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. 1988 Oct;170(10):4669–4674. doi: 10.1128/jb.170.10.4669-4674.1988

Organization of the biosynthesis genes for the peptide antibiotic gramicidin S.

M Krause 1, M A Marahiel 1
PMCID: PMC211506  PMID: 2459107

Abstract

A recombinant bacteriophage containing the intact Bacillus brevis gene for gramicidin S synthetase 1, grsA, and the 5' end of the gramicidin S synthetase 2 gene, grsB, was identified by screening an EMBL3 library with anti-GrsA antibodies. This clone, EMBL315, has a 14-kilobase (kb) insert that hybridizes to the previously isolated 3.9-kb fragment of the grsB gene, which encodes the 155-kilodalton ornithine-activating domain of gramicidin S synthetase 2. Deletion and subcloning experiments with the 14-kb insert located the grsA structural gene and its putative promoter on a 4.5-kb PvuII fragment which encoded the full-length 120-kilodalton protein in Escherichia coli. In addition, hybridization analysis revealed that the 5' end of the grsB gene is located approximately 3 kb from the grsA structural gene. Furthermore, these studies indicated that grsA and grsB are transcribed in opposite orientations.

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Selected References

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  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  3. Carr L. G., Skatrud P. L., Scheetz M. E., 2nd, Queener S. W., Ingolia T. D. Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum. Gene. 1986;48(2-3):257–266. doi: 10.1016/0378-1119(86)90084-3. [DOI] [PubMed] [Google Scholar]
  4. Chater K. F., Bruton C. J. Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered. EMBO J. 1985 Jul;4(7):1893–1897. doi: 10.1002/j.1460-2075.1985.tb03866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fishman S. E., Cox K., Larson J. L., Reynolds P. A., Seno E. T., Yeh W. K., Van Frank R., Hershberger C. L. Cloning genes for the biosynthesis of a macrolide antibiotic. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8248–8252. doi: 10.1073/pnas.84.23.8248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Helfman D. M., Feramisco J. R., Fiddes J. C., Thomas G. P., Hughes S. H. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A. 1983 Jan;80(1):31–35. doi: 10.1073/pnas.80.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. doi: 10.1016/0076-6879(79)68021-7. [DOI] [PubMed] [Google Scholar]
  9. Hopwood D. A., Malpartida F., Kieser H. M., Ikeda H., Duncan J., Fujii I., Rudd B. A., Floss H. G., Omura S. Production of 'hybrid' antibiotics by genetic engineering. Nature. 1985 Apr 18;314(6012):642–644. doi: 10.1038/314642a0. [DOI] [PubMed] [Google Scholar]
  10. Katz E., Demain A. L. The peptide antibiotics of Bacillus: chemistry, biogenesis, and possible functions. Bacteriol Rev. 1977 Jun;41(2):449–474. doi: 10.1128/br.41.2.449-474.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kleinkauf H., von Döhren H. Biosynthesis of peptide antibiotics. Annu Rev Microbiol. 1987;41:259–289. doi: 10.1146/annurev.mi.41.100187.001355. [DOI] [PubMed] [Google Scholar]
  12. Krause M., Marahiel M. A., von Döhren H., Kleinkauf H. Molecular cloning of an ornithine-activating fragment of the gramicidin S synthetase 2 gene from Bacillus brevis and its expression in Escherichia coli. J Bacteriol. 1985 Jun;162(3):1120–1125. doi: 10.1128/jb.162.3.1120-1125.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lipmann F. Bacterial production of antibiotic polypeptides by thiol-linked synthesis on protein templates. Adv Microb Physiol. 1980;21:227–266. doi: 10.1016/s0065-2911(08)60357-4. [DOI] [PubMed] [Google Scholar]
  15. Malpartida F., Hopwood D. A. Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host. 1984 May 31-Jun 6Nature. 309(5967):462–464. doi: 10.1038/309462a0. [DOI] [PubMed] [Google Scholar]
  16. Malpartida F., Hopwood D. A. Physical and genetic characterisation of the gene cluster for the antibiotic actinorhodin in Streptomyces coelicolor A3(2). Mol Gen Genet. 1986 Oct;205(1):66–73. doi: 10.1007/BF02428033. [DOI] [PubMed] [Google Scholar]
  17. Marahiel M. A., Danders W., Krause M., Kleinkauf H. Biological role of gramicidin S in spore functions. Studies on gramicidin-S-negative mutants of Bacillus brevis ATCC9999. Eur J Biochem. 1979 Aug 15;99(1):49–55. doi: 10.1111/j.1432-1033.1979.tb13229.x. [DOI] [PubMed] [Google Scholar]
  18. Marahiel M. A., Krause M., Skarpeid H. J. Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli. Mol Gen Genet. 1985;201(2):231–236. doi: 10.1007/BF00425664. [DOI] [PubMed] [Google Scholar]
  19. Marahiel M. A., Zuber P., Czekay G., Losick R. Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis. J Bacteriol. 1987 May;169(5):2215–2222. doi: 10.1128/jb.169.5.2215-2222.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ohnuki T., Imanaka T., Aiba S. Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance. J Bacteriol. 1985 Oct;164(1):85–94. doi: 10.1128/jb.164.1.85-94.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ramón D., Carramolino L., Patiño C., Sánchez F., Peñalva M. A. Cloning and characterization of the isopenicillin N synthetase gene mediating the formation of the beta-lactam ring in Aspergillus nidulans. Gene. 1987;57(2-3):171–181. doi: 10.1016/0378-1119(87)90120-x. [DOI] [PubMed] [Google Scholar]
  22. Samson S. M., Belagaje R., Blankenship D. T., Chapman J. L., Perry D., Skatrud P. L., VanFrank R. M., Abraham E. P., Baldwin J. E., Queener S. W. Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium. Nature. 1985 Nov 14;318(6042):191–194. doi: 10.1038/318191a0. [DOI] [PubMed] [Google Scholar]
  23. Samson S. M., Chapman J. L., Belagaje R., Queener S. W., Ingolia T. D. Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5705–5709. doi: 10.1073/pnas.84.16.5705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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