CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in . Previous sequence analyses of the gene revealed two possible start codons, an ATG, and a GTG located in-frame 18 bp downstream of the ATG. To determine the true start codon, was expressed, either from the GTG or ATG codon, in . A protein product was only obtained from the ATG construct. Similarly, constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in species were prepared and used to complement a mutant. Bioassays showed that only the ATG construct could complement the mutant to restore cephamycin C production, and Western analysis confirmed the presence of CcaR in the mutant complemented with the ATG construct only. To ensure that expression of from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into , converting the GTG to GTC. The GTC construct still fully complemented a mutant, confirming that ATG is the true start codon. Inspection of the region upstream of by S1 nuclease protection and primer extension analyses indicated the presence of two transcript start points that mapped to residues located 74 and 173 bp upstream of the ATG codon.


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  1. Alexander, D. C. & Jensen, S. E.(1998). Investigation of the Streptomyces clavuligerus cephamycin C gene cluster and its regulation by the CcaR protein. J Bacteriol 180, 4068–4079. [Google Scholar]
  2. Bierman, M., Logan, R., O'Brien, E. T., Seno, E. T., Rao, N. & Schoner, B. E.(1992). Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116, 43–49.[CrossRef] [Google Scholar]
  3. Folcher, M., Gaillard, H., Nguyen, L. T., Nguyen, K. T., Lacroix, P., Bamas-Jacques, N., Rinkel, M. & Thompson, C. J.(2001). Pleiotropic functions of a Streptomyces pristinaespiralis autoregulator receptor in development, antibiotic biosynthesis, and expression of a superoxide dismutase. J Biol Chem 276, 44297–44306.[CrossRef] [Google Scholar]
  4. Hindle, Z. & Smith, C. P.(1984). Substrate induction and catabolite repression of the Streptomyces coelicolor glycerol operon are mediated through the GylR protein. Mol Microbiol 12, 737–745. [Google Scholar]
  5. Jensen, S. E., Westlake, D. W., Bowers, R. J. & Wolfe, S.(1982). Cephalosporin formation by cell-free extracts from Streptomyces clavuligerus. J Antibiot 35, 1351–1360.[CrossRef] [Google Scholar]
  6. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A.(2000).Practical Streptomyces Genetics. Norwich, UK: John Innes Foundation.
  7. Kim, H. S., Lee, Y. J., Lee, C. K., Hwang, Y. I., Lee, I. S., Yu, T. S. & Nihara, T.(2002). Cloning of the gene encoding γ-butyrolactone autoregulator receptor protein from Streptomyces clavuligerus. In 9th International Symposium on the Genetics of Industrial Microorganisms, p. 79. Gyeongju, Korea.
  8. Kovacevic, S., Tobin, M. B. & Miller, J. R.(1990). The β-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus. J Bacteriol 172, 3952–3958. [Google Scholar]
  9. Kyung, Y. S., Hu, W.-S. & Sherman, D. S.(2001). Analysis of temporal and spatial expression of the CcaR regulatory element in the cephamycin C biosynthetic pathway using green fluorescent protein. Mol Microbiol 40, 530–541.[CrossRef] [Google Scholar]
  10. 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 Chemother 43, 1215–1224. [Google Scholar]
  11. 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 Bacteriol 177, 1307–1314. [Google Scholar]
  12. 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 Microbiol 27, 831–843.[CrossRef] [Google Scholar]
  13. Perez-Llarena, F. J., Liras, P., Rodriguez-Garcia, A. & Martin, J. F.(1997). A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both β-lactam compounds. J Bacteriol 179, 2053–2059. [Google Scholar]
  14. Petrich, A. K., Wu, X., Roy, K. L. & Jensen, S. E.(1992). Transcriptional analysis of the isopenicillin N synthase-encoding gene of Streptomyces clavuligerus. Gene 111, 77–84.[CrossRef] [Google Scholar]
  15. Petrich, A. K., Leskiw, B. K., Paradkar, A. S. & Jensen, S. E.(1994). Transcriptional mapping of the genes encoding the early enzymes of the cephamycin biosynthetic pathway of Streptomyces clavuligerus. Gene 142, 41–48.[CrossRef] [Google Scholar]
  16. Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  17. Santamarta, I., Rodriguez-Garcia, A., Perez-Redondo, R., Martin, J. F. & Liras, P.(2002). CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus. J Bacteriol 184, 3106–3113.[CrossRef] [Google Scholar]
  18. Tahlan, K., Park, H.-U. & Jensen, S. E.(2004a). Three unlinked gene clusters are involved in clavam metabolite biosynthesis in Streptomyces clavuligerus. Can J Microbiol (in press). [Google Scholar]
  19. Tahlan, K., Park, H.-U., Wong, A., Beatty, P. H. & Jensen, S. E.(2004b). Two sets of paralogous genes encode the enzymes involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in Streptomyces clavuligerus. Antimicrob Agents Chemother 48, 930–939.[CrossRef] [Google Scholar]
  20. Trepanier, N. K., Jensen, S. E., Alexander, D. C. & Leskiw, B. K.(2002). The positive activator of cephamycin C and clavulanic acid production in Streptomyces clavuligerus is mistranslated in a bldA mutant. Microbiology 148, 643–656. [Google Scholar]
  21. Walters, N. J., Barton, B. & Earl, A. J.(1994). Novel Compounds. International patent WO 94/18326-A 1.
  22. 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 Microbiol 25, 1181–1184.[CrossRef] [Google Scholar]

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