The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae

English version

Autoři: James R. J. Haycocks ^aff001; Gemma Z. L. Warren ^aff001; Lucas M. Walker ^aff001; Jennifer L. Chlebek ^aff002; Triana N. Dalia ^aff002; Ankur B. Dalia ^aff002; David C. Grainger ^aff001
Působiště autorů: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom ^aff001; Department of Biology, Indiana University, Bloomington, IN, United States of America ^aff002
Vyšlo v časopise: The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae. PLoS Genet 15(10): e32767. doi:10.1371/journal.pgen.1008362
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008362

Souhrn

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes.

Klíčová slova:

Deoxyribonucleases – DNA transcription – Genetic footprinting – Population density – RNA polymerase – Sequence motif analysis – Vibrio cholerae – DNA footprinting

Zdroje

1. Nelson EJ1, Harris JB, Morris JG Jr, Calderwood SB, Camilli A. 2009. Cholera transmission: the host, pathogen and bacteriophage dynamic. Nat Rev Microbiol. 2009 7:693–702. doi: 10.1038/nrmicro2204 19756008

2. Nalin DR, Daya V, Reid A, Levine MM, Cisneros L. 1979. Adsorption and growth of Vibrio cholerae on chitin. Infect Immun. 25:768–70. 489131

3. Jude BA, Martinez RM, Skorupski K, Taylor RK. 2009. Levels of the secreted Vibrio cholerae attachment factor GbpA are modulated by quorum-sensing-induced proteolysis. J Bacteriol. 191:6911–6917. doi: 10.1128/JB.00747-09 19734310

4. Yamamoto S, Morita M, Izumiya H, Watanabe H. 2010. Chitin disaccharide (GlcNAc)2 induces natural competence in Vibrio cholerae through transcriptional and translational activation of a positive regulatory gene tfoXVC. Gene. 457:42–9. doi: 10.1016/j.gene.2010.03.003 20302923

5. Yamamoto S, Izumiya H, Mitobe J, Morita M, Arakawa E, Ohnishi M, Watanabe H. 2011. Identification of a chitin-induced small RNA that regulates translation of the tfoX gene, encoding a positive regulator of natural competence in Vibrio cholerae. J Bacteriol. 193:1953–1965. doi: 10.1128/JB.01340-10 21317321

6. Yamamoto S, Mitobe J, Ishikawa T, Wai SN, Ohnishi M, Watanabe H, Izumiya H. 2014. Regulation of natural competence by the orphan two-component system sensor kinase ChiS involves a non-canonical transmembrane regulator in Vibrio cholerae. Mol Microbiol. 91:326–347.

7. Meibom KL, Li XB, Nielsen AT, Wu CY, Roseman S, Schoolnik GK. 2004. The Vibrio cholerae chitin utilization program. Proc Natl Acad Sci USA. 101:2524–2529. doi: 10.1073/pnas.0308707101 14983042

8. Meibom KL, Blokesch M, Dolganov NA, Wu CY, Schoolnik GK. 2005. Chitin induces natural competence in Vibrio cholerae. Science 310:1824–1827. doi: 10.1126/science.1120096 16357262

9. Borgeaud S, Metzger LC, Scrignari T, Blokesch M. 2015. The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer. Science. 347:63–67. doi: 10.1126/science.1260064 25554784

10. Seitz P, Blokesch M. 2013. DNA-uptake machinery of naturally competent Vibrio cholerae. Proc Natl Acad Sci USA. 110:17987–17992. doi: 10.1073/pnas.1315647110 24127573

11. Lo Scrudato M, Blokesch M. 2012. The regulatory network of natural competence and transformation of Vibrio cholerae. PLoS Genet. 8:e1002778. doi: 10.1371/journal.pgen.1002778 22737089

12. Dalia AB, McDonough E, Camilli A. 2014. Multiplex genome editing by natural transformation. Proc Natl Acad Sci USA. 111:8937–8942. doi: 10.1073/pnas.1406478111 24889608

13. Ellison CK, Dalia TN, Vidal Ceballos A, Wang JC, Biais N, Brun YV, Dalia AB. 2018. Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae. Nat. Microbiol. 3:773–780. doi: 10.1038/s41564-018-0174-y 29891864

14. Seitz P, Pezeshgi Modarres H, Borgeaud S, Bulushev RD, Steinbock LJ, Radenovic A, Dal Peraro M, Blokesch M. 2014. ComEA is essential for the transfer of external DNA into the periplasm in naturally transformable Vibrio cholerae cells. PLoS Genet. 10:e1004066. doi: 10.1371/journal.pgen.1004066 24391524

15. Hay AJ, Zhu J. 2015. Host intestinal signal-promoted biofilm dispersal induces Vibrio cholerae colonization. Infect Immun. 83:317–323. doi: 10.1128/IAI.02617-14 25368110

16. Childers BM, Klose KE. 2007. Regulation of virulence in Vibrio cholerae; the ToxR regulon. Future Microbiol 2:335–44. doi: 10.2217/17460913.2.3.335 17661707

17. Whiteley M, Diggle SP, Greenberg EP. 2017. Progress in and promise of bacterial quorum sensing research. Nature. 551:313–320. doi: 10.1038/nature24624 29144467

18. Mukherjee S, Bassler BL. 2019. Bacterial quorum sensing in complex and dynamically changing environments. Nat Rev Microbiol. doi: 10.1038/s41579-019-0186-5 [Epub ahead of print]. 30944413

19. Ng WL, Perez LJ, Wei Y, Kraml C, Semmelhack MF, Bassler BL. 2011. Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems. Mol Microbiol. 79:1407–1417. doi: 10.1111/j.1365-2958.2011.07548.x 21219472

20. Eickhoff MJ, Bassler BL. 2018. SnapShot: Bacterial Quorum Sensing. Cell. 174:1328–1328. doi: 10.1016/j.cell.2018.08.003 30142348

21. De Silva RS, Kovacikova G, Lin W, Taylor RK, Skorupski K, Kull FJ. 2005. Crystal structure of the virulence gene activator AphA from Vibrio cholerae reveals it is a novel member of the winged helix transcription factor superfamily. J Biol Chem. 280:13779–13783. doi: 10.1074/jbc.M413781200 15647287

22. Rutherford ST, van Kessel JC, Shao Y, Bassler BL. 2011. AphA and LuxR/HapR reciprocally control quorum sensing in vibrios. Genes Dev. 25:397–408. doi: 10.1101/gad.2015011 21325136

23. Skorupski K, Taylor RK. 1999. A new level in the Vibrio cholerae ToxR virulence cascade: AphA is required for transcriptional activation of the tcpPH operon. Mol Microbiol. 31:763–771 doi: 10.1046/j.1365-2958.1999.01215.x 10048021

24. Kovacikova G, Lin W, Skorupski K. 2004. Vibrio cholerae AphA uses a novel mechanism for virulence gene activation that involves interaction with the LysR-type regulator AphB at the tcpPH promoter. Mol Microbiol. 53:129–142. doi: 10.1111/j.1365-2958.2004.04121.x 15225309

25. van Kessel JC, Rutherford ST, Shao Y, Utria AF, Bassler BL. 2013. Individual and combined roles of the master regulators AphA and LuxR in control of the Vibrio harveyi quorum-sensing regulon. J Bacteriol. 195:436–443. doi: 10.1128/JB.01998-12 23204455

26. Sun F, Zhang Y, Wang L, Yan X, Tan Y, Guo Z, Qiu J, Yang R, Xia P, Zhou D. 2012. Molecular characterization of direct target genes and cis-acting consensus recognized by quorum-sensing regulator AphA in Vibrio parahaemolyticus. PLoS One. 7:e44210. doi: 10.1371/journal.pone.0044210 22984476

27. Gu D, Liu H, Yang Z, Zhang Y, Wang Q. 2016. Chromatin Immunoprecipitation Sequencing Technology Reveals Global Regulatory Roles of Low-Cell-Density Quorum-Sensing Regulator AphA in the Pathogen Vibrio alginolyticus. J Bacteriol. 198:2985–2999. doi: 10.1128/JB.00520-16 27551022

28. Ng D, Harn T, Altindal T, Kolappan S, Marles JM, Lala R, Spielman I, Gao Y, Hauke CA, Kovacikova G, Verjee Z, Taylor RK, Biais N, Craig L. 2016. The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus. PLoS Pathog. 12:e1006109. doi: 10.1371/journal.ppat.1006109 27992883

29. Nguyen Y, Sugiman-Marangos S, Harvey H, Bell SD, Charlton CL, Junop MS, Burrows LL. 2015. Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin. J Biol Chem. 290:601–611. doi: 10.1074/jbc.M114.616904 25389296

30. Papenfort K, Förstner KU, Cong JP, Sharma CM, Bassler BL. 2015. Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation. Proc Natl Acad Sci USA. 112:E766–75. doi: 10.1073/pnas.1500203112 25646441

31. Herzog R, Peschek N, Fröhlich KS, Schumacher K, Papenfort K. 2019. Three autoinducer molecules act in concert to control virulence gene expression in Vibrio cholerae. Nucleic Acids Res. 47:3171–3183. doi: 10.1093/nar/gky1320 30649554

32. Wu R, Zhao M, Li J, Gao H, Kan B, Liang W. 2015. Direct regulation of the natural competence regulator gene tfoX by cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Vibrios. Sci Rep. 5:14921. doi: 10.1038/srep14921 26442598

33. Schultz SC, Shields GC, Steitz TA. 1991. Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees. Science. 253:1001–1007. doi: 10.1126/science.1653449 1653449

34. Park SC, Kwak YM, Song WS, Hong M, Yoon SI. 2017. Structural basis of effector and operator recognition by the phenolic acid-responsive transcriptional regulator PadR. Nucleic Acids Res. 45:13080–13093. doi: 10.1093/nar/gkx1055 29136175

35. Liang W1, Sultan SZ, Silva AJ, Benitez JA. 2008. Cyclic AMP post-transcriptionally regulates the biosynthesis of a major bacterial autoinducer to modulate the cell density required to activate quorum sensing. FEBS Lett. 582:3744–3750. doi: 10.1016/j.febslet.2008.10.008 18930049

36. Freeman JA, Bassler BL. 1999. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. Mol Microbiol. 31:665–677. doi: 10.1046/j.1365-2958.1999.01208.x 10027982

37. Singh PK, Bartalomej S, Hartmann R, Jeckel H, Vidakovic L, Nadell CD, Drescher K. 2017. Vibrio cholerae Combines Individual and Collective Sensing to Trigger Biofilm Dispersal. Curr Biol. 27:3359–3366. doi: 10.1016/j.cub.2017.09.041 29056457

38. Blokesch M, Schoolnik GK. 2008. The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae. J Bacteriol. 190:7232–7240. doi: 10.1128/JB.00959-08 18757542

39. Gangel H, Hepp C, Müller S, Oldewurtel ER, Aas FE, Koomey M, Maier B. 2014. Concerted spatio-temporal dynamics of imported DNA and ComE DNA uptake protein during gonococcal transformation. PLoS Pathog. 10:e1004043. doi: 10.1371/journal.ppat.1004043 24763594

40. Hepp C, Maier B. 2016. Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism. Proc Natl Acad Sci USA. 113:12467–12472. doi: 10.1073/pnas.1608110113 27791096

41. Pollack-Berti A, Wollenberg MS, Ruby EG. 2010. Natural transformation of Vibrio fischeri requires tfoX and tfoY. Environ Microbiol. 12:2302–11. doi: 10.1111/j.1462-2920.2010.02250.x 21966921

42. Dalia AB, Lazinski DW, Camilli A. 2014. Identification of a membrane-bound transcriptional regulator that links chitin and natural competence in Vibrio cholerae. mBio. 5:e01028–13. doi: 10.1128/mBio.01028-13 24473132

43. Jaskólska M, Stutzmann S, Stoudmann C, Blokesch M. 2018. QstR-dependent regulation of natural competence and type VI secretion in Vibrio cholerae. Nucleic Acids Res. 46:10619–10634. doi: 10.1093/nar/gky717 30102403

44. Lo Scrudato M, Blokesch M. 2013. A transcriptional regulator linking quorum sensing and chitin induction to render Vibrio cholerae naturally transformable. Nucleic Acids Res. 41:3644–3658. doi: 10.1093/nar/gkt041 23382174

45. Kovacikova G, Skorupski K. 2001. Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter. Mol Microbiol. 41:393–407. doi: 10.1046/j.1365-2958.2001.02518.x 11489126

46. Kovacikova G, Lin W, Skorupski K. 2005. Dual regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate-responsive LysR-type regulator AlsR in Vibrio cholerae. Mol Microbiol. 57:420–433. doi: 10.1111/j.1365-2958.2005.04700.x 15978075

47. Häse CC, Mekalanos JJ. 1998. TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA. 95:730–734. doi: 10.1073/pnas.95.2.730 9435261

48. Kovacikova G, Lin W, Skorupski K. 2003. The virulence activator AphA links quorum sensing to pathogenesis and physiology in Vibrio cholerae by repressing the expression of a penicillin amidase gene on the small chromosome. J Bacteriol. 185:4825–4836. doi: 10.1128/JB.185.16.4825-4836.2003 12897002

49. Haycocks JR, Sharma P, Stringer AM, Wade JT, Grainger DC. 2015. The molecular basis for control of ETEC enterotoxin expression in response to environment and host. PLoS Pathog. 11:e1004605. doi: 10.1371/journal.ppat.1004605 25569153

50. Sharma P, Haycocks JRJ, Middlemiss AD, Kettles RA, Sellars LE, Ricci V, Piddock LJV, Grainger DC. 2017. The multiple antibiotic resistance operon of enteric bacteria controls DNA repair and outer membrane integrity. Nat Commun. 8:1444. doi: 10.1038/s41467-017-01405-7 29133912

51. Li H. Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics. 25:1754–1760. doi: 10.1093/bioinformatics/btp324 19451168

52. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 25:2078–2079. doi: 10.1093/bioinformatics/btp352 19505943

53. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Čech M, Chilton J, Clements D, Coraor N, Grüning B, Guerler A, Hillman-Jackson J, Jalili V, Rasche H, Soranzo N, Goecks J, Taylor J, Nekrutenko A, Blankenberg D. 2018. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Research 46:W537–W544. doi: 10.1093/nar/gky379 29790989

54. Ramírez F, Ryan DP, Grüning B, Bhardwaj V, Kilpert F, Richter AS, Heyne S, Dündar F, Manke T. 2016. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44:W160–5. doi: 10.1093/nar/gkw257 27079975

55. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. 2009. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37:W202–8. doi: 10.1093/nar/gkp335 19458158

56. Manneh-Roussel J, Haycocks JRJ, Magán A, Perez-Soto N, Voelz K, Camilli A, Krachler AM, Grainger DC. 2018. cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization. mBio. 9:e00966–18. doi: 10.1128/mBio.00966-18 29991587

57. Burgess RR, Jendrisak JJ. 1975. A procedure for the rapid, large-scale purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 14:4634–4638. doi: 10.1021/bi00692a011 1101952

58. Lamberte LE, Baniulyte G, Singh SS, Stringer AM, Bonocora RP, Stracy M, Kapanidis AN, Wade JT, Grainger DC. 2017. Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase. Nat Microbiol. 2:16249. doi: 10.1038/nmicrobiol.2016.249 28067866

59. Grainger DC, Goldberg MD, Lee DJ, Busby SJW. 2008. Selective repression by Fis and H-NS at the Escherichia coli dps promoter. Mol Microbiol. 68:1366–1377. doi: 10.1111/j.1365-2958.2008.06253.x 18452510

60. Chintakayala K, Singh SS, Rossiter AE, Shahapure R, Dame RT, Grainger DC. 2013. E. coli Fis Protein Insulates the cbpA Gene from Uncontrolled Transcription. PLoS Genet. 9:e1003152. doi: 10.1371/journal.pgen.1003152 23341772

61. Singh SS, Grainger DC. 2013. H-NS Can Facilitate Specific DNA-binding by RNA Polymerase in AT-rich Gene Regulatory Regions. PLoS Genet. 9:e1003589. doi: 10.1371/journal.pgen.1003589 23818873

62. Kolb A, Kotlarz D, Kusano S, Ishihama A. 1995. Selectivity of the Escherichia coli RNA polymerase Eσ38 for overlapping promoters and ability to support CRP activation. Nucleic Acids Res. 23:819–826. doi: 10.1093/nar/23.5.819 7708498

63. Savery NJ, Lloyd GS, Kainz M, Gaal T, Ross W, Ebright RH, et al. 1998. Transcription activation at class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase α subunit. EMBO J. 17:3439–3447. doi: 10.1093/emboj/17.12.3439 9628879

64. Miller JH. 1972. Experiments in molecular genetics. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.