Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Nov;169(11):5046–5053. doi: 10.1128/jb.169.11.5046-5053.1987

Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli.

C Wandersman 1, P Delepelaire 1, S Letoffe 1, M Schwartz 1
PMCID: PMC213906  PMID: 2822661

Abstract

Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three antigenically and structurally distinct proteases, A, B, and C and produces a protease inhibitor, a low-molecular-weight, heat-stable protein which remains mostly intracellular and which binds specifically to the A, B, and C proteases. The structural genes for proteases A, B, and C and for the inhibitor are clustered on a ca. 40-kilobase DNA fragment present in cosmid pEW4. Escherichia coli strains harboring pEW4 secrete the three proteases into the medium during the exponential phase of growth, without intracellular accumulation and in the absence of detectable cell lysis. An 8.5-kilobase EcoRI fragment derived from the cosmid encodes proteases B and C and the inhibitor as well as functions involved in the synthesis or secretion (or both) of the proteases. The inhibitor is not required for protease synthesis or secretion.

Full text

PDF
5050

Images in this article

5047Image
on p.5047
5048Image
on p.5048
5049Image
on p.5049
5050Image
on p.5050
5051Image
on p.5051

Selected References

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

  1. Andro T., Chambost J. P., Kotoujansky A., Cattaneo J., Bertheau Y., Barras F., Van Gijsegem F., Coleno A. Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase. J Bacteriol. 1984 Dec;160(3):1199–1203. doi: 10.1128/jb.160.3.1199-1203.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barth P. T., Grinter N. J. Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulfonamides. J Bacteriol. 1974 Nov;120(2):618–630. doi: 10.1128/jb.120.2.618-630.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Chatterjee A. K., Starr M. P. Genetics of Erwinia species. Annu Rev Microbiol. 1980;34:645–676. doi: 10.1146/annurev.mi.34.100180.003241. [DOI] [PubMed] [Google Scholar]
  6. Chung C. H., Ives H. E., Almeda S., Goldberg A. L. Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases. J Biol Chem. 1983 Sep 25;258(18):11032–11038. [PubMed] [Google Scholar]
  7. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  8. Collmer A., Schoedel C., Roeder D. L., Ried J. L., Rissler J. F. Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase. J Bacteriol. 1985 Mar;161(3):913–920. doi: 10.1128/jb.161.3.913-920.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frey J., Bagdasarian M., Feiss D., Franklin F. C., Deshusses J. Stable cosmid vectors that enable the introduction of cloned fragments into a wide range of gram-negative bacteria. Gene. 1983 Oct;24(2-3):299–308. doi: 10.1016/0378-1119(83)90090-2. [DOI] [PubMed] [Google Scholar]
  10. Hershfield V., Boyer H. W., Yanofsky C., Lovett M. A., Helinski D. R. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3455–3459. doi: 10.1073/pnas.71.9.3455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hirst T. R., Randall L. L., Hardy S. J. Cellular location of heat-labile enterotoxin in Escherichia coli. J Bacteriol. 1984 Feb;157(2):637–642. doi: 10.1128/jb.157.2.637-642.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Keen N. T., Dahlbeck D., Staskawicz B., Belser W. Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli. J Bacteriol. 1984 Sep;159(3):825–831. doi: 10.1128/jb.159.3.825-831.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kotoujansky A., Diolez A., Boccara M., Bertheau Y., Andro T., Coleno A. Molecular cloning of Erwinia chrysanthemi pectinase and cellulase structural genes. EMBO J. 1985 Mar;4(3):781–785. doi: 10.1002/j.1460-2075.1985.tb03697.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Labia R., Barthélémy M. L'enzymogramme des beta-lactamases: adaptation en cel de la méthode iodométrique. Ann Microbiol (Paris) 1979 Oct;130B(3):295–304. [PubMed] [Google Scholar]
  15. Laskowski M., Jr, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem. 1980;49:593–626. doi: 10.1146/annurev.bi.49.070180.003113. [DOI] [PubMed] [Google Scholar]
  16. Michaelis S., Beckwith J. Mechanism of incorporation of cell envelope proteins in Escherichia coli. Annu Rev Microbiol. 1982;36:435–465. doi: 10.1146/annurev.mi.36.100182.002251. [DOI] [PubMed] [Google Scholar]
  17. Michaelis S., Chapon C., D'Enfert C., Pugsley A. P., Schwartz M. Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae. J Bacteriol. 1985 Nov;164(2):633–638. doi: 10.1128/jb.164.2.633-638.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Millet J. Characterization of a protein inhibitor of intracellular protease from Bacillus subtilis. FEBS Lett. 1977 Feb 15;74(1):59–61. doi: 10.1016/0014-5793(77)80752-7. [DOI] [PubMed] [Google Scholar]
  19. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  20. Pugsley A. P. The ins and outs of colicins. Part I: Production, and translocation across membranes. Microbiol Sci. 1984 Oct;1(7):168–175. [PubMed] [Google Scholar]
  21. Reverchon S., Hugouvieux-Cotte-Pattat N., Robert-Baudouy J. Cloning of genes encoding pectolytic enzymes from a genomic library of the phytopathogenic bacterium, Erwinia chrysanthemi. Gene. 1985;35(1-2):121–130. doi: 10.1016/0378-1119(85)90164-7. [DOI] [PubMed] [Google Scholar]
  22. Sabik J. F., Suit J. L., Luria S. E. cea-kil operon of the ColE1 plasmid. J Bacteriol. 1983 Mar;153(3):1479–1485. doi: 10.1128/jb.153.3.1479-1485.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schoonejans E., Toussaint A. Utilization of plasmid pULB113 (RP4::mini-Mu) to construct a linkage map of Erwinia carotovora subsp. chrysanthemi. J Bacteriol. 1983 Jun;154(3):1489–1492. doi: 10.1128/jb.154.3.1489-1492.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wöhner G., Wöber G. Pullulanase, an enzyme of starch catabolism, is associated with the outer membrane of Klebsiella. Arch Microbiol. 1978 Mar;116(3):303–310. doi: 10.1007/BF00417856. [DOI] [PubMed] [Google Scholar]
  25. 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]

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

RESOURCES