Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1993 Apr;61(4):1371–1377. doi: 10.1128/iai.61.4.1371-1377.1993

Genetic and functional characterization of the gene cluster specifying expression of Pseudomonas aeruginosa pili.

T Koga 1, K Ishimoto 1, S Lory 1
PMCID: PMC281373  PMID: 7681046

Abstract

The genetic organization of the gene cluster containing pilA, the structural gene for type IV pilin of Pseudomonas aeruginosa, as well as the accessory genes pilB, pilC, and pilD, has been studied. DNA sequences capable of initiating transcription when fused to a promoterless lacZ gene have been identified in the pilA-pilB and pilB-pilC intergenic regions. Unlike pilA, which requires rpoN (encoding the sigma 54 subunit of RNA polymerase) and products of two regulatory genes, pilS and pilR, expression of pilB, pilC, or pilD did not depend on any of these transcriptional regulators. Moreover, transcription of pilA from the tac promoter in an rpoN mutant background resulted in piliated bacteria, suggesting that the RpoN-based regulatory network is specific for pilA and does not control expression of any other genes necessary for formation of pili. Insertion of the omega fragment containing strong transcriptional terminators into pilB, pilC, and pilD failed to have a polar effect on expression of downstream genes, as determined by the ability of each cloned gene to complement, in trans, the corresponding insertionally inactivated chromosomal copy. Insertions into pilC, however, resulted in decreased synthesis of PilD as determined by quantitation of PilD enzymatic activity in processing prepilin in vitro and by immunoassay. This finding suggests that PilD may require PilC for its optimal stability or correct membrane localization.

Full text

PDF
1371

Images in this article

1374Image
on p.1374
1375Image
on p.1375
1375Image
on p.1375
1375Image
on p.1375

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradley D. E. A pilus-dependent Pseudomonas aeruginosa bacteriophage with a long noncontractile tail. Virology. 1973 Feb;51(2):489–492. doi: 10.1016/0042-6822(73)90448-0. [DOI] [PubMed] [Google Scholar]
  3. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gilligan P. H. Microbiology of airway disease in patients with cystic fibrosis. Clin Microbiol Rev. 1991 Jan;4(1):35–51. doi: 10.1128/cmr.4.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hancock R. E., Nikaido H. Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J Bacteriol. 1978 Oct;136(1):381–390. doi: 10.1128/jb.136.1.381-390.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ishimoto K. S., Lory S. Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1954–1957. doi: 10.1073/pnas.86.6.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ishimoto K. S., Lory S. Identification of pilR, which encodes a transcriptional activator of the Pseudomonas aeruginosa pilin gene. J Bacteriol. 1992 Jun;174(11):3514–3521. doi: 10.1128/jb.174.11.3514-3521.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson K., Parker M. L., Lory S. Nucleotide sequence and transcriptional initiation site of two Pseudomonas aeruginosa pilin genes. J Biol Chem. 1986 Nov 25;261(33):15703–15708. [PubMed] [Google Scholar]
  11. Kustu S., Santero E., Keener J., Popham D., Weiss D. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev. 1989 Sep;53(3):367–376. doi: 10.1128/mr.53.3.367-376.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Leong S. A., Ditta G. S., Helinski D. R. Heme biosynthesis in Rhizobium. Identification of a cloned gene coding for delta-aminolevulinic acid synthetase from Rhizobium meliloti. J Biol Chem. 1982 Aug 10;257(15):8724–8730. [PubMed] [Google Scholar]
  14. Marrs C. F., Schoolnik G., Koomey J. M., Hardy J., Rothbard J., Falkow S. Cloning and sequencing of a Moraxella bovis pilin gene. J Bacteriol. 1985 Jul;163(1):132–139. doi: 10.1128/jb.163.1.132-139.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McKern N. M., O'Donnell I. J., Inglis A. S., Stewart D. J., Clark B. L. Amino acid sequence of pilin from Bacteroides nodosus (strain 198), the causative organism of ovine footrot. FEBS Lett. 1983 Nov 28;164(1):149–153. doi: 10.1016/0014-5793(83)80039-8. [DOI] [PubMed] [Google Scholar]
  16. Meyer T. F., Billyard E., Haas R., Storzbach S., So M. Pilus genes of Neisseria gonorrheae: chromosomal organization and DNA sequence. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6110–6114. doi: 10.1073/pnas.81.19.6110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nunn D. N., Lory S. Components of the protein-excretion apparatus of Pseudomonas aeruginosa are processed by the type IV prepilin peptidase. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):47–51. doi: 10.1073/pnas.89.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nunn D. N., Lory S. Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3281–3285. doi: 10.1073/pnas.88.8.3281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nunn D., Bergman S., Lory S. Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol. 1990 Jun;172(6):2911–2919. doi: 10.1128/jb.172.6.2911-2919.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pier G. B. Pulmonary disease associated with Pseudomonas aeruginosa in cystic fibrosis: current status of the host-bacterium interaction. J Infect Dis. 1985 Apr;151(4):575–580. doi: 10.1093/infdis/151.4.575. [DOI] [PubMed] [Google Scholar]
  21. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Strom M. S., Lory S. Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly. J Biol Chem. 1991 Jan 25;266(3):1656–1664. [PubMed] [Google Scholar]
  25. Strom M. S., Lory S. Cloning and expression of the pilin gene of Pseudomonas aeruginosa PAK in Escherichia coli. J Bacteriol. 1986 Feb;165(2):367–372. doi: 10.1128/jb.165.2.367-372.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Strom M. S., Nunn D., Lory S. Multiple roles of the pilus biogenesis protein pilD: involvement of pilD in excretion of enzymes from Pseudomonas aeruginosa. J Bacteriol. 1991 Feb;173(3):1175–1180. doi: 10.1128/jb.173.3.1175-1180.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Totten P. A., Lory S. Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J Bacteriol. 1990 Dec;172(12):7188–7199. doi: 10.1128/jb.172.12.7188-7199.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Woods D. E., Straus D. C., Johanson W. G., Jr, Berry V. K., Bass J. A. Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells. Infect Immun. 1980 Sep;29(3):1146–1151. doi: 10.1128/iai.29.3.1146-1151.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. von Gabain A., Belasco J. G., Schottel J. L., Chang A. C., Cohen S. N. Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci U S A. 1983 Feb;80(3):653–657. doi: 10.1073/pnas.80.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES