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  • Review Article
  • Published:

Living in the matrix: assembly and control of Vibrio cholerae biofilms

Key Points

  • Biofilms of Vibrio cholerae, the causative agent of cholera, have an important role during the aquatic and intestinal phases of the bacterial life cycle, conferring greater resistance to environmental stresses and increasing infectivity.

  • V. cholerae biofilm formation is a multistep process that begins with initial attachment via the bacterial mannose-sensitive haemagglutinin (MSHA) pili. The key components of a V. cholerae biofilm are secreted by the cell at various times during biofilm formation and include Vibrio polysaccharide (VPS), the biofilm proteins rugosity and biofilm structure modulator A (RbmA), Bap1 and RbmC, and extracellular DNA, all of which are critical for the formation of mature biofilms.

  • Biofilm formation in V. cholerae is controlled by an integrated network of transcriptional regulators. The major transcriptional activators include VpsR, VpsT and AphA, and major transcriptional repressors include HapR and H-NS; alternative RNA polymerase sigma factors, regulatory small RNAs and signalling molecules also function as regulators of this complex process.

  • Nucleotide-based signals play an important part in controlling biofilm formation and include cyclic di-GMP (c-di-GMP), which positively regulates biofilm formation and negatively regulates motility to influence the planktonic-to-biofilm transition. Additionally, cyclic AMP represses biofilm formation, and guanosine tetraphosphate and guanosine pentaphosphate (collectively called (p)ppGpp) enhance biofilm formation.

  • V. cholerae biofilm formation is influenced by a number of fluctuating environmental factors, including nutritional status, shifts in salinity and osmolarity, phosphate limitation, the presence of polyamines, variations in calcium levels, and exposure to indole and bile. The ability of V. cholerae to activate or repress biofilm formation in response to external signals probably contributes to the environmental survival and persistence of the bacterium and demonstrates the complexity of the biofilm regulation programme.

  • New small-molecule therapeutics have emerged that target and disrupt V. cholerae biofilm formation. Such therapeutics include quorum sensing inhibitors, disruptors of c-di-GMP signalling and compounds with unknown molecular targets. Whole-cell phenotypic imaging coupled with cellular-viability measurements have been used to differentiate bactericidal agents and compounds that selectively disrupt biofilm formation without affecting cell survival. These studies have led to the discovery of several compounds that show promise for biofilm inhibition and for the treatment of cholera.

Abstract

Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are associated with biotic and abiotic surfaces and are composed of aggregates of cells that are encased by a self-produced or acquired extracellular matrix. Vibrio cholerae has been studied as a model organism for understanding biofilm formation in environmental pathogens, as it spends much of its life cycle outside of the human host in the aquatic environment. Given the important role of biofilm formation in the V. cholerae life cycle, the molecular mechanisms underlying this process and the signals that trigger biofilm assembly or dispersal have been areas of intense investigation over the past 20 years. In this Review, we discuss V. cholerae surface attachment, various matrix components and the regulatory networks controlling biofilm formation.

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Figure 1: Biofilms in the Vibrio cholerae life cycle.
Figure 2: Building a Vibrio cholerae biofilm.
Figure 3: The regulatory network controlling Vibrio cholerae biofilms.

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References

  1. Charles, R. C. & Ryan, E. T. Cholera in the 21st century. Curr. Opin. Infect. Dis. 24, 472–477 (2011).

    PubMed  Google Scholar 

  2. Islam, M. S. et al. Biofilm acts as a microenvironment for plankton-associated Vibrio cholerae in the aquatic environment of Bangladesh. Microbiol. Immunol. 51, 369–379 (2007).

    CAS  PubMed  Google Scholar 

  3. Faruque, S. M. et al. Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment. Proc. Natl Acad. Sci. USA 103, 6350–6355 (2006). This study demonstrates that the ingestion of metabolically quiescent V. cholerae cells can lead to the formation of in vivo biofilms that are hyperinfective when excreted in patient stool.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Alam, M. et al. Viable but nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in cholera transmission. Proc. Natl Acad. Sci. USA 104, 17801–17806 (2007). This report reveals the crucial role of biofilms in maintaining environmental reservoirs between epidemics as well as the role of host passage in the activation of metabolically quiescent cells.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Matz, C. et al. Biofilm formation and phenotypic variation enhance predation-driven persistence of Vibrio cholerae. Proc. Natl Acad. Sci. USA 102, 16819–16824 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Beyhan, S. & Yildiz, F. H. Smooth to rugose phase variation in Vibrio cholerae can be mediated by a single nucleotide change that targets c-di-GMP signalling pathway. Mol. Microbiol. 63, 995–1007 (2007).

    CAS  PubMed  Google Scholar 

  7. Tamplin, M. L., Gauzens, A. L., Huq, A., Sack, D. A. & Colwell, R. R. Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters. Appl. Environ. Microbiol. 56, 1977–1980 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Rawlings, T. K., Ruiz, G. M. & Colwell, R. R. Association of Vibrio cholerae O1 El Tor and O139 Bengal with the Copepods Acartia tonsa and Eurytemora affinis. Appl. Environ. Microbiol. 73, 7926–7933 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Meibom, K. L. et al. The Vibrio cholerae chitin utilization program. Proc. Natl Acad. Sci. USA 101, 2524–2529 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Li, X. & Roseman, S. The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase. Proc. Natl Acad. Sci. USA 101, 627–631 (2004).

    CAS  PubMed  Google Scholar 

  11. Meibom, K. L., Blokesch, M., Dolganov, N. A., Wu, C.-Y. & Schoolnik, G. K. Chitin induces natural competence in Vibrio cholerae. Science 310, 1824–1827 (2005).

    CAS  PubMed  Google Scholar 

  12. Lipp, E. K., Huq, A. & Colwell, R. R. Effects of global climate on infectious disease: the cholera model. Clin. Microbiol. Rev. 15, 757–770 (2002).

    PubMed  PubMed Central  Google Scholar 

  13. Huq, A. et al. Critical factors influencing the occurrence of Vibrio cholerae in the environment of Bangladesh. Appl. Environ. Microbiol. 71, 4645–4654 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Colwell, R. R. et al. Reduction of cholera in Bangladeshi villages by simple filtration. Proc. Natl Acad. Sci. USA 100, 1051–1055 (2003). This work shows that simple filtration to remove particulates >20 μm could reduce cholera outbreaks by 48%, suggesting that the removal of biofilms can significantly reduce V. cholerae infections.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Bari, S. M. N. et al. Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples. Proc. Natl Acad. Sci. USA 110, 9926–9931 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Colwell, R. R. et al. Viable but non-culturable Vibrio cholerae O1 revert to a cultivable state in the human intestine. World J. Microbiol. Biotechnol. 12, 28–31 (1996).

    CAS  PubMed  Google Scholar 

  17. Tamayo, R., Patimalla, B. & Camilli, A. Growth in a biofilm induces a hyperinfectious phenotype in Vibrio cholerae. Infect. Immun. 78, 3560–3569 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Nielsen, A. T. et al. A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine. PLoS Pathog. 6, e1001102 (2010).

    PubMed  PubMed Central  Google Scholar 

  19. Hang, L. et al. Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae. Proc. Natl Acad. Sci. USA 100, 8508–8513 (2003).

    PubMed  PubMed Central  Google Scholar 

  20. Lombardo, M.-J. et al. An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers. Proc. Natl Acad. Sci. USA 104, 18229–18234 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Fong, J. C. N., Syed, K. A., Klose, K. E. & Yildiz, F. H. Role of Vibrio polysaccharide (vps) genes in VPS production, biofilm formation and Vibrio cholerae pathogenesis. Microbiology 156, 2757–2769 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Utada, A. S. et al. Vibrio cholerae use pili and flagella synergistically to effect motility switching and conditional surface attachment. Nature Commun. 5, 4913 (2014). This paper reveals the unique near-surface motility behaviours and attachment steps of V. cholerae.

    CAS  Google Scholar 

  23. Berke, A., Turner, L., Berg, H. & Lauga, E. Hydrodynamic attraction of swimming microorganisms by surfaces. Phys. Rev. Lett. 101, 038102 (2008).

    PubMed  Google Scholar 

  24. Kojima, S., Yamamoto, K., Kawagishi, I. & Homma, M. The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force. J. Bacteriol. 181, 1927–1930 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Lauga, E., DiLuzio, W. R., Whitesides, G. M. & Stone, H. A. Swimming in circles: motion of bacteria near solid boundaries. Biophys. J. 90, 400–412 (2006).

    CAS  PubMed  Google Scholar 

  26. Watnick, P. I. & Kolter, R. Steps in the development of a Vibrio cholerae El Tor biofilm. Mol. Microbiol. 34, 586–595 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Gibiansky, M. L. et al. Bacteria use type IV pili to walk upright and detach from surfaces. Science 330, 197 (2010).

    CAS  PubMed  Google Scholar 

  28. O'Toole, G. A. & Kolter, R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30, 295–304 (1998).

    CAS  PubMed  Google Scholar 

  29. Zhao, K. et al. Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms. Nature 497, 388–391 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Gloag, E. S. et al. Self-organization of bacterial biofilms is facilitated by extracellular DNA. Proc. Natl Acad. Sci. USA 110, 11541–11546 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Watnick, P. I., Lauriano, C. M., Klose, K. E., Croal, L. & Kolter, R. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol. Microbiol. 39, 223–235 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Lauriano, C. M., Ghosh, C., Correa, N. E. & Klose, K. E. The sodium-driven flagellar motor controls exopolysaccharide expression in Vibrio cholerae. J. Bacteriol. 186, 4864–4874 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Reichhardt, C., Fong, J. C. N., Yildiz, F. & Cegelski, L. Characterization of the Vibrio cholerae extracellular matrix: a top-down solid-state NMR approach. Biochim. Biophys. Acta 1848, 378–383 (2015).

    CAS  PubMed  Google Scholar 

  34. Yildiz, F. H. & Schoolnik, G. K. Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Proc. Natl Acad. Sci. USA 96, 4028–4033 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Yildiz, F., Fong, J., Sadovskaya, I., Grard, T. & Vinogradov, E. Structural characterization of the extracellular polysaccharide from Vibrio cholerae O1 El-Tor. PLoS ONE 9, e86751 (2014). The article reports, for the first time, the chemical structure of the polysaccharide portion of VPS and introduces a new method for the reliable isolation of VPS from extracellular material.

    PubMed  PubMed Central  Google Scholar 

  36. Berk, V. et al. Molecular architecture and assembly principles of Vibrio cholerae biofilms. Science 337, 236–239 (2012). This study uses an in vivo labelling strategy to visualize and characterize the spatial and temporal organization of the major V. cholerae biofilm components — VPS, RbmA, Bap1 and RbmC — revealing unique roles for each within the biofilm.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Fong, J. C. N. & Yildiz, F. H. The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae. J. Bacteriol. 189, 2319–2330 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Fong, J. C. N., Karplus, K., Schoolnik, G. K. & Yildiz, F. H. Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae. J. Bacteriol. 188, 1049–1059 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Nesper, J. et al. Characterization of Vibrio cholerae O1 El tor galU and galE mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation. Infect. Immun. 69, 435–445 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Absalon, C., Van Dellen, K. & Watnick, P. I. A communal bacterial adhesin anchors biofilm and bystander cells to surfaces. PLoS Pathog. 7, e1002210 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Giglio, K. M., Fong, J. C., Yildiz, F. H. & Sondermann, H. Structural basis for biofilm formation via the Vibrio cholerae matrix protein RbmA. J. Bacteriol. 195, 3277–3286 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Maestre-Reyna, M., Wu, W.-J. & Wang, A. H.-J. Structural insights into RbmA, a biofilm scaffolding protein of V. cholerae. PLoS ONE 8, e82458 (2013).

    PubMed  PubMed Central  Google Scholar 

  43. Hollenbeck, E. C. et al. Molecular determinants of mechanical properties of V. cholerae biofilms at the air-liquid interface. Biophys. J. 107, 2245–2252 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Duperthuy, M. et al. Role of the Vibrio cholerae matrix protein Bap1 in cross-resistance to antimicrobial peptides. PLoS Pathog. 9, e1003620 (2013).

    PubMed  PubMed Central  Google Scholar 

  45. Levan, S. R. & Olson, R. Determination of the carbohydrate-binding specificity of lectin-like domains in Vibrio cholerae cytolysin. Biophys. J. 102, 461a–462a (2012).

    Google Scholar 

  46. Johnson, T. L., Fong, J. C. N., Rule, C., Yildiz, F. H. & Sandkvist, M. The type II secretion system delivers matrix proteins for biofilm formation by Vibrio cholerae. J. Bacteriol. 196, 4245–4252 (2014).

    PubMed  PubMed Central  Google Scholar 

  47. Altindis, E., Fu, Y. & Mekalanos, J. J. Proteomic analysis of Vibrio cholerae outer membrane vesicles. Proc. Natl Acad. Sci. USA 111, E1548–E1556 (2014). This research shows that all three major biofilm matrix proteins are associated with OMVs, which implies that OMVs may have an as-yet- uncharacterized role in biofilm formation.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Seper, A. et al. Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation. Mol. Microbiol. 82, 1015–1037 (2011). This work identifies two extracellular nucleases, DNase and Xds, and characterizes their role in the development of biofilm architecture, nutrient acquisition and biofilm dispersal, revealing previously unknown roles for extracellular nucleases and DNA in biofilm formation.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Hay, A. J. & Zhu, J. Host intestinal signal-promoted biofilm dispersal induces Vibrio cholerae colonization. Infect. Immun. 83, 317–323 (2015).

    PubMed  Google Scholar 

  50. Yildiz, F. H., Dolganov, N. A. & Schoolnik, G. K. VpsR, a member of the response regulators of the two-component regulatory systems, is required for expression of vps biosynthesis genes and EPSETr-associated phenotypes in Vibrio cholerae O1 El Tor. J. Bacteriol. 183, 1716–1726 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Zamorano-Sánchez, D., Fong, J. C. N., Kilic, S., Erill, I. & Yildiz, F. H. Identification and characterization of VpsR and VpsT binding sites in Vibrio cholerae. J. Bacteriol. 197, 1221–1235 (2015).

    PubMed  PubMed Central  Google Scholar 

  52. Beyhan, S., Bilecen, K., Salama, S. R., Casper-Lindley, C. & Yildiz, F. H. Regulation of rugosity and biofilm formation in Vibrio cholerae: comparison of VpsT and VpsR regulons and epistasis analysis of vpsT, vpsR, and hapR. J. Bacteriol. 189, 388–402 (2007).

    CAS  PubMed  Google Scholar 

  53. Yildiz, F. H., Liu, X. S., Heydorn, A. & Schoolnik, G. K. Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant. Mol. Microbiol. 53, 497–515 (2004).

    CAS  PubMed  Google Scholar 

  54. Srivastava, D., Harris, R. C. & Waters, C. M. Integration of cyclic di-GMP and quorum sensing in the control of vpsT and aphA in Vibrio cholerae. J. Bacteriol. 193, 6331–6341 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Krasteva, P. V. et al. Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP. Science 327, 866–868 (2010). This article reports the crystal structure of VpsT and demonstrates that VpsT dimerizes and directly binds a c-di-GMP dimer to activate the transcriptional regulation of genes involved in biofilm regulation.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Yang, M., Frey, E. M., Liu, Z., Bishar, R. & Zhu, J. The virulence transcriptional activator AphA enhances biofilm formation by Vibrio cholerae by activating expression of the biofilm regulator VpsT. Infect. Immun. 78, 697–703 (2010).

    CAS  PubMed  Google Scholar 

  57. Casper-Lindley, C. & Yildiz, F. H. VpsT is a transcriptional regulator required for expression of vps biosynthesis genes and the development of rugose colonial morphology in Vibrio cholerae O1 El Tor. J. Bacteriol. 186, 1574–1578 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. He, H., Cooper, J. N., Mishra, A. & Raskin, D. M. Stringent response regulation of biofilm formation in Vibrio cholerae. J. Bacteriol. 194, 2962–2972 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Zhu, J. & Mekalanos, J. J. Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae. Dev. Cell 5, 647–656 (2003).

    CAS  PubMed  Google Scholar 

  60. Hammer, B. K. & Bassler, B. L. Quorum sensing controls biofilm formation in Vibrio cholerae. Mol. Microbiol. 50, 101–104 (2003).

    CAS  PubMed  Google Scholar 

  61. Jobling, M. G. & Holmes, R. K. Characterization of hapR, a positive regulator of the Vibrio cholerae HA/protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene. Mol. Microbiol. 26, 1023–1034 (1997).

    CAS  PubMed  Google Scholar 

  62. Waters, C. M., Lu, W., Rabinowitz, J. D. & Bassler, B. L. Quorum sensing controls biofilm formation in Vibrio cholerae through modulation of cyclic di-GMP levels and repression of vpsT. J. Bacteriol. 190, 2527–2536 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. De Silva, R. S. et al. Crystal structure of the Vibrio cholerae quorum-sensing regulatory protein HapR. J. Bacteriol. 189, 5683–5691 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Liu, Z., Stirling, F. R. & Zhu, J. Temporal quorum-sensing induction regulates Vibrio cholerae biofilm architecture. Infect. Immun. 75, 122–126 (2007).

    CAS  PubMed  Google Scholar 

  65. Ng, W.-L. & Bassler, B. L. Bacterial quorum-sensing network architectures. Annu. Rev. Genet. 43, 197–222 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Tsou, A. M., Liu, Z., Cai, T. & Zhu, J. The VarS/VarA two-component system modulates the activity of the Vibrio cholerae quorum-sensing transcriptional regulator HapR. Microbiology 157, 1620–1628 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Lenz, D. H., Miller, M. B., Zhu, J., Kulkarni, R. V. & Bassler, B. L. CsrA and three redundant small RNAs regulate quorum sensing in Vibrio cholerae. Mol. Microbiol. 58, 1186–1202 (2005).

    CAS  PubMed  Google Scholar 

  68. Lenz, D. H. & Bassler, B. L. The small nucleoid protein Fis is involved in Vibrio cholerae quorum sensing. Mol. Microbiol. 63, 859–871 (2007).

    CAS  PubMed  Google Scholar 

  69. Shikuma, N. J. et al. Overexpression of VpsS, a hybrid sensor kinase, enhances biofilm formation in Vibrio cholerae. J. Bacteriol. 191, 5147–5158 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Liang, W., Pascual-Montano, A., Silva, A. J. & Benitez, J. A. The cyclic AMP receptor protein modulates quorum sensing, motility and multiple genes that affect intestinal colonization in Vibrio cholerae. Microbiology 153, 2964–2975 (2007).

    CAS  PubMed  Google Scholar 

  71. Liu, Z., Hsiao, A., Joelsson, A. & Zhu, J. The transcriptional regulator VqmA increases expression of the quorum-sensing activator HapR in Vibrio cholerae. J. Bacteriol. 188, 2446–2453 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Wang, H., Ayala, J. C., Silva, A. J. & Benitez, J. A. The histone-like nucleoid structuring protein (H-NS) is a repressor of Vibrio cholerae exopolysaccharide biosynthesis (vps) genes. Appl. Environ. Microbiol. 78, 2482–2488 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Stonehouse, E. A., Hulbert, R. R., Nye, M. B., Skorupski, K. & Taylor, R. K. H-NS binding and repression of the ctx promoter in Vibrio cholerae. J. Bacteriol. 193, 979–988 (2011).

    CAS  PubMed  Google Scholar 

  74. Tischler, A. D. & Camilli, A. Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation. Mol. Microbiol. 53, 857–869 (2004). This report identifies and characterizes the phosphodiesterase VieA, and is the first study to demonstrate the significant role c-di-GMP in regulating biofilm formation.

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Römling, U., Galperin, M. Y. & Gomelsky, M. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev. 77, 1–52 (2013).

    PubMed  PubMed Central  Google Scholar 

  76. Beyhan, S., Odell, L. S. & Yildiz, F. H. Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae. J. Bacteriol. 190, 7392–7405 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Pratt, J. T., Tamayo, R., Tischler, A. D. & Camilli, A. PilZ domain proteins bind cyclic diguanylate and regulate diverse processes in Vibrio cholerae. J. Biol. Chem. 282, 12860–12870 (2007).

    CAS  PubMed  Google Scholar 

  78. Sudarsan, N. et al. Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science 321, 411–413 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Liu, X., Beyhan, S., Lim, B., Linington, R. G. & Yildiz, F. H. Identification and characterization of a phosphodiesterase that inversely regulates motility and biofilm formation in Vibrio cholerae. J. Bacteriol. 192, 4541–4552 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Beyhan, S., Tischler, A. D., Camilli, A. & Yildiz, F. H. Transcriptome and phenotypic responses of Vibrio cholerae to increased cyclic di-GMP level. J. Bacteriol. 188, 3600–3613 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Srivastava, D., Hsieh, M.-L., Khataokar, A., Neiditch, M. B. & Waters, C. M. Cyclic di-GMP inhibits Vibrio cholerae motility by repressing induction of transcription and inducing extracellular polysaccharide production. Mol. Microbiol. 90, 1262–1276 (2013).

    CAS  PubMed  Google Scholar 

  82. Shikuma, N. J., Fong, J. C. N. & Yildiz, F. H. Cellular levels and binding of c-di-GMP control subcellular localization and activity of the Vibrio cholerae transcriptional regulator VpsT. PLoS Pathog. 8, e1002719 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Lim, B., Beyhan, S. & Yildiz, F. H. Regulation of Vibrio polysaccharide synthesis and virulence factor production by CdgC, a GGDEF-EAL domain protein, in Vibrio cholerae. J. Bacteriol. 189, 717–729 (2007).

    CAS  PubMed  Google Scholar 

  84. Beyhan, S. & Yildiz, F. H. in The Second Messenger Cyclic Di-GMP (eds Wolfe, A. J. & Visick, K. L.) 253–269 (American Society for Microbiology Press, 2010).

    Google Scholar 

  85. Hammer, B. K. & Bassler, B. L. Regulatory small RNAs circumvent the conventional quorum sensing pathway in pandemic Vibrio cholerae. Proc. Natl Acad. Sci. USA 104, 11145–11149 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Koestler, B. J. & Waters, C. M. Bile acids and bicarbonate inversely regulate intracellular cyclic di-GMP in Vibrio cholerae. Infect. Immun. 82, 3002–3014 (2014).

    PubMed  PubMed Central  Google Scholar 

  87. Cockerell, S. R. et al. Vibrio cholerae NspS, a homologue of ABC-type periplasmic solute binding proteins, facilitates transduction of polyamine signals independent of their transport. Microbiology 160, 832–843 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Liang, W., Silva, A. J. & Benitez, J. A. The cyclic AMP receptor protein modulates colonial morphology in Vibrio cholerae. Appl. Environ. Microbiol. 73, 7482–7487 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Fong, J. C. N. & Yildiz, F. H. Interplay between cyclic AMP-cyclic AMP receptor protein and cyclic di-GMP signaling in Vibrio cholerae biofilm formation. J. Bacteriol. 190, 6646–6659 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Das, B., Pal, R. R., Bag, S. & Bhadra, R. K. Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene. Mol. Microbiol. 72, 380–398 (2009).

    CAS  PubMed  Google Scholar 

  91. Raskin, D. M., Judson, N. & Mekalanos, J. J. Regulation of the stringent response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae. Proc. Natl Acad. Sci. USA 104, 4636–4641 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Song, T. et al. Vibrio cholerae utilizes direct sRNA regulation in expression of a biofilm matrix protein. PLoS ONE 9, e101280 (2014). This study shows that a conserved sRNA, VrrA, binds to and inhibits the translation of rbmC mRNA, thus providing the first example of direct regulation of a biofilm matrix component by an sRNA.

    PubMed  PubMed Central  Google Scholar 

  93. Song, T. et al. A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles. Mol. Microbiol. 70, 100–111 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Mey, A. R., Craig, S. A. & Payne, S. M. Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of ryhB in biofilm formation. Infect. Immun. 73, 5706–5719 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Kaper, J. B., Morris, J. G. & Levine, M. M. Cholera. Clin. Microbiol. Rev. 8, 48–86 (1995).

    CAS  PubMed  Google Scholar 

  96. Balteanu, I. The receptor structure of Vibrio cholerae (V. comma) with observations on variations in cholera and cholera-like organisms. J. Pathol. Bacteriol. 29, 251–277 (1926).

    Google Scholar 

  97. White, P. B. The rugose variant of vibrios. J. Pathol. Bacteriol. 46, 1–6 (1938).

    CAS  Google Scholar 

  98. Peach, K. C. et al. An image-based 384-well high-throughput screening method for the discovery of biofilm inhibitors in Vibrio cholerae. Mol. Biosyst. 7, 1176–1184 (2011). This investigation uses high-throughput epifluorescence microscopy imaging to evaluate a 3080-member small-molecule library and identifies inhibitors of V. cholerae biofilm formation, leading to the discovery of potential inhibitors.

    CAS  PubMed  PubMed Central  Google Scholar 

  99. Heydorn, A. et al. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146, 2395–2407 (2000).

    CAS  PubMed  Google Scholar 

  100. Houot, L., Chang, S., Absalon, C. & Watnick, P. I. Vibrio cholerae phosphoenolpyruvate phosphotransferase system control of carbohydrate transport, biofilm formation, and colonization of the germfree mouse intestine. Infect. Immun. 78, 1482–1494 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  101. Houot, L., Chang, S., Pickering, B. S., Absalon, C. & Watnick, P. I. The phosphoenolpyruvate phosphotransferase system regulates Vibrio cholerae biofilm formation through multiple independent pathways. J. Bacteriol. 192, 3055–3067 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Ymele-Leki, P., Houot, L. & Watnick, P. I. Mannitol and the mannitol-specific enzyme IIB subunit activate Vibrio cholerae biofilm formation. Appl. Environ. Microbiol. 79, 4675–4683 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Kapfhammer, D., Karatan, E., Pflughoeft, K. J. & Watnick, P. I. Role for glycine betaine transport in Vibrio cholerae osmoadaptation and biofilm formation within microbial communities. Appl. Environ. Microbiol. 71, 3840–3847 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. Shikuma, N. J. & Yildiz, F. H. Identification and characterization of OscR, a transcriptional regulator involved in osmolarity adaptation in Vibrio cholerae. J. Bacteriol. 191, 4082–4096 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Shikuma, N. J., Davis, K. R., Fong, J. N. C. & Yildiz, F. H. The transcriptional regulator, CosR, controls compatible solute biosynthesis and transport, motility and biofilm formation in Vibrio cholerae. Environ. Microbiol. 15, 1387–1399 (2013).

    CAS  PubMed  Google Scholar 

  106. Correll, D. L. Phosphorus: a rate limiting nutrient in surface waters. Poult. Sci. 78, 674–682 (1999).

    CAS  PubMed  Google Scholar 

  107. Jahid, I. K., Silva, A. J. & Benitez, J. A. Polyphosphate stores enhance the ability of Vibrio cholerae to overcome environmental stresses in a low-phosphate environment. Appl. Environ. Microbiol. 72, 7043–7049 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  108. Pratt, J. T., McDonough, E. & Camilli, A. PhoB regulates motility, biofilms, and cyclic di-GMP in Vibrio cholerae. J. Bacteriol. 191, 6632–6642 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  109. Von Kruger, W. M. A., Humphreys, S. & Ketley, J. M. A role for the PhoBR regulatory system homologue in the Vibrio cholerae phosphate-limitation response and intestinal colonization. Microbiology 145, 2463–2475 (1999).

    CAS  PubMed  Google Scholar 

  110. Sultan, S. Z., Silva, A. J. & Benitez, J. A. The PhoB regulatory system modulates biofilm formation and stress response in El Tor biotype Vibrio cholerae. FEMS Microbiol. Lett. 302, 22–31 (2010).

    CAS  PubMed  Google Scholar 

  111. Bilecen, K. & Yildiz, F. H. Identification of a calcium-controlled negative regulatory system affecting Vibrio cholerae biofilm formation. Environ. Microbiol. 11, 2015–2029 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. Kierek, K. & Watnick, P. I. The Vibrio cholerae O139 O-antigen polysaccharide is essential for Ca2+-dependent biofilm development in sea water. Proc. Natl Acad. Sci. USA 100, 14357–14362 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Mueller, R. S., Beyhan, S., Saini, S. G., Yildiz, F. H. & Bartlett, D. H. Indole acts as an extracellular cue regulating gene expression in Vibrio cholerae. J. Bacteriol. 191, 3504–3516 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  114. Hung, D. T., Zhu, J., Sturtevant, D. & Mekalanos, J. J. Bile acids stimulate biofilm formation in Vibrio cholerae. Mol. Microbiol. 59, 193–201 (2006).

    CAS  PubMed  Google Scholar 

  115. Ng, W. -L., Perez, L., Cong, J., Semmelhack, M. F. & Bassler, B. L. Broad spectrum pro-quorum-sensing molecules as inhibitors of virulence in vibrios. PLoS Pathog. 8, e1002767 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. Brackman, G. et al. Structure-activity relationship of cinnamaldehyde analogs as inhibitors of AI-2 based quorum sensing and their effect on virulence of Vibrio spp. PLoS ONE 6, e16084 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. Gutierrez, J. A. et al. Transition state analogs of 5′-methylthioadenosine nucleosidase disrupt quorum sensing. Nature Chem. Biol. 5, 251–257 (2009).

    CAS  Google Scholar 

  118. Wei, Y., Perez, L. J., Ng, W.-L., Semmelhack, M. F. & Bassler, B. L. Mechanism of Vibrio cholerae autoinducer-1 biosynthesis. ACS Chem. Biol. 6, 356–365 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. Perez, L. J., Karagounis, T. K., Hurley, A., Bassler, B. L. & Semmelhack, M. F. Highly potent, chemically stable quorum sensing agonists for Vibrio cholerae. Chem. Sci. 5, 151–155 (2014).

    CAS  PubMed  Google Scholar 

  120. Sambanthamoorthy, K. et al. Identification of a novel benzimidazole that inhibits bacterial biofilm formation in a broad-spectrum manner. Antimicrob. Agents Chemother. 55, 4369–4378 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Sambanthamoorthy, K. et al. Identification of small molecules that antagonize diguanylate cyclase enzymes to inhibit biofilm formation. Antimicrob. Agents Chemother. 56, 5202–5211 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  122. Navarro, G. et al. Image-based 384-well high-throughput screening method for the discovery of skyllamycins A to C as biofilm inhibitors and inducers of biofilm detachment in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 58, 1092–1099 (2014).

    PubMed  PubMed Central  Google Scholar 

  123. León, B. et al. Development of quinoline-based disruptors of biofilm formation against Vibrio cholerae. Org. Lett. 15, 1234–1237 (2013).

    PubMed  Google Scholar 

  124. Peach, K. C., Cheng, A. T., Oliver, A. G., Yildiz, F. H. & Linington, R. G. Discovery and biological characterization of the auromomycin chromophore as an inhibitor of biofilm formation in Vibrio cholerae. ChemBioChem 14, 2209–2215 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. Warner, C. J. A., Cheng, A. T., Yildiz, F. H. & Linington, R. G. Development of benzo[1,4]oxazines as biofilm inhibitors and dispersal agents against Vibrio cholerae. Chem. Commun. (Camb.). 51, 1305–1308 (2014).

    PubMed Central  Google Scholar 

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Acknowledgements

The authors thank L. Lang and A. Gurcan for figure preparation. They thank N. Fong for the photographs of V. cholerae colony, biofilm and pellicle. The authors also thank members of our laboratories for critical reading of the manuscript. This work was supported by the US National Institutes of Health (NIH) grants AI102584, AI114261 and AI055987.

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Glossary

Planktonic state

Single drifting cells that inhabit the water column.

Chitin

A β(1→4)-linked homopolymer of N-acetyl-D-glucosamine, found in the exoskeleton of zooplankton and other crustaceans. It is an abundant source of carbon, nitrogen and energy for many microorganisms.

Rugosity

Pertaining to a bacterial colony: a corrugated colony phenotype associated with high levels of biofilm matrix production.

Roaming

A motility mode of surface-skimming Vibrio cholerae cells in which cells move with meandering, gently-curved trajectories that range over large areas of the surface.

Orbiting

A motility mode of surface-skimming Vibrio cholerae cells in which cells move with tightly curved circular trajectories that hover over small areas of the surface.

Flagellum

A motility structure composed of a cytoplasmic basal body that functions as a motor, a rod that extends from the cytoplasm through the membrane, and a long filamentous polymer projecting from the cell.

Flow cell

A piece of equipment that is used for the in vitro culture and examination of bacterial biofilms under hydrodynamic flow conditions.

Radius of gyration

(Rgyr). As used here: a statistical measure of the spatial extent of a bacterial track. It is defined as the root of the mean square distance between each point of a track and the centre of mass of that track. For a perfect circle of radius r, Rgyr = r.

Pili

Proteinaceous filaments that are found on the surface of many bacteria and are often involved in adhesion or motility.

Pellicles

Biofilms formed at an air–liquid interface.

V. cholerae biofilm matrix cluster

(VcBMC). A genetic module comprising the Vibrio polysaccharide 1 (vps-1), rugosity and biofilm structure modulator (rbm) and vps-2 gene clusters, which encode many of the proteins that generate VPS and major biofilm proteins.

Serogroups

Groups of bacterial strains based on the structure of the surface O antigen group.

Cyclic di-GMP

(c-di-GMP). A key signalling molecule that controls the motile-to-biofilm transition and biofilm formation by inhibiting motility and stimulating the synthesis of cell-surface adhesins and/or exopolysaccharides.

TetR regulators

A family of proteins involved in the transcriptional control of several cellular processes, including biofilm formation, pathogenesis, catabolism, antibiotic resistance, and differentiation. TetR family members harbour a helix–turn–helix motif that is highly similar to the DNA-binding motif of TetR (a regulator that controls the expression of tetracycline resistance (tet) genes).

Cyclic AMP

(cAMP). A second-messenger signalling molecule that is involved in the regulation of several cell processes, including cell division, catabolite repression, motility and biofilm formation.

Riboswitches

Regulatory RNA sensors each composed of a structured non-coding RNA that binds to specific small molecules and regulates gene expression.

Stringent response

A bacterial stress response that is triggered by nutritional stress and results in the synthesis of guanosine tetraphosphate and guanosine pentaphosphate, which in turn control anabolic and catabolic processes and thereby regulate growth rate.

(p)ppGpp

The collective term for the two alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are synthesized in response to nutrient limitation and other stress conditions to induce the stringent response and subsequent changes in cell physiology.

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Teschler, J., Zamorano-Sánchez, D., Utada, A. et al. Living in the matrix: assembly and control of Vibrio cholerae biofilms. Nat Rev Microbiol 13, 255–268 (2015). https://doi.org/10.1038/nrmicro3433

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