Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Nov;178(22):6608–6617. doi: 10.1128/jb.178.22.6608-6617.1996

A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion.

J K Gustin 1, E Kessler 1, D E Ohman 1
PMCID: PMC178548  PMID: 8932318

Abstract

The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasAdelta mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.

Full Text

The Full Text of this article is available as a PDF (943.2 KB).

Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. Darzins A., Peters J. E., Galloway D. R. Revised nucleotide sequence of the lasA gene from Pseudomonas aeruginosa PAO1. Nucleic Acids Res. 1990 Nov 11;18(21):6444–6444. doi: 10.1093/nar/18.21.6444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Franklin M. J., Chitnis C. E., Gacesa P., Sonesson A., White D. C., Ohman D. E. Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase. J Bacteriol. 1994 Apr;176(7):1821–1830. doi: 10.1128/jb.176.7.1821-1830.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Freck-O'Donnell L. C., Darzins A. Pseudomonas aeruginosa lasA gene: determination of the transcription start point and analysis of the promoter/regulatory region. Gene. 1993 Jul 15;129(1):113–117. doi: 10.1016/0378-1119(93)90705-8. [DOI] [PubMed] [Google Scholar]
  7. Fukushima J., Yamamoto S., Morihara K., Atsumi Y., Takeuchi H., Kawamoto S., Okuda K. Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase. J Bacteriol. 1989 Mar;171(3):1698–1704. doi: 10.1128/jb.171.3.1698-1704.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldberg J. B., Ohman D. E. Activation of an elastase precursor by the lasA gene product of Pseudomonas aeruginosa. J Bacteriol. 1987 Oct;169(10):4532–4539. doi: 10.1128/jb.169.10.4532-4539.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goldberg J. B., Ohman D. E. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J Bacteriol. 1984 Jun;158(3):1115–1121. doi: 10.1128/jb.158.3.1115-1121.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goldberg J. B., Ohman D. E. Cloning and transcriptional regulation of the elastase lasA gene in mucoid and nonmucoid Pseudomonas aeruginosa. J Bacteriol. 1987 Mar;169(3):1349–1351. doi: 10.1128/jb.169.3.1349-1351.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Häse C. C., Finkelstein R. A. Bacterial extracellular zinc-containing metalloproteases. Microbiol Rev. 1993 Dec;57(4):823–837. doi: 10.1128/mr.57.4.823-837.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Inouye M., Halegoua S. Secretion and membrane localization of proteins in Escherichia coli. CRC Crit Rev Biochem. 1980;7(4):339–371. doi: 10.3109/10409238009105465. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Kessler E., Safrin M. Partial purification and characterization of an inactive precursor of Pseudomonas aeruginosa elastase. J Bacteriol. 1988 Mar;170(3):1215–1219. doi: 10.1128/jb.170.3.1215-1219.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kessler E., Safrin M., Peretz M., Burstein Y. Identification of cleavage sites involved in proteolytic processing of Pseudomonas aeruginosa preproelastase. FEBS Lett. 1992 Mar 16;299(3):291–293. doi: 10.1016/0014-5793(92)80134-3. [DOI] [PubMed] [Google Scholar]
  16. Kessler E., Safrin M. Synthesis, processing, and transport of Pseudomonas aeruginosa elastase. J Bacteriol. 1988 Nov;170(11):5241–5247. doi: 10.1128/jb.170.11.5241-5247.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kessler E., Safrin M. The propeptide of Pseudomonas aeruginosa elastase acts an elastase inhibitor. J Biol Chem. 1994 Sep 9;269(36):22726–22731. [PubMed] [Google Scholar]
  18. Kessler E. beta-lytic endopeptidases. Methods Enzymol. 1995;248:740–756. doi: 10.1016/0076-6879(95)48050-1. [DOI] [PubMed] [Google Scholar]
  19. Li S. L., Norioka S., Sakiyama F. Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus. J Bacteriol. 1990 Nov;172(11):6506–6511. doi: 10.1128/jb.172.11.6506-6511.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McIver K. S., Kessler E., Olson J. C., Ohman D. E. The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol. 1995 Dec;18(5):877–889. doi: 10.1111/j.1365-2958.1995.18050877.x. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. McIver K., Kessler E., Ohman D. E. Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase. J Bacteriol. 1991 Dec;173(24):7781–7789. doi: 10.1128/jb.173.24.7781-7789.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ohman D. E., Chakrabarty A. M. Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun. 1981 Jul;33(1):142–148. doi: 10.1128/iai.33.1.142-148.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ohman D. E., Cryz S. J., Iglewski B. H. Isolation and characterization of Pseudomonas aeruginosa PAO mutant that produces altered elastase. J Bacteriol. 1980 Jun;142(3):836–842. doi: 10.1128/jb.142.3.836-842.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Park S., Galloway D. R. Purification and characterization of LasD: a second staphylolytic proteinase produced by Pseudomonas aeruginosa. Mol Microbiol. 1995 Apr;16(2):263–270. doi: 10.1111/j.1365-2958.1995.tb02298.x. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schad P. A., Iglewski B. H. Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene. J Bacteriol. 1988 Jun;170(6):2784–2789. doi: 10.1128/jb.170.6.2784-2789.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shortridge V. D., Pato M. L., Vasil A. I., Vasil M. L. Physical mapping of virulence-associated genes in Pseudomonas aeruginosa by transverse alternating-field electrophoresis. Infect Immun. 1991 Oct;59(10):3596–3603. doi: 10.1128/iai.59.10.3596-3603.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Silen J. L., Frank D., Fujishige A., Bone R., Agard D. A. Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity. J Bacteriol. 1989 Mar;171(3):1320–1325. doi: 10.1128/jb.171.3.1320-1325.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Silen J. L., McGrath C. N., Smith K. R., Agard D. A. Molecular analysis of the gene encoding alpha-lytic protease: evidence for a preproenzyme. Gene. 1988 Sep 30;69(2):237–244. doi: 10.1016/0378-1119(88)90434-9. [DOI] [PubMed] [Google Scholar]
  34. Thayer M. M., Flaherty K. M., McKay D. B. Three-dimensional structure of the elastase of Pseudomonas aeruginosa at 1.5-A resolution. J Biol Chem. 1991 Feb 15;266(5):2864–2871. doi: 10.2210/pdb1ezm/pdb. [DOI] [PubMed] [Google Scholar]
  35. Toder D. S., Ferrell S. J., Nezezon J. L., Rust L., Iglewski B. H. lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity. Infect Immun. 1994 Apr;62(4):1320–1327. doi: 10.1128/iai.62.4.1320-1327.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Wandersman C. Secretion, processing and activation of bacterial extracellular proteases. Mol Microbiol. 1989 Dec;3(12):1825–1831. doi: 10.1111/j.1365-2958.1989.tb00169.x. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES