will!have!a!downstream!effect!on!CtrA!activity.!Our!results!show!DcbrA!disrupts!the!coordination!between!DNA!replication!and!cell!division,!and!decreases!phosphorylation9dependent!DivK! localization,! suggesting! it! is! a!DivK! kinase.!Currently,! I! am! testing!whether!CbrA!regulates!DivK!directly! using! in& vitro! phosphotransfer! profiling! and!have! successfully! over9expressed! and! affinity9purified! tagged!DivK!and!CbrA.!Finally,! I!am!testing! if!phosphorylation!and!stability!of!CtrA! is!affected!by!DcbrA.!By!characterizing! free9living!cell!cycle!regulation,!I!will!be!able!to!address!how!host!symbiosis!modifies!this!canonical!free9living!cell!cycle.!!


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127!! Combined!S733!and!O718!Isotope!Tracing!of!Intracellular!Sulfur!Metabolism!during!Microbial!Sulfate!Reduction!

!Gilad!Antler1,!Tanja!Bosak2,!Shuhei!Ono2,!Orit!Sivan3,!and!Alexandra!V.!Turchyn1!1! Department! of! Earth! Sciences,! University! of! Cambridge,! Cambridge! CB2! 3EQ,! UK,! 2Department! of! Earth,! Atmospheric! and!Planetary! Sciences,! Massachusetts! Institute! of! Technology,! Cambridge! MA! 02139,USA,! 3Department! of! Geological! and!Environmental!Sciences,!Ben!Gurion!University,!Beer!Sheva!84105,!Israel.!!Sulfate!reduction!(SR)!is!a!key!player!in!the!global!carbon!cycle,!oxidizing!nearly!50%!of!organic!matter!reaching!marine!sediments,!donating! electrons! to! sulfate! and! producing! sulfide.! The! biochemical! pathway! of! SR! includes! several! enzymatic! steps! whose!reversibilities!influence!the!observed!isotopic!discrimination!of!33S!/32S,!34S/32S!and!18O/16O.!Previous!studies!have!shown!that!the!availability!and!type!of!organic!substrate!control!the!magnitude!of!34S/32S!and!33S/32S,!but!the!influence!of!the!electron!donor!on!the!oxygen! isotope!effects! is! less!well!understood.!We!will! report!our!results!of!combined!the!sulfur!and!oxygen! isotope! in!pure!cultures!of!a!marine!Desulfovibrio!sp.!grown!on!different!organic!substrates.!

Our!measurements!show!that!the!fractionations!of!oxygen!and!sulfur! isotopes!correlate!with!the!cell! specific!SR!rate,!with!faster!reduction!rates!yielding!the!smaller!isotopic!effects.!This!relationship!indicates!that!more!intracellular!sulfite!may!be!oxidized!back!to!APS!when!the!flux!of!electrons!from!the!electron!donor!to!sulfite!is!low,!allowing!an!isotopic!exchange!of!oxygen!in!sulfite!and!water.!Our! results! demonstrate! similar! trend! as! in! the! natural! environment,! and! therefore,! can! help! us! understanding! this!process!where!the!nature!of!the!electron!donor!is!elusive.!!!

128!! Identification!of!a!Growth!Factor!for!Marine!Uncultured!Bacteria!

Bijaya!Sharma1,!Anthony!D’Onofrio1,!Eric!Dimise2,!Eric!J.!Stewart1,!Jon!Clardy2,!and!Kim!Lewis1!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1Antimicrobial!Discovery!Center,!Department!of!Biology,!Northeastern!University,!Boston!2Department!of!Biological!Chemistry!and!Molecular!Pharmacology,!Harvard!Medical!School,!Boston!

A! vast! majority! of! bacterial! species! do! not! grow! in! the! laboratory.! Previously,! we! showed! that! siderophores! produced! by!neighboring! bacteria! can! induce! the! growth! of! a! subset! of! uncultured! bacteria.! We! reasoned! that! there! are! other! growth9promoting!factors!required!to!cultivate!the!missing!bacteria.!Using!co9culture!on!media!supplemented!with!soluble!iron!to!bypass!siderophore!dependency,!we!cultivated!an!isolate!from!marine!sand!biofilm,!KLE1215!(97%!similar!to!Flaviramulus&basaltis&by!16s!rDNA! sequence),! which! grows! only! when! it! is! present! at! high! cell! density! or! in! the! presence! of! a! helper! like!Escherichia&coli&or!Micrococcus& luteus.&Upon! testing! deletion! strains! of!E.& coli,&we! found! that! DubiG&strain! of!E.& coli,&which! is! deficient! in!ubiquionone!biosynthesis,!does!not!induce!growth!of!KLE1215.!Here,!we!show!that!menaquinone94!(MK4)!delivered!in!liposomes!induces! the! growth!of! KLE1215.! This! shows! that! quinones! represent! a! new! class! of! growth! factors! for! uncultured!bacteria.! The!requirement! for! liposomes! for! delivery! of! this! hydrophobic! compound! suggests! that! outer! membrane! vesicles! may! serve! as! a!natural!delivery!vehicle!for!quinones.!!!

129!! Dynamic! reprogramming! of! tRNA! modifications! is! linked! to! activation! of! the! Dos! regulon! in! hypoxia7induced!mycobacterial!dormancy!!Yok!Hian!Chionh,1,2!Fabian!Hia,2!Sylvie!Alonso,1,2!Peter!C.!Dedon2,3!1Department!of!Microbiology,!School!of!Medicine,!National!University!of!Singapore;!2Infectious!Disease!Interdisciplinary!Research!Group,! Singapore9MIT! Alliance! for! Research! and! Technology,! Singapore;! 3Department! of! Biological! Engineering,! Massachusetts!Institute!of!Technology,!Cambridge,!MA!!We!seek!to!understand!how!pathogenic!mycobacteria!utilize!RNA!modifications!to!control!translation!to!survive!the!human!innate!immune!response.!Despite!the!availability!of!genomic!and!proteomic!knowledge!on!how!the!invading!pathogen!responds!to!hostile!host! environments,! we! know! relatively! little! about! the! role! of! translation! in! mycobacterial! host9pathogen! interactions.!Tuberculosis,!specifically,!involves!the!formation!of!hypoxic!granulomas!where!pathogenic!mycobacteria!persist!in!a!dormant,!non9replicative! state.! !Using!Mycobacterium&bovis! BCG!as! a!model,!we! first! defined! the! complete! set! of!modified! ribonucleosides! in!tRNA!using!a!novel!platform!consisting!of!multidimensional!HPLC!to!isolate!tRNA!and!chromatography9coupled!mass!spectrometry!(LC9MS)! to! identify! and! quantify! 31! ribonucleosides! in! BCG.! BCG! cells! were! then! subjected! to! gradual! hypoxia! (Wayne),! with!hypoxia9induced! changes! in! the! relative! quantities! of!modified! ribonucleosides! analyzed! by!multivariate! statistical! analysis.! The!analysis!revealed!that!tRNA!modifications!in!mycobacteria!undergo!stress9induced!reprogramming!to!control!selective!translation!of!key!stress!response!genes!as!the!organism!enters!and!exits!hypoxia9induced!non9replicative!state.!One!such!modification,!cmo5U,!


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is!predicted!to!occur!at!the!wobble!position!of!tRNAThrUGU.!!This!tRNA!decodes!codon!ACG,!which!is!significantly!enriched!in!DosR!–!a!

transcription!factor!that!controls!hypoxic!latency!in!BCG!and!Mycobacterium&tuberculosis.!!!

130!! The!role!of!volatiles!as!infochemicals!in!bacterial!inter7specific!interactions!

!Ruth!Schmidt1,!Kees!Hordijk1,!Saskia!Gerards1,!Wietse!de!Boer1,!2!and!Paolina!Garbeva2!1!Department!of!Microbial!Ecology,!NIOO9KNAW,!2!Department!of!Soil!Quality,!Wageningen!University!!The!importance!of!bacterial!interactions!in!microbial!ecology!is!increasingly!recognized.!However,!little!attention!is!paid!to!the!role!of!bacterial!volatiles! in!microbial! interactions.!Gas9filled!pores!are!an! important!constituent!of!most! soils!and! therefore!volatiles!may! have! a! prominent! role! in! the! interactions! between! relatively! distantly! distributed! soil!microorganisms.!! In! order! to! obtain!insight!into!the!importance!of!volatiles!in!soil!microbial!interactions!we!performed!sand!microcosm!experiments.!Petri!dishes!were!designed! to! enable! growth! of! different! microorganisms! in! physically! separated! areas! within! a! common! atmosphere.! We!determined! the! population! dynamics,! behavior! and! gene! expression! of! the! model! soil! isolate! Pseudomonas& fluorescens! Pf091!exposed!to!volatiles!produced!by!different!mono9!and!mixed!cultures!of!several!soil!bacteria.!The!produced!volatiles!were!collected!and! analyzed! by! GC9MS.! Results! show! that! the! composition! of! volatiles! produced! by! different! monocultures! was! different.!Microarray9based!analyses! indicated!strongly!different!responses! in!the!gene!expression!of!P.&fluorescens!Pf091!when!exposed!to!the! volatiles! of! different! soil! bacterial! species.! Differentially! expressed! genes!were!mainly! involved! in! amino! acid! transport! and!metabolism,! signal! transduction! mechanisms,! inorganic! ion! transport! and! metabolism,! cell! motility! and! secretion! and! energy!production! and! conversion.! The! obtained! results! support! the! important! role! of! volatiles! as! infochemicals! in! soil! bacterial!interactions.!!!

131!! Transcriptome!Analysis!of!Adaptation!to!Water!Stress!by!Pseudomonas&fluorescens!Pf071!Growing!in!Soil!

!Douglas!C.!Marshall1,!Stuart!B.!Levy2,!and!Mark!W.!Silby1!1Department! of! Biology,! University! of! Massachusetts! Dartmouth,! 2Department! of! Molecular! Biology! and! Microbiology,! Tufts!University!School!of!Medicine!&

P.& fluorescens& adapts! rapidly! to! environmental! changes! which! contributes! to! its! potential! in! the! biocontrol! of! crop! diseases.!Biocontrol!by!P.&fluorescens&has!variable!success!because!of!a!failure!to!establish/maintain!an!adequate!population!under!natural!soil! conditions.!Water! content! fluctuates! widely! in! soil,! but! microbial! response! mechanisms! to! water! stress! in& situ& is! not! well!studied.!Using!functional!genomics,!the!mechanisms!by!which!P.&fluorescens&Pf091!adapts!to!dehydration!while!growing!in!soil!were!investigated.! Following! dehydration! of! soil,! widespread! gene! expression! changes!were! observed! in!P.& fluorescens,! indicating! an!immediate! stress! response! and! longer9term! adaptive! strategies.! Genes! specifying! alginate! production! were! upregulated! during!water! stress.! Deletion! of! alginate! biosynthesis! genes! algD,! alg44,! and! the! regulatory! gene! algB! decreased! colonization! and!maximum! population! density! in!moist! soil.! After! dehydration,! the! alg&mutant! populations! immediately! declined! at! least! 69fold!greater! than! Pf091,! suggesting! that! Pf091! constitutively! produces! alginate! to! maintain! populations! in! soil! and! upregulates!production!under!dry!conditions.!Increased!alginate!gene!expression!reveals!the!critical!importance!of!alginate!in!the!production!of!protective,!hydrated!microenvironments!in!soils,!where!moisture!fluctuates.!Studies!examining!adaptive!gene!expression!of!Pf091!in!soil!will!improve!the!development!and!deployment!of!effective!biocontrol!agents.!!!

132!! The! high& light>inducible! genes! of! the! marine! cyanobacterium! Prochlorococcus:&& A! diverse! dynamic,! high! copy!number!gene!family!!Jessie!Thompson1*,!Greg!Kettler1*,!Steve!Biller1,!Julie!Miller1,!Lisa!Moore2,!and!Sallie!W.!Chisholm1!1Civil! and! Environmental! Engineering,! MIT,! 2Department! of! Biological! Sciences,! University! of! Southern! Maine.! *These! authors!contributed!equally!to!the!work.!!With!its!extraordinary!abundance!across!the!oceans,!Prochlorococcus!plays!a!profound!role!in!the!cycling!of!nutrients!and!flow!of!energy! in!marine! ecosystems.! This! success! comes! in! part! from! diversity! within! Prochlorococcus,! including! adaptations! to!many!conditions.! The!high& lightBinducible& (hli)! gene! family,! small! chlorophyll9binding! proteins! active! in! the! cyanobacterial! response! to!light!shock!and!other!stress,!is!one!source!of!this!variation.!Hlis!occur!at!varying!copy!number!–!a!genome!may!have!less!than!10!or!more! than! 40! –! with! high! sequence! diversity.! Here! we! examine! their! evolutionary! dynamics,! primarily! through! comparative!genomics.!Using! improved!search!tools! for!annotation,!we!offer!an!updated!picture!of! the!distribution!of!hlis! in!Prochlorococcus!


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and! their! phage.! These! include! a! newly! closed! genome,! from! a! light! shock! tolerant! clade,! targeted! to! study! the! origins! and!mechanisms! behind! recent! expansion! of! hlis! in! this! clade.!Hlis& often! occur! in! tandem! arrays;! in! this! dataset! we! observe! their!expansion!and!contraction!across!lineages.!These!hli&arrays!can!be!in!genomic!islands,!with!neighboring!phage9like!sequences!and!tRNAs,!hallmarks!of!horizontal!gene!transfer.!These!arrangements!support!phylogenetic!evidence!of!mobility!and!hint!at!possible!mechanisms!of!change!in!the!flexible!genome!of!Prochlorococcus.!!!

133!! The!role!of!InlF!in!Listeria&monocytogenes&infection!of!the!brain!!

!Elizabeth!Moritz!Halvorsen!and!Darren!E.!Higgins!Department!of!Microbiology!and!Immunobiology,!Harvard!Medical!School,!Boston,!MA!!&

Listeria&monocytogenes,!an! intracellular!bacterial!pathogen,! is! the! leading!cause!of!death!by!a! food9borne!agent!and!most!often!affects!infants,!pregnant!women,!and!the!elderly.!Of!particular!concern!is!the!ability!of!L.&monocytogenes!to!invade!the!brain,!which!can! lead! to! life9threatening!meningitis.! Infection! studies! in!mice!have! revealed! that! a!L.&monocytogenes! surface!protein,! InlF,! is!required! for! successful! colonization! of! the! brain.! Furthermore,! the! results! of! affinity! chromatography! and! mass! spectrometry!analysis! indicated! that! InlF! interacts! with! a! host! cell! protein,! Vimentin.! Preliminary! results! have! shown! that! in! the! absence! of!Vimentin,! L.& monocytogenes! infection! of! the! brain! in! mice! is! severely! compromised.! We! hypothesize! that! InlF! interacts! with!Vimentin!to!mediate!successful!entry!of!L.&monocytogenes!into!brain!cells.!We!are!using!a!combination!of!in&vivo&mouse!infection!models,&in&vitro&tissue!culture!cell!infections,!and!biochemical!approaches!to!elucidate!how!InlF!interacts!with!Vimentin!to!facilitate!colonization!of!the!brain.!!Since!L.&monocytogenes!is!a!model!intracellular!bacterial!pathogen,!understanding!how!InlF!and!Vimentin!contribute!to!colonization!of!the!brain!may!establish!a!paradigm!that!is!applicable!to!other!intracellular!pathogens.!!!

134!! A! co7cultivation! assay! to! detect! small7molecule7mediated! interactions! between! common!members! of! the! nostril!microbiota!!Kelly!L.!Aldridge1,2,!Michael!S.!Wollenberg1,!and!Katherine!P.!Lemon1,3!1Department!of!Microbiology,!The!Forsyth!Institute,!2Department!of!Microbiology,!University!of!Massachusetts,!Amherst,!3Division!of!Infectious!Diseases,!Boston!Children’s!Hospital,!Harvard!Medical!School.!&

Staphylococcus&aureus!is!a!clinically!important!pathobiont!that!colonizes!the!nostrils!(anterior!nares)!of!~30%!of!the!U.S.!population!at!any!one!time.!In!addition!to!S.&aureus,&a!number!of!other!bacteria!are!present!in!the!nostril!microbiota.!Among!these,!two!of!the!most! common! genera! are! Propionibacterium! and! Corynebacterium.! We! hypothesize! that! small! molecules! produced! by!Propionibacterium! and!Corynebacterium! affect! the! growth! and/or! behavior! of!S.& aureus.! To! test! this! in& vitro,!we!performed! co9culture!assays!using!primary!nostril!isolates.!Previous!work!in!our!lab!demonstrated!that!some!Propionibacterium!isolates!inhibited!the!growth!of!S.&aureus&and!Corynebacterium&sp.!in!an!overlay!assay!for!antibiosis.!The!goal!of!the!current!work!was!to!optimize!an!assay! to!detect!pair9wise! small9molecule9mediated! interactions!between! these!genera!when!grown!side9by9side!under! the! same!conditions.! This! necessitated! accommodation!of! their! differential! growth! rates.! Various! assay! set9ups!were! tested! to! determine!which!was!most!sensitive!for!detecting!potential!interspecies!interactions!mediated!by!diffusible!substances.!Further!investigation!to!identify!the!molecule(s)!responsible!for!these!interactions!will!provide!insights!into!the!chemical!ecology!of!the!microbiota!of!the!human!nostrils.!This!could!lead!to!new!ways!of!preventing/eradicating!S.&aureus&colonization!and&preventing!subsequent!infections.!!!

135!! The!Effect!of!Bisphenol7A!on!Representative!Gut!Microbiota!

!Kimberly!Mohareb1,!Michelle!Wheeler1,!and!Ellen!Fynan1!1Department!of!Biology,!Worcester!State!University!!Bisphenol9A!(BPA)!is!a!chemical!used!to!make!polycarbonate!plastics!and!epoxy!resins.!BPA!can!leach!from!the!polycarbonate!into!liquid!and!solid!foods.!Exposure!of!BPA!to!humans!has!raised!many!questions!about!its!effects!on!the!human!body.!This!experiment!focuses!on!the!effect!of!BPA!on!representative!gut!microbiota:!Lactobacillus&acidophilus&and!Escherichia&coli.!After!growth!media!(Lactobacillus!MRS!or!Mueller!Hinton!agar!plates)!were!inoculated!with!the!bacterial!cultures,!various!concentrations!of!the!control!substance,!ampicillin,!were!pipetted!into!wells.!Following!incubation!at!37°C,!the!zones!of!inhibition!were!measured.!This!served!as!a!basis!of!comparison!for!observing!the!inhibitory!effect!of!BPA!on!these!two!organisms!when!the!same!procedure!was!followed!using! BPA! instead! of! ampicillin.! Both! L.& acidophilus& and! E.& coli! exhibited! no! inhibitory! effect! after! being! exposed! to! various!concentrations!of!BPA.!Future!studies!are!necessary!to!draw!further!conclusions!about!how!BPA!affects!human!gut!microbiota!and!


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ultimately!human!health.!!!

136!! Lipid7dependent! Inhibition! of! the! Pathobiont! Streptococcus& pneumoniae! by! Corynebacterium!Primary! Nostril!Isolates!!Lindsey!Bomar1,!Silvio!Brugger2,!and!Katherine!P.!Lemon1,3!1Department!of!Microbiology,!The!Forsyth! Institute,!Cambridge,!MA!2Present!address:!Department!of!General! Internal!Medicine,!Inselspital,!Bern!University!Hospital,!and!University!of!Bern,!Switzerland!3Division!of!Infectious!Diseases,!Boston!Children’s!Hospital,!Harvard!Medical!School!!Non9pathogenic!Corynebacterium&spp.&are!common!constituents!of!human!nostril/skin!microbiota,!yet!their!function!within!these!communities! is! virtually!unknown.!Data! from!our! lab!and!others! indicate! that! children!without!Streptococcus&pneumoniae&nasal!colonization!have! increased!relative!abundance!of!nasal!Corynebacterium&spp.!Therefore,!we!hypothesize! that!antagonism!exists!between! Corynebacterium! spp.! and! the! pathobiont! S.& pneumoniae.&Our! co9cultivation! assays! with! S.& pneumoniae& and! primary!nostril! isolates! of! Corynebacterium& demonstrated! mutual! antagonism! in! vitro.! Not! unexpectedly,! S.& pneumoniae9produced!hydrogen! peroxide! inhibited! Corynebacterium.& In! co9cultivation! assays! using! lipophilic! (lipid9requiring)! and! non9lipophilic!Corynebacterium& spp.,! the! medium! component! Tween! 80! was! necessary! for! pneumococcal! inhibition.! We! speculate! that!Corynebacterium& spp.! hydrolyze! Tween! 80! (polyethylene! glycol! sorbitan! monooleate)! releasing! oleic! acid,! a! fatty! acid! with!antimicrobial!properties.!Preliminary! results! indicate! that!oleic!acid!mediates!part!of! the!observed!S.&pneumoniae& inhibition.!We!postulate! that! free! fatty! acids! released! by! Corynebacterium& spp.! from! skin! surface! lipids! modify! the! local! habitat! impeding!colonization!by/proliferation!of! pathobionts! such!as!Streptococcus! spp.! and!Staphylococcus&aureus& (reported! in! the! literature! as!susceptible! to! oleic! acid).! Determining! this! will! increase! understanding! of! pathobiont! colonization! dynamics! and! interpersonal!variation!in!pathobiont!carriage,!and!uncover!alternative!approaches!to!prevent/treat!pathobiont!infections.!!!

137!! Expression!of!antibodies!in!SHuffle!

!Michael9Paul! Robinson1,! Julie! Lobstein2,! 3,! Thomas! J.!Mansell1,! Corinna! Tuckey2,! Paul! Riggs2,! Paul! Colussi2,! 4,! Chris! Noren2,! Chris!Taron2,!Matthew!P.!DeLisa1!and!Mehmet!Berkmen2!1School!of!Chemical!and!Biomolecular!Engineering,!Cornell!University,!2New!England!Biolabs,!3Molecular!Partners!AG,!Switzerland,!4Tetragenetics,!Cambridge,!!We! describe! the! design! of! an! engineered! expression! platform! that! permits! biosynthesis! of! full9length! immunoglobulin! G! (IgG)!antibodies! directly! in! the! cytoplasm! of! Escherichia& coli& cells.! This! was! achieved! by! creating! a! synthetic,! bicistronic! expression!plasmid! encoding! full9length! heavy! and! light! chains,! both! of! which! lacked! a! canonical! export! signals! and! thus! were! produced!exclusively! in! the! cytoplasmic! compartment! and! assembled! as! aglycosylated! IgGs.! Proper! folding! and! assembly! of! cytoplasmic!antibodies,!so9called!cytomAbs,!was!facilitated!by!the!use!of!a!specially!engineered!E.&coli&strain!having!an!altered!redox!state!that!permits!the!formation!of!stable!disulfide!bonds!within!its!cytoplasm.!Unlike!all!other!cell9based!methods!for!producing!full9length!IgGs,! our! approach! does! not! hinge! on! secretion! of! IgGs! across! one! or!more! biological!membranes.! By! removing! this! barrier! to!production,! the!amount!of!active!cytomAbs!generated! in! the!cytoplasm!was!49! to!109times!greater! than!that!obtained! following!secretion!of!the!same!IgGs!into!the!periplasm.!We!demonstrated!the!capacity!of!our!system!to!act!as!a!platform!to!design!new!IgGs!by!grafting!epitope!recognition!domains!resulting!in!IgGs!with!new!antigen!binding!capacities.!We!further!remodeled!the!fragment!crystallizable! (Fc)!domain!of!cytomAbs!with!amino!acid!substitutions!that!are!known!to!promote!binding!to!Fc!gamma!receptors!(e.g.,!Fc!gamma!RI)!with!nanomolar!affinity,!demonstrating! that!clinically! relevant!antigen9!and!effector9binding!activities!can!be!achieved!in!the!absence!of!membrane!translocation!and!glycosylation.!!!!!!!!!!!!


77

138!! Genome7wide!mapping!of!the!Bacterial!DNA!Phosphorothioatome!using!Single7Molecule,!Real7Time!Sequencing!

!Bo!Cao1,3,!Tyson!A!Clark2,!Michael!S!DeMott3,!Qiuxiang!Cheng1,!Zixin!Deng1,!Matthew!Boitano2,!Khai!Luong2,!Stephen!W!Turner2,!Delin!You1,!Peter!C!Dedon3,!Jonas!Korlach2!!1State! Key! Laboratory! of! Microbial! Metabolism! and! School! of! Life! Sciences! and! Biotechnology,! Shanghai! Jiao! Tong! University,!Shanghai,! China.! 2Pacific! Biosciences,! Menlo! Park,! California,! USA.! 3Departments! of! Biological! Engineering,! and! Center! for!Environmental!Health!Science,!Massachusetts!Institute!of!Technology,!Cambridge,!Massachusetts,!USA!!!Building!on!the!observation!of!a!DNA!degradation!phenotype!involving!sulfur!incorporation!into!DNA!by!products!of!the!bacterial!dnd!gene!family,!we!recently!discovered!that!dnd!gene!products!incorporate!sulfur!into!the!DNA!backbone!as!a!phosphorothioate!(PT)!modification! in! a! sequence9! and! stereospecific!manner.! PT!modifications! impart! antioxidant! properties! and!participate! in! a!novel! restriction9modification! system! in! bacteria.! However,! the! genome9wide! distribution! of! this! modification! and! its!corresponding!consensus!sequence!has!remained!largely!unknown.!In!this!study,!we!adapt!single!molecule,!real9time!(SMRT)!DNA!sequencing! to! the! detection! of! PT! in! oligonucleotides,! plasmids,! and! genomic! DNA.! We! demonstrate! that! PT! can! be! directly!detected!at! the! level!of! single!base9pair!and!DNA!strand!resolution.!During!sequencing,!PT!generates!distinct!polymerase!kinetic!signatures! that! differ! from! those! of! canonical! nucleotides,! but! only! when! adopting! the! naturally! occurring! Rp! stereochemical!configuration.!Using!this!method,!we!localize!fifteen!PT!sites!on!pBluescript!SK!plus!isolated!from!S.!serovar!Cerro!87.!Interestingly,!quantitative!analysis!of!PT!modifications!on!this!plasmid!reveals!that!each!plasmid!was!modified!only!once.!At!last,!we!describe!the!first!genome9wide!analysis!of!PT!in!Escherichia!coli!B7A,!thus!enhancing!our!understanding!of!this!epigenetic!modification!to!DNA.!!!

139!! An!elegant!solution!for!the!production!of!biofilm!matrix!protein!by!Staphylococcus&aureus!

!Lucy!Foulston!1,!Alexander!Elsholz!1!and!Richard!Losick!1!1!Department!of!Molecular!and!Cellular!Biology,!Harvard!University!!Biofilms!provide!a!physical!protective!barrier!for!bacterial!pathogens,!significantly!increasing!the!severity!of!infections!and!reducing!treatment!options.!Staphylococcus&aureus! is!one!of! the!most!common!causes!of!multi9drug!resistant! infection,! for!which!biofilm!formation!is!a!major!contributing!factor.!

Protein!and!extracellular!DNA!are!essential!components!of!the!biofilm!matrix!formed!by!S.&aureus!HG003! in&vitro.!We!used!quantitative! proteomics! to! compare! proteins! found! under! biofilm! and! non9biofilm! conditions,! through! a! differential! isotopic!labeling!approach!followed!by!two!parallel!methods!that!selectively!enrich!for!cell!surface9associated!proteins.!We!found!that!the!vast! majority! of! these! proteins! are! known! or! predicted! cytoplasmic! proteins,! including! those! involved! in! metabolism! and! cell!growth.!

We! hypothesize! that! release! of! cytoplasmic! proteins,! by! autolysis! within! a! biofilm,! provides! S.& aureus! with! a! generalized!mechanism! for! the! production! of!matrix! protein.! This! elegant!mechanism! allows! the! pathogen! to! expend! very! little! additional!energy!to!produce!matrix!and!makes!biofilm!intrinsically!resilient!to!mutation!and!versatile!for!a!range!of!environmental!conditions.!!!

140!! Pleiotropy!positively!correlates!with!the!direct!effect!of!beneficial!mutations!

!Rachel!Staples1,!David!Morejon1,!and!Vaughn!Cooper1!1Molecular,!Cellular,!and!Biomedical!Sciences,!University!of!New!Hampshire!!Rare,! beneficial! mutations! increasing! fitness! provide! the! foundation! by! which! organisms! adapt! to! their! surroundings.! Theory!predicts! that! the! individual! fitness! effects! of! these! beneficial!mutations! are! exponentially! distributed.!! However,! little! is! known!about! how! adaptive! history! and! the! selective! environment! impact! the! availability! of! beneficial! mutations,! nor! influence! their!indirect! effects! in! alternative! environments.! We! experimentally! evolved! the! cystic! fibrosis! pathogen& Burkholderia& cenocepacia!under! biofilm! and! planktonic! conditions,! collected!mutants,! and! directly!measured! their! fitness! in! a! variety! of! environments! to!quantify! the!direct!and! indirect! (pleiotropic)!effects!of!each.!We! found! that!direct! fitness! in! the! selective!environment!was!best!modeled! by! a! non9exponential! distribution!with! a! clustering! of! high! fitness! values,! indicating! that!mutations! greatly! increasing!fitness!were! readily! available.! !Pleiotropic! fitness!was! generally! positive,! although!mutants! experienced! a! trade9off! under! select!alternative! conditions,! suggesting! that!biofilm9associated!mutations!may!be!more! specific! to! structure!and! cause!narrow!overall!niche! breadth.! Additionally,! the! magnitude! of! direct! and! pleiotropic! fitness! effects! was! strongly! correlated,! suggesting! that!mutations! of! higher! initial! benefit! also! dramatically! influence! fitness! elsewhere,!the! negative! effects! of!which!may! impact! their!success!in!complex,!fluctuating!environments.!

2013!Boston!Bacterial!Meeting!7!Meeting!Attendees!

78

Name! Position! Lab! Institution! Email!Kathrin!Abele! Graduate!Student! Veronica!Godoy! Northeastern!University! [email protected]!

Andres!Abin9Fuentes! Post9Doc!Daniel!IC!Wang!and!Kristala!LJ!Prather!

Massachusetts!Institute!of!Technology! [email protected]!

Neethu!Abraham! Research!Scientist! BASF!LAB! BASF! [email protected]!Ethan!Addicott! Undergraduate!Student! Pringle!Lab! Harvard!University! [email protected]!Kelly!Aldridge! Undergraduate!Student! Lemon!Lab! Forsyth!Institute! [email protected]!Emrah!Altindis! Post9Doc! Mekalanos!Lab! Harvard!Medical!School! [email protected]!Erica!Anderson! Research!Scientist! Excelimmune! Excelimmune! [email protected]!

Fenton!Andrew! Post9Doc! Bernhardt/Rudner!Labs!

Harvard!Medical!School! [email protected]!

Jon!Anthony! Research!Scientist! Meyer!Lab! Boston!College! [email protected]!

Gilad!Antler! Graduate!Student! Department!of!Earth!Sciences! University!of!Cambridge! [email protected]!

Seblewongel!Asrat! Graduate!Student! Ralph!Isberg! Tufts!University!School!of!Medicine! [email protected]!

Elham!Azizi! Graduate!Student!Biomedical!Engineering! Boston!University! [email protected]!

Arianne!Babina! Graduate!Student! Meyer!Lab! Boston!College! [email protected]!

Christina!Baer! Post9Doc! Sassetti!Lab! University!of!Massachusetts!Medical!School! [email protected]!

Amy!Baker! Graduate!Student! O'Toole!Lab! Dartmouth!Medical!School! [email protected]!Pooja!Balani! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Roberto!Balbontín! Post9Doc! Kolter!lab! Harvard!Medical!School! [email protected]!Alicia!Ballok! Graduate!Student! O'Toole! Dartmouth!Medical!School! [email protected]!

Aditya!Bandekar! Graduate!Student! Sassetti!Lab! University!of!Massachusetts!Medical!School,!Worcester! [email protected]!

Amy!Barczak! Post9Doc! Hung!Lab/Rubin!Lab! Massachusetts!General!Hospital! [email protected]!Bonnie!Bassler! Faculty! Bassler! Princeton! [email protected]!Shubham!Basu! Graduate!Student! Kevin!Griffith! University!of!Massachusetts,!Amherst! [email protected]!

Kylynda!Bauer! Undergraduate!Student!Turnbaugh!Laboratory! Harvard!University! [email protected]!

Lisa!Baumoel! Graduate!Student! Morita! University!of!Massachusetts,!Amherst! [email protected]!Jon!Beckwith! Faculty! Beckwith! Harvard!Medical!School! [email protected]!

Christine!Beemelmanns! Post9Doc! Clardy! Harvard!Medical!School! [email protected]!

Michelle!Bellerose! Graduate!Student! Sassetti!Lab!University!of!Massachusetts!Medical!School! [email protected]!

Owen!Bennion! Research!Scientist! Bree!Aldridge!Lab! Tufts!University!School!of!Medicine! [email protected]!Ryan!Benson! Post9Doc! Godoy!Lab! Northeastern!University! [email protected]!Brittany!Berdy! Graduate!Student! Epstein!Lab! Northeastern!University! [email protected]!Alice!Berenson! Undergraduate!Student! Burton!Lab! Harvard!University! [email protected]!Melanie!Berkmen! Faculty! Berkmen!lab! Suffolk!University! [email protected]!Mehmet!Berkmen! Research!Scientist! Berkmen! New!England!Biolabs! [email protected]!Katie!Berry! Post9Doc! Hochschild!Lab! Harvard!Medical!School! [email protected]!Marina!Besprozvannaya! Graduate!Student! Burton!Lab! Harvard!University! [email protected]!

Eric!Binder! Private!Sector! Research!&!Development! Rapid!Micro!Biosystems! [email protected]!

Anthony!Bissell! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!

Adam!Bitzer! Graduate!Student! Silby!Lab! University!of!Massachusetts!Dartmouth!

[email protected]!

Kristina!Boguslawski! Graduate!Student! Griffith! University!of!Massachusetts,!Amherst! [email protected]!Lindsey!Bomar! Post9Doc! Katherine!Lemon! The!Forsyth!Institute! [email protected]!Bijou!Bose! Post9Doc! Burton! Harvard!University! [email protected]!Elsa!Bou!Ghanem! Post9Doc! John!Leong!Lab! Tufts!University! [email protected]!Cara!Boutte! Post9Doc! Rubin! Harvard!School!of!Public!Health! [email protected]!

Chelsea!Boyd! Graduate!Student! O'Toole!Lab! Geisel!School!of!Medicine!at!Dartmouth!

[email protected]!

Dana!Boyd! Faculty! Beckwith! Harvard!Medical!School! [email protected]!Niels!Bradshaw! Post9Doc! Losick! Harvard!University! [email protected]!Chris!Brennan! Undergraduate!Student! Pellock!Lab! Providence!College! [email protected]!Christopher!Brigham! Faculty! Brigham!Laboratory! UMass!Dartmouth! [email protected]!


79

Name! Position! Lab! Institution! Email!Shaun!Brinsmade! Post9Doc! Sonenshein! Tufts!University!School!of!Medicine! [email protected]!

Ilana!Brito! Post9Doc! Eric!Alm!Massachusetts!Institute!of!Technology! [email protected]!

Megan!Brophy! Graduate!Student! Nolan!Lab! Massachusetts!Institute!of!Technology! [email protected]!

Autumn!Brown! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Laura!Brown! Graduate!Student! Susan!T.!Lovett! Brandeis!University! ltbrown@!brandeis.edu!Kevin!Bu! Undergraduate!Student! Balskus!Lab! Harvard!University! [email protected]!Minh!Bui! Undergraduate!Student! Berkmen!lab! Suffolk!University! [email protected]!Briana!Burton! Faculty! Burton! Harvard!University! [email protected]!Julie!Button! Post9Doc! Dutton! Harvard!University! [email protected]!Matthew!Cabeen! Post9Doc! Losick! Harvard!University! [email protected]!Tiziana!Cafarelli! Graduate!Student! Godoy!Laboratory! Northeastern!University! [email protected]!

Stephane!Calmat! Post9Doc! Tania!Baker! Massachusetts!Institute!of!Technology!

[email protected]!

Jodi!Camberg! Faculty! Camberg! University!of!Rhode!Island! [email protected]!Ewen!Cameron! Post9Doc! James!Collins! Boston!University!/!HHMI! [email protected]!Amy!Camp! Faculty! Amy!Camp! Mount!Holyoke!College! [email protected]!Amber!Cantin! Research!Scientist! Novartis! Novartis! [email protected]!

Bo!Cao! Graduate!Student! Biological!Engineering!

Massachusetts!Institute!of!Technology! [email protected]!

Lia!Cardarelli! Post9Doc! Gibbs! Harvard!University! [email protected]!Rachel!Carmody! Post9Doc! Turnbaugh! Harvard!University! [email protected]!Brent!Cezairliyan! Post9Doc! Ausubel! Massachusetts!General!Hospital! [email protected]!Maryam!Chaib!De!Mares! Other! Pringle!Lab!OEB! Harvard!University! [email protected]!

Phoom!Chairatana! Graduate!Student! Nolan!lab! Massachusetts!Institute!of!Technology!

[email protected]!

Pete!Chandrangsu! Post9Doc! John!Helmann! Cornell!University! [email protected]!Seungwoo!Chang! Post9Doc! Joseph!Loparo! Harvard!Medical!School! [email protected]!Michael!Chao! Post9Doc! Waldor! Brigham!and!Women's!Hospital! [email protected]!Christine!Chapman! Research!Scientist! Wai9Leung!Ng! Tufts!University!School!of!Medicine! [email protected]!Claire!Chatelle! Graduate!Student! Berkmen!Lab! New!England!Biolabs! [email protected]!Chao!Chen! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Johnathan!Cheng! Post9Doc! Suzanne!Walker! Harvard!Medical!School! [email protected]!

Diana!Chien! Graduate!Student! Polz!Lab! Massachusetts!Institute!of!Technology!

[email protected]!

Peter!Chien! Faculty! Chien! University!of!Massachusetts,!Amherst! [email protected]!Scott!Chilton! Graduate!Student! Briana!Burton! Harvard!University! [email protected]!Carly!Ching! Graduate!Student! Veronica!Godoy! Northeastern!University! [email protected]!

Yok!Hian!Chionh! Graduate!Student!Singapore9MIT!Alliance!for!Research!and!Technology!

Massachusetts!Institute!of!Technology!

[email protected]!

Brandon!Chiou! Undergraduate!Student! Jean!Huang! Olin!College!of!Engineering! [email protected]!Hannah!Chmura! Undergraduate!Student! Berkmen! New!England!Biolabs! [email protected]!Philicia!Chow! Undergraduate!Student! Huang! Olin!College!of!Engineering! [email protected]!Lauren!Christopher! Research!Scientist! Eric!Rubin! Harvard!School!of!Public!Health! [email protected]!Hattie!Chung! Graduate!Student! Kishony!Lab! Harvard!Medical!School! [email protected]!

Sean!Clarke! Graduate!Student! Alm!Massachusetts!Institute!of!Technology! [email protected]!

Brian!Conlon! Post9Doc! Kim!Lewis!lab! Northeastern!University! [email protected]!Paige!Cote! Undergraduate!Student! Jean!Huang! Olin!College!of!Engineering! [email protected]!Carla!Cugini! Post9Doc! Lemon/Davey! The!Forsyth!Institute! [email protected]!Kurt!Dahlstrom! Graduate!Student! George!O'Toole! Dartmouth!Medical!School! [email protected]!Ankur!Dalia! Post9Doc! Andrew!Camilli!lab! Tufts!University!School!of!Medicine! [email protected]!

Olga!Danilchanka! Post9Doc! Microbiology!and!Immunobiology!


Manoshi!Datta! Graduate!Student! Jeff!Gore!Massachusetts!Institute!of!Technology! [email protected]!

Kim!Davis! Post9Doc! Ralph!Isberg! Tufts!University! [email protected]!


80

Name! Position! Lab! Institution! Email!Dennise!de!Jesus! Graduate!Student! Isberg!Lab! Tufts!University! [email protected]!

Rodolfo!De!la!Vega! Post9Doc!

Laboratory!for!Muskuloskeletal!research!and!Innovation!

Massachusetts!General!Hospital! [email protected]!

Sandra!Dedrick! Other! Tim!van!Opijnen!Lab! Boston!College! [email protected]!Alicia!DeFrancesco! Graduate!Student! Losick! Harvard!University! [email protected]!Patrick!Degnan! Post9Doc! Andrew!Goodman! Yale!University! [email protected]!Padraig!Deighan! Post9Doc! Hochschild! Harvard!Medical!School! [email protected]!Kaila!Deiorio9Haggar! Graduate!Student! Meyer!Lab! Boston!College! [email protected]!Elena!Del!Tordello! Post9Doc! John!Mekanalos! Harvard!Medical!School! [email protected]!

Nicholas!DeLateur! Graduate!Student! Beuning,!Ondrechen!labs! Northeastern!University! [email protected]!

Aaron!DeLoughery! Graduate!Student! losick! Harvard!University! [email protected]!Lynn!Deng! Faculty! DENG! Boston!University!School!of!Medicine! [email protected]!Marcus!Dillon! Graduate!Student! Dr.!Vaughn!Cooper! University!of!New!Hampshire! [email protected]!

Alexander!Dills! Faculty! Silby!Lab!University!of!Massachusetts,!Dartmouth! [email protected]!

Xiaoyuan!Ding! Research!Scientist! Harvard!Medical!School! Microbiotix! [email protected]!

Tobias!Doerr! Post9Doc! Waldor! Harvard!Medical!School! [email protected]!Jamie!Dombach! Graduate!Student! Gibson!lab! University!of!Massachusetts,!Boston! [email protected]!Gabrielle!Dowell! Undergraduate!Student! Dr.!Vaughn!Cooper! University!of!New!Hampshire! [email protected]!

Daniel!Dowling! Post9Doc! Drennan!Massachusetts!Institute!of!Technology! [email protected]!

Juan!Du! Post9Doc! Infectious!Disease!Dept.!


Keith!Eidell! Research!Scientist! Russell! Excelimmune! [email protected]!Crystal!Ellis! Post9Doc! Harris/!LaRocque!lab! Massachusetts!General!Hospital! [email protected]!

Antonia!Elsen! Undergraduate!Student! Student!Researcher! Franklin!W.!Olin!College!of!Engineering! [email protected]!

Alexander!Elsholz! Post9Doc! Losick! Harvard!University! [email protected]!

Helen!(Eleina)!England! Graduate!Student! Grossman! Massachusetts!Institute!of!Technology!

[email protected]!

Ye!Jin!Eun! Post9Doc! Ethan!Garner! Harvard!University! [email protected]!Jaymes!Farrell! Undergraduate!Student! Sandler!Lab! University!of!Massachusetts,!Amherst! [email protected]!Isabel!Fernandez!Escapa! Post9Doc! Katherine!Lemon!Lab! Harvard!Medical!School! [email protected]!Laura!Filkins! Graduate!Student! George!O'Toole! Geisel!Medical!School!at!Dartmouth! [email protected]!Kelly!Fimlaid! Graduate!Student! Graduate!Student! University!of!Vermont! [email protected]!Taryn!Fiorenza! Other! N/A! AdvanDx! [email protected]!Lauren!Fitch! Graduate!Student! Kim!Lewis! Northeastern!University! [email protected]!Laura!Fleck! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Kathleen!Fleming! Graduate!Student! O'Shea! Harvard!University! [email protected]!Eleanor!Fleming! Graduate!Student! Camilli!Lab! Tufts!University!School!of!Medicine! [email protected]!Kenneth!Flynn! Graduate!Student! Vaughn!Cooper!Lab! University!of!New!Hampshire! [email protected]!

Luciane!Fonseca! Graduate!Student! Grossman!Lab! Massachusetts!Institute!of!Technology! [email protected]!

Christopher!Ford! Post9Doc! Regev! The!Broad!Institute! [email protected]!Hannah!Foster! Graduate!Student! Burton! Harvard!University! [email protected]!Lucy!Foulston! Post9Doc! Losick! Harvard!University! foulston@fas,harvard.edu!Yang!Fu! Graduate!Student! Meyer!Lab! Boston!College! [email protected]!Matthew!Fullmer! Graduate!Student! JPeter!Gogarten! University!of!Connecticut!Storrs! [email protected]!

Michael!Funk! Graduate!Student! Drennan!Massachusetts!Institute!of!Technology! [email protected]!

Ellen!Fynan! Faculty! Fynan! Worcester!State!University! [email protected]!

Tantan!Gao! Graduate!Student! Harvard!University!Losick!Lab!

Harvard!University! [email protected]!

Claudette!Gardel! Faculty! Linc!Sonenshein! Tufts!University!School!of!Medicine! [email protected]!Kenneth!Gareau! Research!Scientist! Russell! Excelimmune,!Inc! [email protected]!Ethan!Garner! Faculty! Garner! Harvard!University! [email protected]!


81

Name! Position! Lab! Institution! Email!Anna!Garza9Mayers! Graduate!Student! Marcia!Goldberg! Harvard!Medical!School! [email protected]!Richa!Gawande! Graduate!Student! Fortune! Harvard!School!of!Public!Health! [email protected]!Eddie!Geisinger! Post9Doc! Ralph!Isberg! Tufts!University!School!of!Medicine! [email protected]!Matt!Gerding! Graduate!Student! Matt!Waldor! Harvard!Medical!School! [email protected]!

Sonali!Ghosh! Graduate!Student! Graduate!student! University!of!Connecticut!Health!Center! [email protected]!

Karine!Gibbs! Faculty! Gibbs!Lab! Harvard!University! [email protected]!Carew!Giberson9Chen! Undergraduate!Student! Kahne!Lab! Harvard!University! [email protected]!

Marcus!Gibson! Graduate!Student! Drennan!Lab! Massachusetts!Institute!of!Technology!

[email protected]!

Sarah!Gignoux9Wolfsohn! Graduate!Student! Vollmer!Lab! Northeastern!University! [email protected]!

Molly!Gilligan! Undergraduate!Student! Meyer!Lab! Boston!College! [email protected]!Michael!Gilmore! Faculty! Gilmore!Lab! Harvard!Medical!School! [email protected]!Veronica!Godoy9Carter! Graduate!Student! Godoy!Laboratory! Northeastern!University! [email protected]!

Antonio!Gomes! Graduate!Student!Bioinformatics!Program! Boston!University! [email protected]!

Andy!Goodman! Faculty! Goodman!lab! Yale!University! [email protected]!Daniel!Goodman! Graduate!Student! George!Church! Harvard!Medical!School! [email protected]!Erin!Green! Graduate!Student! Mecsas! Tufts!University!School!of!Medicine! [email protected]!Neil!Greene! Graduate!Student! Camilli! Tufts!University! [email protected]!Jennifer!Greenwich! Graduate!Student! Chai! Northeastern!University! [email protected]!Kevin!Griffith! Faculty! Griffith! University!of!Massachusetts,!Amherst! [email protected]!Daniel!Grubaugh! Graduate!Student! Higgins!Lab,!MBIB! Harvard!Medical!School! [email protected]!

Chen!Gu! Graduate!Student! Dedon!Lab!Massachusetts!Institute!of!Technology! [email protected]!

Marc!Guell! Post9Doc! Church!lab! Harvard!Medical!School! [email protected]!Allison!Guinta! Research!Scientist! Dr.!Losick! BASF! [email protected]!Adam!Hacking! Faculty! Hacking!Lab! Massachusetts!General!Hospital! [email protected]!Henry!J.!Haiser! Post9Doc! Turnbaugh!lab! Harvard!University! [email protected]!Elizabeth!Halvorsen! Post9Doc! Darren!Higgins! Harvard!Medical!School! [email protected]!Kook!Han! Post9Doc! Lory!Lab! Harvard!Medical!School! [email protected]!Feras!Hatahet! Post9Doc! Beckwith! Harvard!Medical!School! [email protected]!Stavroula!Hatzios! Post9Doc! Waldor!Lab! Brigham!and!Women's!Hospital! [email protected]!Lisa!Hawver! Graduate!Student! Dr.!Penny!Beuning! Northeastern!University! [email protected]!Jennifer!Hayashi! Graduate!Student! Morita!Lab! University!of!Massachusetts,!Amherst! [email protected]!

Guixin!(Susan)!He! Faculty! Clinical!Microbiology!Laboratory! University!of!Massachusetts!Lowell! [email protected]!

Matt!Heidtman! Research!Scientist! Glycosyn!LLC! Glycosyn!LLC! [email protected]!John!Helmann! Faculty! Helmann! Cornell!University! [email protected]!Andrew!Hempstead! Graduate!Student! Ralph!Isberg! Tufts!University!School!of!Medicine! [email protected]!Nelly!Henry! Research!Scientist! Kolter!Lab! Harvard!Medical!School! [email protected]!Gary!Heussler! Graduate!Student! George!O'Toole! Dartmouth!Medical!School! [email protected]!Alex!Histed! Graduate!Student! Dr.!Bree!Aldridge! Tufts!University! [email protected]!Linfeng!Huang! Post9Doc! Judy!Lieberman! Children's!Hospital!Boston! [email protected]!Jean!Huang! Faculty! Huang! Olin!College! [email protected]!Taylor!Hunt! Undergraduate!Student! Pellock!Lab! Providence!College! [email protected]!Edel!Hyland! Post9Doc! Murray! Harvard!University! [email protected]!Dervla!Isaac! Post9Doc! Ralph!Isberg! Tufts!University!School!of!Medicine! [email protected]!Vincent!Isabella! Post9Doc! Lewis!Lab! Northeastern!University! [email protected]!Ralph!Isberg! Faculty! Isberg!Lab! Tufts!University!School!of!Medicine! [email protected]!Ayman!Ismail! Post9Doc! Camilli!lab! Tufts!University! [email protected]!Joao!Jaapas! Other! molecular!lab! Boston!University!School!of!Medicine! [email protected]!Huang!Jack! Undergraduate!Student! Gilmore!Lab! Harvard!University! [email protected]!Jacquelin!Jackter! Undergraduate!Student! Amy!Camp! Mount!Holyoke!College! [email protected]!John!Janetzko! Graduate!Student! Walker! Harvard!Medical!School! [email protected]!Jessica!Jarett! Post9Doc! Michael!Lesser!Lab! University!of!New!Hampshire! [email protected]!Peng!Jiao! Research!Scientist! Process!development! Lonza!Biologics! [email protected]!


82

Name! Position! Lab! Institution! Email!

Christopher!Johnson! Post9Doc! Grossman!Massachusetts!Institute!of!Technology! [email protected]!

Kristina!Jonas! Post9Doc! Michael!Laub! Massachusetts!Institute!of!Technology! [email protected]!

Tonee!Jones! Undergraduate!Student! Rubin!Lab! Harvard!School!of!Public!Health! [email protected]!Sarah!Jung! Graduate!Student! ! Tufts!University!School!of!Medicine! [email protected]!Heather!Kamp! Post9Doc! Andrew!Camilli! Tufts!University!School!of!Medicine! [email protected]!Hong!Kang! Other! Huaiqiu!Zhu! Peking!University! [email protected]!James!Kath! Graduate!Student! Loparo!Lab! Harvard!Medical!School! [email protected]!Meghan!Keane! Undergraduate!Student! Pellock!Lab! Providence!College! [email protected]!Iris!Keren! Research!Scientist! Lewis!Lab! Northeastern!University! [email protected]!Augustus!Kesselly! Private!Sector! Discovery! Sanofi!Pasteur! [email protected]!Jemila!Kester! Graduate!Student! Fortune! Harvard!School!of!Public!Health! [email protected]!Karen!Kieser! Graduate!Student! Rubin!Lab! Harvard!School!of!Public!Health! [email protected]!Taehyun!Kim! Post9Doc! Lory!Lab! Harvard!Medical!School! [email protected]!Natalia!Kirienko! Post9Doc! Ausubel! Massachusetts!General!Hospital! [email protected]!Vanja!Klepac9Ceraj! Faculty! Klepac9Ceraj! Wellesley!College! [email protected]!Roberto!Kolter! Faculty! Kolter!lab! Harvard!Medical!School! [email protected]!Veronica!Kos! Post9Doc! Translational!Science! AstraZeneca! [email protected]!

Sriram!Kosuri! Graduate!Student! Drew!Endy!Massachusetts!Institute!of!Technology! [email protected]!

Kristin!Kotewicz! Graduate!Student! Isberg! Tufts!University! [email protected]!Sherry!Kuchma! Research!Scientist! George!O'Toole! Dartmouth!Medical!School! [email protected]!Annette!LaBauve! Graduate!Student! Wargo! University!of!Vermont! [email protected]!Cindy!Lai! Graduate!Student! John!Leong! Tufts!University!School!of!Medicine! [email protected]!Ghee!Chuan!Lai! Graduate!Student! Bernhardt,!Paulsson! Harvard!Medical!School! [email protected]!Hubert!Lam! Private!Sector! Discovery! Sanofi!Pasteur! [email protected]!Cristina!Landeta! Post9Doc! Beckwith!Lab! Harvard!Medical!School! [email protected]!Francois!Lebreton! Post9Doc! Gilmore!lab! Harvard!Medical!School! [email protected]!Soo!Young!Lee! Graduate!Student! Marcia!Goldberg! Harvard!Medical!School! [email protected]!Jeongwook!(John)!Lee! Post9Doc! Dr.!Collins! Boston!University! [email protected]!Henry!Lee! Post9Doc! George!Church! Harvard!Medical!School! [email protected]!Sara!Leiman! Graduate!Student! Losick! Harvard!University! [email protected]!Pushkar!Lele! Post9Doc! Berg!Lab! Harvard!University! [email protected]!Katherine!Lemon! Faculty! Lemon!Lab! Forsyth!and!Boston!Children's! [email protected]!Jessica!Leonard! Undergraduate!Student! Pellock!Lab! Providence!College! [email protected]!Maxime!Leroux! Post9Doc! Steven!Sandler! University!of!Massachusetts,!Amherst! [email protected]!Vivian!Leung! Undergraduate!Student! Rubin!Lab! Harvard!University! [email protected]!Kathryn!Levasseur! Graduate!Student! Dove!Lab! Children's!Hospital!Boston! [email protected]!Kim!Lewis! Faculty! Lewis!Lab! Northeastern!University! [email protected]!Zaoping!Li! Post9Doc! Marcia!Goldberg! Harvard!Medical!School! [email protected]!

Rubing!Liang! Post9Doc!Department!of!Organismal!and!Evolutionary!Biology!

Harvard!University! [email protected]!

Tami!Lieberman! Graduate!Student! Kishony!Lab! Harvard!Medical!School! [email protected]!Ida!Lin! Undergraduate!Student! Godoy!Lab! Northeastern!University! [email protected]!

Lorraine!Ling! Graduate!Student! Tania!A.!Baker! Massachusetts!Institute!of!Technology! [email protected]!

Kristin!Little! Graduate!Student! Gibbs!Lab! Harvard!University! [email protected]!Songnian!Liu! Graduate!Student! Wiatrowski! Clark!University! [email protected]!

Jarukit!Long! Post9Doc! Sassetti!University!of!Massachusetts!Worcester! [email protected]!

Christian!Lorenz! Post9Doc! Lory!Lab! Harvard!Medical!School! [email protected]!Richard!Losick! Faculty! Losick! Harvard!University! [email protected]!Rustin!Lovewell! Graduate!Student! Dr.!Brent!Berwin! Dartmouth!Medical!School! [email protected]!Yun!Luo! Post9Doc! George!O'Toole! Dartmouth!Medical!School! [email protected]!Chu9Yuan!Luo! Graduate!Student! Dr.!Morita! University!of!Massachusetts,!Amherst! [email protected]!An!Luong! Undergraduate!Student! Peter!Turnbaugh!Lab! Harvard!University! [email protected]!Ashley!MacGuire! Graduate!Student! Godoy!Lab! Northeastern!University! [email protected]!


83

Name! Position! Lab! Institution! Email!Subramony!Mahadevan! Research!Scientist! Kolter! Harvard!Medical!School! [email protected]!Mohlopheni!Jackson!Marakalala! Post9Doc! Rubin!Lab,! Harvard!School!of!Public!Health! [email protected]!

Jonathan!Marks! Undergraduate!Student! Balskus!Lab! Harvard!University! [email protected]!

Douglas!Marshall! Graduate!Student! Silby! University!of!Massachusetts!Dartmouth! [email protected]!

Eugenie!Martineau! Undergraduate!Student! Beckwith! Harvard!Medical!School! [email protected]!Revati!Masilamani! Post9Doc! Camilli!Lab! Tufts!University!School!of!Medicine! [email protected]!Sheba!Mathew! Undergraduate!Student! Waldor!Lab! Brigham!and!Women's!Hospital! [email protected]!

Corinne!Maurice! Post9Doc! FAS!Center!for!Systems!Biology! Harvard!University! [email protected]!

Nick!Mazzucca! Undergraduate!Student! Pellock!Lab! Providence!College! [email protected]!

John!McCoy! Private!Sector! Metabolic!Engineering!R&D!

[email protected]!

EmilyKate!McDonough! Graduate!Student! Camilli!Lab! Tufts!University! [email protected]!

Abigail!McGuire! Research!Scientist! Microbial!genome!analysis!group! Broad!Institute! [email protected]!

Brenley!McIntosh! Research!Scientist! Jim!Hu! Texas!A&M! [email protected]!Sarah!McLeod! Private!Sector! AstraZ! AstraZeneca! [email protected]!Jamie!Meadows! Graduate!Student! Matthew!Wargo! University!of!Vermont! [email protected]!Elizabeth!Mearls! Graduate!Student! Lynd,!Lee! Dartmouth!Medical!School! [email protected]!Violetta!Medik! Other! Research! Excelimmune! [email protected]!Brian!Meehan! Post9Doc! Beckwith! Harvard!Medical!School! [email protected]!Alexander!Meeske! Graduate!Student! Rudner! Harvard!Medical!School! [email protected]!

Xavier!Meniche! Post9Doc! Sassetti!lab! University!of!Massachusetts!Medical!School,!Worcester! [email protected]!

Massimo!Merighi! Private!Sector! Metabolic!Engineering!R&D!

[email protected]!

Sylvain!Meylan! Post9Doc! Collins! Boston!University!Biomedical!engineering! [email protected]!

Stephanie!Mitchell! Graduate!Student! Camilli! Tufts!University! [email protected]!Wolfram!Moebius! Post9Doc! Nelson!Lab! Harvard!University! [email protected]!Andrea!Moell! Post9Doc! Waldor! Harvard!Medical!School! [email protected]!Kimberly!Mohareb! Undergraduate!Student! Fynan! Worcester!State!University! [email protected]!Paula!Montero!Llopis! Post9Doc! David!Rudner!Lab! Harvard!Medical!School! [email protected]!

Diana!Morales! Post9Doc!Microbiology!and!Immunobiology!department!


Yasu!Morita! Faculty! Morita! University!of!Massachusetts,!Amherst! [email protected]!Georgeanna!Morton! Undergraduate!Student! Berkmen!Lab! Suffolk!University! [email protected]!

Felix!Moser! Graduate!Student! Voigt! Massachusetts!Institute!of!Technology! [email protected]!

Xiangyu!Mou! Graduate!Student! David!Nelson!Lab! University!of!Rhode!Island! [email protected]!Samir!Moussa! Post9Doc! Suzanne!Walker! Harvard!Medical!School! [email protected]!Melanie!Mueller! Post9Doc! Nelson! Harvard!University! [email protected]!Bianca!Mulaney! Undergraduate!Student! Kolter!Lab! Harvard!University! [email protected]!Matthew!Mulé! Undergraduate!Student! Camilli!Lab! Tufts!University!School!of!Medicine! [email protected]!Paul!Muller! Graduate!Student! Epstein! Northeastern!University! [email protected]!Julia!Murphy! Graduate!Student! Mecsas! Tufts!University!School!of!Medicine! [email protected]!Connor!Murphy! Undergraduate!Student! Isberg!Lab! Tufts!University! [email protected]!Laura!Murphy! Undergraduate!Student! Amy!Camp! Mount!Holyoke!College! [email protected]!

Kendall!Murray! Graduate!Student! Silby!Lab! University!of!Massachusetts,!Dartmouth!

[email protected]!

Anastasia!Murthy! Undergraduate!Student! Berkmen!lab! Suffolk!University! [email protected]!Pallavi!Murugkar! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Alissa!Myrick! Post9Doc! Rubin!Lab! Harvard!School!of!Public!Health! [email protected]!

Sidrotun!Naim! Post9Doc! Nibert!Lab! Surya!University,!Indonesia!and!University!of!Arizona,!Tucson! [email protected]!

Gorbachuk!Nataliya! Undergraduate!Student! Hung!Lab! Harvard!University! [email protected]!Dipti!Nayak! Graduate!Student! Marx! Harvard!University! [email protected]!


84


Asha!Nayar! Research!Scientist!Infection!BioScience,!AstraZeneca!Pharmaceuticals!

AstraZeneca!Pharmaceuticals! [email protected]!

Marta!Neto! Graduate!Student! Kolter!Lab! Harvard!Medical!School! [email protected]!Wai9Leung!Ng! Faculty! Ng! Tufts!University!School!of!Medicine! [email protected]!Peter!Nguyen! Post9Doc! Neel!S.!Joshi! Harvard!University! [email protected]!Ben!Niu! Post9Doc! Kolter!Lab! Harvard!Medical!School! [email protected]!Thomas!Norman! Graduate!Student! Losick! Harvard!University! [email protected]!Matthew!Norton! Graduate!Student! Godoy! Northeastern!University! [email protected]!Amy!Nwaobasi! Undergraduate!Student! Dr.!Steven!Sandler! University!of!Massachusetts,!Amherst! [email protected]!Jennifer!Oghene! Other! Waldor! Brigham!and!Women's!Hospital! [email protected]!Burak!Okumus! Post9Doc! Johan!Paulsson's!Lab! Harvard!Medical!School! [email protected]!

Maghnus!O'Seaghdha! Post9Doc! Wessels! Children's!Hospital!Boston! [email protected]!

Rajat!Kumar!Pal! Graduate!Student! Kevin!Griffith! University!of!Massachusetts,!Amherst! [email protected]!

Ting!Pang! Post9Doc! Rudner!and!Bernhardt! Harvard!Medical!School! [email protected]!

Kadamba!Papavinasasundaram!

Research!Scientist! Sassettilab! UMASS!Medical!School,!Worcester! [email protected]!

Lincoln!Pasquina! Graduate!Student! Suzanne!Walker! Harvard!Medical!School! [email protected]!Vaidehi!Patel! Graduate!Student! John!Helmann! Cornell!University! [email protected]!Sarju!Patel! Graduate!Student! Dr.!Arguello! Worcester!Polytechnic!Institute! [email protected]!

Norton!Peet! Private!Sector!Microbiotix!Chemistry!Department!

Microbiotix! [email protected]!

Shermin!Pei! Graduate!Student! Meyer!Lab! Boston!College! [email protected]!Brett!Pellock! Faculty! Pellock!Lab! Providence!College! [email protected]!

Shaaretha!Pelly! Post9Doc! Gyanu!Lamichhane! Johns!Hopkins!University!School!of!Medicine!

[email protected]!

Jon!Penterman! Post9Doc! Graham!Walker!Lab! Massachusetts!Institute!of!Technology! [email protected]!

Jan!Pero! Other! Myriant!Corporation! Myriant!Corporation! [email protected]!Kyle!Perry! Graduate!Student! Higgins!lab! Harvard!Medical!School! [email protected]!Rita!de!Cassia!Pessotti! Graduate!Student! Kolter!Lab! Harvard!Medical!School! [email protected]!Cara!Pina! Graduate!Student! Dr.!Susan!T.!Lovett! Brandeis!University! [email protected]!Jessica!Polka! Post9Doc! Pamela!Silver! Harvard!Medical!School! [email protected]!Laurent!Potvin9Trottier! Graduate!Student! Johan!Paulsson! Harvard!Medical!School! [email protected]!Michelle!Prew! Undergraduate!Student! Meyer!Lab! Boston!College! [email protected]!

Katherine!Price! Post9Doc! O'Toole! Geisel!School!of!Medicine!at!Dartmouth! [email protected]!

Justin!Pritchard! Post9Doc! Rubin!Lab! Harvard!School!of!Public!Health! [email protected]!Katherine!Prosen! Research!Scientist! H.!Russell! Excelimmune,!Inc.! [email protected]!

Oliver!Purcell! Post9Doc! Lu!lab!Massachusetts!Institute!of!Technology! [email protected]!

Yuan!Qiao! Graduate!Student! Walker! Harvard!Medical!School! [email protected]!Matheus!Quintana!Barreto! Undergraduate!Student! Brigham!Lab! University!of!Massachusetts!

Dartmouth! [email protected]!

Jennifer!Rabb! Post9Doc! Drennan!Massachusetts!Institute!of!Technology! [email protected]!

Mithila!Rajagopal! Graduate!Student! Walker! Harvard!Medical!School! [email protected]!Elisabeth!Raleigh! Research!Scientist! Raleigh! New!England!Biolabs! [email protected]!Vishal!Ramnarine! Research!Scientist! Microbiology! BASF!Corporation! [email protected]!Matthew!Ramsey! Post9Doc! Gilmore!Lab! Harvard!Medical!School! [email protected]!Aviram!Rasouly! Post9Doc! Lory!Lab! Harvard!Medical!School! [email protected]!Hesper!Rego! Post9Doc! Rubin! Harvard!School!of!Public!Health! [email protected]!Eammon!Riley! Research!Scientist! Rudner!Lab! Harvard!Medical!School! [email protected]!

Ardeshir!Rineh! Graduate!Student!Wellman!center!for!photomedicine! Massachusetts!General!Hospital! [email protected]!

Emily!Rittershaus! Graduate!Student! Sassetti! University!of!Massachusetts!Medical!School! [email protected]!


85

Name! Position! Lab! Institution! Email!Sara!Roggensack! Graduate!Student! John!Leong! Tufts!University! [email protected]!Amy!Rohlfing! Graduate!Student! Simon!Dove! Children's!Hospital!Boston! [email protected]!Robert!Rosenberg! Other! 9999999999! SMC! [email protected]!Chloe!Roussel!Rossin! Graduate!Student! Raleigh! New!England!Biolabs! [email protected]!Sarah!Rowe! Post9Doc! Lewis!lab! Northeastern!University! [email protected]!

Jacob!Rubens! Graduate!Student! Timothy!Lu! Massachusetts!Institute!of!Technology! [email protected]!

David!Rudner! Faculty! Rudner!Lab! Harvard!Medical!School! [email protected]!

Hugh!Russell! Private!Sector! Research!and!Development!

Excelimmune,!Inc! [email protected]!

Brandon!Russell! Graduate!Student! Dedon!Laboratory! Massachusetts!Institute!of!Technology! [email protected]!

Jon!Russell! Graduate!Student! Richard!Losick! Harvard!University! [email protected]!Christina!Saak! Graduate!Student! Gibbs!lab! Harvard!University! [email protected]!Tanisha!Saini! Post9Doc! Burton!Lab! Harvard!University! [email protected]!Buck!Samuel! Post9Doc! Gary!Ruvkun! Massachusetts!General!Hospital! [email protected]!Steven!Sandler! Faculty! Sandler! University!of!Massachusetts,!Amherst! [email protected]!John!Santa!Maria! Graduate!Student! Walker! Harvard!Medical!School! [email protected]!Marina!Santiago! Graduate!Student! Suzanne!Walker! Harvard!Medical!School! [email protected]!Karla!Schallies! Graduate!Student! Gibson!Lab! University!of!Massachusetts,!Boston! [email protected]!Kathrin!Schirner! Post9Doc! Suzanne!Walker!Lab! Harvard!Medical!School! [email protected]!Ruth!Schmidt! Graduate!Student! Paolina!Garbeva! NIOO9KNAW! [email protected]!Maia!Schoonmaker! Post9Doc! Lamichhane!Lab! Johns!Hopkins!School!of!Medicine! [email protected]!Kathrin!Schulz! Graduate!Student! Burton!Lab! Harvard!University! [email protected]!Brian!Schuster! Research!Scientist! Eric!Rubin! Harvard!School!of!Public!Health! [email protected]!Sarah!Seaton! Post9Doc! Stuart!Levy! Tufts!University!School!of!Medicine! [email protected]!Kim!Seed! Post9Doc! Camilli!Lab! Tufts!University!School!of!Medicine! [email protected]!Einat!Segev! Post9Doc! Kolter! Harvard!Medical!School! [email protected]!

Charlotte!Seid! Graduate!Student! Grossman!Massachusetts!Institute!of!Technology! [email protected]!

Benedicte!Sempe! Post9Doc! Infecious!Diseases9!Wessels!Lab!

Children's!Hospital!Boston! [email protected]!

Alecia!Septer! Post9Doc! Gibbs! Harvard!University! [email protected]!Mara!Shainheit! Post9Doc! Andrew!Camilli! Tufts!University!School!of!Medicine! [email protected]!Lok!To!Sham! Post9Doc! Thomas!G.!Bernhardt! Harvard!Medical!School! [email protected]!Rebecca!Shapiro! Post9Doc! James!Collins! Boston!University! [email protected]!Bijaya!Sharma! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Onkar!Sharma! Post9Doc! Wessels! Children's!Hospital!Boston! [email protected]!Scarlet!Shell! Post9Doc! Sarah!Fortune! Harvard!School!of!Public!Health! [email protected]!Aimee!Shen! Faculty! Shen!Lab! University!of!Vermont! [email protected]!Jenny!Shih! Undergraduate!Student! Verdine!Lab! Harvard!University! [email protected]!

Mark!Silby! Faculty! Silby! University!of!Massachusetts!Dartmouth!

[email protected]!

Naveen!Sinha! Graduate!Student! Brenner! Harvard!University! [email protected]!

Alexandra!Sirota9Madi! Post9Doc!Sackler!School!of!Physics!and!Astronomy!

Tel9Aviv!University,!Israel! [email protected]!

Viknesh!Sivanathan! Post9Doc! Hochschild! Harvard!Medical!School! [email protected]!Betty!Slinger! Graduate!Student! Michelle!Meyer! Boston!College! [email protected]!

Chris!Smillie! Graduate!Student! Eric!Alm! Massachusetts!Institute!of!Technology!

[email protected]!

Mark!Smith! Graduate!Student! Alm!Massachusetts!Institute!of!Technology! [email protected]!

Mark!Soo! Undergraduate!Student! Michelle!Meyer! Boston!College! [email protected]!Arpana!Sood! Graduate!Student! Mecsas/Isberg! Tufts!University!School!of!Medicine! [email protected]!Skye!Souter! Post9Doc! Lesser!Lab! Massachusetts!General!Hospital! [email protected]!William!Spears! Other! Lesser!lab! Massachusetts!General!Hospital! [email protected]!Rachel!Staples! Research!Scientist! Vaughn!Cooper! University!of!New!Hampshire! [email protected]!Bram!Sterling! Graduate!Student! Briana!Burton! Harvard!University! [email protected]!


86


Kerry9Ann!Stewart! Graduate!Student! Dr.!Peter!Setlow!University!of!Connecticut!Health!Center! [email protected]!

Philip!Strandwitz! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Kyle!Swerdlow! Undergraduate!Student! Berkmen!Lab! Suffolk!University! [email protected]!

Sujata!Syamal! Other! Hacking!Orthopaedic!Lab9MGH! Massachusetts!General!Hospital! [email protected]!

Olive!Tang! Undergraduate!Student! Rudner!Lab! Harvard!Medical!School! [email protected]!Farida!Tanko! Undergraduate!Student! Helmann!Lab! Cornell!University! [email protected]!Tommy!Tashjian! Research!Scientist! Microbiotix! Microbiotix! [email protected]!Elizabeth!Tenorio! Faculty! Linden!Hu! Tufts!University!School!of!Medicine! [email protected]!Alyssa!Theodore! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!

Jessie!Thompson! Graduate!Student! Chisholm! Massachusetts!Institute!of!Technology! [email protected]!

Murray!Tipping! Post9Doc! Karine!Gibbs! Harvard!University! [email protected]!

Amaris!Torres9Delgado! Graduate!Student! HHMI!Massachusetts!Institute!of!Technology! [email protected]!

Heather!Torrey! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Bjorn!Traag! Post9Doc! Losick! Harvard!University! [email protected]!Erin!Troy! Post9Doc! Linden!Hu! Tufts!Medical!Center! [email protected]!Peter!Turnbaugh! Faculty! Turnbaugh! Harvard!University! [email protected]!Michael!Valentino! Post9Doc! Gilmore!Lab! Harvard!Medical!School! [email protected]!Tim!van!Opijnen! Faculty! 9! Boston!College! [email protected]!Daria!Van!Tyne! Post9Doc! Gilmore!Lab! Harvard!Medical!School! [email protected]!

Nicole!Vega! Post9Doc! Jeff!Gore!Massachusetts!Institute!of!Technology! [email protected]!

Alexander!Vickers! Undergraduate!Student! Heather!Wiatrowski! Clark!University! [email protected]!Marissa!Viola! Graduate!Student! Camberg!Lab! University!of!Rhode!Island! [email protected]!Minh!Vong! Graduate!Student! David!Rudner! Harvard!University! [email protected]!Sarah!Wacker! Post9Doc! Losick!Lab! Harvard!University! [email protected]!Abraham!Waldman! Graduate!Student! Balskus!Lab! Harvard!University! [email protected]!

Benjamin!Waldman! Undergraduate!Student! Sandler!Lab!University!of!Connecticut!Health!Center! [email protected]!

Rebecca!Walsh! Graduate!Student! Camilli!Lab! Tufts!University!School!of!Medicine! [email protected]!Xindan!Wang! Post9Doc! David!Rudner! Harvard!Medical!School! [email protected]!Yingjiao!Wang! Graduate!Student! Wiatrowski!lab! Clark!University! [email protected]!Anna!Wang!Erickson! Graduate!Student! Richard!Losick! Harvard!University! [email protected]!Larissa!Wenren! Research!Fellow! Karine!Gibbs! Harvard!University! [email protected]!Michelle!Wheeler! Undergraduate!Student! Fynan! Worcester!State!University! [email protected]!Kathrin!Witt! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Carl!Wivagg! Post9Doc! Ethan!Garner! Harvard!University! [email protected]!Benjamin!Wolfe! Post9Doc! Dutton!(Rachel)!Lab! Harvard!University! [email protected]!Michael!Wollenberg! Post9Doc! Lemon!Lab! The!Forsyth!Institute! [email protected]!

Laurel!Wright! Graduate!Student! Alan!Grossman!Massachusetts!Institute!of!Technology! [email protected]!

Bo!Wu! Other! Production! New!England!Biolabs! [email protected]!Yoshiharu!Yamaichi! Post9Doc! Waldor! Brigham!and!Women's!Hospital! [email protected]!

Lei!Yang! Post9Doc! Voigt!lab! Massachusetts!Institute!of!Technology!

[email protected]!

May!Yang! Undergraduate!Student! Nibert!Lab! Harvard!University! [email protected]!Minmin!Yen! Graduate!Student! Camilli! Tufts!University! [email protected]!Vivian!Yeong! Undergraduate!Student! Silver!Lab! Harvard!University! [email protected]!Yeva!Yue!Shan! Graduate!Student! Lewis!Lab! Northeastern!University! [email protected]!Grace!Yuen! Graduate!Student! Ausubel!Lab! Massachusetts!General!Hospital! [email protected]!Martha!Zepeda! Graduate!Student! Gibbs! Harvard!University! [email protected]!Yanjia!Zhang! Graduate!Student! Eric!Rubin! Harvard!School!of!Public!Health! [email protected]!Wenjing!Zhao! Graduate!Student! Nelson! University!of!Rhode!Island! [email protected]!Heng!Zhao! Graduate!Student! John!Helmann!Lab! Cornell!University! [email protected]!

Huanzhen!Zhu! Undergraduate!Student! Jean!J.!Huang!Franklin!W.!Olin!College!of!Engineering! [email protected]!

!!

!!!

Organizing!Committee!!

Matt!Cabeen,!Harvard!University!Edel!Hyland,!Harvard!University!Lucy!Foulston!Harvard!University!Sara!Leiman,!Harvard!University!Kristin!Little,!Harvard!University!

Thomas!Norman,!Harvard!University!Cara!Boutte,!Harvard!School!of!Public!Health!

Neil!Greene,!Tufts!Medical!School!Stephanie!Mitchell,!Tufts!Medical!School!

Julia!Murphy,!Tufts!Medical!School!Erin!Troy,!Tufts!Medical!School!

Alyssa!Theodore,!Northeastern!University!Kathrin!Witt,!Northeastern!University!

Mike!Mee,!Boston!University!Charlotte!Seid,!MIT!

!!

!Faculty!Sponsors!

!Roberto!Kolter,!Harvard!Medical!School!

Richard!Losick,!Harvard!University!!John!Mekalanos,!Harvard!Medical!School!!

Andrew!Wright,!Tufts!University!School!of!Medicine!!!!!

Financial!Coordinator!!

Massa!Kandakai!MCB!Financial!Office!

Bbm book 2013 final version with cover full bleed

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