Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Mar;179(6):2053–2059. doi: 10.1128/jb.179.6.2053-2059.1997

A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds.

F J Pérez-Llarena 1, P Liras 1, A Rodríguez-García 1, J F Martín 1
PMCID: PMC178932  PMID: 9068654

Abstract

A regulatory gene (ccaR), located within the cephamycin gene cluster of Streptomyces clavuligerus, is linked to a gene (blp) encoding a protein similar to a beta-lactamase-inhibitory protein. Expression of ccaR is required for cephamycin and clavulanic acid biosynthesis in S. clavuligerus. The ccaR-encoded protein resembles the ActII-ORF4, RedD, AfsR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs. Disruption of ccaR by targeted double recombination resulted in the loss of the ability to synthesize cephamycin and clavulanic acid. Complementation of the disrupted mutant with ccaR restored production of both secondary metabolites. ccaR was expressed as a monocistronic transcript at 24 and 48 h in S. clavuligerus cultures (preceding the phase of antibiotic accumulation), but no transcript hybridization signals were observed at 72 or 96 h. This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis. Amplification of ccaR in S. clavuligerus resulted in a two- to threefold increase in the production of cephamycin and clavulanic acid.

Full Text

The Full Text of this article is available as a PDF (560.3 KB).

Selected References

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

  1. Anzai H., Murakami T., Imai S., Satoh A., Nagaoka K., Thompson C. J. Transcriptional regulation of bialaphos biosynthesis in Streptomyces hygroscopicus. J Bacteriol. 1987 Aug;169(8):3482–3488. doi: 10.1128/jb.169.8.3482-3488.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bibb M. 1995 Colworth Prize Lecture. The regulation of antibiotic production in Streptomyces coelicolor A3(2). Microbiology. 1996 Jun;142(Pt 6):1335–1344. doi: 10.1099/13500872-142-6-1335. [DOI] [PubMed] [Google Scholar]
  3. Brown A. G., Butterworth D., Cole M., Hanscomb G., Hood J. D., Reading C., Rolinson G. N. Naturally-occurring beta-lactamase inhibitors with antibacterial activity. J Antibiot (Tokyo) 1976 Jun;29(6):668–669. doi: 10.7164/antibiotics.29.668. [DOI] [PubMed] [Google Scholar]
  4. Coque J. J., Enguita F. J., Martín J. F., Liras P. A two-protein component 7 alpha-cephem-methoxylase encoded by two genes of the cephamycin C cluster converts cephalosporin C to 7-methoxycephalosporin C. J Bacteriol. 1995 Apr;177(8):2230–2235. doi: 10.1128/jb.177.8.2230-2235.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coque J. J., Liras P., Laiz L., Martín J. F. A gene encoding lysine 6-aminotransferase, which forms the beta-lactam precursor alpha-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans. J Bacteriol. 1991 Oct;173(19):6258–6264. doi: 10.1128/jb.173.19.6258-6264.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coque J. J., Liras P., Martín J. F. Genes for a beta-lactamase, a penicillin-binding protein and a transmembrane protein are clustered with the cephamycin biosynthetic genes in Nocardia lactamdurans. EMBO J. 1993 Feb;12(2):631–639. doi: 10.1002/j.1460-2075.1993.tb05696.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coque J. J., Martín J. F., Calzada J. G., Liras P. The cephamycin biosynthetic genes pcbAB, encoding a large multidomain peptide synthetase, and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in Acremonium chrysogenum and Penicillium chrysogenum. Mol Microbiol. 1991 May;5(5):1125–1133. doi: 10.1111/j.1365-2958.1991.tb01885.x. [DOI] [PubMed] [Google Scholar]
  8. Coque J. J., Martín J. F., Liras P. Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus. Mol Gen Genet. 1993 Jan;236(2-3):453–458. doi: 10.1007/BF00277148. [DOI] [PubMed] [Google Scholar]
  9. Coque J. J., Pérez-Llarena F. J., Enguita F. J., Fuente J. L., Martín J. F., Liras P. Characterization of the cmcH genes of Nocardia lactamdurans and Streptomyces clavuligerus encoding a functional 3'-hydroxymethylcephem O-carbamoyltransferase for cephamycin biosynthesis. Gene. 1995 Aug 30;162(1):21–27. doi: 10.1016/0378-1119(95)00308-s. [DOI] [PubMed] [Google Scholar]
  10. Doran J. L., Leskiw B. K., Aippersbach S., Jensen S. E. Isolation and characterization of a beta-lactamase-inhibitory protein from Streptomyces clavuligerus and cloning and analysis of the corresponding gene. J Bacteriol. 1990 Sep;172(9):4909–4918. doi: 10.1128/jb.172.9.4909-4918.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fernández-Moreno M. A., Caballero J. L., Hopwood D. A., Malpartida F. The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces. Cell. 1991 Aug 23;66(4):769–780. doi: 10.1016/0092-8674(91)90120-n. [DOI] [PubMed] [Google Scholar]
  12. Furuya K., Hutchinson C. R. The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis. J Bacteriol. 1996 Nov;178(21):6310–6318. doi: 10.1128/jb.178.21.6310-6318.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garcia-Dominguez M., Martin J. F., Mahro B., Demain A. L., Liras P. Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus. Appl Environ Microbiol. 1987 Jun;53(6):1376–1381. doi: 10.1128/aem.53.6.1376-1381.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gramajo H. C., Takano E., Bibb M. J. Stationary-phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated. Mol Microbiol. 1993 Mar;7(6):837–845. doi: 10.1111/j.1365-2958.1993.tb01174.x. [DOI] [PubMed] [Google Scholar]
  15. Horinouchi S., Beppu T. Autoregulatory factors and communication in actinomycetes. Annu Rev Microbiol. 1992;46:377–398. doi: 10.1146/annurev.mi.46.100192.002113. [DOI] [PubMed] [Google Scholar]
  16. Horinouchi S., Kito M., Nishiyama M., Furuya K., Hong S. K., Miyake K., Beppu T. Primary structure of AfsR, a global regulatory protein for secondary metabolite formation in Streptomyces coelicolor A3(2). Gene. 1990 Oct 30;95(1):49–56. doi: 10.1016/0378-1119(90)90412-k. [DOI] [PubMed] [Google Scholar]
  17. Janssen G. R., Bibb M. J. 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. Gene. 1993 Feb 14;124(1):133–134. doi: 10.1016/0378-1119(93)90774-w. [DOI] [PubMed] [Google Scholar]
  18. Kieser T., Melton R. E. Plasmid pIJ699, a multi-copy positive-selection vector for Streptomyces. Gene. 1988 May 15;65(1):83–91. doi: 10.1016/0378-1119(88)90419-2. [DOI] [PubMed] [Google Scholar]
  19. Kovacevic S., Miller J. R. Cloning and sequencing of the beta-lactam hydroxylase gene (cefF) from Streptomyces clavuligerus: gene duplication may have led to separate hydroxylase and expandase activities in the actinomycetes. J Bacteriol. 1991 Jan;173(1):398–400. doi: 10.1128/jb.173.1.398-400.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kovacevic S., Weigel B. J., Tobin M. B., Ingolia T. D., Miller J. R. Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase. J Bacteriol. 1989 Feb;171(2):754–760. doi: 10.1128/jb.171.2.754-760.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kumar C. V., Coque J. J., Martín J. F. Efficient Transformation of the Cephamycin C Producer Nocardia lactamdurans and Development of Shuttle and Promoter-Probe Cloning Vectors. Appl Environ Microbiol. 1994 Nov;60(11):4086–4093. doi: 10.1128/aem.60.11.4086-4093.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kumar V., de la Fuente J. L., Leitão A. L., Liras P., Martín J. F. Effect of amplification or targeted disruption of the beta-lactamase gene of Nocardia lactamdurans on cephamycin biosynthesis. Appl Microbiol Biotechnol. 1996 Jun;45(5):621–628. doi: 10.1007/s002530050739. [DOI] [PubMed] [Google Scholar]
  23. Leskiw B. K., Lawlor E. J., Fernandez-Abalos J. M., Chater K. F. TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2461–2465. doi: 10.1073/pnas.88.6.2461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Madduri K., Hutchinson C. R. Functional characterization and transcriptional analysis of the dnrR1 locus, which controls daunorubicin biosynthesis in Streptomyces peucetius. J Bacteriol. 1995 Mar;177(5):1208–1215. doi: 10.1128/jb.177.5.1208-1215.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Madduri K., Stuttard C., Vining L. C. Cloning and location of a gene governing lysine epsilon-aminotransferase, an enzyme initiating beta-lactam biosynthesis in Streptomyces spp. J Bacteriol. 1991 Feb;173(3):985–988. doi: 10.1128/jb.173.3.985-988.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Martin J. F., Demain A. L. Control of antibiotic biosynthesis. Microbiol Rev. 1980 Jun;44(2):230–251. doi: 10.1128/mr.44.2.230-251.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martín M. F., Liras P. Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu Rev Microbiol. 1989;43:173–206. doi: 10.1146/annurev.mi.43.100189.001133. [DOI] [PubMed] [Google Scholar]
  28. Narva K. E., Feitelson J. S. Nucleotide sequence and transcriptional analysis of the redD locus of Streptomyces coelicolor A3(2). J Bacteriol. 1990 Jan;172(1):326–333. doi: 10.1128/jb.172.1.326-333.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Paradkar A. S., Jensen S. E. Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus. J Bacteriol. 1995 Mar;177(5):1307–1314. doi: 10.1128/jb.177.5.1307-1314.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Strynadka N. C., Jensen S. E., Johns K., Blanchard H., Page M., Matagne A., Frère J. M., James M. N. Structural and kinetic characterization of a beta-lactamase-inhibitor protein. Nature. 1994 Apr 14;368(6472):657–660. doi: 10.1038/368657a0. [DOI] [PubMed] [Google Scholar]
  31. Stutzman-Engwall K. J., Otten S. L., Hutchinson C. R. Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J Bacteriol. 1992 Jan;174(1):144–154. doi: 10.1128/jb.174.1.144-154.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Takano E., Gramajo H. C., Strauch E., Andres N., White J., Bibb M. J. Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2). Mol Microbiol. 1992 Oct;6(19):2797–2804. doi: 10.1111/j.1365-2958.1992.tb01459.x. [DOI] [PubMed] [Google Scholar]
  33. Ward J. M., Hodgson J. E. The biosynthetic genes for clavulanic acid and cephamycin production occur as a 'super-cluster' in three Streptomyces. FEMS Microbiol Lett. 1993 Jun 15;110(2):239–242. doi: 10.1111/j.1574-6968.1993.tb06326.x. [DOI] [PubMed] [Google Scholar]
  34. Wright F., Bibb M. J. Codon usage in the G+C-rich Streptomyces genome. Gene. 1992 Apr 1;113(1):55–65. doi: 10.1016/0378-1119(92)90669-g. [DOI] [PubMed] [Google Scholar]
  35. Yu H., Serpe E., Romero J., Coque J. J., Maeda K., Oelgeschläger M., Hintermann G., Liras P., Martín J. F., Demain A. L. Possible involvement of the lysine epsilon-aminotransferase gene (lat) in the expression of the genes encoding ACV synthetase (pcbAB) and isopenicillin N synthase (pcbC) in Streptomyces clavuligerus. Microbiology. 1994 Dec;140(Pt 12):3367–3377. doi: 10.1099/13500872-140-12-3367. [DOI] [PubMed] [Google Scholar]

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

RESOURCES