1887

Abstract

Mu transposition complexes were used for transposon mutagenesis of strain PA68. Mu DNA transposition complexes were assembled with MuA transposase and an artificial mini-Mu transposon , and introduced into by electroporation. Eight mutants deficient in twitching motility were isolated. Southern blotting confirmed that the insertions had occurred as single events. DNA sequencing of the region flanking the insertion in the twitching-motility mutants revealed that the mini-Mu transposon had inserted into six different genes, , , , , and . Four of these have previously been proven to be needed for twitching motility, whereas the and genes have not previously been shown to be required for twitching motility. The twitching-motility defect in the mutant was partially complemented by providing the gene , and the defect in the mutant was fully complemented when was provided. A mutant and a mutant were constructed by gene replacement in the PAO1 strain. These mutants were deficient in twitching motility, showing that both the and the gene are involved in twitching motility.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27131-0
2004-08-01
2019-11-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/8/mic1502653.html?itemId=/content/journal/micro/10.1099/mic.0.27131-0&mimeType=html&fmt=ahah

References

  1. Aldridge, P. & Jenal, U. ( 1999; ). Cell cycle-dependent degradation of a flagellar motor component requires a novel-type response regulator. Mol Microbiol 32, 379–391.[CrossRef]
    [Google Scholar]
  2. Allet, B. ( 1979; ). Mu insertion duplicates a 5 base pair sequence at host-inserted site. Cell 16, 123–130.[CrossRef]
    [Google Scholar]
  3. Alm, R. A. & Mattick, J. S. ( 1995; ). Identification of a gene, pilV, required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa, whose product possesses a pre-pilin-like leader sequence. Mol Microbiol 16, 485–496.[CrossRef]
    [Google Scholar]
  4. 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]
  5. Arora, S. K., Dasgupta, N., Lory, S. & Ramphal, R. ( 2000; ). Identification of two distinct types of flagellar cap proteins, FliD, in Pseudomonas aeruginosa. Infect Immun 68, 1474–1479.[CrossRef]
    [Google Scholar]
  6. Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Howe, K. L. & Sonnhammer, E. L. ( 2000; ). The Pfam protein families database. Nucleic Acids Res 28, 263–266.[CrossRef]
    [Google Scholar]
  7. Beatson, S. A., Whitchurch, C. B., Sargent, J. L., Levesque, R. C. & Mattick, J. S. ( 2002; ). Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J Bacteriol 184, 3605–3613.[CrossRef]
    [Google Scholar]
  8. Bradley, D. E. ( 1980; ). A function of Pseudomonas aeruginosa PAO pili: twitching motility. Can J Microbiol 26, 146–154.[CrossRef]
    [Google Scholar]
  9. Cai, B., Han, Y., Liu, B., Ren, Y. & Jiang, S. ( 2003; ). Isolation and characterization of an atrazine-degrading bacterium from industrial wastewater in China. Lett Appl Microbiol 36, 272–276.[CrossRef]
    [Google Scholar]
  10. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. ( 1999; ). Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322.[CrossRef]
    [Google Scholar]
  11. Croft, L., Beatson, S. A., Whitchurch, C. B., Huang, B., Blakeley, R. L. & Mattick, J. S. ( 2000; ). An interactive web-based Pseudomonas aeruginosa genome database: discovery of new genes, pathways and structures. Microbiology 146, 2351–2364.
    [Google Scholar]
  12. 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]
  13. Halkides, C. J., Zhu, X., Phillion, D. P., Matsumura, P. & Dahlquist, F. W. ( 1998; ). Synthesis and biochemical characterization of an analogue of CheY-phosphate, a signal transduction protein in bacterial chemotaxis. Biochemistry 37, 13674–13680.[CrossRef]
    [Google Scholar]
  14. Hecht, G. B. & Newton, A. ( 1995; ). Identification of a novel response regulator required for the swarmer-to-stalked-cell transition in Caulobacter crescentus. J Bacteriol 177, 6223–6229.
    [Google Scholar]
  15. Huang, B., Whitchurch, C. B. & Mattick, J. S. ( 2003; ). FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa. J Bacteriol 185, 7068–7076.[CrossRef]
    [Google Scholar]
  16. Kang, P. J., Hauser, A. R., Apodaca, G., Fleiszig, S. M., Wiener-Kronish, J., Mostov, K. & Engel, J. N. ( 1997; ). Identification of Pseudomonas aeruginosa genes required for epithelial cell injury. Mol Microbiol 24, 1249–1262.[CrossRef]
    [Google Scholar]
  17. Lamberg, A., Nieminen, S., Qiao, M. & Savilahti, H. ( 2002; ). Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage Mu. Appl Environ Microbiol 68, 705–712.[CrossRef]
    [Google Scholar]
  18. Lane, D. J. ( 1991; ). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 371–375. Edited by E. Stackebrandt & M. Goodfellow. Chester, UK: Wiley.
  19. Mah, T. F. & O'Toole, G. A. ( 2001; ). Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 9, 34–38.[CrossRef]
    [Google Scholar]
  20. Martin, P. R., Hobbs, M., Free, P. D., Jeske, Y. & Mattick, J. S. ( 1993; ). Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa. Mol Microbiol 9, 857–868.[CrossRef]
    [Google Scholar]
  21. Mattick, J. S. ( 2002; ). Type IV pili and twitching motility. Annu Rev Microbiol 56, 289–314.[CrossRef]
    [Google Scholar]
  22. McBride, M. J. ( 2001; ). Bacterial gliding motility: multiple mechanisms for cell movement over surfaces. Annu Rev Microbiol 55, 49–75.[CrossRef]
    [Google Scholar]
  23. 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]
  24. Rashid, M. H. & Kornberg, A. ( 2000; ). Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97, 4885–4890.[CrossRef]
    [Google Scholar]
  25. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  26. Schultz, J., Copley, R. R., Doerks, T., Ponting, C. P. & Bork, P. ( 2000; ). smart: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res 28, 231–234.[CrossRef]
    [Google Scholar]
  27. 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]
  28. Shan, Z., Xu, H., Shi, X., Nie, Z., Yu, Y., Zhang, X., Bai, Y., Qiao, M. & Gao, C. ( 2004; ). The study of optimal conditions of electroporation in Pseudomonas aeruginosa. Acta Genetica Sinica 31, 39–44.
    [Google Scholar]
  29. Simpson, D. A. & Speert, D. P. ( 2000; ). RpmA is required for nonopsonic phagocytosis of Pseudomonas aeruginosa. Infect immun 68, 2493–2502.[CrossRef]
    [Google Scholar]
  30. Smith, A. W. & Iglewski, B. H. ( 1989; ). Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res 17, 10509.[CrossRef]
    [Google Scholar]
  31. Wall, D. & Kaiser, D. ( 1999; ). Type IV pili and cell motility. Mol Microbiol 32, 1–10.[CrossRef]
    [Google Scholar]
  32. Ward, M. J. & Zusman, D. R. ( 1997; ). Regulation of directed motility in Myxococcus xanthus. Mol Microbiol 24, 885–893.[CrossRef]
    [Google Scholar]
  33. Whitchurch, C. B., Erova, T. E., Emery, J. A., Sargent, J. L., Harris, J. M., Semmler, A. B. T., Young, M. D., Mattick, J. S. & Wozniak, D. J. ( 2002; ). Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for Type IV fimbria-mediated twitching motility. J Bacteriol 184, 4544–4554.[CrossRef]
    [Google Scholar]
  34. Whiteley, M., Bangera, M. G., Bumgarner, R. E., Parsek, M. R., Teitzel, G. M., Lory, S. & Greenberg, E. P. ( 2001; ). Gene expression in Pseudomonas aeruginosa biofilms. Nature 413, 860–864.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27131-0
Loading
/content/journal/micro/10.1099/mic.0.27131-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error