1887

Abstract

The gene encoding BLIP, a eta-actamase-nhibitory rotein, was disrupted in wild-type and in a clavulanic acid non-producing mutant. The resulting BLIP mutant and BLIP/clavulanic acid double mutant showed no residual proteinaceous β-lactamase-inhibitory activity, indicating that only a single β-lactamase-inhibitory protein exists in . The lack of any proteinaceous β-lactamase-inhibitory activity in the and / mutants also indicates that BLP, the BLIP-like protein, encoded by does not possess β-lactamase-inhibitory activity despite its similarity to BLIP. The mutant and the / double mutant did not show any aberrant growth morphology, sporulation defects, or alterations in cephamycin C production or penicillin G resistance when compared to wild-type or to the single mutant. Mutants bearing the gene disruption did show an elevated level of production of clavam-2-carboxylate and hydroxymethyl clavam as well as clavulanic acid. This phenomenon was observed in the middle stages of production of these clavams but was not detected during maximum production. The production of BLIP was also determined to be down-regulated in a mutant, lacking the pathway-specific transcriptional regulator required for production of cephamycin C and clavulanic acid. Sequencing of the regions flanking the gene showed the presence of a partial open reading frame that encodes a DNA-binding protein, and several open reading frames apparently involved in the production of an ABC transporter.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-2-325
2001-02-01
2020-01-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/2/1470325a.html?itemId=/content/journal/micro/10.1099/00221287-147-2-325&mimeType=html&fmt=ahah

References

  1. Aharonowitz Y., Demain A. L. 1978; Carbon catabolite regulation of cephamycin production in Streptomyces clavuligerus. Antimicrob Agents Chemother14:159–164[CrossRef]
    [Google Scholar]
  2. Aidoo K. A., Wong A., Alexander D. C., Rittamer R. A., Jensen S. E.. 1994; Cloning, sequencing and disruption of a gene involved in clavulanic acid biosynthesis. Gene147:41–46[CrossRef]
    [Google Scholar]
  3. Alexander D. C., Jensen S. E.. 1998; Investigation of the Streptomyces clavuligerus cephamycin C gene cluster and its regulation by the CcaR protein. J Bacteriol180:4068–4079
    [Google Scholar]
  4. Baggaley K. H., Brown A. G., Schofield C. J.. 1997; Chemistry and biosynthesis of clavulanic acid and other clavams. Nat Prod Rep14:309–333[CrossRef]
    [Google Scholar]
  5. Bailey C. R., Winstanley D. J.. 1986; Inhibition of restriction in Streptomyces clavuligerus by heat treatment. J Gen Microbiol132:2945–2947
    [Google Scholar]
  6. Butler J. S., Springer M., Grunberg-Manago M.. 1987; AUU to AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo. Proc Natl Acad Sci USA84:4022–4025[CrossRef]
    [Google Scholar]
  7. Chung C. T., Niemela S.L., Miller R. H.. 1989; One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA86:2172–2175[CrossRef]
    [Google Scholar]
  8. Daniels L., Hanson R. S., Phillips J. A.. 1994; Chemical analysis. In Methods for General and Molecular Bacteriology pp.534–535Edited by Gerhardt P., Murray R. G. E., Wood W. A., Krieg N. R.. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  9. Doran J. L., Leskiw B. K., Aippersbach S., Jensen S. E.. 1990; Isolation and characterization of a β-lactamase-inhibitory protein from Streptomyces clavuligerus and cloning and analysis of the corresponding gene. J Bacteriol172:4909–4918
    [Google Scholar]
  10. Fath M. J., Kolter R.. 1993; ABC transporters: bacterial exporters. Microbiol Rev57:995–1017
    [Google Scholar]
  11. Hata T., Omura S., Iwaw Y., Ohno H., Takeshima T., Yamaguchi N.. 1972; Studies on penicillinase inhibitors produced by microorganisms. J Antibiot25:473–474[CrossRef]
    [Google Scholar]
  12. Hopwood D. A., Bibb M. J., Chater K. F..7 other authors 1985; Genetic Manipulation of Streptomyces: a Laboratory Manual Norwich, UK: The John Innes Foundation;
    [Google Scholar]
  13. Janssen G. R., Bibb M. J.. 1993; Derivatives of pUC18 that have BglII sites flanking a modified multiple cloning site and that retain the ability to identify recombinant clones by visual screening of Escherichia coli colonies. Gene124:133–134[CrossRef]
    [Google Scholar]
  14. Kang S. G., Park H. U., Lee H. S., Kim H. T., Lee K. J.. 2000; New β-lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus AMF19 and its roles on the morphological differentiation. J Biol Chem275:16851–16856[CrossRef]
    [Google Scholar]
  15. Katz E., Thompson C. J., Hopwood D. A.. 1983; Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol129:2703–2714
    [Google Scholar]
  16. Kim M. K., Lee K. J.. 1994; Characteristics of β-lactamase inhibiting proteins from Streptomyces exfoliatus SMF19. Appl Environ Microbiol60:1029–1032
    [Google Scholar]
  17. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathic character of a protein. J Mol Biol157:105–132[CrossRef]
    [Google Scholar]
  18. Mendez C., Salas J. A. 1998; ABC transporters in antibiotic-producing actinomycetes. FEMS Microbiol Lett158:1–8[CrossRef]
    [Google Scholar]
  19. Mosher R. H., Paradkar A. S., Anders C., Barton B., Jensen S. E.. 1999; Genes specific for the biosynthesis of clavam metabolites antipodal to clavulanic acid are clustered with the gene for clavaminate synthase 1 in Streptomyces clavuligerus. Antimicrob Agents Chemother43:1215–1224
    [Google Scholar]
  20. Ogawara H., Horikawa S.. 1980; Penicillin-binding proteins of Streptomyces cacaoi, Streptomyces olivaceus and Streptomyces clavuligerus. Antimicrob Agents Chemother8:402–408
    [Google Scholar]
  21. Paradkar A. S., Jensen S. E.. 1995; Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus. J Bacteriol177:1307–1314
    [Google Scholar]
  22. Paradkar A. S., Petrich A. K., Leskiw B. K., Aidoo K. A., Jensen S. E.. 1994; Transcriptional analysis and heterologous expression of the gene encoding β-lactamase inhibitor protein (BLIP) from Streptomyces clavuligerus. Gene144:31–36[CrossRef]
    [Google Scholar]
  23. Paradkar A. S., Aidoo K. A., Jensen S. E.. 1998; A pathway-specific transcriptional activator regulates late steps of clavulanic acid biosynthesis in Streptomyces clavuligerus. Mol Microbiol27:831–843[CrossRef]
    [Google Scholar]
  24. Park H. U., Lee K. J.. 1998; Cloning and heterologous expression of the gene for BLIP-II, a β-lactamase-inhibitory protein from Streptomyces exfoliatus SMF-19. Microbiology144:2161–2167[CrossRef]
    [Google Scholar]
  25. Perez-Llarena F. J., Liras P., Rodriguez-Garcia A., Martin J. F.. 1997a; A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both β-lactam compounds. J Bacteriol179:2053–2059
    [Google Scholar]
  26. Perez-Llarena F., Martin J. F., Galleni M., Coque J. J., Fuente J. L., Frere J. M., Liras P.. 1997b; The bla gene of the cephamycin cluster of Streptomyces clavuligerus encodes a class A β-lactamase of low enzymatic activity. J Bacteriol179:6035–6040
    [Google Scholar]
  27. Pruess D. L., Kellett M.. 1983; Ro 22-5417, a new clavam antibiotic from Streptomyces clavuligerus, discovery and biological activity. J Antibiot36:208–212[CrossRef]
    [Google Scholar]
  28. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  29. Stuttard C.. 1982; Temperate phages of Streptomyces venezuelae: lysogeny and host specificity shown by phages SV1 and SV2. J Gen Microbiol128:115–121
    [Google Scholar]
  30. Vieira J., Messing J.. 1987; Production of single-stranded plasmid DNA. Methods Enzymol153:3–11
    [Google Scholar]
  31. Wietzorrek A., Bibb M.. 1997; A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR-like DNA-binding fold. Mol Microbiol25:1181–1184[CrossRef]
    [Google Scholar]
  32. Wright F., Bibb M. J. 1992; Codon usage in the G+C-rich Streptomyces genome. Gene113:55–65[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-2-325
Loading
/content/journal/micro/10.1099/00221287-147-2-325
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error