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. 1992 Apr;174(7):2361–2366. doi: 10.1128/jb.174.7.2361-2366.1992

Production of active Serratia marcescens metalloprotease from Escherichia coli by alpha-hemolysin HlyB and HlyD.

Y Suh 1, M J Benedik 1
PMCID: PMC205859  PMID: 1551853

Abstract

Serratia marcescens produces an abundant extracellular metalloprotease. The gene for this protease had previously been cloned and expressed in Escherichia coli, in which no functional protease could be found. However, the protease gene carries the LXGGXGND repeat motif found in alpha-hemolysin and other proteins secreted by homologous systems. Using a dual-plasmid complementation system, we show that the alpha-hemolysin hlyB and hlyD transport determinants are sufficient to allow secretion and activation of a functional metalloprotease species from E. coli, as are the comparable protease secretion functions of Erwinia chrysanthemi. However, strains expressing protease with the hlyBD transport system are unstable and rapidly lose the ability to produce functional protease.

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Selected References

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  1. Aiyappa P. S., Harris J. O. The extracellular metalloprotease of Serratia marcescens: I. Purification and characterization. Mol Cell Biochem. 1976 Nov 30;13(2):95–100. doi: 10.1007/BF01837059. [DOI] [PubMed] [Google Scholar]
  2. Ball T. K., Saurugger P. N., Benedik M. J. The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli. Gene. 1987;57(2-3):183–192. doi: 10.1016/0378-1119(87)90121-1. [DOI] [PubMed] [Google Scholar]
  3. Ball T. K., Wasmuth C. R., Braunagel S. C., Benedik M. J. Expression of Serratia marcescens extracellular proteins requires recA. J Bacteriol. 1990 Jan;172(1):342–349. doi: 10.1128/jb.172.1.342-349.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blight M. A., Holland I. B. Structure and function of haemolysin B,P-glycoprotein and other members of a novel family of membrane translocators. Mol Microbiol. 1990 Jun;4(6):873–880. doi: 10.1111/j.1365-2958.1990.tb00660.x. [DOI] [PubMed] [Google Scholar]
  5. Braun V., Schmitz G. Excretion of a protease by Serratia marcescens. Arch Microbiol. 1980 Jan;124(1):55–61. doi: 10.1007/BF00407028. [DOI] [PubMed] [Google Scholar]
  6. Braunagel S. C., Benedik M. J. The metalloprotease gene of Serratia marcescens strain SM6. Mol Gen Genet. 1990 Jul;222(2-3):446–451. doi: 10.1007/BF00633854. [DOI] [PubMed] [Google Scholar]
  7. Bromke B. J., Hammel J. M. Regulation of extracellular protease formation by Serratia marcescens. Can J Microbiol. 1979 Jan;25(1):47–52. doi: 10.1139/m79-008. [DOI] [PubMed] [Google Scholar]
  8. Dahler G. S., Barras F., Keen N. T. Cloning of genes encoding extracellular metalloproteases from Erwinia chrysanthemi EC16. J Bacteriol. 1990 Oct;172(10):5803–5815. doi: 10.1128/jb.172.10.5803-5815.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Delepelaire P., Wandersman C. Protease secretion by Erwinia chrysanthemi. Proteases B and C are synthesized and secreted as zymogens without a signal peptide. J Biol Chem. 1989 May 25;264(15):9083–9089. [PubMed] [Google Scholar]
  10. Economou A., Hamilton W. D., Johnston A. W., Downie J. A. The Rhizobium nodulation gene nodO encodes a Ca2(+)-binding protein that is exported without N-terminal cleavage and is homologous to haemolysin and related proteins. EMBO J. 1990 Feb;9(2):349–354. doi: 10.1002/j.1460-2075.1990.tb08117.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gilson L., Mahanty H. K., Kolter R. Genetic analysis of an MDR-like export system: the secretion of colicin V. EMBO J. 1990 Dec;9(12):3875–3884. doi: 10.1002/j.1460-2075.1990.tb07606.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Givskov M., Olsen L., Molin S. Cloning and expression in Escherichia coli of the gene for extracellular phospholipase A1 from Serratia liquefaciens. J Bacteriol. 1988 Dec;170(12):5855–5862. doi: 10.1128/jb.170.12.5855-5862.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Glaser P., Sakamoto H., Bellalou J., Ullmann A., Danchin A. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 1988 Dec 1;7(12):3997–4004. doi: 10.1002/j.1460-2075.1988.tb03288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gray L., Baker K., Kenny B., Mackman N., Haigh R., Holland I. B. A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium. J Cell Sci Suppl. 1989;11:45–57. doi: 10.1242/jcs.1989.supplement_11.4. [DOI] [PubMed] [Google Scholar]
  15. Highlander S. K., Engler M. J., Weinstock G. M. Secretion and expression of the Pasteurella haemolytica Leukotoxin. J Bacteriol. 1990 May;172(5):2343–2350. doi: 10.1128/jb.172.5.2343-2350.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hines D. A., Saurugger P. N., Ihler G. M., Benedik M. J. Genetic analysis of extracellular proteins of Serratia marcescens. J Bacteriol. 1988 Sep;170(9):4141–4146. doi: 10.1128/jb.170.9.4141-4146.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Itakura K., Hirose T., Crea R., Riggs A. D., Heyneker H. L., Bolivar F., Boyer H. W. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science. 1977 Dec 9;198(4321):1056–1063. doi: 10.1126/science.412251. [DOI] [PubMed] [Google Scholar]
  18. Jones J. D., Grady K. L., Suslow T. V., Bedbrook J. R. Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens. EMBO J. 1986 Mar;5(3):467–473. doi: 10.1002/j.1460-2075.1986.tb04235.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Létoffé S., Delepelaire P., Wandersman C. Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi. Mol Microbiol. 1989 Jan;3(1):79–86. doi: 10.1111/j.1365-2958.1989.tb00106.x. [DOI] [PubMed] [Google Scholar]
  21. Létoffé S., Delepelaire P., Wandersman C. Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions. J Bacteriol. 1991 Apr;173(7):2160–2166. doi: 10.1128/jb.173.7.2160-2166.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Létoffé S., Delepelaire P., Wandersman C. Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli alpha-haemolysin. EMBO J. 1990 May;9(5):1375–1382. doi: 10.1002/j.1460-2075.1990.tb08252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nakahama K., Yoshimura K., Marumoto R., Kikuchi M., Lee I. S., Hase T., Matsubara H. Cloning and sequencing of Serratia protease gene. Nucleic Acids Res. 1986 Jul 25;14(14):5843–5855. doi: 10.1093/nar/14.14.5843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nicaud J. M., Mackman N., Gray L., Holland I. B. Characterisation of HlyC and mechanism of activation and secretion of haemolysin from E. coli 2001. FEBS Lett. 1985 Aug 5;187(2):339–344. doi: 10.1016/0014-5793(85)81272-2. [DOI] [PubMed] [Google Scholar]
  25. Schmitz G., Braun V. Cell-bound and secreted proteases of Serratia marcescens. J Bacteriol. 1985 Mar;161(3):1002–1009. doi: 10.1128/jb.161.3.1002-1009.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Strathdee C. A., Lo R. Y. Cloning, nucleotide sequence, and characterization of genes encoding the secretion function of the Pasteurella haemolytica leukotoxin determinant. J Bacteriol. 1989 Feb;171(2):916–928. doi: 10.1128/jb.171.2.916-928.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  28. Wandersman C., Delepelaire P., Letoffe S., Schwartz M. Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli. J Bacteriol. 1987 Nov;169(11):5046–5053. doi: 10.1128/jb.169.11.5046-5053.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wandersman C., Delepelaire P. TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4776–4780. doi: 10.1073/pnas.87.12.4776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yanagida N., Uozumi T., Beppu T. Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli. J Bacteriol. 1986 Jun;166(3):937–944. doi: 10.1128/jb.166.3.937-944.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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