Small RNA Control of Quorum Sensing Part I Goodmorning. I’d like to thank the organizers, Mogens in particular, for inviting me to this meeting. I have already learned a lot and I’m sure that will continue throughout the week. Today, I want to tell you about two areas of research that I am interested in, one is about how bacteria communicate with each other to make decisions as a community, a process called quorum sensing. The other is about small regulatory, noncoding RNAs, which is a relatively new mechanism of gene regulation in bacteria. Of course the reas on that I want to talk about these two topics is that in the Vibrio species, which I work on, small RNAs are central to the control of cell-to-cell communication. Quorum The number of Members required to be present to conduct official business (218 in the House, 51 in the Senate) Sine Lo Svenningsen Bassler Lab, Princeton University Geilo School 2007

Quorum sensing bacteria exist in one of two behavioral modes Individual cell mode Social mode

Examples of quorum sensing regulated behaviors Biofilm formation Virulence factor production Bioluminescence Conjugation Competence

Quorum sensing is accomplished by the detection of signaling molecules Low [AI] High [AI]

Gram-negative quorum sensing systems LuxI/R-type quorum sensing systems

Acyl-Homoserine Lactone Autoinducers V. fischeri/LuxI P. aeruginosa/LasI P. aeruginosa/RhlI A. tumefaciens/TraI V. harveyi/LuxM

Gram-positive quorum sensing systems

Oligopeptide Autoinducers

Quorum sensing regulates bioluminescence in Vibrio harveyi

The Vibrio harveyi Quorum Sensing Circuit LuxM LuxS

The Vibrio harveyi Quorum Sensing Circuit LuxM LuxS LuxM LuxS

AI-2 Inter-species communication

Bacteria That Contain luxS Neisseria gonorrhoeae Neisseria meningitidis Pasteurella multocida Pasteurella trehalosi Porphyromonas gingivalis Proteus mirabilis Salmonella paratyphi Salmonella typhi Salmonella typhimurium Shewanella putrefaciens Shigella flexneri Staphylococcus aureus Staphylococcus epidermidis Streptococcus gordonii Streptococcus mutans Streptococcus pneumoniae Streptococcus pyogenes Vibrio cholerae Vibrio harveyi Vibrio parahaemolyticus Vibrio anguillarum Vibrio vulnificus Yersinia pestis Actinobacillus actinomycetemcomitans Actinobacillus pleuropneumoniae Actinobacillus suis Bacillus anthracis Bacillus halodurans Bacillus subtilis Borrelia burgdorferi Campylobacter jejuni Clostridium acetobutylicum Clostridium difficile Clostridium perfringens Deinococcus radiodurans Escherichia coli MG1655 Escherichia coli O157:H7 Enterococcus faecalis Haemophilus influenzae Helicobacter pylori Klebsiella pneumoniae Lactococcus lactis Leuconostoc oenos Listeria monocytogenes Mannheimia haemolytica

LuxS resides with genes for SAM utilization pfs metK luxS

SAM Utilization Met MetK SAM Methyltransferases SAH Pfs SRH LuxS 4,5-Dihydroxy- 2,3 pentanedione + Homocysteine

The LuxS Reaction SRH LuxS 4,5-Dihydroxy- 2,3-pentanedione + Homocysteine

The Vibrio harveyi Quorum Sensing Circuit LuxM LuxS LuxM LuxS

Crystal structure of LuxP

Crystal revealed AI-2 structure

AI-2 Formation in V. harveyi LsrB ligand

The Salmonella Lsr Operon lsrA lsrC lsrD lsrB lsrF lsrG lsrE LsrB LsrC LsrA LsrB AI-2 LsrD periplasm cytoplasm

Crystal structure of Salmonella LsrB Apo-LsrB Holo-LsrB

Crystal structure of Salmonella LsrB

V. harveyi and S. typhimurium AI-2s

A Chemical System of Interconverting Molecules LuxS product LuxP ligand LsrB ligand

A Chemical System of Interconverting Molecules LuxS product LuxP ligand LsrB ligand

Targets of AI-2 Actinobacillus actinomycetemcomitans Periodontal virulence, Mixed-species biofilms Bacillus anthracis Growth Bacillus cereus Biofilms Bacillus subtilis Development Borrelia burgdorferi Virulence Campylobacter jejuni Motility, Toxins Clostridium difficile Virulence, Toxins Clostridium perfringens Virulence, Toxins Escherichia coli K12 AI-2 transport, Biofilms, Cell division, DNA processing, Iron uptake Escherichia coli EHEC O157:H7 Virulence, Type III Secretion , Motility Escherichia coli EPEC Motility, Type III Secretion Haemophilus influenzae Virulence, Cell invasion Helicobacter pylori Motility, Biofilms Klebsiella pneumoniae Biofilms Lactobacillus reuteri Biofilms in mice Listeria monocytogenes Biofilms Mannheimia haemolytica Virulence, Encapsulation, Adhesion Neisseria meningitidis Virulence, Bacteremia Photorhabdus luminescens Antibiotic production Porphyromonas gingivalis Virulence, Proteases Hemin acquisition, Stress response Salmonella typhi Biofilms Salmonella typhimurium AI-2 transport, Biofilms Serratia marcescens Virulence in C. elegans, Hemolysin, Antibiotic production Shigella flexneri VirB (virulence factor) Staphylococcus epidermis Virulence, Biofilms Streptococcus gordonii Mixed-species biofilms Streptococcus mutans Virulence, Biofilms, Competence, Stress response Streptococcus pneumoniae Virulence, Persistence, Competence Streptococcus pyogenes Virulence factors, Protease, Hemolysin Vibrio anguillarum Protease Vibrio cholerae Virulence, Toxins, Biofilms Vibrio fischeri Bioluminescence, Colonization Vibrio harveyi Bioluminescence, Biofilms, Siderophore, Virulence, Type III Secretion, Protease

The Vibrio harveyi Quorum Sensing Circuit

Random Promoter-GFP Library A Screen For Quorum Sensing Regulated Genes Differential Fluorescence Induction (DFI) Sort for GFP+ (+AI or -AI) FACS: Flourescence activated cell sorting AI- V. harveyi Random Promoter-GFP Library Plate Colonies +AI -AI Screen

Quorum Sensing-Controlled Genes in V. harveyi Qrr4 luxR repressor - VP1482-putative response regulator - VPA0567-sigma cross reacting protein + Qrr3 luxR repressor - Qrr2 luxR repressor - VP0699-GGDEFprotein - VP2888-GGDEF protein - VP1698-Type III secretion operon - VP2214-VacJ lipoprotein - VPA0166-putative outer membrane protein + VPA0248-OmpA + VP0047-peptide ABC transporter - VP1105-FtsK - VP2258-FlaA-first gene of flagellar operon - VP2235-FlhA flagellar operon - VPA0818-conserved hypothetical - VP0748-5-nucleotidase precursor - VP2180-adenine phosphotransferase - VV0153-putative hydroxyacylglutathione hydrolase - VP0325-malate dehydrogenase - VP2561-enolase + VP2599-fructose-bisphosphate aldolase - VP0497-formate acetyl transferase-related - downstream of VPA1399-maltose ABC transporter - inside gene VP2499-sugar fermentation protein - anti-sense of VP1525-spermidine, putresceine ABC transporter - internal segment of VP1556-phage f237 ORF5 - anti-sense to VPA1200-nitrate reductase, cytochrome c-type protein - inside gene VP0477-peptide ABC transporter  - VP1243-hypothetical - VPA0137-hypothetical - VP1147-conserved hypothetical - VPA1525-hypothetical - VPA1318-hypothetical - VP1240-hypothetical - 14 genes no homology to anything in the database 7+/7- REGULATORY STRUCTURAL METABOLIC POSSIBLE sRNA HYPOTHETICAL UNKNOWN Class Designation Regulation

The Vibrio harveyi Quorum Sensing Circuit 50 New Targets

Differential Responses of Target Genes to Autoinducer Inputs in V Differential Responses of Target Genes to Autoinducer Inputs in V. harveyi 400 300 * Fluorescence 200 100 324 244 295 257 334 212 266 282 284 352 312 270 341 249 227 234 253 342 262 335 260 222 250 259 207 258 272 214 343 317 303 345 301 219 337 307 245 248 201 242 349 340 291 338 308 247 275 268 204 205 353 276 208 209 364 251 368 330 267 269 252 255 350 254 AI Regulated Promoter no AI , AI-2 , AI-1 , AI-2+AI-1

Three Classes of Autoinducer Regulated Genes 120 100 no AI AI-2 AI-1 AI-1 + AI-2 80 Normalized Fluorescence 60 40 20 338 (FtsK) Class 3 317 (unknown) Class 1 275 (GGDEF) Class 2

Autoinducer Regulation of LuxR Production 1010 109 108 Relative Light Units 107 106 105 104 No AI AI-2 AI-1 AI-2, AI-1 % LuxR 1.6 6.8 26 100

The Vibrio harveyi Quorum Sensing Circuit Class 3 High affinity Class 2 Medium affinity Class 1 Low affinity

Small RNA Control of Quorum Sensing Part II

The quorum sensing circuit of Vibrio cholerae: Low Cell Density High Cell Density O.M. O.M. System 1 System 1 System 2 System 2 CAI CAI CAI CAI - - - - I I I I AI AI AI AI - - - - 2 2 2 2 LuxP LuxP I.M. I.M. HI HI HI HI CqsA CqsA CqsA CqsA CqsS CqsS LuxQ LuxQ LuxQ LuxQ LuxS LuxS LuxS LuxS DI DI DI DI P P P P LuxU LuxU H2 H2 Other Other inputs inputs LuxO LuxO D2 D2 + + σ σ 54 54 Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR

LuxO-type transcription factors work with σ54 Low Cell Density High Cell Density O.M. O.M. System 1 System 1 System 2 System 2 CAI CAI CAI CAI - - - - I I I I AI AI AI AI - - - - 2 2 2 2 LuxP LuxP I.M. I.M. HI HI HI HI CqsA CqsA CqsA CqsA CqsS CqsS LuxQ LuxQ LuxQ LuxQ LuxS LuxS LuxS LuxS DI DI DI DI P P P P LuxU LuxU H2 H2 Other Other inputs inputs LuxO LuxO D2 D2 + + σ σ 54 54 Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR

Repression of HapR is indirect Low Cell Density HI DI LuxP CqsA LuxS H2 HapR P D2 CAI - I AI 2 CqsS LuxQ System 1 System 2 LuxU LuxO σ 54 + I.M. O.M. X High Cell Density O.M. O.M. System 1 System 1 System 2 System 2 CAI CAI CAI CAI - - - - I I I I AI AI AI AI - - - - 2 2 2 2 LuxP LuxP I.M. I.M. HI HI HI HI CqsA CqsA CqsA CqsA CqsS CqsS LuxQ LuxQ LuxQ LuxQ LuxS LuxS LuxS LuxS DI DI DI DI P P P P LuxU LuxU H2 H2 LuxO LuxO D2 D2 + + σ σ 54 54 X Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR

Genetic screen to identify “X” in Vibrio cholerae Mutagenesis with mini-Mu transposon luxO D47E “dark”

The screen yielded Hfq Hexameric, RNA-binding protein. Homology to eukaryotic Sm proteins. Many small RNAs depend on Hfq for action.

Regulation by small RNAs Storz et al. 2004

The role of Hfq in small RNA regulation Storz et al. 2004

Hypothesis: Hfq and a small RNA repress HapR Low Cell Density High Cell Density O.M. O.M. O.M. O.M. System 1 System 1 System 2 System 2 System 1 System 1 System 2 System 2 CAI CAI - - I I AI AI - - 2 2 CAI CAI CAI CAI - - - - I I I I AI AI AI AI - - - - 2 2 2 2 LuxP LuxP LuxP LuxP HI HI HI HI I.M. I.M. I.M. I.M. P P P P HI HI HI HI CqsA CqsA CqsS CqsS LuxQ LuxQ LuxS LuxS CqsA CqsA CqsA CqsA CqsS CqsS LuxQ LuxQ LuxQ LuxQ LuxS LuxS LuxS LuxS DI DI DI DI DI DI DI DI P P P P P P LuxU LuxU H2 H2 LuxU LuxU H2 H2 LuxO LuxO P P LuxO LuxO + + σ σ 54 54 D2 D2 + + σ σ 54 54 D2 D2 sRNA + Hfq? Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR HapR HapR

Screen for the sRNA: A computational approach s54 Promoter Intergenic Region Rho-Independent Terminator Conserved in the Vibrios

The screen yielded four homologous sRNAs V. cholerae Qrr1 Qrr2 Qrr3 Qrr4 hapR,, complement RBS Start +1 Qrr1 Qrr2 Qrr4 Qrr3 Qrr: Quorum regulatory RNA

Hypothesis: Hfq and a small RNA repress HapR Low Cell Density High Cell Density O.M. O.M. O.M. O.M. System 1 System 1 System 2 System 2 System 1 System 1 System 2 System 2 CAI CAI - - I I AI AI - - 2 2 CAI CAI CAI CAI - - - - I I I I AI AI AI AI - - - - 2 2 2 2 LuxP LuxP LuxP LuxP HI HI HI HI I.M. I.M. I.M. I.M. P P P P HI HI HI HI CqsA CqsA CqsS CqsS LuxQ LuxQ LuxS LuxS CqsA CqsA CqsA CqsA CqsS CqsS LuxQ LuxQ LuxQ LuxQ LuxS LuxS LuxS LuxS DI DI DI DI DI DI DI DI P P P P P P LuxU LuxU H2 H2 LuxU LuxU H2 H2 LuxO LuxO P P LuxO LuxO + + σ σ 54 54 D2 D2 + + σ σ 54 54 D2 D2 sRNA + Hfq? Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR HapR HapR

All four Qrr sRNAs are regulated by LuxO 1010 ΔluxO luxO D47E 109 qrr-lux expression 108 107 106 qrr1 qrr2 qrr3 qrr4 qrr1 qrr2 qrr3 qrr4

Hypothesis: Hfq and a small RNA repress HapR Low Cell Density High Cell Density O.M. O.M. O.M. O.M. System 1 System 1 System 2 System 2 System 1 System 1 System 2 System 2 CAI CAI - - I I AI AI - - 2 2 CAI CAI CAI CAI - - - - I I I I AI AI AI AI - - - - 2 2 2 2 LuxP LuxP LuxP LuxP HI HI HI HI I.M. I.M. I.M. I.M. P P P P HI HI HI HI CqsA CqsA CqsS CqsS LuxQ LuxQ LuxS LuxS CqsA CqsA CqsA CqsA CqsS CqsS LuxQ LuxQ LuxQ LuxQ LuxS LuxS LuxS LuxS DI DI DI DI DI DI DI DI P P P P P P LuxU LuxU H2 H2 LuxU LuxU H2 H2 LuxO LuxO P P LuxO LuxO + + σ σ 54 54 D2 D2 + + σ σ 54 54 D2 D2 sRNA + Hfq? Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR HapR HapR

All four Qrr sRNAs must be deleted to eliminate quorum sensing 10 11 WT 1.00E+10 10 10 luxO 1.00E+09 10 9 qrr1+ 1.00E+08 10 8 qrr2+ RLU V.c. 1.00E+07 10 7 qrr3+ 1.00E+06 10 6 qrr4+ 1.00E+05 10 5 4 sRNA- 1.00E+04 10 4 0.0001 0.001 0.01 0.1 1 10 100 0.0001 0.001 0.01 0.1 1 10 100 OD600 Lenz et al. 2004

The quorum sensing circuit of Vibrio cholerae. Low Cell Density HI DI LuxP CqsA LuxS H2 Qrr sRNAs + Hfq HapR hapR mRNA P D2 qrr1 - 4 CAI I AI 2 σ 54 CqsS LuxQ Pathway 1 Pathway 2 I.M. O.M. LuxU LuxO Activated genes OFF High Cell Density O.M. Pathway 1 Pathway 2 CAI - I AI - 2 LuxP I.M. HI HI CqsA CqsS LuxQ LuxS DI DI P P LuxU H2 LuxO D2 σ X 54 qrr1 - 4 Hfq hapR mRNA Repressed genes ON Activated genes ON Repressed genes OFF HapR

Regulation by small RNAs Storz et al. 2004

Qrr1-4 decrease the stability of hapR mRNA wildtype ∆hfq ∆qrr1-4 0 5 10 30 60 0 5 10 30 60 0 5 10 30 60 Minutes after rif. hapR mRNA Total RNA

The quorum sensing circuit of Vibrio cholerae. Low Cell Density High Cell Density O.M. O.M. Pathway 1 Pathway 2 Pathway 1 Pathway 2 CAI - I AI - 2 CAI - I AI - 2 LuxP LuxP I.M. I.M. HI HI HI HI CqsA CqsS P P LuxQ LuxS CqsA CqsS LuxQ LuxS DI DI DI DI P P P LuxU H2 LuxU H2 LuxO P LuxO X D2 σ 54 qrr1 - 4 D2 σ 54 qrr1 - 4 Hfq Qrr sRNAs + Hfq hapR mRNA hapR mRNA Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR

hapR mRNA and Qrr sRNAs accumulate reciprocally

The quorum sensing circuit of Vibrio cholerae. Low Cell Density High Cell Density O.M. O.M. Pathway 1 Pathway 2 Pathway 1 Pathway 2 CAI - I AI - 2 CAI - I AI - 2 LuxP LuxP I.M. I.M. HI HI HI HI CqsA CqsS P P LuxQ LuxS CqsA CqsS LuxQ LuxS DI DI DI DI P P P LuxU H2 LuxU H2 LuxO P LuxO X D2 σ 54 qrr1 - 4 D2 σ 54 qrr1 - 4 Hfq Qrr sRNAs + Hfq hapR mRNA hapR mRNA Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR

Qrr1-4 levels vary proportionally with hapR levels.

The quorum sensing circuit of Vibrio cholerae. Low Cell Density High Cell Density O.M. O.M. Pathway 1 Pathway 2 Pathway 1 Pathway 2 CAI - I AI - 2 CAI - I AI - 2 LuxP LuxP I.M. I.M. HI HI HI HI CqsA CqsS P P LuxQ LuxS CqsA CqsS LuxQ LuxS DI DI DI DI P P P LuxU H2 LuxU H2 LuxO P LuxO X D2 σ 54 qrr1 - 4 D2 σ 54 qrr1 - 4 Hfq Qrr sRNAs + Hfq hapR mRNA hapR mRNA Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR

HapR activates transcription of the qrr promoters

HapR protein, and not the 5’UTR, activates qrr4 qrr4-lux HapR protein, and not the 5’UTR, activates qrr4

The quorum sensing circuit of Vibrio cholerae. Low Cell Density High Cell Density O.M. O.M. Pathway 1 Pathway 2 Pathway 1 Pathway 2 CAI - I AI - 2 CAI - I AI - 2 LuxP LuxP I.M. I.M. HI HI HI HI CqsA CqsS P P LuxQ LuxS CqsA CqsS LuxQ LuxS DI DI DI DI P P P LuxU H2 LuxU H2 LuxO P LuxO X D2 σ 54 qrr1 - 4 D2 σ 54 qrr1 - 4 Hfq Qrr sRNAs + Hfq hapR mRNA hapR mRNA Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR

HapR feedback is only relevant in the presence of LuxO-P Pqrr4-lux WT, pHapR WT, pVector luxO D47A, pHapR luxO D47A, pVector RLU/OD600/mL OD600

The quorum sensing circuit of Vibrio cholerae. Low Cell Density High Cell Density O.M. O.M. Pathway 1 Pathway 2 Pathway 1 Pathway 2 CAI - I AI - 2 CAI - I AI - 2 LuxP LuxP I.M. I.M. HI HI HI HI CqsA CqsS P P LuxQ LuxS CqsA CqsS LuxQ LuxS DI DI DI DI P P P LuxU H2 LuxU H2 LuxO P LuxO X D2 σ 54 qrr1 - 4 D2 σ 54 qrr1 - 4 Hfq Qrr sRNAs + Hfq hapR mRNA hapR mRNA Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR

Possible effects of the feedback loop The HapR feedback could: Accelerate the transition from social mode to individual cell mode. Allow the four Qrr sRNAs to compensate for each other. ? ?

Does hapR accelerate the transition? High cell density cultures Used medium “High cell density” wash Wildtype Fresh medium “Low cell density” Used medium “High cell density” wash ∆hapR Fresh medium “Low cell density”

HapR accelerates the transition to low cell density mode. qrr1-4 Wildtype ∆hapR fresh used fresh used hapR Minutes after wash 0 2 5 10 30 0 2 5 10 30 0 2 5 10 30 0 2 5 10 30 Qrr4 hapR mRNA 5S RNA

Possible effects of the feedback loop The HapR feedback could: Accelerate the transition from social mode to individual cell mode. Allow the four Qrr sRNAs to compensate for each other. ?

The quorum sensing circuit of Vibrio cholerae. Low Cell Density High Cell Density O.M. O.M. Pathway 1 Pathway 2 Pathway 1 Pathway 2 CAI - I AI - 2 CAI - I AI - 2 LuxP LuxP I.M. I.M. HI HI HI HI CqsA CqsS P P LuxQ LuxS CqsA CqsS LuxQ LuxS DI DI DI DI P P P LuxU H2 LuxU H2 LuxO P LuxO X D2 σ 54 qrr1 - 4 D2 σ 54 qrr1 - 4 Hfq Qrr sRNAs + Hfq hapR mRNA hapR mRNA Activated genes OFF Repressed genes ON Activated genes ON Repressed genes OFF HapR HapR

Bassler Lab members: Jian-Ping Cong Acknowledgements Bonnie Bassler Derrick Lenz Kenny Mok Conrad Shebelut Yunzhou Wei My thesis committee: Tom Silhavy Ned Wingreen Bassler Lab members: Jian-Ping Cong Mike Federle Brian Hammer Doug Higgins Wai-Leung Ng Audra Pompeani Danielle Swem Lee Swem Kim Tu Chris Waters Julie Wu