The Small RNA Chaperone Hfq and Multiple Small RNAs Control Quorum Sensing in Vibrio harveyi and...
-
Upload
clarissa-gardner -
Category
Documents
-
view
220 -
download
0
Transcript of The Small RNA Chaperone Hfq and Multiple Small RNAs Control Quorum Sensing in Vibrio harveyi and...
The Small RNA Chaperone Hfq andMultiple Small RNAs Control Quorum Sensing in Vibrio harveyi and Vibrio
cholerae
Derrick H. Lenz,1,3 Kenny C. Mok, Brendan N. Lilley,1,4 Rahul V.
Kulkarni, Ned S. Wingreen,1,2 and Bonnie
1Department of Molecular Biology Princeton University Princeton, New Jersey 08544 2NEC Laboratories America, Inc.
4 Independence Way Princeton, New Jersey 08540
IntroductionIntroduction
Quorum SensingQuorum SensingRegulation system that recognizes a Regulation system that recognizes a
signal produced by the bacteriumsignal produced by the bacteriumSignals are autoinducersSignals are autoinducers
Senses the concentration of a particular Senses the concentration of a particular speciesspecies
Can control either activators or Can control either activators or repressorsrepressors
IntroductionIntroduction
AutoinducersAutoinducersCan promote intraspecies or Can promote intraspecies or
interspecies communicationinterspecies communicationControlsControls
BioluminescenceBioluminescenceSiderophore productionSiderophore productionColony morphologyColony morphologyMetalloprotease productionMetalloprotease productionType III secretionType III secretion
Quorum Sensing CircuitsQuorum Sensing Circuits
Autoinducers Al-1 & Al-2 Autoinducers Al-1 & Al-2 produced by synthases produced by synthases LuxM & LuxSLuxM & LuxS
LuxN & LuxPQ detection of LuxN & LuxPQ detection of autoinducersautoinducers
Systems converge to LuxUSystems converge to LuxU Transmission of signal to Transmission of signal to
LuxOLuxO LuxO requires LuxO requires σσ54 54 to to
functionfunction
LuxR required for LuxR required for expressionexpression
Figure 1. (A) Two quorum-sensing Figure 1. (A) Two quorum-sensing systems function in parallel to systems function in parallel to regulate gene expression in regulate gene expression in V. V. harveyiharveyi. Pentagons and triangles . Pentagons and triangles represent Al-1 and Al-2, represent Al-1 and Al-2, respectively.respectively.
Quorum Sensing CircuitQuorum Sensing Circuit
Autoinducers CAl-1 & Al-2 Autoinducers CAl-1 & Al-2 produced by synthases produced by synthases CqsA & LuxSCqsA & LuxS
CqsS & LuxPQ detection of CqsS & LuxPQ detection of autoinducersautoinducers
Third system not yet Third system not yet identifiedidentified
Systems converge to LuxOSystems converge to LuxO LuxO requires LuxO requires σσ54 54 to to
functionfunction
HapR required for HapR required for expressionexpression
Figure 1. (B) Three quorum-sensing systems function in Figure 1. (B) Three quorum-sensing systems function in parallel to regulate gene expression in parallel to regulate gene expression in V. choleraeV. cholerae. The . The functions making up the third circuit (denoted System 3) functions making up the third circuit (denoted System 3) remain to be identified. Diamonds and triangles remain to be identified. Diamonds and triangles represent Cal-1 and Al-2, respectively. In both circuits, represent Cal-1 and Al-2, respectively. In both circuits, phosphate flows in the direction indicated by the arrows phosphate flows in the direction indicated by the arrows at low cell density and in the opposite direction at high at low cell density and in the opposite direction at high cell density.cell density.
Circuit OperationCircuit Operation
Low Cell DensityLow Cell DensityLow concentration of autoinducerLow concentration of autoinducerSensors act as kinasesSensors act as kinasesTransfer of phosphate via LuxU to LuxOTransfer of phosphate via LuxU to LuxOLuxO-P is active and negatively LuxO-P is active and negatively
regulates regulates luxlux
Circuit OperationCircuit Operation
High Cell DensityHigh Cell DensityHigh Concentration of autoinducersHigh Concentration of autoinducersSensors act as phosphatasesSensors act as phosphatasesPhosphate flow is reversedPhosphate flow is reversedDephosphorylation and inactivation of Dephosphorylation and inactivation of
LuxOLuxOLuxR/HapR bind to LuxR/HapR bind to lux lux promoter and promoter and
activate transcriptionactivate transcription
Revealing Quorum Sensing Revealing Quorum Sensing Repressor HfqRepressor Hfq
LuxO D47E used to identify quorum sensing LuxO D47E used to identify quorum sensing repressorrepressor Site of phosphorylation is alteredSite of phosphorylation is altered LuxO D47E protein locked and mimics LuxO-PLuxO D47E protein locked and mimics LuxO-P
40,000 transposon insertion mutants generated, 40,000 transposon insertion mutants generated, 85 were bright85 were bright
82 contained transposon insertions in either 82 contained transposon insertions in either luxOluxO or or rpoNrpoN, genes encoding , genes encoding σσ5454
Three did not have mutations of these genesThree did not have mutations of these genes A A V. harveyiV. harveyi genomic cosmid library was genomic cosmid library was
introduced into one mutant w/o luxO or rpoN introduced into one mutant w/o luxO or rpoN mutations (BNL211)mutations (BNL211)
Revealing Quorum Sensing Revealing Quorum Sensing Repressor HfqRepressor Hfq
All cosmids w/ dark phenotype contained overlapping All cosmids w/ dark phenotype contained overlapping regions of DNAregions of DNA
Cosmid pBNL2014 mutated w/ Tn5Cosmid pBNL2014 mutated w/ Tn5lacZ lacZ to find to find luxlux repression regionrepression region
Region cloned and sequencedRegion cloned and sequenced Found to contain gene hfqFound to contain gene hfqFigure 2AFigure 2A
(A) The (A) The hfqhfq locus in locus in V. harveyiV. harveyi, , miaAmiaA and and hflChflC were not fully were not fully sequenced (unsequenced regions are denoted by light-sequenced (unsequenced regions are denoted by light-colored shading).colored shading).
Requirement for Quorum Requirement for Quorum Sensing RepressionSensing Repression
Question: Is Question: Is hfqhfq required for quorum required for quorum sensing repression?sensing repression?
Strains/mutantsStrains/mutantsWild-type (WT)Wild-type (WT)luxOluxOluxOluxO D47E D47EhfqhfqluxOluxO D47E, D47E, hfqhfq
Figure 2. (B) Bioluminescence assays Figure 2. (B) Bioluminescence assays for for V. harveyiV. harveyi strains are: BB120 strains are: BB120 (WT, squares), JAF78 ((WT, squares), JAF78 (luxO::cmluxO::cmrr, , diamonds), JAF548 (diamonds), JAF548 (luxOluxO D47E, open D47E, open triangles), BNL258 (triangles), BNL258 (hfqhfq::Tn5lacZ, ::Tn5lacZ, circles), and BNL211 (circles), and BNL211 (luxOluxO D47E, D47E, hfqhfq::Mini-Mu::Mini-MulacZlacZ, closed triangles). , closed triangles). Relative light units for Relative light units for V. harveyiV. harveyi are are defined as counts mindefined as counts min-1-1 ml ml-1-1 x 10 x 1033/cfu /cfu mlml-1-1..
Figure 2. (C) Bioluminescence Figure 2. (C) Bioluminescence assays for assays for V. choleraeV. cholerae strains are: strains are: MM227 (WT, squares), MM349 MM227 (WT, squares), MM349 ((luxO, diamonds), BH48 (luxO, diamonds), BH48 (luxOluxO D47E, open triangles), DL2078 D47E, open triangles), DL2078 ((hfqhfq, circles), and DL2378 (, circles), and DL2378 (luxOluxO D47E, D47E, hfqhfq, closed triangles). , closed triangles). Relative light units for V. cholerae Relative light units for V. cholerae are defined as counts minare defined as counts min-1-1 mlml-1-1/OD/OD600nm600nm..
In (B) and (C), the dotted lines represent the limit of detection for light.
Regulation of Virulence Regulation of Virulence GenesGenes
Western blotting of Western blotting of TcpA production was TcpA production was measured to show measured to show that Hfq is not that Hfq is not restricted to nonnative restricted to nonnative luxlux target in target in V. V. choleraecholerae
Wild type: TcpA presentWild type: TcpA present luxOluxO: TcpA absent: TcpA absent luxOluxO D47E: TcpA present in D47E: TcpA present in
high levelshigh levels hapRhapR: TcpA present in high : TcpA present in high
levelslevels HfqHfq: low TcpA production: low TcpA production luxOluxO D47E, hfq & D47E, hfq & hapRhapR, ,
hfq: Hfq acts downstream hfq: Hfq acts downstream of LuxO and upstream of of LuxO and upstream of HapR HapR
Figure 2. (D) V. cholerae strains analyzed for TcpA production by Western blot are: C6706str2 (WT), MM307 (luxO), BH38 (luxO D47E), MM194 (hapR), DL2066(hfq), DL2146 (luxO D47E, hfq), and DL2607 (hapR, hfq)
Hfq IS Required for Quorum Hfq IS Required for Quorum Sensing RepressionSensing Repression
PredictionsPredictionsQuorum sensing repression occurs Quorum sensing repression occurs
posttranscriptionallyposttranscriptionallyThere must be one or more sRNA There must be one or more sRNA
involvedinvolvedAt low cell density, the LuxO-P- At low cell density, the LuxO-P- σσ54 54
complex activates the transcription of complex activates the transcription of the gene(s) encoding the sRNA(s)the gene(s) encoding the sRNA(s)
Prediction 1. Quorum sensing Prediction 1. Quorum sensing repression occurs posttranscriptionallyrepression occurs posttranscriptionally
Northern blots used to determine the effect of hfq Northern blots used to determine the effect of hfq mutations on mutations on luxRluxR and and hapRhapR mRNA stability. mRNA stability.
Rifampicin added to terminate transcriptionRifampicin added to terminate transcriptionFigure 3. Hfq Regulates the Expression of Figure 3. Hfq Regulates the Expression of luxRluxR and and hapRhapR Posttranscriptionally Posttranscriptionally
(A) Non-steady-state Northern blots were used to analyze luxR/hapR transcript stability in the following: V. harveyi JAF548 (luxO D47E) and BNL211 (luxO D47E, hfq::Mini-MulacZ); and V. cholerae BH38 (luxO D47E) and DL2146 (luxO D47E, Δhfq)
Prediction 1. Quorum sensing Prediction 1. Quorum sensing repression occurs posttranscriptionallyrepression occurs posttranscriptionally
Western blots show that the increased stability of Western blots show that the increased stability of the luxR and hapR mRNAs in the the luxR and hapR mRNAs in the hfqhfq mutants mutants lead to increased levels of the LuxR and HapR lead to increased levels of the LuxR and HapR proteinsproteins
Low cell density, Hfq destabilizes the luxR and Low cell density, Hfq destabilizes the luxR and hapR mRNAhapR mRNA
(B) Western blots on lysates of V. harveyi BB120 (WT), JAF548 (luxO D47E), BNL258 (hfq::Tn5lacZ), BNL211 (luxO D47E, hfq::Mini-MulacZ), and V. cholerae C6706str2 (WT), BH38 (luxO D47E), DL2066 (Δhfq), DL2146 (luxO D47E, Δhfq) measured LuxR and HapR protein, respectively.
LuxO-P Regulation of LuxO-P Regulation of hapRhapR is is Posttranscriptional and Requires Posttranscriptional and Requires
HfqHfqConstructed chromosomal Constructed chromosomal hapR-lacZhapR-lacZ
transcriptional, translational, and transcriptional, translational, and promoter fusionspromoter fusions
Measured their activities in V. Measured their activities in V. cholerae strainscholerae strainsWild-typeWild-typeluxOluxO D47E D47E hfqhfq luxOluxO D47E D47E
• The transcriptional and translational are repressed in the luxO D47E strain, and repression requires Hfq.• LuxO D47E does not repress the hapR-lacZ promoter fusion.• Results suggest that LuxO-P regulation of hapR is posttranscriptional
Identification of sRNAs using Identification of sRNAs using BioinformaticsBioinformatics
ParametersParametersUpstream region of the sRNA locus must Upstream region of the sRNA locus must
contain a contain a σσ5454 binding site binding siteAssumed sRNAs have Rho-independent Assumed sRNAs have Rho-independent
terminatorsterminatorsRestricted search to regions between Restricted search to regions between
annotated genesannotated genessRNAs must be conserved in sRNAs must be conserved in V. choleraeV. cholerae, ,
V. parahaemolyticusV. parahaemolyticus, and , and V. vulnificusV. vulnificus
Identification of sRNAs using Identification of sRNAs using BioinformaticsBioinformatics
Two techniques used to find potential Two techniques used to find potential σσ54 54
binding sitesbinding sites PATSERPATSER
Weight matrix constructed w/ compiled set of approx. Weight matrix constructed w/ compiled set of approx. 180 σ180 σ54 54 binding sites from multiple bacterial speciesbinding sites from multiple bacterial species
Includes all binding sites upstream of genes in V. Includes all binding sites upstream of genes in V. cholerae known to be regulated by σcholerae known to be regulated by σ5454
Upstream regions of known V. cholerae σUpstream regions of known V. cholerae σ5454 genes were extractedgenes were extracted
Using CONSENSUS searched for 16 bp motif Using CONSENSUS searched for 16 bp motif Aligned set of binding sites used to construct σAligned set of binding sites used to construct σ5454
weight matrix weight matrix
Identification of sRNAs using Identification of sRNAs using BioinformaticsBioinformatics
Four intergenic regionsFour intergenic regions Conservation across the specified vibrio Conservation across the specified vibrio
genomesgenomes Contained Rho-independent terminatorsContained Rho-independent terminators
Figure 5Figure 5
(A) Multiple sequence alignment of the qrr genes encoding the sRNAs identified in V. cholerae, V. parahaemolyticus, V. vulnificus, and V. harveyi.
Identification of sRNAs using Identification of sRNAs using BioinformaticsBioinformatics
V. parahaemolyticus V. parahaemolyticus && V. vulnificus V. vulnificusFive intergenic regionsFive intergenic regions
Conservation across the specified vibrio Conservation across the specified vibrio genomesgenomes
Contained Rho-independent terminatorsContained Rho-independent terminators
V. harveyiV. harveyi is most closely related to is most closely related to V. parahaemolyticusV. parahaemolyticusAssumed that Assumed that V. harveyiV. harveyi has five sRNAs has five sRNAs
Identification of sRNAs using Identification of sRNAs using BioinformaticsBioinformatics
RNAFOLDRNAFOLD Prediction of secondary structuresPrediction of secondary structures
Qrr 2, Qrr3, & Qrr4 very similarQrr 2, Qrr3, & Qrr4 very similar Loop composition variable, stem conservedLoop composition variable, stem conserved
(B) Lowest-energy secondary-structural predictions for the Qrr sRNAs identified in V. cholerae. Bold typeface indicates regions conserved across all sRNAs in V. cholerae, V. parahaemolyticus, and V. vulnificus.
Identification of sRNAs using Identification of sRNAs using BioinformaticsBioinformatics
Using LALIGNUsing LALIGN Aligned complement of Aligned complement of hapRhapR untranslated untranslated
upstream region with Qrr 1-4upstream region with Qrr 1-4 Aligned complement of Aligned complement of luxRluxR untranslated untranslated
upstream region with Qrr 1upstream region with Qrr 1
(C) Alignment of the complement of the hapR UTR with a portion of the Qrr sRNAs identified in V. cholerae. (D) Alignment of the complement of the luxR UTR with a portion of the Qrr1 identified in V. harveyi.
LuxO-P- σ54 Controls the LuxO-P- σ54 Controls the Expression of the sRNA lociExpression of the sRNA loci
Question: Are Question: Are sRNAs regulated sRNAs regulated by LuxO-P- by LuxO-P- σσ5454?? Northern blot used Northern blot used
to quantify to quantify transcript levelstranscript levels
hapRhapR++: qrr4 is : qrr4 is regulatedregulated
hapRhapR--: qrr2 & qrr3 : qrr2 & qrr3 are regulatedare regulated
Unable to detect Unable to detect qrr1qrr1
Detection of Qrr4 Detection of Qrr4 from V. harveyifrom V. harveyi Expression induced Expression induced
by LuxO D47Eby LuxO D47E
Figure 6. (A) V. cholerae C6706str2 (WT), MM307 (ΔluxO), BH38 (luxO D47E), BH76 (ΔrpoN) was probed for sRNAs Qrr1, Qrr2, Qrr3, and Qrr4, and V. cholerae rpsL is shown as the loading control. (B) RNA isolated from V. harveyi qrr1 and for sRNA Qrr4 with a probe made against V. cholerae qrr4. V. harveyi rspL is shown as the loading control.
LuxO-P- σ54 Controls the LuxO-P- σ54 Controls the Expression of the sRNA lociExpression of the sRNA loci
qrr1 transcriptional qrr1 transcriptional reporterreporter Fusion of upstream Fusion of upstream
region of region of V. V. choleraecholerae qrr1 to qrr1 to luciferase operonluciferase operon
Results indicate Results indicate qrr1 is regulated qrr1 is regulated by LuxO-P- by LuxO-P- σσ5454
(C) Single time point RLU for V. cholerae strains DL3212 (luxO) and DL3213 (luxO D47E) containing the qrr1-lux transcriptional fusion in trans.
sRNAs Involved in Quorum sRNAs Involved in Quorum Sensing RepressionSensing Repression
Individual roles of Individual roles of sRNAssRNAs Presence of any one Presence of any one
sRNA expresses sRNA expresses density-dependent density-dependent bioluminescence bioluminescence similar to WTsimilar to WT
Deletion of all Deletion of all sRNAs eliminates sRNAs eliminates quorum sensing quorum sensing repressionrepression
Figure 7. (A) Bioluminescence assays were performed on V. cholerae.
sRNAs Involved in Quorum sRNAs Involved in Quorum Sensing RepressionSensing Repression
Overexpression of Overexpression of one sRNA results in one sRNA results in quorum sensing quorum sensing repressionrepression
Epistasis testEpistasis test The four sRNAs in The four sRNAs in
V. cholerae are V. cholerae are epistatic to LuxO-P epistatic to LuxO-P in regulation of in regulation of tcpAtcpA
(B) Single time point RLU for V. cholerae strains. Western blots probed for hapR and TcpA from V. cholerae.
Accumulation RateAccumulation Rate
If rate of synthesis If rate of synthesis of sRNA exceeds of sRNA exceeds that of its target, that of its target, sRNA can sRNA can accumulate in the accumulate in the cellcell
If rate of synthesis If rate of synthesis of a target of a target exceeds that of its exceeds that of its regulatory sRNA, regulatory sRNA, the message can the message can accumulate in the accumulate in the cellcell
ConclusionConclusion
Hfq is an RNA chaperone for a large number of sRNAsHfq is an RNA chaperone for a large number of sRNAs Presence of multiple sRNAs is important in fine tuning Presence of multiple sRNAs is important in fine tuning
the transition between low to high cell density by the transition between low to high cell density by allowing the influence of additional regulatory inputs.allowing the influence of additional regulatory inputs.
Simultaneous inactivation of all four sRNAs is Simultaneous inactivation of all four sRNAs is necessary to eliminate Hfq-mediated quorum sensing necessary to eliminate Hfq-mediated quorum sensing repressionrepression
Overexpression of only one sRNA is sufficient for Overexpression of only one sRNA is sufficient for repressionrepression
Simultaneous presence of multiple autoinducers is Simultaneous presence of multiple autoinducers is required to reverse the direction of phosphoflow required to reverse the direction of phosphoflow through the system and initiate the transition through the system and initiate the transition between low to high cell density.between low to high cell density.
ReferencesReferences
Lenz, D. H., K. C. Mok, B. N. Lilley, R. V. Lenz, D. H., K. C. Mok, B. N. Lilley, R. V. Kulkarni, N. S. Wingreen, and B. L. Bassler. Kulkarni, N. S. Wingreen, and B. L. Bassler. 2004. The small RNA chaperone hfq and 2004. The small RNA chaperone hfq and multiple small RNAs control quorum multiple small RNAs control quorum sensing in sensing in V. harveyiV. harveyi and and V. choleraeV. cholerae. . Cell. Cell. 118118:69-82.:69-82.
Salyers, A. A., and D. D. Whitt. 2001. Salyers, A. A., and D. D. Whitt. 2001. Microbiology: Diversity, Disease, and the Microbiology: Diversity, Disease, and the EnvironmentEnvironment, pp. 107. Fitzgerald Science , pp. 107. Fitzgerald Science Press, Inc., Bethesda.Press, Inc., Bethesda.