Identification of a novel gene, , involved in twitching motility in

The GenBank accession number for the sequence determined in this work is U93274.

Free

Abstract

Transposon mutagenesis was used to identify a new locus required for twitching motility in . Four Tn-B21 mutants which lacked twitching motility and a fifth which exhibited impaired motility were found to map to the same I restriction fragment at approximately 40 min on the genome. Cloning and sequencing studies showed that all five transposon insertions occurred within the same 28 kb ORF, which was termed . The product of this gene has a putative peptidoglycan-binding domain, predicted transmembrane domains, a highly acidic C terminus and anomalous electrophoretic migration, indicating unusual primary or secondary structure. The genome also possesses a paralogue of . Homologues of were also found in the sequenced genomes of the other type-IV-fimbriated bacteria , , and , but not in those of other bacteria which lack type IV fimbriae. A homologue was also found in the genome sequence of , along with many other homologues of type IV fimbrial genes, indicating that this bacterium is also likely to produce type IV fimbriae. Wild-type twitching motility was restored to mutants by complementation in a dosage-dependent manner. Overexpression of resulted in an unusual phenotype where the cells were massively elongated and migrated in large convoys at the periphery of the colony. It is suggested that FimV may be involved in remodelling of the peptidoglycan layer to enable assembly of the type IV fimbrial structure and machinery.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-6-1321
2000-06-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/6/1461321a.html?itemId=/content/journal/micro/10.1099/00221287-146-6-1321&mimeType=html&fmt=ahah

References

  1. Akrim M., Bally M., Ball G., Tommassen J., Teerink H., Filloux A., Lazdunski A. 1993; Xcp-mediated protein secretion in Pseudomonas aeruginosa: identification of two additional genes and evidence for regulation of xcp gene expression. Mol Microbiol 10:431–443 [CrossRef]
    [Google Scholar]
  2. 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 prepilin-like leader sequence. Mol Microbiol 16:485–496 [CrossRef]
    [Google Scholar]
  3. Alm R. A., Mattick J. S. 1996; Identification of two genes with prepilin-like leader sequences required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa. J Bacteriol 178:3809–3817
    [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. Alm R. A., Bodero A. J., Free P. D., Mattick J. S. 1996; Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa. J Bacteriol 178:46–53
    [Google Scholar]
  6. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., 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]
  7. Arps P. J., Winkler M. E. 1987; Structural analysis of the Escherichia coli K-12 hisT operon by using a kanamycin resistance cassette. J Bacteriol 169:1061–1070
    [Google Scholar]
  8. Bally M., Filloux A., Akrim M., Ball G., Lazdunski A., Tommassen J. 1992; Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol 6:1121–1131 [CrossRef]
    [Google Scholar]
  9. Barnett T. C., Kirov S. M., Strom M. S., Sanderson K. 1997; Aeromonas spp. possess at least two distinct type IV pilus families. Microb Pathog 23:241–247 [CrossRef]
    [Google Scholar]
  10. Bodey G. P., Bolivar R., Fainstein V., Jadeja L. 1983; Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5:279–313 [CrossRef]
    [Google Scholar]
  11. Bradley D. E. 1980; A function of Pseudomonas aeruginosa PAO pili: twitching motility. Can J Microbiol 26:146–154 [CrossRef]
    [Google Scholar]
  12. Brunschwig E., Darzins A. 1992; A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa. Gene 111:35–41 [CrossRef]
    [Google Scholar]
  13. 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]
  14. 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]
  15. Dalrymple B., Mattick J. S. 1987; An analysis of the organization and evolution of type 4 fimbrial (MePhe) subunit proteins. J Mol Evol 25:261–269 [CrossRef]
    [Google Scholar]
  16. 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]
  17. Darzins A. 1994; Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus biosythesis and twitching motility: sequence similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus. Mol Microbiol 11:137–153 [CrossRef]
    [Google Scholar]
  18. Dorr J., Hurek T., Reinhold-Hurek B. 1998; Type IV pili are involved in plant–microbe and fungus–microbe interactions. Mol Microbiol 30:7–17 [CrossRef]
    [Google Scholar]
  19. Engvall E., Perlmann P. 1972; Enzyme-linked immunoabsorbent assay, Elisa. III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J Immunol 109:128–135
    [Google Scholar]
  20. Filloux A., Bally M., Ball G., Akrim M., Tommasson J., Lazdunski A. 1990; Protein secretion in Gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria. EMBO J 9:4323–4329
    [Google Scholar]
  21. Folkhard W., Marvin D. A., Watts T. H., Paranchych W. 1981; Structure of polar pili from Pseudomonas aeruginosa strains K and O. J Mol Biol 149:79–93 [CrossRef]
    [Google Scholar]
  22. Forest K. T., Tainer J. A. 1997; Type-4 pilus-structure: outside to inside and top to bottom – a minireview. Gene 192:165–169 [CrossRef]
    [Google Scholar]
  23. Hazlett L. D., Moon M. M., Singh A., Berk R. S., Rudner X. L. 1991; Analysis of adhesion, piliation, protease production and ocular infectivity of several P. aeruginosa strains. Curr Eye Res 10:351–362 [CrossRef]
    [Google Scholar]
  24. Hoang T. T., Schweizer H. P. 1997; Identification and genetic characterization of the Pseudomonas aeruginosa leuB gene encoding 3-isopropylmalate dehydrogenase. Mol Gen Genet 254:166–170 [CrossRef]
    [Google Scholar]
  25. Hoang T. T., Williams S., Schweizer H. P., Lam J. S. 1997; Molecular genetic analysis of the region containing the essential Pseudomonas aeruginosa asd gene encoding aspartate-β-semialdehyde dehydrogenase. Microbiology 143:899–907 [CrossRef]
    [Google Scholar]
  26. Hobbs M., Dalrymple B. P., Cox P. T., Livingston S. P., Delaney S. F., Mattick J. S. 1991; Organization of the fimbrial gene region of Bacteroides nodosus: class I and class II strains. Mol Microbiol 5:543–560 [CrossRef]
    [Google Scholar]
  27. Hobbs M., Collie E. S. R., Free P. D., Livingston S. P., Mattick J. S. 1993; PilS and PilR, a two-component transcriptional regulatory system controlling transcription of type 4 fimbriae in Pseudomonas aeruginosa. Mol Microbiol 7:669–682 [CrossRef]
    [Google Scholar]
  28. Joris B., Englebert S., Chu C. P., Kariyama R., Daneo-Moore L., Shockman G. D., Ghuysen J. M. 1992; Modular design of the Enterococcus hirae muramidase-2 and Streptococcus faecalis autolysin. FEMS Microbiol Lett 91:257–264 [CrossRef]
    [Google Scholar]
  29. Kizil G., Todd I., Atta M., Borriello S. P., Ait-Tahar K., Ala’Aldeen D. A. 1999; Identification and characterization of TspA, a major CD4(+) T-cell- and B-cell-stimulating Neisseria-specific antigen. Infect Immun 67:3533–3541
    [Google Scholar]
  30. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  31. Liles M. R., Viswanathan V. K., Cianciotto N. P. 1998; Identification and temperature regulation of Legionella pneumophila genes involved in type IV pilus biogenesis and type II protein secretion. Infect Immun 66:1776–1782
    [Google Scholar]
  32. 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]
  33. Mattick J. S., Bills M. M., Anderson B. J., Dalrymple B., Mott M. R., Egerton J. R. 1987; Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa. J Bacteriol 169:33–41
    [Google Scholar]
  34. Mattick J. S., Hobbs M., Cox P. T., Dalrymple B. P. 1993; Molecular biology of the fimbriae of Dichelobacter (prev. Bacteroides) nodosus. In Genetics and Molecular Biology of Anaerobic Bacteria pp. 517–545Edited by Sebald M. New York: Springer;
    [Google Scholar]
  35. Mongkolsuk S., Vattanaviboon P., Rabibhadana S., Kiatpapan P. 1993; Versatile gene cassette plasmids to facilitate the construction of generalized and specialized cloning vectors. Gene 124:131–132 [CrossRef]
    [Google Scholar]
  36. Morales V. M., Backman A., Bagdasarian M. 1991; A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97:39–47 [CrossRef]
    [Google Scholar]
  37. Murata T. 1996; The trpF nucleotide sequence and its promoter analysis in Pseudomonas aeruginosa. Microbiol Immunol 40:107–114 [CrossRef]
    [Google Scholar]
  38. Nakai K., Horton P. 1999; psort: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–36 [CrossRef]
    [Google Scholar]
  39. 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]
  40. Paranchych W., Frost L. S. 1988; The physiology and biochemistry of pili. Adv Microb Physiol 29:53–114
    [Google Scholar]
  41. Parge H. E., Bernstein S. L., Deal C. D.9 other authors 1990; Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae. J Biol Chem 265:2278–2285
    [Google Scholar]
  42. Pepe C. M., Eklund M. W., Strom M. S. 1996; Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion. Mol Microbiol 19:857–869 [CrossRef]
    [Google Scholar]
  43. Ponting C. P., Schultz J., Milpetz F., Bork P. 1999; smart: identification and annotation of domains from signalling and extracellular protein sequences. Nucleic Acids Res 27:229–232 [CrossRef]
    [Google Scholar]
  44. Potera C. 1999; Forging a link between biofilms and disease. Science 283:1837–1839 [CrossRef]
    [Google Scholar]
  45. Ratnaningsih E., Dharmsthiti S., Krishnapillai V., Morgan A., Sinclair M., Holloway B. W. 1990; A combined physical and genetic map of Pseudomonas aeruginosa PAO. J Gen Microbiol 136:2351–2357 [CrossRef]
    [Google Scholar]
  46. 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]
  47. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  48. Sato H., Okinaga K., Saito H. 1988; Role of pili in the pathogenesis of Pseudomonas aeruginosa burn infection. Microbiol Immunol 32:131–139 [CrossRef]
    [Google Scholar]
  49. Schultz J., Milpetz F., Bork P., Ponting C. P. 1998; smart, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA 95:5857–5864 [CrossRef]
    [Google Scholar]
  50. Schweizer H. P. 1991; EscherichiaPseudomonas shuttle vectors derived from pUC18/19. Gene 97:109–112 [CrossRef]
    [Google Scholar]
  51. Schweizer H. P. 1992; Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6:1195–1204 [CrossRef]
    [Google Scholar]
  52. 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]
  53. Simon R., Priefer U., Pühler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1:784–791 [CrossRef]
    [Google Scholar]
  54. Stone B. J., Abu Kwaik Y. 1998; Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells. Infect Immun 66:1768–1775
    [Google Scholar]
  55. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354 [CrossRef]
    [Google Scholar]
  56. 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]
  57. Watson A. A., Alm R. A., Mattick J. S. 1996a; Construction of improved vectors for protein production in Pseudomonas aeruginosa. Gene 172:163–164 [CrossRef]
    [Google Scholar]
  58. Watson A. A., Mattick J. S., Alm R. A. 1996b; Functional expression of heterologous type 4 fimbriae in Pseudomonas aeruginosa. Gene 175:143–150 [CrossRef]
    [Google Scholar]
  59. West S. E. H., Iglewski B. H. 1988; Codon usage in Pseudomonas aeruginosa. Nucleic Acids Res 16:9323–9334 [CrossRef]
    [Google Scholar]
  60. Wu S. S., Kaiser D. 1995; Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus. Mol Microbiol 18:547–558 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-6-1321
Loading
/content/journal/micro/10.1099/00221287-146-6-1321
Loading

Data & Media loading...

Most cited Most Cited RSS feed