1887

Abstract

Twitching motility is a form of bacterial translocation over firm surfaces that requires retractile type IV pili. Microscopic colonies of strains AW1, K60 and GMI1000 growing on the surface of a rich medium solidified with 16% agar appeared to exhibit twitching motility, because early on they divided into motile ‘rafts’ of cells and later developed protruding ‘spearheads’ at their margins. Individual motile bacteria were observed only when they were embedded within masses of other cells. Varying degrees of motility were observed for 33 of 35 strains of in a selected, diverse collection. Timing was more important than culture conditions for observing motility, because by the time wild-type colonies were easily visible by eye (about 48 h) this activity ceased and the spearheads were obscured by continued bacterial multiplication. In contrast, inactivation of PhcA, a transcriptional regulator that is essential for to cause plant disease, resulted in colonies that continued to expand for at least several additional days. Multiple strains with mutations in regulatory genes important for virulence were tested, but all exhibited wild-type motility. Many of the genes required for production of functional type IV pili, and hence for twitching motility, are conserved among unrelated bacteria, and , and orthologues were identified in . Colonies of and mutants did not develop spearheads or rafts, confirming that the movement of cells that had been observed was due to twitching motility. Compared to the wild-type parents, both and mutants caused slower and less severe wilting on susceptible tomato plants. This is the first report of twitching motility by a phytopathogenic bacterium, and the first example where type IV pili appear to contribute significantly to plant pathogenesis.

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2001-12-01
2019-12-16
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References

  1. Allen, C., Gay, J. & Simon-Buela, L. ( 1997; ). A regulatory locus, pehSR, controls polygalacturonase production and other virulence functions in Ralstonia solanacearum. Mol Plant-Microbe Interact 9, 1054-1064.
    [Google Scholar]
  2. Alm, R. A. & Mattick, J. S. ( 1997; ). Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa. Gene 192, 89-98.[CrossRef]
    [Google Scholar]
  3. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Webb, M. & Lipman, D. J. ( 1997; ). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.[CrossRef]
    [Google Scholar]
  4. Araud-Razou, I., Vasse, J., Montrozier, H., Etchebar, C. & Trigalet, A. ( 1998; ). Detection and visualization of the major acidic exopolysaccharide of Ralstonia solanacearum and its role in tomato root infection and vascular colonization. Eur J Plant Pathol 104, 795-809.[CrossRef]
    [Google Scholar]
  5. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1989). Short Protocols in Molecular Biology. New York: Green Publishing Associates and Wiley-Interscience.
  6. Bertolla, F., van Gijsegem, F., Nesme, X. & Simonet, P. ( 1997; ). Conditions for natural transformation of Ralstonia solanacearum. Appl Environ Microbiol 63, 4965-4968.
    [Google Scholar]
  7. Bradley, D. E. ( 1980; ). A function of Pseudomonas aeruginosa PAO polar pili: twitching motility. Can J Microbiol 26, 146-154.[CrossRef]
    [Google Scholar]
  8. Brito, B., Marenda, M., Barberis, P., Boucher, C. & Genin, S. ( 1999; ). prhJ and hrpG, two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum. Mol Microbiol 31, 237-251.[CrossRef]
    [Google Scholar]
  9. Brumbley, S. M. & Denny, T. P. ( 1990; ). Cloning of phcA from wild-type Pseudomonas solanacearum, a gene that when mutated alters expression of multiple traits that contribute to virulence. J Bacteriol 172, 5677-5685.
    [Google Scholar]
  10. Carney, B. F. & Denny, T. P. ( 1990; ). A cloned avirulence gene from Pseudomonas solanacearum determines incompatibility on Nicotiana tabacum at the host species level. J Bacteriol 172, 4836-4843.
    [Google Scholar]
  11. Chen, W. P. & Kuo, T. T. ( 1993; ). A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Res 21, 2260.[CrossRef]
    [Google Scholar]
  12. Clough, S. J., Schell, M. A. & Denny, T. P. ( 1994; ). Evidence for involvement of a volatile extracellular factor in Pseudomonas solanacearum virulence gene expression. Mol Plant-Microbe Interact 7, 621-630.[CrossRef]
    [Google Scholar]
  13. Clough, S. J., Flavier, A. B., Schell, M. A. & Denny, T. P. ( 1997a; ). Differential expression of virulence genes and motility in Ralstonia (Pseudomonas) solanacearum during exponential growth. Appl Environ Microbiol 63, 844-850.
    [Google Scholar]
  14. Clough, S. J., Lee, K.-E., Schell, M. A. & Denny, T. P. ( 1997b; ). A two-component system in Ralstonia (Pseudomonas) solanacearum modulates production of PhcA-regulated virulence factors in response to 3-hydroxypalmitic acid methyl ester. J Bacteriol 179, 3639-3648.
    [Google Scholar]
  15. Cook, D. & Sequeira, L. ( 1991; ). Genetic and biochemical characterization of a Pseudomonas solanacearum gene cluster required for extracellular polysaccharide production and for virulence. J Bacteriol 173, 1654-1662.
    [Google Scholar]
  16. Cook, D., Barlow, E. & Sequeira, L. ( 1989; ). Genetic diversity of Pseudomonas solanacearum: detection of restriction fragment length polymorphisms with DNA probes that specify virulence and the hypersensitive response. Mol Plant-Microbe Interact 2, 113-121.[CrossRef]
    [Google Scholar]
  17. Cornelis, G. R. & van Gijsegem, F. ( 2000; ). Assembly and function of type III secretory systems. Annu Rev Microbiol 54, 735-774.[CrossRef]
    [Google Scholar]
  18. Darzins, A. ( 1993; ). The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY. J Bacteriol 175, 5934-5944.
    [Google Scholar]
  19. Denny, T. P., Makini, F. W. & Brumbley, S. M. ( 1988; ). Characterization of Pseudomonas solanacearum Tn5 mutants deficient in extracellular polysaccharide. Mol Plant-Microbe Interact 1, 215-223.[CrossRef]
    [Google Scholar]
  20. Denny, T. P., Carney, B. F. & Schell, M. A. ( 1990; ). Inactivation of multiple virulence genes reduces the ability of Pseudomonas solanacearum to cause wilt symptoms. Mol Plant-Microbe Interact 3, 293-300.[CrossRef]
    [Google Scholar]
  21. van Doorn, J., Boonekamp, P. M. & Oudega, B. ( 1994; ). Partial characterization of fimbriae of Xanthomonas campestris pv. hyacinthi. Mol Plant-Microbe Interact 7, 334-344.[CrossRef]
    [Google Scholar]
  22. Fernandez, L. A. & Berenguer, J. ( 2000; ). Secretion and assembly of regular surface structures in Gram-negative bacteria. FEMS Microbiol Rev 24, 21-44.[CrossRef]
    [Google Scholar]
  23. Flavier, A. B., Ganova-Raeva, L. M., Schell, M. A. & Denny, T. P. ( 1997; ). Hierarchical autoinduction in Ralstonia solanacearum: control of acyl-homoserine lactone production by a novel autoregulatory system responsive to 3-hydroxypalmitic acid methyl ester. J Bacteriol 179, 7089-7097.
    [Google Scholar]
  24. Flavier, A. B., Schell, M. A. & Denny, T. P. ( 1998; ). An RpoS (σS) homologue regulates acylhomoserine lactone-dependent autoinduction in Ralstonia solanacearum. Mol Microbiol 28, 475-486.[CrossRef]
    [Google Scholar]
  25. Fuerst, J. A. & Hayward, A. C. ( 1969; ). Surface appendages similar to fimbriae (pili) on Pseudomonas species. J Gen Microbiol 58, 227-237.[CrossRef]
    [Google Scholar]
  26. Fussenegger, M., Rudel, T., Barten, R., Ryll, R. & Meyer, T. F. ( 1997; ). Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae – a review. Gene 192, 125-134.[CrossRef]
    [Google Scholar]
  27. Galan, J. E., Ginocchio, C. & Costeas, P. ( 1992; ). Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J Bacteriol 174, 4338-4349.
    [Google Scholar]
  28. Genin, S. & Boucher, C. A. ( 1994; ). A superfamily of proteins involved in different secretion pathways in Gram-negative bacteria: modular structure and specificity of the N-terminal domain. Mol Gen Genet 243, 112-118.[CrossRef]
    [Google Scholar]
  29. van Gijsegem, F., Gough, C., Zischek, C. & 7 other authors ( 1995; ). The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex. Mol Microbiol 15, 1095–1114.[CrossRef]
    [Google Scholar]
  30. van Gijsegem, F., Vasse, J., Camus, J. C., Marenda, M. & Boucher, C. ( 2000; ). Ralstonia solanacearum produces Hrp-dependent pili that are required for PopA secretion but not for attachment of bacteria to plant cells. Mol Microbiol 36, 249-260.[CrossRef]
    [Google Scholar]
  31. Hayward, A. C. ( 2000; ). Ralstonia solanacearum. In Encyclopedia of Microbiology , pp. 32-42. Edited by J. Lederberg. San Diego, CA:Academic Press.
  32. Henrichsen, J. ( 1972; ). Bacterial surface translocation: a survey and a classification. Bacteriol Rev 36, 478-503.
    [Google Scholar]
  33. Henrichsen, J. ( 1975a; ). The occurence of twitching motility among gram-negative bacteria. Acta Pathol Microbiol Scand Sect B 83, 171-178.
    [Google Scholar]
  34. Henrichsen, J. ( 1975b; ). The influence of changes in the environment on twitching motility. Acta Pathol Microbiol Scand Sect B 83, 179-186.
    [Google Scholar]
  35. Henrichsen, J. ( 1983; ). Twitching motility. Annu Rev Microbiol 37, 81-93.[CrossRef]
    [Google Scholar]
  36. Henrichsen, J. & Blom, J. ( 1975; ). Examination of fimbriation of some Gram-negative rods with and without twitching and gliding motility. Acta Pathol Microbiol Scand Sect B 83, 161-170.
    [Google Scholar]
  37. Huang, Q. & Allen, C. ( 1997; ). An exo-poly-α-d-galacturonosidase, PehB, is required for wild-type virulence of Ralstonia solanacearum . J Bacteriol 179, 7369-7378.
    [Google Scholar]
  38. Huang, J., Carney, B. F., Denny, T. P., Weissinger, A. K. & Schell, M. A. ( 1995; ). A complex network regulates expression of eps and other virulence genes of Pseudomonas solanacearum. J Bacteriol 177, 1259-1267.
    [Google Scholar]
  39. Hueck, C. J. ( 1998; ). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62, 379-433.
    [Google Scholar]
  40. Kagami, Y., Ratliff, M., Surber, M., Martinez, A. & Nunn, D. N. ( 1998; ). Type II protein secretion by Pseudomonas aeruginosa: genetic suppression of a conditional mutation in the pilin-like component XcpT by the cytoplasmic component XcpR. Mol Microbiol 27, 221-233.[CrossRef]
    [Google Scholar]
  41. Kang, Y., Huang, J. Z., Mao, G. Z., He, L. Y. & Schell, M. A. ( 1994; ). Dramatically reduced virulence of mutants of Pseudomonas solanacearum defective in export of extracellular proteins across the outer membrane. Mol Plant-Microbe Interact 7, 370-377.[CrossRef]
    [Google Scholar]
  42. Kang, Y., Saile, E., Schell, M. A. & Denny, T. P. ( 1999; ). Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum. Appl Environ Microbiol 65, 2356-2362.
    [Google Scholar]
  43. 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]
  44. Kitten, T. & Willis, D. K. ( 1996; ). Suppression of a sensor kinase-dependent phenotype in Pseudomonas syringae by ribosomal proteins L35 and L20. J Bacteriol 178, 1548-1555.
    [Google Scholar]
  45. Lory, S. & Strom, M. S. ( 1997; ). Structure-function relationship of type-IV prepilin peptidase of Pseudomonas aeruginosa – a review. Gene 192, 117-121.[CrossRef]
    [Google Scholar]
  46. McGarvey, J. A., Denny, T. P. & Schell, M. A. ( 1999; ). Spatial-temporal and quantitative analysis of growth and EPS I production by Ralstonia solanacearum in resistant and susceptible tomato cultivars. Phytopathology 89, 1233-1239.[CrossRef]
    [Google Scholar]
  47. Marenda, M., Brito, B., Callard, D., Genin, S., Barberis, P., Boucher, C. & Arlat, M. ( 1998; ). PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells. Mol Microbiol 27, 437-453.[CrossRef]
    [Google Scholar]
  48. Matthysse, A. G., Stretton, S., Dandie, C., McClure, N. C. & Goodman, A. E. ( 1996; ). Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiol Lett 145, 87-94.[CrossRef]
    [Google Scholar]
  49. Merz, A. J., So, M. & Sheetz, M. P. ( 2000; ). Pilus retraction powers bacterial twitching motility. Nature 407, 98-102.[CrossRef]
    [Google Scholar]
  50. Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  51. Nunn, D. ( 1999; ). Bacterial type II protein export and pilus biogenesis: more than just homologies? Trends Cell Biol 9, 402-408.[CrossRef]
    [Google Scholar]
  52. 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]
  53. Ojanen-Reuhs, T., Kalkkinen, N., Westerlund-Wikstrom, B., van Doorn, J., Haahtela, K., Nurmiaholassila, E. L., Wengelnik, K., Bonas, U. & Korhonen, T. K. ( 1997; ). Characterization of the fimA gene encoding bundle-forming fimbriae of the plant pathogen Xanthomonas campestris pv. vesicatoria. J Bacteriol 179, 1280-1290.
    [Google Scholar]
  54. Pugsley, A. P. ( 1993; ). The complete general secretory pathway in Gram-negative bacteria. Microbiol Rev 57, 50-108.
    [Google Scholar]
  55. Roine, E., Raineri, D. M., Romantschuk, M., Wilson, M. & Nunn, D. N. ( 1998; ). Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000. Mol Plant-Microbe Interact 11, 1048-1056.[CrossRef]
    [Google Scholar]
  56. Saile, E., Schell, M. A. & Denny, T. P. ( 1997; ). Role of extracellular polysaccharide and endoglucanase in root invasion and colonization of tomato plants by Ralstonia solanacearum. Phytopathology 87, 1264-1271.[CrossRef]
    [Google Scholar]
  57. Schell, M. A. ( 2000; ). Control of virulence and pathogenicity genes of Ralstonia solanacearum by an elaborate sensory array. Annu Rev Phytopathol 38, 263-292.[CrossRef]
    [Google Scholar]
  58. Schweizer, H. P. ( 1993; ). Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. BioTechniques 15, 831-833.
    [Google Scholar]
  59. Semmler, A. B. T., Whitchurch, C. B. & Mattick, J. S. ( 1999; ). A re-examination of twitching motility in Pseudomonas aeruginosa. Microbiology 145, 2863-2873.
    [Google Scholar]
  60. Simpson, A. J. G., Reinach, F. C., Arruda, P. & 112 other authors ( 2000; ). The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406, 151–159.[CrossRef]
    [Google Scholar]
  61. Skerker, J. M. & Berg, H. C. ( 2001; ). Direct observation of extension and retraction of type IV pili. Proc Natl Acad Sci USA 98, 6901-6904.[CrossRef]
    [Google Scholar]
  62. Staskawicz, B., Dahlbeck, D., Keen, N. & Napoli, C. ( 1987; ). Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol 169, 5789-5794.
    [Google Scholar]
  63. Stemmer, W. P. C. & Sequeira, L. ( 1987; ). Fimbriae of phytopathogenic and symbiotic bacteria. Phytopathology 77, 1633-1639.[CrossRef]
    [Google Scholar]
  64. Strom, M. S. & Lory, S. ( 1993; ). Structure-function and biogenesis of the type IV pili. Annu Rev Microbiol 47, 565-596.[CrossRef]
    [Google Scholar]
  65. Su, W. C., Tung, S. Y., Yang, M. K. & Kuo, T. T. ( 1999; ). The pilA gene of Xanthomonas campestris pv. citri is required for infection by the filamentous phage cf. Mol Gen Genet 262, 22-26.[CrossRef]
    [Google Scholar]
  66. Sun, H., Zusman, D. R. & Shi, W. Y. ( 2000; ). Type IV pilus of Myxococcus xanthus is a motility apparatus controlled by the frz chemosensory system. Curr Biol 10, 1143-1146.[CrossRef]
    [Google Scholar]
  67. Tans-Kersten, J., Guan, Y. F. & Allen, C. ( 1998; ). Ralstonia solanacearum pectin methylesterase is required for growth on methylated pectin but not for bacterial wilt virulence. Appl Environ Microbiol 64, 4918-4923.
    [Google Scholar]
  68. Tans-Kersten, J., Huang, H. Y. & Allen, C. ( 2001; ). Ralstonia solanacearum needs motility for invasive virulence on tomato. J Bacteriol 183, 3597-3605.[CrossRef]
    [Google Scholar]
  69. Titarenko, E., Lopez-Solanilla, E., Garcia-Olmedo, F. & Rodriguez-Palenzuela, P. ( 1997; ). Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco. J Bacteriol 179, 6699-6704.
    [Google Scholar]
  70. Wall, D. & Kaiser, D. ( 1999; ). Type IV pili and cell motility. Mol Microbiol 32, 1-10.[CrossRef]
    [Google Scholar]
  71. Wall, D., Kolenbrander, P. E. & Kaiser, D. ( 1999; ). The Myxococcus xanthus pilQ (sglA) gene encodes a secretin homolog required for type IV pilus biogenesis, social motility, and development. J Bacteriol 181, 24-33.
    [Google Scholar]
  72. Wolfgang, M., Park, H. S., Hayes, S. F., van Putten, J. P. M. & Koomey, M. ( 1998; ). Suppression of an absolute defect in Type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae. Proc Natl Acad Sci USA 95, 14973-14978.[CrossRef]
    [Google Scholar]
  73. Wolfgang, M., van Putten, J. P., Hayes, S. F., Dorward, D. & Koomey, M. ( 2000; ). Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili. EMBO J 19, 6408-6418.[CrossRef]
    [Google Scholar]
  74. Young, D. H., Stemmer, W. P. C. & Sequeira, L. ( 1985; ). Reassembly of a fimbrial hemagglutinin from Pseudomonas solanacearum after purification of the subunit by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Appl Environ Microbiol 50, 605-610.
    [Google Scholar]
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