Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Aug;63(8):2977–2982. doi: 10.1128/aem.63.8.2977-2982.1997

Amy as a reporter gene for promoter activity in Nocardia lactamdurans: comparison of promoters of the cephamycin cluster.

V K Chary 1, J L de la Fuente 1, P Liras 1, J F Martin 1
PMCID: PMC168596  PMID: 9251185

Abstract

Promoter probe vectors containing the pA origin of replication and the Streptomyces griseus promoterless amy gene (encoding alpha-amylase) as reporter have been constructed to study transcription initiation regions in Nocardia lactamdurans. In some of the promoter probe vectors the phage fd terminator has been introduced to avoid readthrough expression from upstream sequences. By using these vectors, four different transcription initiation regions of the cephamycin gene cluster have been studied in N. lactamdurans. The bla gene encoding a beta-lactamase has a relatively strong promoter. Two other separate promoters corresponding to the lat and cefD genes (encoding, respectively, lysine-6-aminotransferase and isopenicillin N-epimerase) showed weak transcription initiation ability. These two promoters are arranged in a bidirectional transcription initiation region located in the center of the cephamycin gene cluster. The cmcH gene (encoding 3-hydroxymethylcephem carbamoyltransferase) upstream region did not contain a functional promoter, suggesting that cmcH is transcribed as a part of a polycistronic mRNA. The native amy promoter is used very efficiently in N. lactamdurans, resulting in secretion of high levels of extracellular alpha-amylase.

Full Text

The Full Text of this article is available as a PDF (408.3 KB).

Selected References

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

  1. Band L., Yansura D. G., Henner D. J. Construction of a vector for cloning promoters in Bacillus subtilis. Gene. 1983 Dec;26(2-3):313–315. doi: 10.1016/0378-1119(83)90204-4. [DOI] [PubMed] [Google Scholar]
  2. Blanco J., Coque J. J., Martín J. F. Characterization of the secA gene of Streptomyces lividans encoding a protein translocase which complements and Escherichia coli mutant defective in the ATPase activity of SecA. Gene. 1996 Oct 17;176(1-2):61–65. doi: 10.1016/0378-1119(96)00220-x. [DOI] [PubMed] [Google Scholar]
  3. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  4. Cadenas R. F., Gil J. A., Martín J. F. Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans. Appl Microbiol Biotechnol. 1992 Dec;38(3):362–369. doi: 10.1007/BF00170087. [DOI] [PubMed] [Google Scholar]
  5. Clayton T. M., Bibb M. J. Streptomyces promoter-probe plasmids that utilise the xylE gene of Pseudomonas putida. Nucleic Acids Res. 1990 Feb 25;18(4):1077–1077. doi: 10.1093/nar/18.4.1077. [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., 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]
  8. 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]
  9. Garcia-Gonzalez M. D., Martin J. F., Vigal T., Liras P. Characterization, expression in Streptomyces lividans, and processing of the amylase of Streptomyces griseus IMRU 3570: two different amylases are derived from the same gene by an intracellular processing mechanism. J Bacteriol. 1991 Apr;173(8):2451–2458. doi: 10.1128/jb.173.8.2451-2458.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ginther C. L. Sporulation and the production of serine protease and cephamycin C by Streptomyces lactamdurans. Antimicrob Agents Chemother. 1979 Apr;15(4):522–526. doi: 10.1128/aac.15.4.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Martín J. F., Liras P. Enzymes involved in penicillin, cephalosporin and cephamycin biosynthesis. Adv Biochem Eng Biotechnol. 1989;39:153–187. doi: 10.1007/BFb0051954. [DOI] [PubMed] [Google Scholar]
  15. Pérez-Llarena F. J., Liras P., Rodríguez-García A., Martín J. F. A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds. J Bacteriol. 1997 Mar;179(6):2053–2059. doi: 10.1128/jb.179.6.2053-2059.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rosenberg M., Chepelinsky A. B., McKenney K. Studying promoters and terminators by gene fusion. Science. 1983 Nov 18;222(4625):734–739. doi: 10.1126/science.6356355. [DOI] [PubMed] [Google Scholar]
  17. Strohl W. R. Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 1992 Mar 11;20(5):961–974. doi: 10.1093/nar/20.5.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Vigal T., Gil J. A., Daza A., García-González M. D., Martín J. F. Cloning, characterization and expression of an alpha-amylase gene from Streptomyces griseus IMRU3570. Mol Gen Genet. 1991 Feb;225(2):278–288. doi: 10.1007/BF00269860. [DOI] [PubMed] [Google Scholar]
  19. Vigal T., Gil J. A., Daza A., García-González M. D., Villadas P., Martín J. F. Effects of replacement of promoters and modification of the leader peptide region of the amy gene of Streptomyces griseus on synthesis and secretion of alpha-amylase by Streptomyces lividans. Mol Gen Genet. 1991 Dec;231(1):88–96. doi: 10.1007/BF00293826. [DOI] [PubMed] [Google Scholar]
  20. Virolle M. J., Gagnat J. Sequences involved in growth-phase-dependent expression and glucose repression of a Streptomyces alpha-amylase gene. Microbiology. 1994 May;140(Pt 5):1059–1067. doi: 10.1099/13500872-140-5-1059. [DOI] [PubMed] [Google Scholar]
  21. de Boer H. A. A versatile plasmid system for the study of prokaryotic transcription signals in Escherichia coli. Gene. 1984 Oct;30(1-3):251–255. doi: 10.1016/0378-1119(84)90129-x. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES