Presentation is loading. Please wait.

Presentation is loading. Please wait.

The Small RNA Chaperone Hfq and Multiple Small RNAs Control Quorum Sensing in Vibrio harveyi and Vibrio cholerae Derrick H. Lenz, 1,3 Kenny C. Mok, Brendan.

Similar presentations


Presentation on theme: "The Small RNA Chaperone Hfq and Multiple Small RNAs Control Quorum Sensing in Vibrio harveyi and Vibrio cholerae Derrick H. Lenz, 1,3 Kenny C. Mok, Brendan."— Presentation transcript:

1 The Small RNA Chaperone Hfq and Multiple 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 1 Department of Molecular Biology Princeton University Princeton, New Jersey NEC Laboratories America, Inc. 4 Independence Way Princeton, New Jersey 08540

2 Introduction Quorum Sensing Quorum Sensing Regulation system that recognizes a signal produced by the bacterium Regulation system that recognizes a signal produced by the bacterium Signals are autoinducers Signals are autoinducers Senses the concentration of a particular species Senses the concentration of a particular species Can control either activators or repressors Can control either activators or repressors

3 Introduction Autoinducers Autoinducers Can promote intraspecies or interspecies communication Can promote intraspecies or interspecies communication Controls Controls Bioluminescence Bioluminescence Siderophore production Siderophore production Colony morphology Colony morphology Metalloprotease production Metalloprotease production Type III secretion Type III secretion

4 Quorum Sensing Circuits Autoinducers Al-1 & Al-2 produced by synthases LuxM & LuxS Autoinducers Al-1 & Al-2 produced by synthases LuxM & LuxS LuxN & LuxPQ detection of autoinducers LuxN & LuxPQ detection of autoinducers Systems converge to LuxU Systems converge to LuxU Transmission of signal to LuxO Transmission of signal to LuxO LuxO requires σ 54 to function LuxO requires σ 54 to function LuxR required for expression LuxR required for expression Figure 1. (A) Two quorum-sensing systems function in parallel to regulate gene expression in V. harveyi. Pentagons and triangles represent Al-1 and Al-2, respectively. Figure 1. (A) Two quorum-sensing systems function in parallel to regulate gene expression in V. harveyi. Pentagons and triangles represent Al-1 and Al-2, respectively.

5 Quorum Sensing Circuit Autoinducers CAl-1 & Al-2 produced by synthases CqsA & LuxS Autoinducers CAl-1 & Al-2 produced by synthases CqsA & LuxS CqsS & LuxPQ detection of autoinducers CqsS & LuxPQ detection of autoinducers Third system not yet identified Third system not yet identified Systems converge to LuxO Systems converge to LuxO LuxO requires σ 54 to function LuxO requires σ 54 to function HapR required for expression HapR required for expression Figure 1. (B) Three quorum-sensing systems function in parallel to regulate gene expression in V. cholerae. The functions making up the third circuit (denoted System 3) remain to be identified. Diamonds and triangles represent Cal-1 and Al-2, respectively. In both circuits, phosphate flows in the direction indicated by the arrows at low cell density and in the opposite direction at high cell density. Figure 1. (B) Three quorum-sensing systems function in parallel to regulate gene expression in V. cholerae. The functions making up the third circuit (denoted System 3) remain to be identified. Diamonds and triangles represent Cal-1 and Al-2, respectively. In both circuits, phosphate flows in the direction indicated by the arrows at low cell density and in the opposite direction at high cell density.

6 Circuit Operation Low Cell Density Low Cell Density Low concentration of autoinducer Low concentration of autoinducer Sensors act as kinases Sensors act as kinases Transfer of phosphate via LuxU to LuxO Transfer of phosphate via LuxU to LuxO LuxO-P is active and negatively regulates lux LuxO-P is active and negatively regulates lux

7 Circuit Operation High Cell Density High Cell Density High Concentration of autoinducers High Concentration of autoinducers Sensors act as phosphatases Sensors act as phosphatases Phosphate flow is reversed Phosphate flow is reversed Dephosphorylation and inactivation of LuxO Dephosphorylation and inactivation of LuxO LuxR/HapR bind to lux promoter and activate transcription LuxR/HapR bind to lux promoter and activate transcription

8 Revealing Quorum Sensing Repressor Hfq LuxO D47E used to identify quorum sensing repressor LuxO D47E used to identify quorum sensing repressor Site of phosphorylation is altered Site of phosphorylation is altered LuxO D47E protein locked and mimics LuxO-P LuxO D47E protein locked and mimics LuxO-P 40,000 transposon insertion mutants generated, 85 were bright 40,000 transposon insertion mutants generated, 85 were bright 82 contained transposon insertions in either luxO or rpoN, genes encoding σ contained transposon insertions in either luxO or rpoN, genes encoding σ 54 Three did not have mutations of these genes Three did not have mutations of these genes A V. harveyi genomic cosmid library was introduced into one mutant w/o luxO or rpoN mutations (BNL211) A V. harveyi genomic cosmid library was introduced into one mutant w/o luxO or rpoN mutations (BNL211)

9 Revealing Quorum Sensing Repressor Hfq All cosmids w/ dark phenotype contained overlapping regions of DNA All cosmids w/ dark phenotype contained overlapping regions of DNA Cosmid pBNL2014 mutated w/ Tn5lacZ to find lux repression region Cosmid pBNL2014 mutated w/ Tn5lacZ to find lux repression region Region cloned and sequenced Region cloned and sequenced Found to contain gene hfq Found to contain gene hfq Figure 2A (A) The hfq locus in V. harveyi, miaA and hflC were not fully sequenced (unsequenced regions are denoted by light-colored shading).

10 Requirement for Quorum Sensing Repression Question: Is hfq required for quorum sensing repression? Question: Is hfq required for quorum sensing repression? Strains/mutants Strains/mutants Wild-type (WT) Wild-type (WT) luxO luxO luxO D47E luxO D47E hfq hfq luxO D47E, hfq luxO D47E, hfq

11 Figure 2. (B) Bioluminescence assays for V. harveyi strains are: BB120 (WT, squares), JAF78 (  luxO::cm r, diamonds), JAF548 (luxO D47E, open triangles), BNL258 (hfq::Tn5lacZ, circles), and BNL211 (luxO D47E, hfq::Mini-MulacZ, closed triangles). Relative light units for V. harveyi are defined as counts min -1 ml -1 x 10 3 /cfu ml -1. Figure 2. (B) Bioluminescence assays for V. harveyi strains are: BB120 (WT, squares), JAF78 (  luxO::cm r, diamonds), JAF548 (luxO D47E, open triangles), BNL258 (hfq::Tn5lacZ, circles), and BNL211 (luxO D47E, hfq::Mini-MulacZ, closed triangles). Relative light units for V. harveyi are defined as counts min -1 ml -1 x 10 3 /cfu ml -1. Figure 2. (C) Bioluminescence assays for V. cholerae strains are: MM227 (WT, squares), MM349 (  luxO, diamonds), BH48 (luxO D47E, open triangles), DL2078 (  hfq, circles), and DL2378 (luxO D47E,  hfq, closed triangles). Relative light units for V. cholerae are defined as counts min -1 ml -1 /OD 600nm. Figure 2. (C) Bioluminescence assays for V. cholerae strains are: MM227 (WT, squares), MM349 (  luxO, diamonds), BH48 (luxO D47E, open triangles), DL2078 (  hfq, circles), and DL2378 (luxO D47E,  hfq, closed triangles). Relative light units for V. cholerae are defined as counts min -1 ml -1 /OD 600nm. In (B) and (C), the dotted lines represent the limit of detection for light.

12 Regulation of Virulence Genes Western blotting of TcpA production was measured to show that Hfq is not restricted to nonnative lux target in V. cholerae Western blotting of TcpA production was measured to show that Hfq is not restricted to nonnative lux target in V. cholerae Wild type: TcpA present Wild type: TcpA present luxO: TcpA absent luxO: TcpA absent luxO D47E: TcpA present in high levels luxO D47E: TcpA present in high levels hapR: TcpA present in high levels hapR: TcpA present in high levels Hfq: low TcpA production Hfq: low TcpA production luxO D47E, hfq & hapR, hfq: Hfq acts downstream of LuxO and upstream of HapR luxO D47E, hfq & hapR, hfq: Hfq acts downstream of LuxO and upstream of 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)

13 Hfq IS Required for Quorum Sensing Repression Predictions Predictions Quorum sensing repression occurs posttranscriptionally Quorum sensing repression occurs posttranscriptionally There must be one or more sRNA involved There must be one or more sRNA involved At low cell density, the LuxO-P- σ 54 complex activates the transcription of the gene(s) encoding the sRNA(s) At low cell density, the LuxO-P- σ 54 complex activates the transcription of the gene(s) encoding the sRNA(s)

14 Prediction 1. Quorum sensing repression occurs posttranscriptionally Northern blots used to determine the effect of hfq mutations on luxR and hapR mRNA stability. Northern blots used to determine the effect of hfq mutations on luxR and hapR mRNA stability. Rifampicin added to terminate transcription Rifampicin added to terminate transcription Figure 3. Hfq Regulates the Expression of luxR and hapR 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)

15 Prediction 1. Quorum sensing repression occurs posttranscriptionally Western blots show that the increased stability of the luxR and hapR mRNAs in the hfq mutants lead to increased levels of the LuxR and HapR proteins Western blots show that the increased stability of the luxR and hapR mRNAs in the hfq mutants lead to increased levels of the LuxR and HapR proteins Low cell density, Hfq destabilizes the luxR and hapR mRNA Low cell density, Hfq destabilizes the luxR and hapR 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.

16 LuxO-P Regulation of hapR is Posttranscriptional and Requires Hfq Constructed chromosomal hapR-lacZ transcriptional, translational, and promoter fusions Constructed chromosomal hapR-lacZ transcriptional, translational, and promoter fusions Measured their activities in V. cholerae strains Measured their activities in V. cholerae strains Wild-type Wild-type luxO D47E luxO D47E hfq hfq luxO D47E luxO D47E

17 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

18 Identification of sRNAs using Bioinformatics Parameters Parameters Upstream region of the sRNA locus must contain a σ 54 binding site Upstream region of the sRNA locus must contain a σ 54 binding site Assumed sRNAs have Rho-independent terminators Assumed sRNAs have Rho-independent terminators Restricted search to regions between annotated genes Restricted search to regions between annotated genes sRNAs must be conserved in V. cholerae, V. parahaemolyticus, and V. vulnificus sRNAs must be conserved in V. cholerae, V. parahaemolyticus, and V. vulnificus

19 Identification of sRNAs using Bioinformatics Two techniques used to find potential σ 54 binding sites Two techniques used to find potential σ 54 binding sites PATSER PATSER Weight matrix constructed w/ compiled set of approx. 180 σ 54 binding sites from multiple bacterial species Weight matrix constructed w/ compiled set of approx. 180 σ 54 binding sites from multiple bacterial species Includes all binding sites upstream of genes in V. cholerae known to be regulated by σ 54 Includes all binding sites upstream of genes in V. cholerae known to be regulated by σ 54 Upstream regions of known V. cholerae σ 54 genes were extracted Upstream regions of known V. cholerae σ 54 genes were extracted Using CONSENSUS searched for 16 bp motif Using CONSENSUS searched for 16 bp motif Aligned set of binding sites used to construct σ 54 weight matrix Aligned set of binding sites used to construct σ 54 weight matrix

20 Identification of sRNAs using Bioinformatics Four intergenic regions Four intergenic regions Conservation across the specified vibrio genomes Conservation across the specified vibrio genomes Contained Rho-independent terminators Contained Rho-independent terminators Figure 5 (A) Multiple sequence alignment of the qrr genes encoding the sRNAs identified in V. cholerae, V. parahaemolyticus, V. vulnificus, and V. harveyi.

21 Identification of sRNAs using Bioinformatics V. parahaemolyticus & V. vulnificus V. parahaemolyticus & V. vulnificus Five intergenic regions Five intergenic regions Conservation across the specified vibrio genomes Conservation across the specified vibrio genomes Contained Rho-independent terminators Contained Rho-independent terminators V. harveyi is most closely related to V. parahaemolyticus V. harveyi is most closely related to V. parahaemolyticus Assumed that V. harveyi has five sRNAs Assumed that V. harveyi has five sRNAs

22 Identification of sRNAs using Bioinformatics RNAFOLD RNAFOLD Prediction of secondary structures Prediction of secondary structures Qrr 2, Qrr3, & Qrr4 very similar Qrr 2, Qrr3, & Qrr4 very similar Loop composition variable, stem conserved Loop 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.

23 Identification of sRNAs using Bioinformatics Using LALIGN Using LALIGN Aligned complement of hapR untranslated upstream region with Qrr 1-4 Aligned complement of hapR untranslated upstream region with Qrr 1-4 Aligned complement of luxR untranslated upstream region with Qrr 1 Aligned complement of luxR untranslated upstream 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.

24 LuxO-P- σ54 Controls the Expression of the sRNA loci Question: Are sRNAs regulated by LuxO-P- σ 54 ? Question: Are sRNAs regulated by LuxO-P- σ 54 ? Northern blot used to quantify transcript levels Northern blot used to quantify transcript levels hapR + : qrr4 is regulated hapR + : qrr4 is regulated hapR - : qrr2 & qrr3 are regulated hapR - : qrr2 & qrr3 are regulated Unable to detect qrr1 Unable to detect qrr1 Detection of Qrr4 from V. harveyi Detection of Qrr4 from V. harveyi Expression induced by LuxO D47E Expression induced by 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.

25 LuxO-P- σ54 Controls the Expression of the sRNA loci qrr1 transcriptional reporter qrr1 transcriptional reporter Fusion of upstream region of V. cholerae qrr1 to luciferase operon Fusion of upstream region of V. cholerae qrr1 to luciferase operon Results indicate qrr1 is regulated by LuxO- P- σ 54 Results indicate qrr1 is regulated by LuxO- P- σ 54 (C) Single time point RLU for V. cholerae strains DL3212 (luxO) and DL3213 (luxO D47E) containing the qrr1-lux transcriptional fusion in trans.

26 sRNAs Involved in Quorum Sensing Repression Individual roles of sRNAs Individual roles of sRNAs Presence of any one sRNA expresses density-dependent bioluminescence similar to WT Presence of any one sRNA expresses density-dependent bioluminescence similar to WT Deletion of all sRNAs eliminates quorum sensing repression Deletion of all sRNAs eliminates quorum sensing repression Figure 7. (A) Bioluminescence assays were performed on V. cholerae.

27 sRNAs Involved in Quorum Sensing Repression Overexpression of one sRNA results in quorum sensing repression Overexpression of one sRNA results in quorum sensing repression Epistasis test Epistasis test The four sRNAs in V. cholerae are epistatic to LuxO-P in regulation of tcpA The four sRNAs in V. cholerae are epistatic to LuxO-P in regulation of tcpA (B) Single time point RLU for V. cholerae strains. Western blots probed for hapR and TcpA from V. cholerae.

28 Accumulation Rate If rate of synthesis of sRNA exceeds that of its target, sRNA can accumulate in the cell If rate of synthesis of sRNA exceeds that of its target, sRNA can accumulate in the cell If rate of synthesis of a target exceeds that of its regulatory sRNA, the message can accumulate in the cell If rate of synthesis of a target exceeds that of its regulatory sRNA, the message can accumulate in the cell

29 Conclusion Hfq is an RNA chaperone for a large number of sRNAs Hfq is an RNA chaperone for a large number of sRNAs Presence of multiple sRNAs is important in fine tuning the transition between low to high cell density by allowing the influence of additional regulatory inputs. Presence of multiple sRNAs is important in fine tuning the transition between low to high cell density by allowing the influence of additional regulatory inputs. Simultaneous inactivation of all four sRNAs is necessary to eliminate Hfq-mediated quorum sensing repression Simultaneous inactivation of all four sRNAs is necessary to eliminate Hfq-mediated quorum sensing repression Overexpression of only one sRNA is sufficient for repression Overexpression of only one sRNA is sufficient for repression Simultaneous presence of multiple autoinducers is required to reverse the direction of phosphoflow through the system and initiate the transition between low to high cell density. Simultaneous presence of multiple autoinducers is required to reverse the direction of phosphoflow through the system and initiate the transition between low to high cell density.

30 References Lenz, D. H., K. C. Mok, B. N. Lilley, R. V. Kulkarni, N. S. Wingreen, and B. L. Bassler The small RNA chaperone hfq and multiple small RNAs control quorum sensing in V. harveyi and V. cholerae. Cell. 118: Lenz, D. H., K. C. Mok, B. N. Lilley, R. V. Kulkarni, N. S. Wingreen, and B. L. Bassler The small RNA chaperone hfq and multiple small RNAs control quorum sensing in V. harveyi and V. cholerae. Cell. 118: Salyers, A. A., and D. D. Whitt Microbiology: Diversity, Disease, and the Environment, pp Fitzgerald Science Press, Inc., Bethesda. Salyers, A. A., and D. D. Whitt Microbiology: Diversity, Disease, and the Environment, pp Fitzgerald Science Press, Inc., Bethesda.


Download ppt "The Small RNA Chaperone Hfq and Multiple Small RNAs Control Quorum Sensing in Vibrio harveyi and Vibrio cholerae Derrick H. Lenz, 1,3 Kenny C. Mok, Brendan."

Similar presentations


Ads by Google