1887

Abstract

pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The xanthan polysaccharide and extracellular enzymes produced by this organism are virulence factors, the expression of which is upregulated by Clp (CRP-like protein) and DSF (diffusible signal factor), which is synthesized by RpfF. It is also known that biofilm formation/dispersal, regulated by the effect of controlled synthesis of DSF on cell–cell signalling, is required for virulence. Furthermore, a deficiency in DSF causes cell aggregation with concomitant production of a gum-like substance that can be dispersed by addition of DSF or digested by exogenous endo--1,4-mannanase expressed by Xcc. In this study, Western blotting of proteins from a mutant (XcMopB) showed Xcc MopB to be the major outer-membrane protein (OMP); Xcc MopB shared over 97 % identity with homologues from other members of . Similarly to the mutant, XcMopB formed aggregates with simultaneous production of a gummy substance, but these aggregates could not be dispersed by DSF or endo--1,4-mannanase, indicating that different mechanisms were involved in aggregation. In addition, XcMopB showed surface deformation, altered OMP composition, impaired xanthan production, increased sensitivity to stressful conditions including SDS, elevated temperature and changes in pH, reduced adhesion and motility and defects in pathogenesis. The finding that the major OMP is required for pathogenicity is unprecedented in phytopathogenic bacteria.

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2010-09-01
2019-12-06
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References

  1. Afkar, E., Reguera, G., Schiffer, M. & Lovley, D. R. ( 2005; ). A novel Geobacteraceae-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe(III) and Mn(IV) oxides in Geobacter sulfurreducens. BMC Microbiol 5, 41.[CrossRef]
    [Google Scholar]
  2. Barabote, R. D., Johnson, O. L., Zetina, E., San Francisco, S. K., Fralick, J. A. & San Francisco, M. J. ( 2003; ). Erwinia chrysanthemi tolC is involved in resistance to antimicrobial plant chemicals and is essential for phytopathogenesis. J Bacteriol 185, 5772–5778.[CrossRef]
    [Google Scholar]
  3. Becker, A., Katzen, F., Puhler, A. & Ielpi, L. ( 1998; ). Xanthan gum biosynthesis and application: a biochemical/genetic perspective. Appl Microbiol Biotechnol 50, 145–152.[CrossRef]
    [Google Scholar]
  4. Bishop, R. E. ( 2008; ). Structural biology of membrane-intrinsic beta-barrel enzymes: sentinels of the bacterial outer membrane. Biochim Biophys Acta 1778, 1881–1896.[CrossRef]
    [Google Scholar]
  5. Bourgault, R. & Bewley, J. D. ( 2002; ). Gel diffusion assays for endo-beta-mannanase and pectin methylesterase can underestimate enzyme activity due to proteolytic degradation: a remedy. Anal Biochem 300, 87–93.[CrossRef]
    [Google Scholar]
  6. Browning, D. F., Whitworth, D. E. & Hodgson, D. A. ( 2003; ). Light-induced carotenogenesis in Myxococcus xanthus: functional characterization of the ECF sigma factor CarQ and antisigma factor CarR. Mol Microbiol 48, 237–251.[CrossRef]
    [Google Scholar]
  7. Chan, J. W. & Goodwin, P. H. ( 1999; ). The molecular genetics of virulence of Xanthomonas campestris. Biotechnol Adv 17, 489–508.[CrossRef]
    [Google Scholar]
  8. Chao, N. X., Wei, K., Chen, Q., Meng, Q. L., Tang, D. J., He, Y. Q., Lu, G. T., Jiang, B. L., Liang, X. X. & other authors ( 2008; ). The rsmA-like gene rsmA(Xcc) of Xanthomonas campestris pv. campestris is involved in the control of various cellular processes, including pathogenesis. Mol Plant Microbe Interact 21, 411–423.[CrossRef]
    [Google Scholar]
  9. Choi, C. H., Lee, J. S., Lee, Y. C., Park, T. I. & Lee, J. C. ( 2008; ). Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells. BMC Microbiol 8, 216.[CrossRef]
    [Google Scholar]
  10. Chou, F. L., Chou, H. C., Lin, Y. S., Yang, B. Y., Lin, N. T., Weng, S. F. & Tseng, Y. H. ( 1997; ). The Xanthomonas campestris gumD gene required for synthesis of xanthan gum is involved in normal pigmentation and virulence in causing black rot. Biochem Biophys Res Commun 233, 265–269.[CrossRef]
    [Google Scholar]
  11. de Mot, R. & Vanderleyden, J. ( 1994a; ). The C-terminal sequence conservation between OmpA-related outer membrane proteins and MotB suggests a common function in both gram-positive and gram-negative bacteria, possibly in the interaction of these domains with peptidoglycan. Mol Microbiol 12, 333–334.[CrossRef]
    [Google Scholar]
  12. de Mot, R. & Vanderleyden, J. ( 1994b; ). A conserved surface-exposed domain in major outer membrane proteins of pathogenic Pseudomonas and Branhamella species shares sequence homology with the calcium-binding repeats of the eukaryotic extracellular matrix protein thrombospondin. Mol Microbiol 13, 379–380.[CrossRef]
    [Google Scholar]
  13. Dharmapuri, S. & Sonti, R. V. ( 1999; ). A transposon insertion in the gumG homologue of Xanthomonas oryzae pv. oryzae causes loss of extracellular polysaccharide production and virulence. FEMS Microbiol Lett 179, 53–59.[CrossRef]
    [Google Scholar]
  14. Dow, J. M., Crossman, L., Findlay, K., He, Y. Q., Feng, J. X. & Tang, J. L. ( 2003; ). Biofilm dispersal in Xanthomonas campestris is controlled by cell-cell signaling and is required for full virulence to plants. Proc Natl Acad Sci U S A 100, 10995–11000.[CrossRef]
    [Google Scholar]
  15. Dums, F., Dow, J. M. & Daniels, M. J. ( 1991; ). Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Mol Gen Genet 229, 357–364.[CrossRef]
    [Google Scholar]
  16. Dye, D. W., Bradbury, J. F., Goto, M., Hayward, A. C., Lelliott, R. A. & Schroth, M. N. ( 1980; ). International standards for naming pathovars of phytopathogenic bacteria and a list of pathovar names and pathotype strains. Rev Plant Pathol 59, 153–168.
    [Google Scholar]
  17. Fu, J. F. & Tseng, Y. H. ( 1990; ). Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey. Appl Environ Microbiol 56, 919–923.
    [Google Scholar]
  18. Gotoh, N., Wakebe, H., Yoshihara, E., Nakae, T. & Nishino, T. ( 1989; ). Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane. J Bacteriol 171, 983–990.
    [Google Scholar]
  19. Hanahan, D. ( 1983; ). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[CrossRef]
    [Google Scholar]
  20. He, Y. W., Xu, M., Lin, K., Ng, Y. J., Wen, C. M., Wang, L. H., Liu, Z. D., Zhang, H. B., Dong, Y. H. & other authors ( 2006; ). Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions. Mol Microbiol 59, 610–622.[CrossRef]
    [Google Scholar]
  21. He, Y. W., Ng, A. Y., Xu, M., Lin, K., Wang, L. H., Dong, Y. H. & Zhang, L. H. ( 2007; ). Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network. Mol Microbiol 64, 281–292.[CrossRef]
    [Google Scholar]
  22. Hsiao, Y. M., Fang, M. C., Sun, P. F. & Tseng, Y. H. ( 2009; ). Clp and RpfF up-regulate transcription of pelA1 gene encoding the major pectate lyase in Xanthomonas campestris pv. campestris. J Agric Food Chem 57, 6207–6215.[CrossRef]
    [Google Scholar]
  23. Hsiao, Y. M., Liu, Y. F., Fang, M. C. & Tseng, Y. H. ( 2010; ). Transcriptional regulation and molecular characterization of the manA gene encoding the biofilm dispersing enzyme mannan endo-1,4-beta-mannosidase in Xanthomonas campestris. J Agric Food Chem 58, 1653–1663.[CrossRef]
    [Google Scholar]
  24. Jackson, D. W., Suzuki, K., Oakford, L., Simecka, J. W., Hart, M. E. & Romeo, T. ( 2002; ). Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli. J Bacteriol 184, 290–301.[CrossRef]
    [Google Scholar]
  25. Katzen, F., Ferreiro, D. U., Oddo, C. G., Ielmini, M. V., Becker, A., Puhler, A. & Ielpi, L. ( 1998; ). Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence. J Bacteriol 180, 1607–1617.
    [Google Scholar]
  26. Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. ( 1988; ). Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70, 191–197.[CrossRef]
    [Google Scholar]
  27. Khan, N. A., Shin, S., Chung, J. W., Kim, K. J., Elliott, S., Wang, Y. & Kim, K. S. ( 2003; ). Outer membrane protein A and cytotoxic necrotizing factor-1 use diverse signaling mechanisms for Escherichia coli K1 invasion of human brain microvascular endothelial cells. Microb Pathog 35, 35–42.[CrossRef]
    [Google Scholar]
  28. Koebnik, R., Locher, K. P. & Van Gelder, P. ( 2000; ). Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol Microbiol 37, 239–253.[CrossRef]
    [Google Scholar]
  29. Kostakioti, M., Newman, C. L., Thanassi, D. G. & Stathopoulos, C. ( 2005; ). Mechanisms of protein export across the bacterial outer membrane. J Bacteriol 187, 4306–4314.[CrossRef]
    [Google Scholar]
  30. Lee, M. C., Weng, S. F. & Tseng, Y. H. ( 2003; ). Flagellin gene fliC of Xanthomonas campestris is upregulated by transcription factor Clp. Biochem Biophys Res Commun 307, 647–652.[CrossRef]
    [Google Scholar]
  31. Lin, H. M. & Tseng, Y. H. ( 1979; ). Exopolysaccharide synthesis in Xanthomonas oryzae. Proc Natl Sci Counc Repub China 3, 279–284.
    [Google Scholar]
  32. Lin, H. M., Tseng, H. C., Wang, C. J., Chyau, C. C., Liao, K. K., Peng, P. L. & Chou, F. P. ( 2007; ). Induction of autophagy and apoptosis by the extract of Solanum nigrum Linn in HepG2 cells. J Agric Food Chem 55, 3620–3628.[CrossRef]
    [Google Scholar]
  33. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    [Google Scholar]
  34. Miller, V. L., Farmer, J. J., III, Hill, W. E. & Falkow, S. ( 1989; ). The ail locus is found uniquely in Yersinia enterocolitica serotypes commonly associated with disease. Infect Immun 57, 121–131.
    [Google Scholar]
  35. Miller, V. L., Bliska, J. B. & Falkow, S. ( 1990; ). Nucleotide sequence of the Yersinia enterocolitica ail gene and characterization of the Ail protein product. J Bacteriol 172, 1062–1069.
    [Google Scholar]
  36. Miller, V. L., Beer, K. B., Loomis, W. P., Olson, J. A. & Miller, S. I. ( 1992; ). An unusual pagC : TnphoA mutation leads to an invasion- and virulence-defective phenotype in salmonellae. Infect Immun 60, 3763–3770.
    [Google Scholar]
  37. Moore, J., Bailey, S. E., Benmechernene, Z., Tzitzilonis, C., Griffiths, N. J., Virji, M. & Derrick, J. P. ( 2005; ). Recognition of saccharides by the OpcA, OpaD, and OpaB outer membrane proteins from Neisseria meningitidis. J Biol Chem 280, 31489–31497.[CrossRef]
    [Google Scholar]
  38. Ojanen, T., Helander, I. M., Haahtela, K., Korhonen, T. K. & Laakso, T. ( 1993; ). Outer membrane proteins and lipopolysaccharides in pathovars of Xanthomonas campestris. Appl Environ Microbiol 59, 4143–4151.
    [Google Scholar]
  39. Page, R. D. M. ( 1996; ). TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
    [Google Scholar]
  40. Pal, U., Yang, X., Chen, M., Bockenstedt, L. K., Anderson, J. F., Flavell, R. A., Norgard, M. V. & Fikrig, E. ( 2004; ). OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J Clin Invest 113, 220–230.[CrossRef]
    [Google Scholar]
  41. Prasadarao, N. V., Wass, C. A., Weiser, J. N., Stins, M. F., Huang, S. H. & Kim, K. S. ( 1996; ). Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells. Infect Immun 64, 146–153.
    [Google Scholar]
  42. Rawling, E. G., Brinkman, F. S. & Hancock, R. E. ( 1998; ). Roles of the carboxy-terminal half of Pseudomonas aeruginosa major outer membrane protein OprF in cell shape, growth in low-osmolarity medium, and peptidoglycan association. J Bacteriol 180, 3556–3562.
    [Google Scholar]
  43. Ray, S. K., Rajeshwari, R., Sharma, Y. & Sonti, R. V. ( 2002; ). A high-molecular-weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non-fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence. Mol Microbiol 46, 637–647.[CrossRef]
    [Google Scholar]
  44. Rigano, L. A., Siciliano, F., Enrique, R., Sendin, L., Filippone, P., Torres, P. S., Questa, J., Dow, J. M., Castagnaro, A. P. & other authors ( 2007; ). Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri. Mol Plant Microbe Interact 20, 1222–1230.[CrossRef]
    [Google Scholar]
  45. Rigden, D. J. & Galperin, M. Y. ( 2004; ). The DxDxDG motif for calcium binding: multiple structural contexts and implications for evolution. J Mol Biol 343, 971–984.[CrossRef]
    [Google Scholar]
  46. Rojas, C. M., Ham, J. H., Deng, W. L., Doyle, J. J. & Collmer, A. ( 2002; ). HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings. Proc Natl Acad Sci U S A 99, 13142–13147.[CrossRef]
    [Google Scholar]
  47. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    [Google Scholar]
  48. Sidhu, V. K., Vorholter, F. J., Niehaus, K. & Watt, S. A. ( 2008; ). Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris. BMC Microbiol 8, 87.[CrossRef]
    [Google Scholar]
  49. Slater, H., Alvarez-Morales, A., Barber, C. E., Daniels, M. J. & Dow, J. M. ( 2000; ). A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris. Mol Microbiol 38, 986–1003.
    [Google Scholar]
  50. Swings, J. G. & Civerolo, E. L. ( 1993; ). Xanthomonas. London. : Chapman & Hall.
    [Google Scholar]
  51. Tadayyon, M., Gittins, J. R., Pratt, J. M. & Broome-Smith, J. K. ( 1994; ). Expression of membrane proteins in Escherichia coli. In Membrane Protein Expression Systems: a User's Guide, pp. 29–83. Edited by Gould, G. W.. London. : Portland Press.
    [Google Scholar]
  52. Tang, J. L., Liu, Y. N., Barber, C. E., Dow, J. M., Wootton, J. C. & Daniels, M. J. ( 1991; ). Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris. Mol Gen Genet 226, 409–417.
    [Google Scholar]
  53. Teng, C. H., Xie, Y., Shin, S., Di Cello, F., Paul-Satyaseela, M., Cai, M. & Kim, K. S. ( 2006; ). Effects of ompA deletion on expression of type 1 fimbriae in Escherichia coli K1 strain RS218 and on the association of E. coli with human brain microvascular endothelial cells. Infect Immun 74, 5609–5616.[CrossRef]
    [Google Scholar]
  54. Vieira, J. & Messing, J. ( 1991; ). New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100, 189–194.[CrossRef]
    [Google Scholar]
  55. Vorholter, F. J., Schneiker, S., Goesmann, A., Krause, L., Bekel, T., Kaiser, O., Linke, B., Patschkowski, T., Ruckert, C. & other authors ( 2008; ). The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis. J Biotechnol 134, 33–45.[CrossRef]
    [Google Scholar]
  56. Wang, Y. ( 2002; ). The function of OmpA in Escherichia coli. Biochem Biophys Res Commun 292, 396–401.[CrossRef]
    [Google Scholar]
  57. Wang, T. W. & Tseng, Y. H. ( 1992; ). Electrotransformation of Xanthomonas campestris by RF DNA of filamentous phage phi Lf. Lett Appl Microbiol 14, 65–68.[CrossRef]
    [Google Scholar]
  58. Wengelnik, K., Marie, C., Russel, M. & Bonas, U. ( 1996; ). Expression and localization of HrpA1, a protein of Xanthomonas campestris pv. vesicatoria essential for pathogenicity and induction of the hypersensitive reaction. J Bacteriol 178, 1061–1069.
    [Google Scholar]
  59. William, P. H. ( 1980; ). Black rot: a continuing threat to world crucifers. Plant Dis 64, 736–742.[CrossRef]
    [Google Scholar]
  60. Woodruff, W. A. & Hancock, R. E. ( 1989; ). Pseudomonas aeruginosa outer membrane protein F: structural role and relationship to the Escherichia coli OmpA protein. J Bacteriol 171, 3304–3309.
    [Google Scholar]
  61. Yang, B. Y. & Tseng, Y. H. ( 1988; ). Production of exopolysaccharide and levels of protease and pectinase activity in pathogenic and non-pathogenic strains of Xanthomonas campestris pv. campestris. Bot Bull Acad Sin 29, 93–99.
    [Google Scholar]
  62. Yang, C. H., Gavilanes-Ruiz, M., Okinaka, Y., Vedel, R., Berthuy, I., Boccara, M., Chen, J. W., Perna, N. T. & Keen, N. T. ( 2002; ). hrp genes of Erwinia chrysanthemi 3937 are important virulence factors. Mol Plant Microbe Interact 15, 472–480.[CrossRef]
    [Google Scholar]
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