1887

Abstract

Biofilm formation enhances the survival and persistence of the facultative human pathogen in natural ecosystems and its transmission during seasonal cholera outbreaks. A major component of the biofilm matrix is the polysaccharide (VPS), which is essential for development of three-dimensional biofilm structures. The genes are clustered in two regions, the I cluster (, , VC0916–27) and the II cluster (, VC0934–39), separated by an intergenic region containing the gene cluster that encodes biofilm matrix proteins. In-frame deletions of the clusters and genes encoding matrix proteins drastically altered biofilm formation phenotypes. To determine which genes within the gene clusters are required for biofilm formation and VPS synthesis, we generated in-frame deletion mutants for all the genes. Many of these mutants exhibited reduced capacity to produce VPS and biofilms. Infant mouse colonization assays revealed that mutants lacking either clusters or (encoding secreted matrix protein RbmA) exhibited a defect in intestinal colonization compared to the wild-type. Understanding the roles of the various gene products will aid in the biochemical characterization of the VPS biosynthetic pathway and elucidate how gene products contribute to VPS biosynthesis, biofilm formation and virulence in .

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2010-09-01
2019-09-24
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References

  1. Alam, M., Sultana, M., Nair, G. B., Sack, R. B., Sack, D. A., Siddique, A. K., Ali, A., Huq, A. & Colwell, R. R. ( 2006; ). Toxigenic Vibrio cholerae in the aquatic environment of Mathbaria, Bangladesh. Appl Environ Microbiol 72, 2849–2855.[CrossRef]
    [Google Scholar]
  2. Aravind, L. & Koonin, E. V. ( 1998; ). Phosphoesterase domains associated with DNA polymerases of diverse origins. Nucleic Acids Res 26, 3746–3752.[CrossRef]
    [Google Scholar]
  3. Bao, Y., Lies, D. P., Fu, H. & Roberts, G. P. ( 1991; ). An improved Tn7-based system for the single-copy insertion of cloned genes into chromosomes of gram-negative bacteria. Gene 109, 167–168.[CrossRef]
    [Google Scholar]
  4. Beyhan, S. & Yildiz, F. H. ( 2007; ). 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.[CrossRef]
    [Google Scholar]
  5. Beyhan, S., Bilecen, K., Salama, S. R., Casper-Lindley, C. & Yildiz, F. H. ( 2007; ). 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.[CrossRef]
    [Google Scholar]
  6. Broza, M., Gancz, H., Halpern, M. & Kashi, Y. ( 2005; ). Adult non-biting midges: possible windborne carriers of Vibrio cholerae non-O1 non-O139. Environ Microbiol 7, 576–585.[CrossRef]
    [Google Scholar]
  7. Campbell, J. A., Davies, G. J., Bulone, V. & Henrissat, B. ( 1997; ). A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem J 326, 929–939.
    [Google Scholar]
  8. Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V. & Henrissat, B. ( 2009; ). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37, D233–D238.[CrossRef]
    [Google Scholar]
  9. Colwell, R. R., Huq, A., Islam, M. S., Aziz, K. M., Yunus, M., Khan, N. H., Mahmud, A., Sack, R. B., Nair, G. B. & other authors ( 2003; ). Reduction of cholera in Bangladeshi villages by simple filtration. Proc Natl Acad Sci U S A 100, 1051–1055.[CrossRef]
    [Google Scholar]
  10. de Lorenzo, V. & Timmis, K. N. ( 1994; ). Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235, 386–405.
    [Google Scholar]
  11. Enos-Berlage, J. L. & McCarter, L. L. ( 2000; ). Relation of capsular polysaccharide production and colonial cell organization to colony morphology in Vibrio parahaemolyticus. J Bacteriol 182, 5513–5520.[CrossRef]
    [Google Scholar]
  12. Faruque, S. M., Albert, M. J. & Mekalanos, J. J. ( 1998; ). Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae. Microbiol Mol Biol Rev 62, 1301–1314.
    [Google Scholar]
  13. Faruque, S. M., Biswas, K., Udden, S. M., Ahmad, Q. S., Sack, D. A., Nair, G. B. & Mekalanos, J. J. ( 2006; ). Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment. Proc Natl Acad Sci U S A 103, 6350–6355.[CrossRef]
    [Google Scholar]
  14. Fong, J. C. & Yildiz, F. H. ( 2007; ). The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae. J Bacteriol 189, 2319–2330.[CrossRef]
    [Google Scholar]
  15. Fong, J. C. & Yildiz, F. H. ( 2008; ). Interplay between cyclic AMP-cyclic AMP receptor protein and cyclic di-GMP signaling in Vibrio cholerae biofilm formation. J Bacteriol 190, 6646–6659.[CrossRef]
    [Google Scholar]
  16. Fong, J. C., Karplus, K., Schoolnik, G. K. & Yildiz, F. H. ( 2006; ). 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.[CrossRef]
    [Google Scholar]
  17. Franco, A. V., Liu, D. & Reeves, P. R. ( 1998; ). The Wzz (Cld) protein in Escherichia coli: amino acid sequence variation determines O-antigen chain length specificity. J Bacteriol 180, 2670–2675.
    [Google Scholar]
  18. Fullner, K. J. & Mekalanos, J. J. ( 1999; ). Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae. Infect Immun 67, 1393–1404.
    [Google Scholar]
  19. Halpern, M., Broza, Y. B., Mittler, S., Arakawa, E. & Broza, M. ( 2004; ). Chironomid egg masses as a natural reservoir of Vibrio cholerae non-O1 and non-O139 in freshwater habitats. Microb Ecol 47, 341–349.
    [Google Scholar]
  20. Hang, L., John, M., Asaduzzaman, M., Bridges, E. A., Vanderspurt, C., Kirn, T. J., Taylor, R. K., Hillman, J. D., Progulske-Fox, A. & other authors ( 2003; ). Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae. Proc Natl Acad Sci U S A 100, 8508–8513.[CrossRef]
    [Google Scholar]
  21. Herrero, M., de Lorenzo, V. & Timmis, K. N. ( 1990; ). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172, 6557–6567.
    [Google Scholar]
  22. Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K. & Molin, S. ( 2000; ). Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146, 2395–2407.
    [Google Scholar]
  23. Hung, D. T., Zhu, J., Sturtevant, D. & Mekalanos, J. J. ( 2006; ). Bile acids stimulate biofilm formation in Vibrio cholerae. Mol Microbiol 59, 193–201.[CrossRef]
    [Google Scholar]
  24. Huo, A., Xu, B., Chowdhury, M. A., Islam, M. S., Montilla, R. & Colwell, R. R. ( 1996; ). A simple filtration method to remove plankton-associated Vibrio cholerae in raw water supplies in developing countries. Appl Environ Microbiol 62, 2508–2512.
    [Google Scholar]
  25. Huq, A., Small, E. B., West, P. A., Huq, M. I., Rahman, R. & Colwell, R. R. ( 1983; ). Ecological relationships between Vibrio cholerae and planktonic crustacean copepods. Appl Environ Microbiol 45, 275–283.
    [Google Scholar]
  26. Huq, A., Huq, S. A., Grimes, D. J., O'Brien, M., Chu, K. H., Capuzzo, J. M. & Colwell, R. R. ( 1986; ). Colonization of the gut of the blue crab (Callinectes sapidus) by Vibrio cholerae. Appl Environ Microbiol 52, 586–588.
    [Google Scholar]
  27. Huq, A., Colwell, R. R., Chowdhury, M. A., Xu, B., Moniruzzaman, S. M., Islam, M. S., Yunus, M. & Albert, M. J. ( 1995; ). Coexistence of Vibrio cholerae O1 and O139 Bengal in plankton in Bangladesh. Lancet 345, 1249
    [Google Scholar]
  28. Kaper, J. B., Morris, J. G., Jr & Levine, M. M. ( 1995; ). Cholera. Clin Microbiol Rev 8, 48–86.
    [Google Scholar]
  29. Kawamura, T., Ishimoto, N. & Ito, E. ( 1979; ). Enzymatic synthesis of uridine diphosphate N-acetyl-d-mannosaminuronic acid. J Biol Chem 254, 8457–8465.
    [Google Scholar]
  30. Kawamura, T., Ichihara, N., Sugiyama, S., Yokota, H., Ishimoto, N. & Ito, E. ( 1985; ). Biosynthesis of UDP-N-acetyl-d-glucosaminuronic acid and UDP-N-acetyl-d-mannosaminuronic acid in Micrococcus luteus. J Biochem 98, 105–116.
    [Google Scholar]
  31. Kennelly, P. J. & Potts, M. ( 1999; ). Life among the primitives: protein O-phosphatases in prokaryotes. Front Biosci 4, D372–D385.[CrossRef]
    [Google Scholar]
  32. Kierek, K. & Watnick, P. I. ( 2003; ). Environmental determinants of Vibrio cholerae biofilm development. Appl Environ Microbiol 69, 5079–5088.[CrossRef]
    [Google Scholar]
  33. Lauriano, C. M., Ghosh, C., Correa, N. E. & Klose, K. E. ( 2004; ). The sodium-driven flagellar motor controls exopolysaccharide expression in Vibrio cholerae. J Bacteriol 186, 4864–4874.[CrossRef]
    [Google Scholar]
  34. Lee, S. H., Butler, S. M. & Camilli, A. ( 2001; ). Selection for in vivo regulators of bacterial virulence. Proc Natl Acad Sci U S A 98, 6889–6894.[CrossRef]
    [Google Scholar]
  35. Lin, W., Kovacikova, G. & Skorupski, K. ( 2007; ). The quorum sensing regulator HapR downregulates the expression of the virulence gene transcription factor AphA in Vibrio cholerae by antagonizing Lrp- and VpsR-mediated activation. Mol Microbiol 64, 953–967.[CrossRef]
    [Google Scholar]
  36. Lombardo, M. J., Michalski, J., Martinez-Wilson, H., Morin, C., Hilton, T., Osorio, C. G., Nataro, J. P., Tacket, C. O., Camilli, A. & other authors ( 2007; ). An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers. Proc Natl Acad Sci U S A 104, 18229–18234.[CrossRef]
    [Google Scholar]
  37. Marolda, C. L., Vicarioli, J. & Valvano, M. A. ( 2004; ). Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit. Microbiology 150, 4095–4105.[CrossRef]
    [Google Scholar]
  38. Marolda, C. L., Tatar, L. D., Alaimo, C., Aebi, M. & Valvano, M. A. ( 2006; ). Interplay of the Wzx translocase and the corresponding polymerase and chain length regulator proteins in the translocation and periplasmic assembly of lipopolysaccharide O antigen. J Bacteriol 188, 5124–5135.[CrossRef]
    [Google Scholar]
  39. Morona, J. K., Paton, J. C., Miller, D. C. & Morona, R. ( 2000; ). Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae. Mol Microbiol 35, 1431–1442.
    [Google Scholar]
  40. Morona, J. K., Morona, R., Miller, D. C. & Paton, J. C. ( 2002; ). Streptococcus pneumoniae capsule biosynthesis protein CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase. J Bacteriol 184, 577–583.[CrossRef]
    [Google Scholar]
  41. Nivens, D. E., Ohman, D. E., Williams, J. & Franklin, M. J. ( 2001; ). Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J Bacteriol 183, 1047–1057.[CrossRef]
    [Google Scholar]
  42. Pier, G. B., Coleman, F., Grout, M., Franklin, M. & Ohman, D. E. ( 2001; ). Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis. Infect Immun 69, 1895–1901.[CrossRef]
    [Google Scholar]
  43. Rashid, M. H., Rajanna, C., Zhang, D., Pasquale, V., Magder, L. S., Ali, A., Dumontet, S. & Karaolis, D. K. ( 2004; ). Role of exopolysaccharide, the rugose phenotype and VpsR in the pathogenesis of epidemic Vibrio cholerae. FEMS Microbiol Lett 230, 105–113.[CrossRef]
    [Google Scholar]
  44. Sau, S., Bhasin, N., Wann, E. R., Lee, J. C., Foster, T. J. & Lee, C. Y. ( 1997; ). The Staphylococcus aureus allelic genetic loci for serotype 5 and 8 capsule expression contain the type-specific genes flanked by common genes. Microbiology 143, 2395–2405.[CrossRef]
    [Google Scholar]
  45. Soulat, D., Vaganay, E., Duclos, B., Genestier, A. L., Etienne, J. & Cozzone, A. J. ( 2002; ). Staphylococcus aureus contains two low-molecular-mass phosphotyrosine protein phosphatases. J Bacteriol 184, 5194–5199.[CrossRef]
    [Google Scholar]
  46. Su, X. D., Taddei, N., Stefani, M., Ramponi, G. & Nordlund, P. ( 1994; ). The crystal structure of a low-molecular-weight phosphotyrosine protein phosphatase. Nature 370, 575–578.[CrossRef]
    [Google Scholar]
  47. Tischler, A. D. & Camilli, A. ( 2005; ). Cyclic diguanylate regulates Vibrio cholerae virulence gene expression. Infect Immun 73, 5873–5882.[CrossRef]
    [Google Scholar]
  48. Vasseur, P., Vallet-Gely, I., Soscia, C., Genin, S. & Filloux, A. ( 2005; ). The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation. Microbiology 151, 985–997.[CrossRef]
    [Google Scholar]
  49. Vincent, C., Doublet, P., Grangeasse, C., Vaganay, E., Cozzone, A. J. & Duclos, B. ( 1999; ). Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb. J Bacteriol 181, 3472–3477.
    [Google Scholar]
  50. Vincent, C., Duclos, B., Grangeasse, C., Vaganay, E., Riberty, M., Cozzone, A. J. & Doublet, P. ( 2000; ). Relationship between exopolysaccharide production and protein-tyrosine phosphorylation in gram-negative bacteria. J Mol Biol 304, 311–321.[CrossRef]
    [Google Scholar]
  51. Watnick, P. I., Lauriano, C. M., Klose, K. E., Croal, L. & Kolter, R. ( 2001; ). The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol Microbiol 39, 223–235.[CrossRef]
    [Google Scholar]
  52. Whitfield, C. ( 1995; ). Biosynthesis of lipopolysaccharide O antigens. Trends Microbiol 3, 178–185.[CrossRef]
    [Google Scholar]
  53. Whitfield, C. ( 2006; ). Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu Rev Biochem 75, 39–68.[CrossRef]
    [Google Scholar]
  54. Yildiz, F. H. & Schoolnik, G. K. ( 1999; ). 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 U S A 96, 4028–4033.[CrossRef]
    [Google Scholar]
  55. Yildiz, F. H., Dolganov, N. A. & Schoolnik, G. K. ( 2001; ). 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.[CrossRef]
    [Google Scholar]
  56. Zhu, J. & Mekalanos, J. J. ( 2003; ). Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae. Dev Cell 5, 647–656.[CrossRef]
    [Google Scholar]
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