the PRIMER - DOE Joint Genome...

Few stop to consider the consequencesof their daily ablutions, the washing ofclothes, the watering of lawns, and theflush of a toilet. However, wastewatertreatment—one of the cornerstones ofmodern civilization—is

inside this issue2. Finishers Convene in NM

Spot Awards 3. Termites in Costa Rica 4. Profile: Erika Lindquist5. Plant Pathogens Decoded

OPA Recipients6. Young Investigator Winner8. Spotlight on Safety9. Hazards of Being a Microbiologist10. All About Webfeeds11. Eukaryotic Finishing at Stanford12. Symbiotic Tree Fungus17. New CSP Targets19. Pichia stipitis20. Aspergillus niger

PRIMERthe

October 2006 Volume 3 Issue 2

First Tree Genome Is Published:Poplar Holds Promise as RenewableBioenergy ResourceWood from a common tree may one dayfigure prominently in meeting trans-portation fuel needs, according toscientists whose research on the fast-growing poplar tree is featured on thecover of the September 15, 2006,edition of the journal Science (vol. 313,No. 5793).

The article, highlighting the analysisof the first complete DNA sequence ofa tree, the black cottonwood, orPopulus trichocarpa, lays groundworkthat may lead to the development of

trees as an ideal “feedstock” for a newgeneration of biofuels such as cellulosicethanol. The research is the result of afour-year scientific and technical effort,led by the U.S. Department of Energy’sJoint Genome Institute (DOE JGI) andOak Ridge National Laboratory (ORNL),and including the University of BritishColumbia; Genome Canada; UmeåUniversity, Sweden; Ghent University,Belgium; and 30 other institutions fromaround the world,

“Biofuels could (cont. on page 6)

No Guts, No Worries—Wonder Worm Enlists Full-Service Microbes forTransportation, Energy, & Waste ManagementResearchers have now characterizedthe unique lifestyle of a gutless wormthat commutes through marine sedi-ments powered by a community ofsymbiotic microbial specialists harboredjust under its skin, obviating the needfor digestive and excretory systems.

Using DNA sequencing and otherdiagnostic techniques, scientists fromthe U.S. Department of Energy’s JointGenome Institute (DOE JGI) havedescribed this complex worm/microbecollaboration in a species of marineoligochaete worm isolated off of thecoast of Elba, the Mediterranean islandof Napoleon’s exile. Their results arepublished in the September 17 editionof the journal Nature (http://www.nature.com/nature/journal/vaop/ncur-

rent/abs/nature05192.html).The worm, Olavius algarvensis, has

no mouth to take in food, but does notgo hungry, thanks to the goodwill of itshardworking bacterial tenants. In thetransaction, the worm shuttles thebacteria to optimal energy sources itencounters wending its way between theupper oxygen-rich and the lower oxygen-depleted coastal sediments. Inexchange, fixed carbon, all requiredamino acids and vitamins are synthe-sized by the subcuticular communitiesof microbial symbionts, providing theirhost with ample nutrition.

On the other end of the digestiveequation, such waste products asammonium and urea, generated by theworm’s metabolism, (cont. on page 14)

(cont. on page 15)

Better Sludge ThroughMetagenomics—Researchers Seek toMaster WastewaterTreatment Failures

2007 JGI Users MeetingMarch 28-30, 2007

Walnut Creek Marriott

For more information,contact Marsha Fenner

[email protected], 925-296-5781

Hold the Date

October 2006 Vol. 3, Issue 2

2 / THE PRIMER

BY REBECCA E. MCINTOSH

The tedious, sometimes frustrating jobof finishing was given well-deservedpraise at the recent “Finishing in theFuture” workshop, when keynotespeaker Julian Parkhill of the SangerInstitute in Cambridge, England,explained exactly why finishing is soimportant: Even though shotgunsequence can yield about 99.9 percentaccuracy, tiny changes such as singlebase knockouts can have drasticphenotypic effects on an organism. Forexample, Y. pestis, the causative agentof plague, does not have a flagellum,but its genome includes the blueprintsfor one. A single-point mutation hasbeen found to inactivate the regulatorfor the entire flagellum biosynthesismechanism, leaving the organism non-motile.

In the past, finishing has been atopic of discussion at many sequencingmeetings and workshops, but Finishingin the Future, held on May 4–5 in SantaFe, New Mexico, was the first workshopsolely dedicated to finishing accom-plishments and techniques. It washosted by the Los Alamos NationalLaboratory (LANL) and was organized byChris Detter (LANL), David Bruce(LANL), Patrick Chain (LLNL), Cliff Han(LANL), Alla Lapidus (DOE JGI), andJeremy Schmutz (Stanford HGC).

Julian Parkhill’s keynote addressgave an overview of the value offinishing as well as some recent accom-plishments in the field. Representativesfrom many of the major sequencingcenters talked about their own finishingprotocols and techniques andcompared notes on which are moresuccessful and on problems finisherstend to face. Participants also attendeda poster session and numerous roundtable discussions on such topics as

technology development, computationalfinishing methods, and laboratoryfinishing methods.

As the essence of finishing is toproblem-solve or troubleshoot throughsections of sequence that were notinitially sequenced correctly, many of thetalks focused on protocols for specificproblems (i.e., hard GC stops and highlyrepetitive sequences). Many partici-pants found it helpful that the work-shops allowed so much time forinformation sharing. Alla Lapidus notedthat participants requested protocolsfrom DOE JGI, and that JGI alsorequested a few from TIGR and Baylor.

Finishing in the Future also allowedparticipants to explore broader issues,such as the difficulty of transformingfinishing into a high-throughput format.

Members of the Sanger Institutedemonstrated their automation of thefinishing process using robotics, butshowed that completely eliminating thehuman component and making theprocess cost-efficient still are majorhurdles. However, Cliff Han explainedthat the future of finishing will need toincorporate automation in processcontrol and data analysis, and in wetlab protocols, to meet the needs of thescientific community. For instance,understanding metagenomes willrequire finishing enormous amounts ofsequence that would stretch currentfinishing labs to the max. Moreover,future dreams of finishing largegenomes and even eukaryotes willdepend on finding ways to efficientlytake advantage of the skill of techni-cians as well as the speed of robots.

If you would like to learn more aboutthe Finishing in the Future meeting visitthe website http://finishingfuture.lanl.gov/index.html or, to get involved with nextyear’s event, contact Chris Detter,[email protected].

Finishers Convene in Santa Fe

Samuel Pitluck - For submitting DOEJGI’s DNA sequence data toGenBank, following through on theever-changing requirements for datasubmission.

Steve Wilson - For developing theSafety Track system, which hasvastly improved JGI’s ability to trackand learn from safety incidents.

Wendell Hom - For his excellent workin completing the Octopus proposalin record time.

Ron McKeever - For his outstandingperformance during the FY 06 JGIproperty audit.

Duncan Scott - For exceeding expec-tations in the development of a newinoc list planning and reportingsystem, yielding a major positiveimpact on the JGI production line.

Jason Baumohl - For greatlyexceeding customer expectations inthe development of a new platingprocess management system.

The Finishing Team - For their effortsin establishing the DOE JointGenome Institute as a leader inmicrobial genome finishing—accounting for nearly a quarter of allfinished microbial genomes world-wide. Pictured are Alex Copeland,Steve Lowry, Eugene Goltsman, PaulRichardson, Ben Horowitz VasanthSingan, and Alla Lapidus. Pat Kaleand Stephan Trong are not pictured.

SPOT AwardRecipients

number of other fascinatingtermite species were discov-ered, usually by JaredLeadbetter, a lead investi-gator from Caltech, whoseems to have a sixthsense when it comes tofinding termites. A dark-purple ground-dwellingtermite was uncovered atthe base of a plant andappears to feed on verydecayed and blackened leaflitter. This could be ofinterest for bioenergyproduction, since thehindgut community may beadapted to degrading lignin,tannins, and humic acids notdegraded by other termitegenera such as Nasutitermes. Thepurple termite proved to be a veryaggressive species, and the soldiershave large, powerful jaws and a longunicorn-like “nasute” from whichterpenes are fired at enemies. A secondNasutitermes species with a jet blackhead was found to have built a coveredpathway up the trunk of a tree thatextended for over 50 feet, presumablyleading to their nest (the tree was too

In late May, members of the DOE JGIMicrobial Ecology Program headedsouth for a second metagenomicsampling trip to Costa Rica. As before,the subject of the expedition was thehumble termite and the explorers werenot disappointed. The rainy season wasjust beginning in Costa Rica and therewas a good chance that the field tripswould be rained out. However, theweather gods smiled upon the expedi-tion and thousands of termites weresuccessfully collected and brought backto the remote lab for processing.

A key target for the sampling tripwas the same genus collected lastyear, Nasutitermes, in which hundredsof wood-hydrolyzing enzymes were iden-tified. This time around, a different partof the hindgut was targeted—the alka-line P1 section—in the hope that asimilar number of enzymes would bediscovered that were optimized for highpH. These enzymes would be veryuseful in industrial cellulosic ethanolproduction where an alkaline pretreat-ment is used. In addition, samples ofthe termite hindguts were taken forinvestigating viruses and gene expres-sion (mRNA), and for microscopy.

In addition to the target species, a


THE PRIMER / 3

tall to spot the nest). The pathway wasso established that moss had grownover it, providing the termites with asecure and discrete channel forforaging in the undergrowth below.

The termite project is a collabora-tion between DOE JGI, Caltech, the SanDiego-based biotechnology companyDiversa, and INBIO, the NationalBiodiversity Institute of Costa Rica.

Small Game Hunting inCosta Rica

Falk Warnecke (foreground) and Eric Matson, post-docfrom collaborator Jared Leadbetter's Caltech lab, securethe termite materials under the watchful eye of PhilHugenholtz. (Photo by Hector Garcia Martin)

Termites in hand. A termite nest.


4 / THE PRIMER

Sometimes, a career path isn’t astraight line. Sometimes it requiressearching and trying many things untilyou discover what’s right for you. In thecase of Erika Lindquist, her career as ascientist was just that—a journey.Erika’s journey included jumping out ofairplanes, creating and testing detona-tors, fighting in a war, and spendingendless hours in a scientific laboratorybefore she decided to focus andbecome a scientist. At present, Erica isthe point person for DOE JGI’s ESTand cDNA projects. All of her adven-tures and experiences made her abetter scientist by providing her withpatience, persistence, and a differentperspective.

Erika Lindquist was destined to bean engineer. Her father was a fusionengineer and project manager at LLNLwho exposed her to math and scienceat an early age. He taught her the basicmethods for problem solving andencouraged her to learn from hermistakes. It was easy for a child whohad a naturally inquisitive mind andloved to solve problems. In fact, Erikaexcelled in math and science, and herfavorite subject at San Ramon HighSchool was physics. It was no surpriseto her family when Erika decided tostudy Electrical Engineering at SantaClara University.

Away from the watchful eye of herfamily and the sheltered life in Alamo,California, she found that the Universityhad many interesting distractions. Thefirst was ROTC. She was excited to takean elective class in rappelling. Theclass was sponsored by ROTC and shewas invited to an advanced ROTCtraining course held at Fort Knox,Kentucky, where she learned marks-

manship with an M16 and M60,survival techniques, grenade launching,orienteering, and leadership skills.Prepared for anything when shereturned to school in the fall, she wasoffered and accepted an ROTCscholarship for the remaining two yearsof school.

After graduating from Santa Clara,Erika completed her ROTC training byattending jump school and officertraining school. She was commissionedas a Lieutenant in the Army NationalGuard and assigned to a Military Policeunit in Pittsburg. She also accepted aposition as an electrical explosive engi-neer at Reynolds Industry Systems.Reynolds created precision secondaryexplosives for highly specific and reli-able uses such as airbag releasecartridges and mining. Her primary taskwas to design firing systems and testexplosive detonators. It was an inter-esting and challenging job in the begin-ning, but the work was in awell-developed field that required littleproblem solving.

Motivated to discover a new career,Erika decided to go back to school tostudy environmental engineering atHumboldt State University. Part of thepreparatory coursework required her totake biology, bacteriology, and genetics.“I found these courses fascinating andloved the hands-on lab work.” She thendecided to pursue a graduate degree inMicrobiology at Berkeley. However,before she was able to attend, DesertStorm began and Erika’s unit wasdeployed to Saudi Arabia.

She didn’t think about the serious-ness of the situation until her unit tookoff from Travis AFB in a C-130 filled withHumvees. “I’ll always remember the

scene, sitting on the plane looking atthe somber faces of my colleagues—some of whom were crying.” Until thatmoment, her career in the military wasmore like a hobby. One weekend amonth she was allowed to play withguns and drive Humvees. As an officer,Erika was required to play a leadershiprole in her unit. “I learned that therewere many types of leaders: the loud,fearless Patton type and the not so in-your-face, quiet and patient type.”

Erika’s MP unit was responsible formanaging POWs, and it followed thetroops advancing into Iraq. “Outside ofthe Republican Guard units, the Iraqiinfantry were not well-trained, nor caredfor, therefore many soldiers gave uphoping that the Americans would clotheand feed them.” Her unit spent sixmonths caring for the Iraqi soldiers intemporary POW camps in the desert.

Erika returned to school with arenewed focus. She

Erika Lindquist—Point PersonD O E J G I F A C E S

(cont. on page 16)


THE PRIMER / 5

Bill CannanSenior Recruiter, HR

Cindy ChoiResearch Associate, Genome Technologies

Mansi ChovatiaSenior Research Technician, Production

Julianna ChowResearch Associate, Genomic Tech.

Alicia ClumResearch Associate, Genome Tech.

Nicholas EattockBiosciences Tech 1, Production

Rob EganSoftware Developer 3, Informatics

Gerald IlogResearch Tech, Production

Megan KennedyBiosciences Tech 1, Production

Duane KubischtaSystems Analyst 3, Production

Kathleen LailSequencing Supervisor, Production

Michael LeePrincipal Safety Technician, EH&S

Aiyesha MaStudent Assistant, Quality Assurance Group

Gladys McCayStudent Assistant BBEI, Genome Tech.

Yanan MengResearch Tech, Production

Bonnie MillenbaughAdministrative Specialist IV, Operations

Mary Ann PedrazaSenior Research Associate, Genome Tech.

Michael PintorResearch Tech, Production

Nicole ShapiroSenior Research Technician, Production

Gurjot “Jay” SinghResearch Technician, Production

Hui SunSenior Research Associate, Genome Tech.

Zalak TrivediSenior Research Technician, Production

Mario VelazquezResearch Technician, Production

Chris WongPorduction Manager

Andy YuenBiosciences Technician, Production

Michael ZhangCSE Trainee, Informatics

Tao ZhangSenior Research Associate,Genetic Analysis Programs

JGI NewEmployees

By comparing the complete genomesequences of two plant-killingpathogens, Phytophthora ramorum andPhytophthora sojae, and related organ-isms, researchers from DOE JGI, withthe Virginia Bioinformatics Institute(VBI) and others, have uncovered crucialaspects of the disease-causing mecha-nisms of sudden oak death (SOD) andsoybean root rot disease. Results of afour-year, $4 million multi-agency projectsupported by DOE, the US Departmentof Agriculture (USDA), and the NationalScience Foundation (NSF), appear in theSept 1, 2006, edition of Science (vol.313, No. 5791).

“What is extraordinary about thePhytophthora genomes is that almosthalf of the genes contained in themshow signs of rapid adaptation. Wespeculate that the rapidly changinggenes are being driven to evolve by

pressure from the defense systems ofthe pathogens' host plants,” said VBIProfessor Brett Tyler, the project's coor-dinator and lead author of the Sciencepaper. “The unprecedented level ofgenetic flexibility in these organismsgives us insights into how thesepathogens have become successful. Atthe same time, it has helped us identifyweak points in the organisms that canbe targeted to control them.”

DOE JGI and VBIDecode Plant-Killing Pathogens

Harris Shapiro - This OPA recognizesyour outstanding leadership andcommitment in ensuring the JGIsequencing pipeline continues assem-bling genomes.Simon Minovitsky - In recognition ofyour successful development of theVISTA Enhancer database.Genome Assembly Team: SergeDusheyko and Jasmyn Pangilinan - ThisOPA recognizes the extraordinary effortsof Serge Dusheko and Jasmyn Pangilianin keeping the Sequencing Pipelineoperational and ensuring critical JGIsequencing goals were met for 2005.Venonat LIMS Team: Dana Alcivare,Patrick Hajek, Aaron Porter, JasonBaumohl, Arkady Voloshin - This OPArecognizes the team’s innovation and

dedication for migrating the VenonatLIMS to a modern platform, defying allconventional wisdom that it would beimpossible. Their work ensures a stablefuture for this critical information system.GeneLib Team : Erika Lindquist and EdKirton - This OPA recognizes theoutstanding accomplishments of Dr.Erika Lindquist and Ed Kirton for devel-oping and implementing the “GeneLib”program, an essential tool forsequencing and the R&D programs.

To nominate your colleagues forsuch honors, see:• LBNL SPOTs/OPAs - http://www.jgi-

psf.org/HR/awards.html• LLNL Awards - http://www.jgi-

psf.org/HR/forms_templates/llnl/BIOAwardsGuidelines2006.pdf

OPA Recipients

Typical spores formed by the vegetativebody, or hyphae, of P. ramorum. MatteoGarbelotto Lab - UC Berkeley.


6 / THE PRIMER

provide a majoranswer to ourenergy needs bygiving the UnitedStates a home-grown, environ-mentally friendlieralternative toimported oil,” saidDOE’s UnderSecretary forScience, Dr.Raymond L.Orbach. “Fine-tuning plants forbiofuels produc-tion is one of thekeys to making biofuels economicallyviable and cost-effective. This research,employing the latest genomic technolo-gies, is an important step on the roadto developing practical, biologically-based substitutes for gasoline andother fossil fuels.”

“Biofuels are not only attractive for

Poplar Holds Promise as RenewableBioenergy Resourcecont. from page 1

their potential to cut reliance on oilimports but also their reduced environ-mental impact,” said Dr. Gerald A.Tuskan, ORNL and DOE JGI researcherand lead author of the Science study.“Biofuels emit fewer pollutants thanfossil fuels such as gasoline. In addi-tion, poplar and

At the 11th International Symposium on Microbial Ecology in Vienna, August 20-25, the International Society for Microbial Ecology (ISME) presented The YoungInvestigator Award to DOE JGI’s own Phil Hugenholtz. The award (a glass plaquewith an embedded solid gold coin) recognizes scientists who have made signifi-cant contributions to microbial ecology. In his acceptance remarks, Phil thankedthe Society for its timely selection because “…next year, I’m officially a middle-aged investigator.” He went on to say that he was indebted to his past andpresent collaborators “…because research, much like a microbial community, isall about teamwork.”

Phil Hugenholtz—ISME YoungInvestigator Award Winner

(cont. on next page)

DOE JGI staff and guests gathered on September 14 to celebrate thepublication of the first tree genome in the journal Science by plantinga black cottonwood, or poplar, of the same type sequenced, Populustrichocarpa. At planting, the sapling measured 50.5 inches. ThePrimer will continue to monitor its growth.

Former Secretary of State GeorgeShultz with DOE JGI SequencingDepartment Head Susan Lucas (left)and Computational Genomics ProgramHead Dan Rokhsar (middle). NowThomas W. and Susan B. FordDistinguished Fellow at the HooverInstitution, Mr. Shultz visited DOE JGIin April, along with Sidney Drell,senior fellow and professor of theoret-ical physics (emeritus) at theStanford Linear Accelerator Center(SLAC), and Lucy Shapiro and HarleyMcAdams from the StanfordUniversity Medical Center.

George ShultzVisits PGF


THE PRIMER / 7

related plants are vital managers ofatmospheric carbon. Trees storecaptured carbon dioxide in their leaves,branches, stems, and roots. Thisnatural process provides opportunitiesto improve carbon removal from the airby producing trees that effectivelyshuttle and store more carbon belowground in their roots and the soil.Moreover, bioenergy crops re-absorbcarbon dioxide emitted when biofuelsare consumed, creating a cycle that isessentially carbon neutral.”

Poplar’s extraordinarily rapidgrowth—it can grow a dozen feet ayear—and its relatively compactgenome size of 480 million nucleotideunits—40 times smaller than thegenome of pine—are among the manyfeatures that led researchers to targetpoplar as a model crop for biofuelsproduction.

Among its major discoveries, thepoplar project identified over 45,000protein-coding genes—more than anyother organism sequenced to date andabout double those in the human

Chris Wong joined the PGF staff in July. He got his start in science in an unenvi-able position in Genentech’s Process Development Group where he conductedmedia screening for fermentation. Genentech farmed out Chris as a resource toIDEC and Immunex before he moved on to Cell Genesys, where he helped buildout a facility from 5,000 to 100,000 square feet, and a staff of four to 60. Hiswife Deanna, a tax attorney, does forecast flow planning for The Gap. They havetwo kids, Brian (four and a half) and Abigail (10 months).

Chris Wong—DOE JGIProduction Manager

genome (which is six times larger thanthe poplar’s). The research team identi-fied 93 genes associated with theproduction of cellulose, hemicelluloseand lignin, the building blocks of plantcell walls. The biopolymers celluloseand hemicellulose are the most abun-dant organic materials on earth. Oncebroken down into sugars by enzymaticaction, they can be fermented anddistilled as fuel-quality ethanol andother liquid fuels.

Poplar is the most complex genometo be sequenced and assembled by asingle public sequencing facility, andonly the third plant to have its genomecompletely sequenced and published.The first two were a tiny weed,Arabidopsis thaliana, and rice. Native tothe Pacific coast from San Diego toAlaska, Populus trichocarpa is one ofthe tallest broadleaf hardwood trees inthe western US. The sequenced DNAwas isolated from a specimen collectedalong the banks of the Nisqually River inWashington State.

The poplar project supports a

broader DOE drive, under the BushAdministration’s Advanced EnergyInitiative, to accelerate research intobiofuels production. In August, DOEannounced it would spend $250 millionover five years to establish and operatetwo new bioenergy research centers.The DOE-supported research focuseson both plants and microbes in seekingnew biotechnology-based, cost-effectivemethods of producing fuels from plantmatter (biomass).

DOE scientists see a future in whichvast poplar farms in the PacificNorthwest, upper Midwest, and portionsof the southeastern U.S. provide asteady supply of cellulose-rich treebiomass which specialized biorefinerieswould transform into fuels like ethanol.Other regions might specialize indifferent “energy crops,” such asswitchgrass and willow, suited to theirclimates and soil conditions. A largequantity of biofuel might also beproduced from agricultural and forestrywaste.


8 / THE PRIMER

SPOTLIGHT ON SAFETY—ERGONOMICS

Ergonomics is about getting a goodmatch between the work we do andwhat our bodies can handle. Whenthere’s a mismatch, it can lead toaches and pains, inefficiencies, andeven to serious injuries if not promptlyaddressed. Unfortunately, ergonomics-related problems are now the mostcommon type of injury at DOE JGI, andfor this reason, improving ergonomicshas become a major focus here.

A major step that DOE JGI hastaken to address ergonomic injurieswas to obtain the services of UCSFsenior ergonomics consultant IraJanowitz, who now holds regular officehours at the PGF to provide advice andassistance on ergonomics issues. Ira’srole is to support the existing programat DOE JGI and add analysis, training,and advice as needed. According toJanowitz, “Good ergonomics is notbased on forcing employees to followrules, but is a problem-solving processbest carried out with the involvement ofthose who actually do the work.”Everyone’s involvement is critical inaddressing ergonomic issues.

In addition, Ira is working with theDOE JGI EH&S staff to arrange for thecreation of an ergonomic equipmentdemonstration room, which will besupplied with several different computermice, keyboards, and other equipmentto make the time employees spend atthe computer a little bit more comfort-able. The selection of these devicesand chairs will combine existing LBNLand LLNL standards with recommenda-tions by Janowitz, and will giveemployees the opportunity to test theseout in one centralized location. EH&Shopes to have several different types ofchairs that employees will be able tobring to their work area and evaluate onsite, thereby reducing the lag time

currently experienced through the LBNLergonomic chair loaner program. Thisdemonstration room will be located inBuilding 400, room 405. Some exam-ples of ergonomic equipment that arecurrently available for trial are:

• Goldtouch Key Ovation keyboard

• Microsoft Natural ErgonomicKeyboard 4000

• Logitech Marble mouse

• Evoluent Vertical mouse (left andright handed)

• Humanscale Whale mouseRemember, we can reduce the risk

of ergonomics-related injuries throughactivities such as taking the time toadjust our workstation and frequent“stretch for recovery” breaks that takeus out of our working posture. If youhave any pain or discomfort, it’s impor-tant that you report it early to Ira or theDOE JGI EH&S staff (Michael Lee orStephen Franaszek) and have yourcurrent workstation ergonomically evalu-ated. Ira is easily contacted [email protected] or 925-351-1142, and ergonomic evaluations canbe directly requested through the LBNLERGO Database website:https://isswprod.lbl.gov/Ergo/Login.asp.

Ergonomically-correct Nicole Shapiro

Safety LessonLearned

Recently, a DOE JGI employee sustaineda chemical burn while returning a bottleof phenol to a chemical storagecabinet. The container cap was looseand the solution spilled onto his arm.We should all learn from this incident.Ensure that the following precautionsare taken while handling corrosives:• Store them below shoulder level

(about 4 feet) in an appropriatelocation, away from incompatiblechemicals. Liquid corrosives shouldhave secondary containment.

• When finished using them, tightenthe cap and immediately store thecontainer.

• Use appropriate personal protectiveequipment (PPE) while handlingcorrosives (as a minimum, tightfitting chemical goggles, lab coat,gloves and closed-toe shoes).

• Before handling corrosives, get thenecessary training and know thehazards of these materials. MaterialSafety Data Sheets (MSDS) aregood info sources.

• Know the location of the nearestemergency eyewash/shower andwhat to do if exposed to corrosivechemicals.Contact DOE JGI Safety Officer

Stephen Franaszek([email protected]; (925)296-5807)or ES&H Tech Michael Lee([email protected]; (925)296-5649) if youhave questions or need assistance.

Phenol


THE PRIMER / 9

THE HAZARDS OF BEING A MICROBIOLOGISTBY BRUCE BALFOUR

When the average person hears thatyou’re a microbiologist, they usuallypicture someone in a lab coat withthick glasses who never leaves thelaboratory. However, microbial samplesdon’t collect themselves, and environ-mental samples have to be collectedfrom…the environment. While toiletbowls and kitchen sinks provide fruitfulenvironments for many lines ofresearch, many biologists are notcontent to while away their hoursslaving over a hot thermal cycler or arecalcitrant centrifuge. The adventurousresearcher leaves his lab to lead a lifeon the edge, traveling to exotic loca-tions in microbe-filled aircraft cabins,drinking bottled water in foreign coun-tries, delivering lectures in front ofpeople who don’t speak the samelanguage, and going to extreme meas-ures to collect extremophiles. Even ifyou don’t wear a biocontainment suit towork, sample collection can behazardous.Hot Enough for You?What’s black and red and white allover? That’s right, a biologist collectingmicrobial samples from a hot spring inYellowstone National Park. It’s an idyllicsetting, high in the mountains under aclear blue sky, with birds chirping andsmall animals watching from the forest,and boiling thermal pools with the odorof rotten eggs that clears your sinusesbetter than nasal spray. The smell, ofcourse, is the result of anaerobicbacteria converting some of thedissolved sulfur in the water tohydrogen sulfide. David Mead ofLucigen braved the bad smells andboiling waters to collect bacterialsamples for metagenomic analysis atthe DOE Joint Genome Institute (DOE

JGI), and he reports losing severaltoenails in the process. On anothersample collection trip in the back-country of Yellowstone, Phil Hugenholtzof DOE JGI recalls: “The park rangerswarned us that if buffalo charged us,not to try to outrun them. They wouldwin. The trick was to get under a tree,where you were less likely to get killed.”It Smells Like VictorySpeaking of bad smells, Phil Hugenholtzwas also drawn to the glamorous envi-ronment of wastewater treatment plantsso that he could study the microbialpopulation structure in sludge. Whilehanging from catwalks over sewagetanks to collect sludge samples inAustralia and Wisconsin, he recallsbeing impressed by the strong immune

systems of the people who work atwastewater treatment facilities. Thecold that he got after his collection tripwas probably just a coincidence.Attacked by TermitesTermites are like little hydrogen facto-ries, where wood goes in one end andhydrogen comes out the other. Termitesaccomplish this task by working withthe 200 different species of microbesthat inhabit their guts, where theenzymes produce hydrogen as abyproduct of the fermentation process.However, to study the microbes insidethe termite, you have to catch themwhere they live—in the dense jungles ofCosta Rica. Falk Warnecke from DOEJGI, and Jared Leadbetter from theCalifornia Institute of (cont. on page 18)

Top left: Kamchatka boiling pot hits 97C--boiling at that altitude. Top right: David Meadcollects samples at a hot spring. By the end of the day the acid from the hot spring haddissolved the knees of his pants. Bottom left: The bears were ambivalent about sharingtheir favorite watering hole. Bottom right: Dr. Gennadi Karpov, deputy director of theInstitute of Volcanology and Seismology in Petropavlovsk-Kamchatsky, local connection tothe many volcanoes on the Kamchatka peninsula.


10 / THE PRIMER

Webfeeds: What Are They?

BY RENÉ PERRIER

The Internet canbe overwhelming.First, there’s thechallenge offinding goodcontent, and thenthere’s the chal-lenge of keepingup with (andsorting through)

the never-ending flood of new content.The same is true (on a smaller scale)for our intranet.

Search engines such as Googlehelp address the first challenge (seearticle in The Primer, Volume 3, Issue 1),but a new kind of online publishing,webfeeds, makes it simple to keeptrack of the latest content published byyour favorite web sites.What is a Webfeed?

In a nutshell, a webfeed (also calledRSS feed) is a way for web sites tocontinuously “feed” you announce-ments of their latest content, with linksto each new content item.

To instantly learn what’s new on aweb site, just check its feed. Whenyou’re trying to follow developments at

dozens or hundreds of sites, your timesavings can be enormous.

The DOE JGI internal web site(http://www.jgi-psf.org/) updates itscontent infrequently. By using this site’swebfeed, you automatically get notifiedwhen a page has been added orupdated.How to Get Started

To read webfeeds, you need a soft-ware tool called an aggregator (alsocalled a feed reader). Your aggregatormaintains a list of your favoritewebfeeds, checks them regularly forupdates, and displays updated contents(or a summary of their contents). Themain categories of aggregators are:Online

An online aggregator is a web siteservice selling or providing free aggrega-tion services. Big Internet companieslike Google (Google Reader) and Yahoo!(My Yahoo!) provide such services, aswell as companies specializing only inwebfeeds, like Bloglines or Newsgator.Online aggregators are often describedas personalized newspapers since theyallow their users to organize their newsfeeds on a single page.

The advantage of an online aggre-gator is that it is available from

anywhere (work, home, cyber cafe). Thedisadvantage is that it will not provideaccess to feeds that are located on anintranet (like the JGI internal web site,the JGI blog server, or the JGI wiki).Standalone

Standalone aggregators run on asingle computer to perform one task:tracking and displaying webfeeds.Unless you need a tool that is special-ized in specific types of feeds likepodcasts or vodcasts (aka videopod-casts), this is not the best option.

Integrated SupportSome web browsers and e-mail

applications provide integrated supportfor webfeeds either by default (likeSafari or Thunderbird—the e-mail appli-cation from Mozilla, which is alsoapproved by the LBNL IT division—or byadding some extension (like Wizz RSSor Sage for Firefox). These allowreceiving feeds from an intranet and arenicely integrated with applications thatwe already use. In fact, when usingThunderbird, the integration is so welldone the news feeds look like e-mails.Local (PGF) users can find more infoabout webfeeds at: http://wiki.jgi-psf.org/cgi-bin/twiki/view/Main/WhatAreWebFeeds.

Softball Season Roundup:G-Nomes Grab Second Place!The G-Nomes finished in 7th place but managed to make it tothe championship game for the second year in a row. This year’steam members were (back row) Jason Baumohl, Steve Wilson,Jarrod Chapman, Harris Shapiro, Chris Hack, David Pletcher,Victor Hepa, (front row) Katrin Brand, Kecia Duffy-Wei, JenniferKuehl, Beth Lacerna, Susan Lucas, Khela Weiler. Not pictured:Jeremy Brand, Steve Paltiel, Brian Simison, Mark Taylor, DanielleMihalkanin, Simona Necula, Elena Shapiro, Kristen Taylor.

René Perrier


THE PRIMER / 11

Eukaryotic Finishing & Whole-GenomeImprovement at Stanford

BY JEREMY SCHMUTZ

I’m often asked, “Why are yousequencing the X genome?” Thesedays, the X often refers to obscuresquiggly creatures that most Americansdon’t even know exist. This is not howit has always been at the StanfordHuman Genome Center (SHGC). Whenwe began working with DOE JGI on thefinishing and assembly of human chro-mosomes 5,16, and 19 in 1999, theanswer to “Why are you sequencing thehuman genome?” was straightforward. Iwas even able to explain to my grand-mother the potential benefits in beingable to screen for, diagnose, and treatgenetically inherited diseases. For ourcurrent projects, however, it is difficultto explain that Nectria haematococca isa fungus that has potential bioremedia-tion uses as it degrades lignins andhydrocarbon chains, or that

Ostreococcus lucimarinus is a marinealgae, a phytoplankton, that plays asignificant role in global carbon-fixationin the ocean. When I propose theseanswers, they inevitably lead to adiscussion (rather one-sided) where Iexplain the global carbon cycle, or usingliving organisms to reclaim and cleanhuman pollutants from soil, or how it ispossible to derive at least some of ourenergy needs from growing plants.Needless to say, eventually people loseinterest as I try to explain “what we aredoing at the Center these days.”

What we are actually doing thesedays is working on whole-genomeshotgun assemblies (WGSA) after thedraft sequencing stage. We still finishclones that are shotgun sequenced atthe DOE JGI PGF just like in the days ofthe human genome, but most of oureffort is focused on evaluating WGSAand developing strategies for improving

the quality of the eukaryotic genomicsequences produced by DOE JGI. At thistime, it is particularly important toproduce useful genomic sequences thatmeet the needs of their respectiveresearch communities because themajority of their exposure to genomicsequence has been with the finishedhuman, near-complete mouse, orfinished bacterial genomes. What thismeans is that the average user of aDOE JGI genome has become accus-tomed to carrying out functional assaysfrom the genomic sequence alone,without having to further refine theunderlying genomic sequence. Theyalso expect to be able to identify all ofthe coding regions in the genome andto have genes that are co-localized on achromosome appear together in thegenomic sequence.

At the SHGC, we endeavor toaccomplish four goals (cont. on page 18)

Group photo: Left to Right; Back row: Jane Grimwood, Michael Young, Kevin Wu, Min Kim, Dave Flowers, James Retterer, Yee Man Chan.Middle Row: Jennifer Chan, Eva Bajorek, Hanz Olonan, Maria Gomez, Ataya Sathirachinda, Catherine Eastman, Xia Liu, Jeremy Schmutz.Front Row: Chenier Caoile, Mirian Denys, Lucia Ramirez, Shelia Hilario, Joan Yang, Ming Tsai, Nu Vo. Unpictured: Richard Myers, StaceyBlack, Dea Fotopulos, Sally Fuess, Liz Jackson, Jui Mei Lee, Lajinia Miller, Jennifer Okamoto.


12 / THE PRIMER

DOE JGI Sequences, Releases Genome ofSymbiotic Tree Fungus The DNA sequence of Laccaria bicolor,a fungus that forms a beneficialsymbiosis with trees and inhabits oneof the most ecologically and commer-cially important microbial niches inNorth American and Eurasian forests,has been determined by DOE JGI. Thecomplete Laccaria genome sequencewas announced July 23 at the FifthInternational Conference on Mycorrhizain Granada, Spain, by an internationalconsortium composed of DOE JGI, OakRidge National Laboratory (ORNL),France’s National Institute forAgricultural Research (INRA), theUniversity of Alabama in Huntsville(UAH), Ghent University in Belgium, andadditional groups in Germany, Sweden,and France.

“The Laccaria genome sequencewill provide the global research commu-nity with a critical resource to developfaster-growing trees for producing morebiomass that can be converted to fuels,and for trees capable of capturing morecarbon from the atmosphere,” said DOEJGI Director Eddy Rubin.

“The woody tissues of trees act asone of the world’s most importantterrestrial sinks for CO2, making treesan important stabilizer of carbon in theearth’s environment,” said FrancisMartin, INRA’s Laccaria project leader.“The steady rise of global atmosphericCO2 concentrations suggests that weare on the trajectory for serious environ-mental problems. This situation couldbe eased by modeling and activelymanaging the complex relationshipsbetween trees and fungi,” said Martin.

Key factors behind the ability oftrees to generate large amounts ofbiomass, or store carbon, reside in the

way that they interact withsoil microbes known asmycorrhizal fungi, whichexcel at procuring neces-sary, but scarce, nutrientssuch as phosphate andnitrogen. When Laccariabicolor partners with plantroots, a mycorrhizal root iscreated, resulting in amutualistic relationshipthat significantly benefitsboth organisms. Thefungus within the root isprotected from competitionwith other soil microbesand gains preferentialaccess to carbohydrateswithin the plant.

Such mycorrhizae arecritical to terrestrialecosystems, Martin said,since some 85 percent ofall plant species, includingtrees, depend on suchinteractions to thrive.Mycorrhizae significantlyimprove photosyntheticcarbon assimilation byplants and are estimated to fix morephosphate and nitrogen than the world’schemical fertilizer industry produces.

“The study and management ofsuch relationships holds immensepotential for the agriculture, forestry,and horticulture industries, as well asfar-reaching implications for landmanagement policies and the impact ofglobal climate change on plants,” saidGopi Podila of UAH.

“This unique research opportunityenables us to advance the under-standing of how functional genomics of

this symbiosis enhances biomassproduction and carbon management,particularly through the interaction withthe poplar tree, also sequenced by DOEJGI,” Rubin said. “We can now harnessthe interaction between these speciesand identify the factors involved inbiomass production by characterizingthe changes that occur between the twogenomes as the tree and the funguscollaborate to generate biomass. It alsohelps us to understand the interactionbetween these two symbionts within thecontext of the changing global climate.”

Fruiting bodies of the symbiotic fungus Laccaria bicolorassociated to seedlings of Douglas fir. Photo © INRA


THE PRIMER / 13

Los Alamos Finishes Landmark 50th Genome

BY KATHERINE HARRINGTON

As DOE’s Joint Genome Institute (DOEJGI) celebrated finishing its 100thgenome last month, the DOE JGI LANLteam celebrated the fact that 50 ofthose 100 genomes were finished inLos Alamos.

The 50th genome, finished in July,was that of the microorganismPolaromonas naphthalenivorans, whichis known for its ability to degrade naph-thalene, a carcinogen commonly foundat DOE's energy production sites.Genetic information about Polaromonasnaphthalenivorans will help researcherswho are seeking insight into how naph-thalene-degrading microbes could beused to manage contaminated ecolog-ical and industrial environments as wellas to understand how organismssurvive in highly contaminated environ-ments.

There have been 354 microbialgenomes sequenced worldwide sincescientists finished the first microbialgenome in 1996. The finishing processinvolves filling gaps that remain afterthe draft sequencing phase, whichoccurs at the Production GenomicsFacility (PGF) in Walnut Creek,California. The genomes that DOE JGIselects to finish are selected on thebasis of their potential to advanceresearch in the Department of Energy’scarbon cycling, bioremediation, andbioenergy mission areas.

Since 2003, when DOE JGI LANLfirst began finishing sequences, thetechnology and chemistry techniqueshave evolved significantly, resulting inhigher-quality data and faster produc-tion rates. When the project began, the

team was only able to produce onegenome a year—now they are able tofinish about one a week. The DOE JGILANL team also developed a databasethat has improved the quality of thedata and the efficiency with which theyare able to finish genomes.

As the team at DOE JGI LANLcontinues to increase efficiency andautomation in sequencing simple micro-bial genomes, such as bacteria, theywill be able to tackle more complexgenomes, including yeast and otherfungi. “That’s what is on the horizon forus” said David Sims of the GenomicSequencing and Computational BiologyGroup at DOE JGI LANL.

Of the 50 finished, some of theorganisms Los Alamos has sequencedare used in bioremediation to clean upoil spills or other such pollutants fromthe environment. For instance,Pseudoalteromonas atlantica has beenstudied off the coasts of California, NewJersey and Florida, and has been shownto absorb twenty to forty percent oftrace metal lead in ocean environments.Having the finished sequence ofP. atlantica, researchers will be able tobetter understand the microbe’sfunction in controlling toxic metalconcentrations.

Microbes might also help carboncycling researchers better understandthe fate of greenhouse gases and mayhelp them develop techniques forreducing the buildup of these gases inthe atmosphere.

One microbe, Sphingopyxisalaskensis, is found in ogliotrophic(nutrient deficient) waters near Alaska,the North Sea, and the North Pacific. Itis able to survive in these nutrient-defi-cient waters because of its ability tofeed on atmospheric carbon.Researchers are interested in studyingthese types of microbes in order topredict how various ecosystems mightrespond to nutrient deficiencies causedby global warming.

Microbes useful in bioenergy workare attractive for their ability to convertorganic matter into renewable energy.Syntrophomonas wolfei Gottingen, whichrecently finished at Los Alamos,produces hydrogen and methane asbyproducts of its metabolism. Both ofthese materials might be burned asclean fuels or used in fuel cells. Bystudying these microbes, scientists alsohave begun developing technology toimitate their processes to create renew-able, less expensive energy sources.

Trichoderma reeseiPhoto: Irma Salovuori, VTT Biotechnology

Polaromonas naphthalenivorans


14 / THE PRIMER

Mighty microbes, thriving in even theworld’s most extreme environments,have learned over billions of years toperform myriad biological functions.Those lessons are among the secretsencoded in their DNA sequence, andthis information can be applied tonational needs for clean renewablesources of energy and the repair ofdamaged environments.

On May 23, at the largest gatheringof microbiologists in the world, the106th general meeting of the AmericanSociety for Microbiology (ASM) inOrlando, Florida, ASM President StanleyMaloy announced that the scientificcommunity has embarked on the thirdgolden age of microbiology, one thatrequires a blend of old and newapproaches—including DNA sequencing.

DOE JGI Finishes 100th Microbial Genome,Tapping the Power of the Unseen World

Maloy highlighted the most recentmilestone achieved by the Departmentof Energy Joint Genome Institute (DOEJGI): its finishing 100 microbialgenomes—a diverse portfolio of thevast and mostly uncharacterized micro-scopic world.

“DNA sequencing is mining a partic-ularly productive vein in expanding thefrontier of microbiology. Especiallywhere, through conventional culturemethods, we are unable to shed light onthe metabolic profiles of these microor-ganisms and their environmental impli-

are taken up by these symbionts—notonly aiding the host in the removal ofthese toxic waste products, but

Olavius algarvensiscont. from page 1

also conserving valuablenitrogen, further main-taining the microbialcommunity.

“It’s an excellentexample of outsourcingenergy and wastemanagement, where thisworm and the microbesliving under its skin areenjoying a mutually bene-ficial relationship,” saidEddy Rubin, DOE JGIDirector. The work wasconducted by postdoc-toral fellow Tanja Woykeand colleagues from the

Rubin lab at DOE JGI and LawrenceBerkeley National Laboratory, andcollaborators led by Nicole Dubilier,

Head of the Symbiosis Research Groupin the Department of Molecular Ecologyat the Max Planck Institute for MarineMicrobiology in Bremen, Germany.

“The microbes, floating around inthe sea, strike up a bargain with theworm—in exchange for housing, themicrobes take care of energy productionand handling the waste,” Rubin said.

The team uncovered the uniquemethod of waste managementemployed by Olavius algarvensis bymetagenomics, a strategy pioneered byDOE JGI and its collaborators. Thistechnique entails isolating, sequencing,and characterizing DNA extracteddirectly from environmental samples toobtain a profile of the microbial commu-nity residing in a particular environment.This is the first (cont. on next page)

cations, DNA sequencing provides us awelcome set of tools.”

DOE JGI has sequenced over 380organisms, more than any other institu-tion in the world. As microbes range insize from typically five million to tens ofmillions of letters of code, severalmicrobes could be sequenced in oneday at DOE JGI’s Production GenomicsFacility (PGF). However, the sequencingprocess is an iterative one, requiringsix- to eight-times coverage of eachgenome. The term, “finished,” refers tothe rigorous standard of accuracyestablished for the Human GenomeProject—tolerating no more than onemistake in 50,000 letters of geneticcode without gaps.

The DOE JGI 100 list can be foundat http://genome.jgi-psf.org/microbial.

Olavius algarvensis

Methanosarcina barkeriFusaro, a methanogenicArchaebacterium. FromKevin Sowers, Universityof Maryland Biotech-nology Institute (UMBI)

of the study and post-doctoral fellowwith Phil Hugenholtz in the DOE JGI’sMicrobial Ecology Program. “With thisinformation now available, there areopportunities to bioengineer theprocess to make it more reliable.”

Removing phosphorus is best doneby an environmentally friendly processknown as enhanced biological phos-phorus removal (EBPR). Theresearchers obtained a nearly completegenetic blueprint for a key player in thisprocess, the bacterial speciesAccumulibacter phosphatis.

Activated-sludge

(http://www.nature.com/nbt/journal/vaop/ncurrent/abs/nbt1247.html).

The metagenomic strategyentails generating DNA sequence infor-mation directly from samples of sewagesludge to provide a blueprint of thegenes and hence the metabolic possi-bilities of the wastewater environment.

“This is a first step in a muchbroader strategy employing a systemsbiology approach to the study of micro-bial communities with the goal ofdesigning predictive models to under-stand how these communities function,”said Hector Garcia Martin, lead author


THE PRIMER / 15

instance of such a symbiotic relation-ship being analyzed by using a metage-nomic shotgun sequencing approach,heralding a renaissance in symbiosisresearch.

In this experiment, Woyke donnedscuba equipment and dove into theMediterranean to sift through tons ofsediment to uncover enough of theworms and their microbial contents,which, like the vast preponderance ofmicrobial life, cannot be grown in thelaboratory.

What they found particularly unusualabout this worm/bacteria relationshipwas that several different microbescame into play, unlike most symbioticinteractions between larger organismsand microbes where usually only onemicrobial species is present.

For the study in question, a total of204 million bases, or units of geneticcode, were generated. Through aprocess known as “binning,” a protocoldeveloped by collaborating author FrankOliver Glöckner from the Max PlankInstitute, these sequences were then

organized by specific DNA signatures toreveal the identities of particular micro-bial species.

“By sequencing the genomes of themicrobes we were able to discern howthey cope with the needs of the wormand why such a diversity of microbes iswarranted,” Rubin said. It turns out thatthe worm isn’t just housing for themicrobes, but is also transportation.The worm burrows into the sedimentand as it heads for different environ-ments, each with different chemicalconstituents, provides energy adaptedto the capabilities of a particular groupof microbes. As the worm doesn’t havea mouth, the microbes use chemicalenergy from the sediment, perfusingthrough the skin, to convert organicmaterial into the stuff that nourishesthe worm.

“It’s not unlike a car with a hybridmotor that can run on both electricityand gas depending on the situation,”Rubin said. “In certain places the wormis powered by specific bacteria that canexploit the chemical energy abundant at

Olavius algarvensiscont. from page 14

the largest microbially-mediatedbiotechnology process on the planet.When it works, it is a microbialsymphony in tune with humanity. Whenit fails, the consequences can be dire.DOE JGI researchers and collaboratorsat the University of Wisconsin-Madisonand the Advanced WastewaterManagement Centre, University ofQueensland, Australia, have publishedthe first metagenomic study of an acti-vated sludge wastewater treatmentprocess. The research appeared onlinein the September 24 edition of thejournal Nature Biotechnology (cont. on next page)

Better Sludgecont. from page 1

a specific location, while in other strata,where a different chemical energysource is abundant, the worm switchesits energy production to residentbacteria that can exploit that availableenergy source.”

The work was conducted under theauspices of the DOE JGI’s CommunitySequencing Program (CSP).

Tanja Woyke, lead author on theO. algavensis Nature paper.


16 / THE PRIMER

applied to graduate school at Berkeleyand accepted a position with RichardStephens, whose lab focused onChlamydia, the world’s leading cause ofpreventable blindness. She emergedfrom Berkeley as a newly-minted PhDscientist, and knew she wanted to focuson bioinformatics. She started herscientific career working on gene anno-tation at Celera Genomics, and latermoved to Incyte Genomics as a projectleader for splice-variant editing andannotation. Following that, she set upthe Biowatch Program in the Bay Area,which was sponsored by theDepartment of Homeland Security andmanaged by the Centers for DiseaseControl. The program was to implementa system designed by LLNL to monitorfor and respond to airborne biological

pathogens on a daily basis. “Although Ithoroughly enjoyed working in biode-fense, when Dee Catino, DOE JGIhuman resources head, called regardingan open position at JGI, I jumped at thechance to interview.”

Erika is currently the cDNA and ESTgroup lead, working for Paul Richardson.The position is responsible for allcomponents of cDNA projects, includingdefinition of the project, discussionswith the collaborator, sequencing, andpublication of the data in GenBank. Partof this process is informatics, which iswhere Harris Shapiro’s group comes in.

“I love working at JGI. I believe inour mission and what we do. In partic-ular, coming from industry wheregenomics companies sold access toinformation, JGI provides free access to

Erika Lindquist at her post during OperationDesert Storm.

Lindquistcont. from page 4

wastewater treat-ment is usedthroughout theworld to purify tril-lions of gallons ofsewage annually.Many treatmentplants usespecializedbacteria toremove phos-phorus, to protectlakes and riversfrom eutrophication,a deterioration of water quality charac-terized by excessive algae blooms.Accumulibacter play a vital role inwastewater management, accumulatingmassive amounts of phosphorus insidetheir cells.

“Engineers and microbiologists havebeen trying for 35 years to grow this

all researchers. Collaborators consis-tently communicate how appreciativethey are and how important the JGI-provided information is to theirresearch. I’m proud to be a contributingpart of the JGI team, and every day Ifeel honored to be working here!”

Erika and her partner Mayumi live ina floating home in Alameda, acrossfrom Jack London Square.

microbe, with no success,” said TrinaMcMahon, Assistant Professor,Department of Civil and EnvironmentalEngineering, University of Wisconsin-Madison, and one of the study’sauthors. “Remarkably, through metage-nomic techniques, we were able toisolate and acquire the genomesequence of Accumulibacter phosphatiswithout a pure culture of the organism,which, like most microbes, eludes labo-ratory culture. We expect that clueshidden in the genome will lead todomestication of this mysteriousorganism, enabling further studies of itshabits and lifestyle.

“The genome sequence will alsoenable biologists and engineers tounderstand why and how these organ-isms accumulate phosphorus, and itwill lead to major advances in opti-mizing and controlling the EBPR waste-

water treatment process,” McMahonsaid. “In particular, it makes possiblefurther research into why some waste-water treatment plants occasionally fail.These failures often result in seriouspollution of lakes, rivers, and estu-aries.”

When things go wrong, the environ-ment can be inundated with untreatedphosphorous, carbon, and nitrogen,necessitating costly and environmen-tally taxing remedies and exposing thepublic to potential disease hazards. Thescale is daunting—more than 31 billiongallons of wastewater are treated dailyin the U.S. alone. Even a marginalimprovement in the process wouldtranslate into huge savings and spellrelief for environmental engineers.

The work was conducted under theauspices of the DOE JGI’s CommunitySequencing Program (CSP).

Hector Garcia Martin

Better Sludgecont. from page 15


THE PRIMER / 17

mental benefits that are associ-ated with its cultivation,” saidChris Somerville, Professor ofBiological Sciences at StanfordUniversity and Director of theCarnegie Institution’sDepartment of Plant Biology. “Ienvision that switchgrass willbe an important feedstock for theemerging lignocellulose-to-ethanolindustry. An enhanced understanding ofgene structure and diversity at themolecular level may lead to newapproaches to enhance both biomassproductivity and feedstock quality forbioenergy production.”

In complement to switchgrass, DOEJGI will be sequencing Brachypodiumdistachyon, a temperate grass modelsystem with a simple genome moreamenable to sequencing. This choiceresponds to the urgent need for devel-oping grasses into superior energycrops while improving grain crops andforage grasses for food production.Brachypodium will be undertaken via atwo-pronged strategy: the first, a whole-genome shotgun sequencing approach,is a collaboration between John Vogel atthe USDA laboratory, colleagues at theJohn Innes Centre in England, and theUniversity of Minnesota; and thesecond, an expressed gene sequencingeffort, is led by Todd Mockler and JeffChang at Oregon State University, withTodd Michael of The Salk Institute forBiological Studies, and Samuel Hazenfrom The Scripps Research Institute.

Another major CSP project is theselection of cassava (Manihotesculenta), an excellent energy sourceand food for approximately one billionpeople around the planet. Its rootscontain 20 to 40 percent starch, from

Energy-Rich Portfolio ofNew Sequencing TargetsBioenergy crop plants switchgrass andcassava, other important agriculturalcommodities such as cotton, andmicrobes geared to break down plantmaterial to render biofuels, round outthe roster of more than 40 projects tobe tackled by DOE JGI over the nextyear. Drawing submissions from DOEJGI’s more than 400-strong usercommunity, the genomes of theseorganisms will be sequenced and char-acterized as part of the CommunitySequencing Program (CSP).

The CSP selections echo recom-mendations outlined in the “Breakingthe Biological Barriers to CellulosicEthanol” report issued by DOE on July7 (http://www.doe.gov/news/3804.htm).

In his 2006 State of the UnionAddress, President George W. Bushspecifically cited the promise of switch-grass as a bioenergy crop. A tall peren-nial grass, and a dominant species ofthe North American prairie, switchgrass(Panicum virgatum) is particularlycompelling because of its relatively lowproduction costs, minimal nutrient andpesticide requirements, perennialgrowth habit, and its ability to adapt toa broad range of growing conditions.The net energy gain for ethanol produc-tion from switchgrass is exceptionallyfavorable, coupled with low greenhousegas emissions. The switchgrass project,which entails sequencing the gene tran-scripts, or Expressed Sequence Tags(ESTs) of the plant, is led by ChristianTobias and researchers at the U.S.Department of Agriculture WesternRegional Research Center in Albany,California, and Gautam Sarath at theUniversity of Nebraska, Lincoln.

“Switchgrass has enormous poten-tial as an energy crop, and environ-

which ethanol can be derived, making itan attractive and strategic source ofrenewable energy. Cassava grows indiverse environments, from extremelydry to humid climates, acidic to alkalinesoils, from sea level to high altitudes,and in nutrient-poor soil.

The cassava project will extendbroad benefits to its vast researchcommunity, including a better under-standing of starch and protein biosyn-thesis, root storage, and stresscontrols. It will also enable cropimprovements for related plants,including the rubber tree and castorbean.

The cassava project is led by ClaudeM. Fauquet, Director of the InternationalLaboratory for Tropical AgriculturalBiotechnology and colleagues at theDanforth Plant Science Center in St.Louis, and includes contributions fromthe USDA laboratory in Fargo, NorthDakota; Washington University St.Louis; University of Chicago; TheInstitute for Genomic Research (TIGR);Missouri Botanical Garden; the BroadInstitute; Ohio State University; theInternational Center for TropicalAgriculture (CIAT) in Cali, Colombia; andthe Smithsonian Institution.

For a full list of the CSP 2007sequencing projects, see:http://www.jgi.doe.gov/sequencing/cspseqplans2007.html.

A farmer tends his cassava field.


18 / THE PRIMER

Technology, traveled over 3,000 milesand hiked through lush rainforests toaccost the local insects, but the tableswere turned when the insects accostedthem. Seeking Nasutitermes corniger, aspecial “higher” wood-feeding termitedevoid of protozoa that would compli-cate DNA samples, the researchershunted for likely volunteers among the

local insect population. The basketball-sized termite nests were easy to find,and so were the angry occupants—upto a million termites in a single nest.Nasute termite soldiers have nozzle-shaped heads that squirt a clear stickyfluid intended to immobilize the threatto the colony. Warnecke was neverimmobilized when he hacked into a nest

with a DOE JGI genome. The first is toincrease the long-range contiguity (scaf-fold contiguity). Ideally, we would likeevery eukaryotic genome sequence toaccurately reflect the number andorganization of chromosomes that theactual creature of interest contains.However, WGSA usually are in manyscaffolds: these range from the smallerside of the simple yeast Pichia stipitiswith 16 scaffolds to the tree Populustrichocarpa with 22,000 or more scaf-folds. We improve scaffold contiguity bysequencing clones that extend from theends of the scaffolds and attempting tojoin scaffolds with this new sequence.We also sequence into telomeres andcentromeres to provide a boundary forthe euchromatic sequence. We inte-grate mapping data from genetic mapsor optical maps, when it is available, toreinforce sequence joins and createchromosome-size genomic pieces. Wehave also recently begun sequencingBAC end libraries from genomes to addjumbo-size linking clones to DOE JGIeukaryotic genome assemblies.

The second goal is to improve andfix the base pair accuracy of thegenome (sequence accuracy). Thisincludes correcting false joins withinscaffolds, validating all joins across thescaffolds, resolving repetitive sequence,closing all sequence gaps, and raising

the quality of and verifying poor-qualitybase pairs. This is usually referred toas sequence finishing.

The third goal is to integrate orphangene sequences into the assembly.Usually, a WGSA is missing a minimumof 5% of known coding sequences, andthis number can go as high as 15%,depending on the complexity of thegenomic sequence organization and theavailable amount of gene-basedevidence. These missing genes cansometimes be found in very largecaptured sequence gaps, which werecover with clone-based sequencing,but they also appear in repetitivesequence arrays and duplications.Using the EST resources developed atthe DOE JGI PGF, we locate as many ofthese genes as possible and representthe surrounding genomic sequence inthe finished genome release. The finalgoal is to provide the resources neces-sary for the research communities touse genomic sequence effectively. Weprovide additional genomic resourcessuch as EST sequences, full-lengthcDNA sequencing, genomic mappingassistance and targeted-clone finishingfor DOE JGI genome projects, includingquality control sequences to aid ingenome assembly.

For now, as DOE JGI focuses onbiological energy sources, I finally have

something that is easy to describe.Because everyone back in Illinois,where I am from, knows what asoybean is, and how expensive it is tofill their truck’s gas tank, they see theconnection and how our work at SHGCfits into their daily lives.

Stanfordcont. from page 11

Hazardscont. from page 9

with his machete, but it was an excitingexperience nonetheless. Leadbettersaid that these soldiers “run up yourarms and spray you with what smellslike lime, which is why we let the post-docs do the sample collection.”Bears Collect Samples, TooTamas Torok and Glen Dahlbacka of theLawrence Berkeley (cont. on next page)

Julita MadejskaScientific Technician to Biomedical Scientist

Cailyn SpurrellBiosciences Technician 2-Shift Lead to

Biomedical ScientistMatt Zane

Biosciences Technician 1 toBiosciences Technician 2-Shift Lead

Eric AbbottBiosciences Technician 1 toBiosciences Technician 2

Edwin KimStudent Assistant to

Computer Systems Engineer TraineeSanna Anwar

Student Assistant to Senior ResearchTechnician

David RobinsonSenior Research Technician to

Research AssociateMiranda Harmon-Smith

Scientific Technician to Sequencing SupervisorChris Daum

Senior Scientific Technician toSequencing Supervisor

Susan LucasProduction Department Headto Production Division Leader

Arshi KhanBiosciences Technician 1 to

Biosciences Technician 2-Shift LeadSusannah Tringe

Special Postdoctoral Fellow toBiologist Scientist

Promotions

National Laboratory were drawn to thehot springs, fumaroles, and acid lakesof Siberia’s Kamchatka Peninsula toseek undiscovered microbes.Unfortunately, the local brown bears—about 6,000 of them, some of thelargest in the world—are also drawn tothe hot springs and geysers becausethey like to keep warm. The localInstitute of Volcanology provides armedescorts to protect researchers from thebears that roam the landscape, butTorok says that evenings are the mostdangerous period—“when they’rehungry.” At night, the bears watched theresearchers through the windows oftheir remote cabin. Who doesn’t like a


THE PRIMER / 19

DOE JGI has recently released thecomplete genomic sequence of theyeast Pichia stipitis, which is known asone of the best native microbes for thefermentation of xylose to ethanol.Completion of the P. stipitis genome hasgreatly advanced our knowledge of howto genetically modify this yeast andother microbes for improved xyloseutilization.

Xylose is a major component of agri-cultural residues and fast-growing hard-woods that could be used forbioconversion. Its economic use is crit-ical for the conversion of biomass intorenewable fuels. Fermentation of cornstarch presently produces more thanfour billion gallons of fuel ethanol peryear. This could be increased to about80 billion gallons, or 40 percent ofautomotive fuels, if the problem of

Pichia stipitis morphology under various conditions. (A) Pichia stipitis growing exponentiallywith bud scars; (B) P. stipitis hat-shaped spores seen from top and side; (C) Pseudomyceliaformed under carbon-limited continuous culture. Photo by Thomas Kuster, USDA, ForestProducts Laboratory

Pichia stipitis Genome Released

Hazardscont. from page 18

lignocellulose conversion can be solved.Yeasts are especially useful for

industrial ethanol fermentation becausethey have thick cell walls that facilitateprocessing and they resist infection bybacteria and viruses. Genomic se-quencing has revealed features in addi-tion to xylose fermentation that make P.stipitis important for bioconversion.

Pichia stipitis is an unusual yeastthat is found in the gut of passalidbeetles. In nature, it apparently helpsthese insects survive on rotted wood. Inthe laboratory, it has been adapted forthe commercial production of ethanolfrom byproducts of wood processingand paper manufacture. A recent start-up company, Xethanol, has licensed theuse of genetically modified P. stipitis forthe fermentation of hemicellulosicsugars to ethanol.

midnight snack?Data Mining Where Nobody Can HearYou ScreamWhen you’re studying ways to clean uptoxic waste sites, the answers may lie inthe deep gold mines of South Africa,where microbes living at high tempera-tures and pressures can tolerate anenvironment rich in uranium and organiccarbon. When gold prices warrant theeffort, miners will descend over threemiles beneath the earth, and it’s hotdown there. Jim Fredrickson of thePacific Northwest National Laboratorysays he only had to go down about twomiles to collect the anaerobic microbeshe was seeking, where the temperature

in the mine shafts was about 60degrees Celsius (140 degreesFahrenheit). For added excitement,mining-induced earthquakes wouldoccasionally rock the shafts.Commenting on the conditions deep inthe mine, he said they were “as closeto hell as I care to get.”

At the end of the day, it seems thatmicrobiologists should be happy just toavoid being boiled alive or eaten by abear, but when they get back to theirlabs, they have to face the greatesthazard of all—the paperwork requiredto maintain their funding.


20 / THE PRIMER

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CONTACT_____________________________David Gilbert, [email protected](925) 296-5643

CSO 12457

DOE JGI and theFungalBiotechnologyTeam of theChemical andBiological ProcessDevelopmentGroup at Pacific

Northwest National Laboratory (PNNL)have released the first public sequenceof Aspergillus niger, an important fila-mentous fungus used to manufactureindustrial enzymes and organic acids.Scott Baker, Senior Research Scientist(PNNL) and Igor Grigoriev (DOE JGI),made a joint announcement of thepublic release of the A. niger genomesequence (www.jgi.doe.gov/aspergillus)at the 3rd Meeting of the Asperglllus

Aspergillus niger Genome Sequence ReleasedGenome Community in Vienna, Austria.The announcement of the completedsequence included an invitation toparticipate in the annotation jamboreefollowing the European Conference onFungal Genetics in Vienna. On April 13,2006 an international team of scien-tists began the process of annotatingmore than 11,000 genes.

The filamentous fungus A. niger hasbeen used for nearly 100 years byindustry to make organic acids, notablycitric acid, and several biomass-processing enzymes. At PNNL, A. nigeris used as a model microbial system tomake value-added products from ligno-cellulosic biomass to augment theproduct portfolio of the anticipatedbiorefinery industry. The sequence of

this organism provides information thatwill accelerate the rate of progresstoward the Office of Biomass Programgoal to enable the development ofeconomical processes for convertingbiomass to fuel ethanol. Just as thepetroleum refinery produces a diversityof products from oil, filamentous fungiwill be used to convert complexbiomass via their specific hydrolyzingenzymes and/or co-products, such asorganic acids and platform moleculesfor the biopolymer industry. Thesequencing of A. niger is an example ofthe collaboration between the Office ofScience (OBER), the EERE Office ofBiomass Program, and PNNL, all ofwhich provided direct or indirect supportof this project.

DOE JGI was one of the recipients ofthe Central Contra Costa SanitaryDistrict 15th Annual PollutionPrevention Awards. This award recog-nizes a local institution’s outstandingjob in protecting our environment byintegrating effective pollution preventionstrategies into its business practices.DOE JGI Safety Coordinator StephenFranaszek accepted the award for DOEJGI at Central San’s September 21board meeting in Martinez.

DOE JGI Wins Pollution Prevention AwardStephen Franaszekreceives pollution preven-tion award from CentralContra Costa SanitaryDistrict’s Mario Menesini(left), President of theBoard of Directors, andTim Potter, SourceControl Superintendent.

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