1887

Abstract

, a causative agent of meningitis and septicaemia, expresses type IV pili, a feature correlating with the uptake of exogenous DNA from the environment by natural transformation. The outer membrane complex PilQ, through which pili are extruded and retracted, has previously been shown to bind DNA in its pore region. In order to further elucidate how DNA is transported across the membranes, we searched for DNA binding proteins within the meningococcal inner membrane. Inner membrane fractions from a panel of neisserial strains were subjected to a solid-phase overlay assay with DNA substrates, and MS was subsequently employed to identify proteins that bind DNA. A number of DNA binding components were detected, including the pilus biogenesis component PilG, the competence protein ComL, and the cell division ATP-binding protein FtsE, as well as two hypothetical proteins. The DNA binding activity of these components was not dependent on the presence of the neisserial DNA uptake sequence. Null mutants, corresponding to each of the proteins identified, were constructed to assess their phenotypes. Only mutants defective in pilus biogenesis were non-competent and non-piliated. The DNA binding activity of the pilus biogenesis components PilQ and PilG and the phenotypes of their respective null mutants suggest that these proteins are directly involved as players in natural transformation, and not only indirectly, through pilus biogenesis.

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2009-03-01
2019-12-08
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References

  1. Achtman, M., Wall, R. A., Bopp, M., Kusecek, B., Morelli, G., Saken, E. & Hassan-King, M. ( 1991; ). Variation in class 5 protein expression by serogroup A meningococci during a meningitis epidemic. J Infect Dis 164, 375–382.[CrossRef]
    [Google Scholar]
  2. Alm, E. J., Huang, K. H., Price, M. N., Koche, R. P., Keller, K., Dubchak, I. L. & Arkin, A. P. ( 2005; ). The MicrobesOnline Web site for comparative genomics. Genome Res 15, 1015–1022.[CrossRef]
    [Google Scholar]
  3. Ambur, O. H., Frye, S. A. & Tønjum, T. ( 2007; ). New functional identity for the DNA uptake sequence in transformation and its presence in transcriptional terminators. J Bacteriol 189, 2077–2085.[CrossRef]
    [Google Scholar]
  4. Assalkhou, R., Balasingham, S., Collins, R. F., Frye, S. A., Davidsen, T., Benam, A. V., Bjørås, M., Derrick, J. P. & Tønjum, T. ( 2007; ). The outer membrane secretin PilQ from Neisseria meningitidis binds DNA. Microbiology 153, 1593–1603.[CrossRef]
    [Google Scholar]
  5. Balasingham, S. V., Collins, R. F., Assalkhou, R., Homberset, H., Frye, S. A., Derrick, J. P. & Tønjum, T. ( 2007; ). Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis. J Bacteriol 189, 5716–5727.[CrossRef]
    [Google Scholar]
  6. Bernatchez, S., Francis, F. M., Salimnia, H., Beveridge, T. J., Li, H. & Dillon, J. A. ( 2000; ). Genomic, transcriptional and phenotypic analysis of ftsE and ftsX of Neisseria gonorrhoeae. DNA Res 7, 75–81.[CrossRef]
    [Google Scholar]
  7. Bieber, D., Ramer, S. W., Wu, C. Y., Murray, W. J., Tobe, T., Fernandez, R. & Schoolnik, G. K. ( 1998; ). Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli. Science 280, 2114–2118.[CrossRef]
    [Google Scholar]
  8. Biswas, G. D., Sox, T., Blackman, E. & Sparling, P. F. ( 1977; ). Factors affecting genetic transformation of Neisseria gonorrhoeae. J Bacteriol 129, 983–992.
    [Google Scholar]
  9. Blake, M. S., MacDonald, C. M. & Klugman, K. P. ( 1989; ). Colony morphology of piliated Neisseria meningitidis. J Exp Med 170, 1727–1736.[CrossRef]
    [Google Scholar]
  10. Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M. C., Estreicher, A., Gasteiger, E., Martin, M. J., Michoud, K., O'Donovan, C. & other authors ( 2003; ). The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res 31, 365–370.[CrossRef]
    [Google Scholar]
  11. Bøvre, K. ( 1964; ). Studies on transformation in Moraxella and organisms assumed to be related to Moraxella. 1. A method for quantitative transformation in Moraxella and Neisseria, with streptomycin resistance as the genetic marker. Acta Pathol Microbiol Scand 61, 457–473.
    [Google Scholar]
  12. Bøvre, K., Bergan, T. & Frøholm, L. O. ( 1970; ). Electron microscopical and serological characteristics associated with colony type in Moraxella nonliquefaciens. Acta Pathol Microbiol Scand [B] Microbiol Immunol 78, 765–779.
    [Google Scholar]
  13. Bradley, D. E. ( 1974; ). The adsorption of Pseudomonas aeruginosa pilus-dependent bacteriophages to a host mutant with nonretractile pili. Virology 58, 149–163.[CrossRef]
    [Google Scholar]
  14. Brinton, C. C., Bryan, J., Dillon, J. A., Guerina, N., Jacobson, L. J., Labik, A., Lee, S., Levine, A., Lim, S. & other authors ( 1978; ). Uses of pili in gonorrhoea control: role of bacterial pili in disease, purification and properties of gonococcal pili, and progress in the development of a gonococcal pilus vaccine for gonorrhoeae. In Immunobiology of Neisseria gonorrhoeae, pp. 155–178. Edited by G. E. Brooks, E. C. Gotschlich, K. H. Homes, W. D. Sawyer & F. E. Young. Washington, DC: American Society for Microbiology.
  15. Brossay, L., Paradis, G., Fox, R., Koomey, M. & Hebert, J. ( 1994; ). Identification, localization, and distribution of the PilT protein in Neisseria gonorrhoeae. Infect Immun 62, 2302–2308.
    [Google Scholar]
  16. Cannon, J. G., Black, W. J., Nachamkin, I. & Stewart, P. W. ( 1984; ). Monoclonal antibody that recognizes an outer membrane antigen common to the pathogenic Neisseria species but not to most nonpathogenic Neisseria species. Infect Immun 43, 994–999.
    [Google Scholar]
  17. Caugant, D. A., Frøholm, L. O., Bøvre, K., Holten, E., Frasch, C. E., Mocca, L. F., Zollinger, W. D. & Selander, R. K. ( 1986; ). Intercontinental spread of a genetically distinctive complex of clones of Neisseria meningitidis causing epidemic disease. Proc Natl Acad Sci U S A 83, 4927–4931.[CrossRef]
    [Google Scholar]
  18. Chen, I. & Gotschlich, E. C. ( 2001; ). ComE, a competence protein from Neisseria gonorrhoeae with DNA binding activity. J Bacteriol 183, 3160–3168.[CrossRef]
    [Google Scholar]
  19. Collins, R. F., Davidsen, L., Derrick, J. P., Ford, R. C. & Tønjum, T. ( 2001; ). Analysis of the PilQ secretin from Neisseria meningitidis by transmission electron microscopy reveals a dodecameric quaternary structure. J Bacteriol 183, 3825–3832.[CrossRef]
    [Google Scholar]
  20. Collins, R. F., Frye, S. A., Balasingham, S., Ford, R. C., Tønjum, T. & Derrick, J. P. ( 2005; ). Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ. J Biol Chem 280, 18923–18930.[CrossRef]
    [Google Scholar]
  21. Comolli, J. C., Hauser, A. R., Waite, L., Whitchurch, C. B., Mattick, J. S. & Engel, J. N. ( 1999; ). Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia. Infect Immun 67, 3625–3630.
    [Google Scholar]
  22. Corbin, B. D., Wang, Y., Beuria, T. K. & Margolin, W. ( 2007; ). Interaction between cell division proteins FtsE and FtsZ. J Bacteriol 189, 3026–3035.[CrossRef]
    [Google Scholar]
  23. Crowe, B. A., Wall, R. A., Kusecek, B., Neumann, B., Olyhoek, T., Abdillahi, H., Hassan-King, M., Greenwood, B. M., Poolman, J. T. & Achtman, M. ( 1989; ). Clonal and variable properties of Neisseria meningitidis isolated from cases and carriers during and after an epidemic in The Gambia, West Africa. J Infect Dis 159, 686–700.[CrossRef]
    [Google Scholar]
  24. Davidsen, T., Rødland, E. A., Lagesen, K., Seeberg, E., Rognes, T. & Tønjum, T. ( 2004; ). Biased distribution of DNA uptake sequences towards genome maintenance genes. Nucleic Acids Res 32, 1050–1058.[CrossRef]
    [Google Scholar]
  25. Davidsen, T., Koomey, M. & Tønjum, T. ( 2007a; ). Microbial genome dynamics in CNS pathogenesis. Neuroscience 145, 1375–1387.[CrossRef]
    [Google Scholar]
  26. Davidsen, T., Tuven, H. K., Bjørås, M., Rødland, E. A. & Tønjum, T. ( 2007b; ). Genetic interactions of DNA repair pathways in the pathogen Neisseria meningitidis. J Bacteriol 189, 5728–5737.[CrossRef]
    [Google Scholar]
  27. Dehio, C., Gray-Owen, S. D. & Meyer, T. F. ( 1998; ). The role of neisserial Opa proteins in interactions with host cells. Trends Microbiol 6, 489–495.[CrossRef]
    [Google Scholar]
  28. de Leeuw, E., Graham, B., Phillips, G. J., ten Hagen-Jongman, C. M., Oudega, B. & Luirink, J. ( 1999; ). Molecular characterization of Escherichia coli FtsE and FtsX. Mol Microbiol 31, 983–993.[CrossRef]
    [Google Scholar]
  29. Facius, D. & Meyer, T. F. ( 1993; ). A novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation. Mol Microbiol 10, 699–712.[CrossRef]
    [Google Scholar]
  30. Filip, C., Fletcher, G., Wulff, J. L. & Earhart, C. F. ( 1973; ). Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol 115, 717–722.
    [Google Scholar]
  31. Fisette, P. L., Ram, S., Andersen, J. M., Guo, W. & Ingalls, R. R. ( 2003; ). The Lip lipoprotein from Neisseria gonorrhoeae stimulates cytokine release and NF-κB activation in epithelial cells in a Toll-like receptor 2-dependent manner. J Biol Chem 278, 46252–46260.[CrossRef]
    [Google Scholar]
  32. Fleckenstein, B., Qiao, S. W., Larsen, M. R., Jung, G., Roepstorff, P. & Sollid, L. M. ( 2004; ). Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. J Biol Chem 279, 17607–17616.[CrossRef]
    [Google Scholar]
  33. Frasch, C. E. & Chapman, S. S. ( 1972; ). Classification of Neisseria meningitidis group B into distinct serotypes. I. Serological typing by a microbactericidal method. Infect Immun 5, 98–102.
    [Google Scholar]
  34. Frasch, C. E. & Mocca, L. F. ( 1978; ). Heat-modifiable outer membrane proteins of Neisseria meningitidis and their organization within the membrane. J Bacteriol 136, 1127–1134.
    [Google Scholar]
  35. Friedrich, A., Prust, C., Hartsch, T., Henne, A. & Averhoff, B. ( 2002; ). Molecular analyses of the natural transformation machinery and identification of pilus structures in the extremely thermophilic bacterium Thermus thermophilus strain HB27. Appl Environ Microbiol 68, 745–755.[CrossRef]
    [Google Scholar]
  36. Frøholm, L. O., Jyssum, K. & Bøvre, K. ( 1973; ). Electron microscopical and cultural features of Neisseria meningitidis competence variants. Acta Pathol Microbiol Scand [B] Microbiol Immunol 81, 525–537.
    [Google Scholar]
  37. Frye, S. A., Assalkhou, R., Collins, R. F., Ford, R. C., Petersson, C., Derrick, J. P. & Tonjum, T. ( 2006; ). Topology of the outer membrane secretin PilQ from Neisseria meningitidis. Microbiology 152, 3751–3764.[CrossRef]
    [Google Scholar]
  38. Fussenegger, M., Facius, D., Meier, J. & Meyer, T. F. ( 1996; ). A novel peptidoglycan-linked lipoprotein (ComL) that functions in natural transformation competence of Neisseria gonorrhoeae. Mol Microbiol 19, 1095–1105.[CrossRef]
    [Google Scholar]
  39. Gill, D. R. & Salmond, G. P. ( 1987; ). The Escherichia coli cell division proteins FtsY, FtsE and FtsX are inner membrane-associated. Mol Gen Genet 210, 504–508.[CrossRef]
    [Google Scholar]
  40. Goodman, S. D. & Scocca, J. J. ( 1988; ). Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proc Natl Acad Sci U S A 85, 6982–6986.[CrossRef]
    [Google Scholar]
  41. Henrichsen, J., Frøholm, L. O. & Bøvre, K. ( 1972; ). Studies on bacterial surface translocation. 2. Correlation of twitching motility and fimbriation in colony variants of Moraxella nonliquefaciens, M. bovis, and M. kingii. Acta Pathol Microbiol Scand [B] Microbiol Immunol 80, 445–452.
    [Google Scholar]
  42. Hitchcock, P. J. ( 1989; ). Unified nomenclature for pathogenic Neisseria species. Clin Microbiol Rev 2 (Suppl.), S64–S65.
    [Google Scholar]
  43. Hitchcock, P. J., Hayes, S. F., Mayer, L. W., Shafer, W. M. & Tessier, S. L. ( 1985; ). Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics. J Exp Med 162, 2017–2034.[CrossRef]
    [Google Scholar]
  44. Holten, E. ( 1979; ). Serotypes of Neisseria meningitidis isolated from patients in Norway during the first six months of 1978. J Clin Microbiol 9, 186–188.
    [Google Scholar]
  45. Hulo, N., Bairoch, A., Bulliard, V., Cerutti, L., Cuche, B. A., de Castro, E., Lachaize, C., Langendijk-Genevaux, P. S. & Sigrist, C. J. ( 2008; ). The 20 years of PROSITE. Nucleic Acids Res 36, D245–D249.
    [Google Scholar]
  46. Jyssum, K. & Lie, S. ( 1965; ). Genetic factors determining competence in transformation of Neisseria meningitidis. 1. A permanent loss of competence. Acta Pathol Microbiol Scand 63, 306–316.
    [Google Scholar]
  47. Koomey, M. ( 1998; ). Competence for natural transformation in Neisseria gonorrhoeae: a model system for studies of horizontal gene transfer. APMIS Suppl 84, 56–61.
    [Google Scholar]
  48. Koomey, J. M. & Falkow, S. ( 1987; ). Cloning of the recA gene of Neisseria gonorrhoeae and construction of gonococcal recA mutants. J Bacteriol 169, 790–795.
    [Google Scholar]
  49. Lorenz, M. G. & Wackernagel, W. ( 1990; ). Natural genetic transformation of Pseudomonas stutzeri by sand-adsorbed DNA. Arch Microbiol 154, 380–385.
    [Google Scholar]
  50. Masson, L. & Holbein, B. E. ( 1983; ). Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis. J Bacteriol 154, 728–736.
    [Google Scholar]
  51. Mathis, L. S. & Scocca, J. J. ( 1984; ). On the role of pili in transformation of Neisseria gonorrhoeae. J Gen Microbiol 130, 3165–3173.
    [Google Scholar]
  52. Mattick, J. S. ( 2002; ). Type IV pili and twitching motility. Annu Rev Microbiol 56, 289–314.[CrossRef]
    [Google Scholar]
  53. McGuinness, B. T., Clarke, I. N., Lambden, P. R., Barlow, A. K., Poolman, J. T., Jones, D. M. & Heckels, J. E. ( 1991; ). Point mutation in meningococcal por A gene associated with increased endemic disease. Lancet 337, 514–517.[CrossRef]
    [Google Scholar]
  54. Menard, R., Sansonetti, P. J. & Parsot, C. ( 1993; ). Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol 175, 5899–5906.
    [Google Scholar]
  55. Merz, A. J., Enns, C. A. & So, M. ( 1999; ). Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells. Mol Microbiol 32, 1316–1332.[CrossRef]
    [Google Scholar]
  56. Merz, A. J., So, M. & Sheetz, M. P. ( 2000; ). Pilus retraction powers bacterial twitching motility. Nature 407, 98–102.[CrossRef]
    [Google Scholar]
  57. Mietzner, T. A., Luginbuhl, G. H., Sandstrom, E. & Morse, S. A. ( 1984; ). Identification of an iron-regulated 37,000-Dalton protein in the cell envelope of Neisseria gonorrhoeae. Infect Immun 45, 410–416.
    [Google Scholar]
  58. Ochman, H., Lawrence, J. G. & Groisman, E. A. ( 2000; ). Lateral gene transfer and the nature of bacterial innovation. Nature 405, 299–304.[CrossRef]
    [Google Scholar]
  59. O'Toole, G. A. & Kolter, R. ( 1998; ). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30, 295–304.[CrossRef]
    [Google Scholar]
  60. Pannekoek, Y., van Putten, J. P. & Dankert, J. ( 1992; ). Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae. J Bacteriol 174, 6928–6937.
    [Google Scholar]
  61. Pujol, C., Eugene, E., Marceau, M. & Nassif, X. ( 1999; ). The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion. Proc Natl Acad Sci U S A 96, 4017–4022.[CrossRef]
    [Google Scholar]
  62. Rice, P., Longden, I. & Bleasby, A. ( 2000; ). EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16, 276–277.[CrossRef]
    [Google Scholar]
  63. Schmidt, K. L., Peterson, N. D., Kustusch, R. J., Wissel, M. C., Graham, B., Phillips, G. J. & Weiss, D. S. ( 2004; ). A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli. J Bacteriol 186, 785–793.[CrossRef]
    [Google Scholar]
  64. Stone, B. J. & Kwaik, Y. A. ( 1999; ). Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili. J Bacteriol 181, 1395–1402.
    [Google Scholar]
  65. Swanson, J. ( 1973; ). Studies on gonococcus infection. IV. Pili: their role in attachment of gonococci to tissue culture cells. J Exp Med 137, 571–589.[CrossRef]
    [Google Scholar]
  66. Swanson, J., Kraus, S. J. & Gotschlich, E. C. ( 1971; ). Studies on gonococcus infection. I. Pili and zones of adhesion: their relation to gonococcal growth patterns. J Exp Med 134, 886–906.[CrossRef]
    [Google Scholar]
  67. Tønjum, T., Weir, S., Bøvre, K., Progulske-Fox, A. & Marrs, C. F. ( 1993; ). Sequence divergence in two tandemly located pilin genes of Eikenella corrodens. Infect Immun 61, 1909–1916.
    [Google Scholar]
  68. Tønjum, T., Freitag, N. E., Namork, E. & Koomey, M. ( 1995; ). Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria. Mol Microbiol 16, 451–464.[CrossRef]
    [Google Scholar]
  69. Tønjum, T., Caugant, D. A., Dunham, S. A. & Koomey, M. ( 1998; ). Structure and function of repetitive sequence elements associated with a highly polymorphic domain of the Neisseria meningitidis PilQ protein. Mol Microbiol 29, 111–124.[CrossRef]
    [Google Scholar]
  70. Treangen, T. J., Ambur, O. H., Tonjum, T. & Rocha, E. P. ( 2008; ). The impact of the neisserial DNA uptake sequences on genome evolution and stability. Genome Biol 9, R60 [CrossRef]
    [Google Scholar]
  71. Voulhoux, R., Bos, M. P., Geurtsen, J., Mols, M. & Tommassen, J. ( 2003; ). Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299, 262–265.[CrossRef]
    [Google Scholar]
  72. Wolfgang, M., Lauer, P., Park, H. S., Brossay, L., Hebert, J. & Koomey, M. ( 1998; ). PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol Microbiol 29, 321–330.[CrossRef]
    [Google Scholar]
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