Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Aug;64(8):3154–3160. doi: 10.1128/iai.64.8.3154-3160.1996

Characterization of elastase-deficient clinical isolates of Pseudomonas aeruginosa.

A N Hamood 1, J Griswold 1, J Colmer 1
PMCID: PMC174201  PMID: 8757847

Abstract

Elastase production in Pseudomonas aeruginosa is regulated by the lasR, lasI, rhlR, and rhlI genes. Recently, we have analyzed several clinical isolates of P. aeruginosa for the production of elastase and other extracellular virulence factors. Four of these isolates (CIT1, CIW5, CIW7, and CIW8) produced no elastolytic activity. We have characterized these isolates with respect to their elastase-deficient phenotype. Elastase was detected by immunoblotting experiments using elastase-specific antiserum. We also determined the presence of IasB and IasR mRNAs by Northern (RNA) blot hybridization experiments using lasB and lasR internal probes, respectively. None of the four elastase-deficient strains produced either the elastase protein or the lasB mRNA. Complementation experiments (using plasmids carrying either the lasB or the lasR gene) were conducted to determine if the isolates carry defective lasB or lasR genes. The presence of either a lasB or a lasR plasmid in CIW7 and CIW8 resulted in the production of very low levels of elastase and lasB mRNA. Neither elastase nor lasB mRNA was detected in CIT1 and CIW5 carrying the lasB plasmid. The presence of the lasR plasmid in CIT1 and CIW5 resulted in the production of lasB mRNA and elastase protein in CIW5 only. All elastase-deficient strains produced detectable levels of lasR mRNA which were enhanced in the presence of the lasR plasmid. The Pseudomonas autoinducer (which is encoded by lasI) was also produced by all strains. CIT1 produced both hemolysin and alkaline protease but was defective in pyocyanin production. These results suggest that (i) CIT1 may contain a defect in a lasB-regulatory gene, (ii) CIW5 carries a defect within lasR, and (iii) the defect in isolates CIW7 and CIW8 affects the efficiency of lasB transcription.

Full Text

The Full Text of this article is available as a PDF (336.3 KB).

Selected References

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

  1. Bagdasarian M., Timmis K. N. Host: vector systems for gene cloning in Pseudomonas. Curr Top Microbiol Immunol. 1982;96:47–67. doi: 10.1007/978-3-642-68315-2_4. [DOI] [PubMed] [Google Scholar]
  2. Berka R. M., Gray G. L., Vasil M. L. Studies of phospholipase C (heat-labile hemolysin) in Pseudomonas aeruginosa. Infect Immun. 1981 Dec;34(3):1071–1074. doi: 10.1128/iai.34.3.1071-1074.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bever R. A., Iglewski B. H. Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J Bacteriol. 1988 Sep;170(9):4309–4314. doi: 10.1128/jb.170.9.4309-4314.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bjorn M. J., Sokol P. A., Iglewski B. H. Influence of iron on yields of extracellular products in Pseudomonas aeruginosa cultures. J Bacteriol. 1979 Apr;138(1):193–200. doi: 10.1128/jb.138.1.193-200.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blackwood L. L., Stone R. M., Iglewski B. H., Pennington J. E. Evaluation of Pseudomonas aeruginosa exotoxin A and elastase as virulence factors in acute lung infection. Infect Immun. 1983 Jan;39(1):198–201. doi: 10.1128/iai.39.1.198-201.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bodey G. P., Bolivar R., Fainstein V., Jadeja L. Infections caused by Pseudomonas aeruginosa. Rev Infect Dis. 1983 Mar-Apr;5(2):279–313. doi: 10.1093/clinids/5.2.279. [DOI] [PubMed] [Google Scholar]
  7. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  8. Brint J. M., Ohman D. E. Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family. J Bacteriol. 1995 Dec;177(24):7155–7163. doi: 10.1128/jb.177.24.7155-7163.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cicmanec J. F., Holder I. A. Growth of Pseudomonas aeruginosa in normal and burned skin extract: role of extracellular proteases. Infect Immun. 1979 Aug;25(2):477–483. doi: 10.1128/iai.25.2.477-483.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Farinha M. A., Kropinski A. M. Construction of broad-host-range vectors for general cloning and promoter selection in Pseudomonas and Escherichia coli. Gene. 1989 Apr 30;77(2):205–210. doi: 10.1016/0378-1119(89)90068-1. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Frank D. W., Iglewski B. H. Kinetics of toxA and regA mRNA accumulation in Pseudomonas aeruginosa. J Bacteriol. 1988 Oct;170(10):4477–4483. doi: 10.1128/jb.170.10.4477-4483.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuqua W. C., Winans S. C., Greenberg E. P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol. 1994 Jan;176(2):269–275. doi: 10.1128/jb.176.2.269-275.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gambello M. J., Iglewski B. H. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol. 1991 May;173(9):3000–3009. doi: 10.1128/jb.173.9.3000-3009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gambello M. J., Kaye S., Iglewski B. H. LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. Infect Immun. 1993 Apr;61(4):1180–1184. doi: 10.1128/iai.61.4.1180-1184.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Goebel W., Hedgpeth J. Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol. 1982 Sep;151(3):1290–1298. doi: 10.1128/jb.151.3.1290-1298.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hamood A. N., Griswold J. A., Duhan C. M. Production of extracellular virulence factors by Pseudomonas aeruginosa isolates obtained from tracheal, urinary tract, and wound infections. J Surg Res. 1996 Mar;61(2):425–432. doi: 10.1006/jsre.1996.0140. [DOI] [PubMed] [Google Scholar]
  19. Hamood A. N., Griswold J. DNA hybridization analysis of the Pseudomonas aeruginosa elastase gene (lasB) from different clinical isolates. Can J Microbiol. 1995 Oct;41(10):910–917. doi: 10.1139/m95-125. [DOI] [PubMed] [Google Scholar]
  20. Hamood A. N., Olson J. C., Vincent T. S., Iglewski B. H. Regions of toxin A involved in toxin A excretion in Pseudomonas aeruginosa. J Bacteriol. 1989 Apr;171(4):1817–1824. doi: 10.1128/jb.171.4.1817-1824.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hedstrom R. C., Funk C. R., Kaper J. B., Pavlovskis O. R., Galloway D. R. Cloning of a gene involved in regulation of exotoxin A expression in Pseudomonas aeruginosa. Infect Immun. 1986 Jan;51(1):37–42. doi: 10.1128/iai.51.1.37-42.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Holloway B. W., Krishnapillai V., Morgan A. F. Chromosomal genetics of Pseudomonas. Microbiol Rev. 1979 Mar;43(1):73–102. doi: 10.1128/mr.43.1.73-102.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kessler E., Safrin M., Olson J. C., Ohman D. E. Secreted LasA of Pseudomonas aeruginosa is a staphylolytic protease. J Biol Chem. 1993 Apr 5;268(10):7503–7508. [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. 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]
  26. Latifi A., Winson M. K., Foglino M., Bycroft B. W., Stewart G. S., Lazdunski A., Williams P. Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol Microbiol. 1995 Jul;17(2):333–343. doi: 10.1111/j.1365-2958.1995.mmi_17020333.x. [DOI] [PubMed] [Google Scholar]
  27. McIver K. S., Olson J. C., Ohman D. E. Pseudomonas aeruginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing, and secretion. J Bacteriol. 1993 Jul;175(13):4008–4015. doi: 10.1128/jb.175.13.4008-4015.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Meighen E. A. Molecular biology of bacterial bioluminescence. Microbiol Rev. 1991 Mar;55(1):123–142. doi: 10.1128/mr.55.1.123-142.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nicas T. I., Iglewski B. H. Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S. Infect Immun. 1984 Aug;45(2):470–474. doi: 10.1128/iai.45.2.470-474.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nicas T. I., Iglewski B. H. The contribution of exoproducts to virulence of Pseudomonas aeruginosa. Can J Microbiol. 1985 Apr;31(4):387–392. doi: 10.1139/m85-074. [DOI] [PubMed] [Google Scholar]
  31. Ohman D. E., Sadoff J. C., Iglewski B. H. Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization. Infect Immun. 1980 Jun;28(3):899–908. doi: 10.1128/iai.28.3.899-908.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Olsen R. H., DeBusscher G., McCombie W. R. Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol. 1982 Apr;150(1):60–69. doi: 10.1128/jb.150.1.60-69.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Olson J. C., Ohman D. E. Efficient production and processing of elastase and LasA by Pseudomonas aeruginosa require zinc and calcium ions. J Bacteriol. 1992 Jun;174(12):4140–4147. doi: 10.1128/jb.174.12.4140-4147.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Passador L., Cook J. M., Gambello M. J., Rust L., Iglewski B. H. Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science. 1993 May 21;260(5111):1127–1130. doi: 10.1126/science.8493556. [DOI] [PubMed] [Google Scholar]
  35. Pearson J. P., Gray K. M., Passador L., Tucker K. D., Eberhard A., Iglewski B. H., Greenberg E. P. Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):197–201. doi: 10.1073/pnas.91.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Peters J. E., Galloway D. R. Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity. J Bacteriol. 1990 May;172(5):2236–2240. doi: 10.1128/jb.172.5.2236-2240.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Peters J. E., Park S. J., Darzins A., Freck L. C., Saulnier J. M., Wallach J. M., Galloway D. R. Further studies on Pseudomonas aeruginosa LasA: analysis of specificity. Mol Microbiol. 1992 May;6(9):1155–1162. doi: 10.1111/j.1365-2958.1992.tb01554.x. [DOI] [PubMed] [Google Scholar]
  38. Rust L., Pesci E. C., Iglewski B. H. Analysis of the Pseudomonas aeruginosa elastase (lasB) regulatory region. J Bacteriol. 1996 Feb;178(4):1134–1140. doi: 10.1128/jb.178.4.1134-1140.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schad P. A., Bever R. A., Nicas T. I., Leduc F., Hanne L. F., Iglewski B. H. Cloning and characterization of elastase genes from Pseudomonas aeruginosa. J Bacteriol. 1987 Jun;169(6):2691–2696. doi: 10.1128/jb.169.6.2691-2696.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schultz D. R., Miller K. D. Elastase of Pseudomonas aeruginosa: inactivation of complement components and complement-derived chemotactic and phagocytic factors. Infect Immun. 1974 Jul;10(1):128–135. doi: 10.1128/iai.10.1.128-135.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Toder D. S., Gambello M. J., Iglewski B. H. Pseudomonas aeruginosa LasA: a second elastase under the transcriptional control of lasR. Mol Microbiol. 1991 Aug;5(8):2003–2010. doi: 10.1111/j.1365-2958.1991.tb00822.x. [DOI] [PubMed] [Google Scholar]
  42. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vasil M. L., Kabat D., Iglewski B. H. Structure-activity relationships of an exotoxin of Pseudomonas aeruginosa. Infect Immun. 1977 Apr;16(1):353–361. doi: 10.1128/iai.16.1.353-361.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. West S. E., Kaye S. A., Hamood A. N., Iglewski B. H. Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A. Infect Immun. 1994 Mar;62(3):897–903. doi: 10.1128/iai.62.3.897-903.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wick M. J., Frank D. W., Storey D. G., Iglewski B. H. Identification of regB, a gene required for optimal exotoxin A yields in Pseudomonas aeruginosa. Mol Microbiol. 1990 Mar;4(3):489–497. doi: 10.1111/j.1365-2958.1990.tb00615.x. [DOI] [PubMed] [Google Scholar]
  46. Wilson R., Pitt T., Taylor G., Watson D., MacDermot J., Sykes D., Roberts D., Cole P. Pyocyanin and 1-hydroxyphenazine produced by Pseudomonas aeruginosa inhibit the beating of human respiratory cilia in vitro. J Clin Invest. 1987 Jan;79(1):221–229. doi: 10.1172/JCI112787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wolz C., Hellstern E., Haug M., Galloway D. R., Vasil M. L., Döring G. Pseudomonas aeruginosa LasB mutant constructed by insertional mutagenesis reveals elastolytic activity due to alkaline proteinase and the LasA fragment. Mol Microbiol. 1991 Sep;5(9):2125–2131. doi: 10.1111/j.1365-2958.1991.tb02142.x. [DOI] [PubMed] [Google Scholar]
  48. Wood R. E. Pseudomonas: the compromised host. Hosp Pract. 1976 Aug;11(8):91–100. doi: 10.1080/21548331.1976.11706983. [DOI] [PubMed] [Google Scholar]
  49. Woods D. E., Cryz S. J., Friedman R. L., Iglewski B. H. Contribution of toxin A and elastase to virulence of Pseudomonas aeruginosa in chronic lung infections of rats. Infect Immun. 1982 Jun;36(3):1223–1228. doi: 10.1128/iai.36.3.1223-1228.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wretlind B., Sjöberg L., Wadström T. Protease-deficient mutants of Pseudomonas aeruginosa: pleiotropic changes in activity of other extracellular enzymes. J Gen Microbiol. 1977 Dec;103(2):329–336. doi: 10.1099/00221287-103-2-329. [DOI] [PubMed] [Google Scholar]
  51. 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