Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Jan;179(1):180–186. doi: 10.1128/jb.179.1.180-186.1997

Use of rpsL for dominance selection and gene replacement in Streptomyces roseosporus.

T J Hosted 1, R H Baltz 1
PMCID: PMC178677  PMID: 8981996

Abstract

We developed a gene replacement system using the rpsL gene of Streptomyces roseosporus and demonstrated its utility by constructing a deletion in the S. roseosporus glnA gene. A 1.3-kb BamHI fragment that hybridized to the Mycobacterium smegmatis rpsL gene was subcloned from an S. roseosporus cosmid library and sequenced. Plasmid pRHB514 containing the rpsL gene conferred streptomycin sensitivity (Sm(S)) to the Sm(r) S. roseosporus TH149. The temperature-sensitive plasmid pRHB543 containing rpsL and the S. roseosporus glnA gene disrupted with a hygromycin resistance (Hm(r)) gene was introduced into S. roseosporus TH149, and recombinants containing single and double crossovers were obtained after a temperature increase. Southern hybridization analysis revealed that single crossovers occurred in the glnA or rpsL genes and that double crossovers resulted in replacement of the chromosomal glnA gene with the disrupted glnA. Glutamine synthetase activity was undetectable in the recombinant containing the disrupted glnA gene.

Full Text

The Full Text of this article is available as a PDF (278.6 KB).

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Baltz R. H. Genetic recombination in Streptomyces fradiae by protoplast fusion and cell regeneration. J Gen Microbiol. 1978 Jul;107(1):93–102. doi: 10.1099/00221287-107-1-93. [DOI] [PubMed] [Google Scholar]
  3. Baltz R. H., Hosted T. J. Molecular genetic methods for improving secondary-metabolite production in actinomycetes. Trends Biotechnol. 1996 Jul;14(7):245–250. doi: 10.1016/0167-7799(96)10034-2. [DOI] [PubMed] [Google Scholar]
  4. Bender R. A., Janssen K. A., Resnick A. D., Blumenberg M., Foor F., Magasanik B. Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. J Bacteriol. 1977 Feb;129(2):1001–1009. doi: 10.1128/jb.129.2.1001-1009.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bibb M. J., Findlay P. R., Johnson M. W. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984 Oct;30(1-3):157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
  6. Bierman M., Logan R., O'Brien K., Seno E. T., Rao R. N., Schoner B. E. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene. 1992 Jul 1;116(1):43–49. doi: 10.1016/0378-1119(92)90627-2. [DOI] [PubMed] [Google Scholar]
  7. Boeck L. D., Fukuda D. S., Abbott B. J., Debono M. Deacylation of A21978C, an acidic lipopeptide antibiotic complex, by Actinoplanes utahensis. J Antibiot (Tokyo) 1988 Aug;41(8):1085–1092. doi: 10.7164/antibiotics.41.1085. [DOI] [PubMed] [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Don R. H., Cox P. T., Wainwright B. J., Baker K., Mattick J. S. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991 Jul 25;19(14):4008–4008. doi: 10.1093/nar/19.14.4008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fisher S. H. Glutamine synthesis in Streptomyces--a review. Gene. 1992 Jun 15;115(1-2):13–17. doi: 10.1016/0378-1119(92)90534-v. [DOI] [PubMed] [Google Scholar]
  11. Hillemann D., Dammann T., Hillemann A., Wohlleben W. Genetic and biochemical characterization of the two glutamine synthetases GSI and GSII of the phosphinothricyl-alanyl-alanine producer, streptomyces viridochromogenes Tü494. J Gen Microbiol. 1993 Aug;139(8):1773–1783. doi: 10.1099/00221287-139-8-1773. [DOI] [PubMed] [Google Scholar]
  12. Hosted T. J., Baltz R. H. Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes. Microbiology. 1996 Oct;142(Pt 10):2803–2813. doi: 10.1099/13500872-142-10-2803. [DOI] [PubMed] [Google Scholar]
  13. Kenney T. J., Churchward G. Cloning and sequence analysis of the rpsL and rpsG genes of Mycobacterium smegmatis and characterization of mutations causing resistance to streptomycin. J Bacteriol. 1994 Oct;176(19):6153–6156. doi: 10.1128/jb.176.19.6153-6156.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kimura M., Kimura J. The complete amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus. FEBS Lett. 1987 Jan 1;210(1):91–96. doi: 10.1016/0014-5793(87)81304-2. [DOI] [PubMed] [Google Scholar]
  15. LEDERBERG J. Streptomycin resistance; a genetically recessive mutation. J Bacteriol. 1951 May;61(5):549–550. doi: 10.1128/jb.61.5.549-550.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lindahl L., Zengel J. M. Ribosomal genes in Escherichia coli. Annu Rev Genet. 1986;20:297–326. doi: 10.1146/annurev.ge.20.120186.001501. [DOI] [PubMed] [Google Scholar]
  17. Mazodier P., Petter R., Thompson C. Intergeneric conjugation between Escherichia coli and Streptomyces species. J Bacteriol. 1989 Jun;171(6):3583–3585. doi: 10.1128/jb.171.6.3583-3585.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nair J., Rouse D. A., Bai G. H., Morris S. L. The rpsL gene and streptomycin resistance in single and multiple drug-resistant strains of Mycobacterium tuberculosis. Mol Microbiol. 1993 Nov;10(3):521–527. doi: 10.1111/j.1365-2958.1993.tb00924.x. [DOI] [PubMed] [Google Scholar]
  19. Ohama T., Yamao F., Muto A., Osawa S. Organization and codon usage of the streptomycin operon in Micrococcus luteus, a bacterium with a high genomic G + C content. J Bacteriol. 1987 Oct;169(10):4770–4777. doi: 10.1128/jb.169.10.4770-4777.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Penyige A., Kálmánczhelyi A., Sipos A., Ensign J. C., Barabás G. Modification of glutamine synthetase in Streptomyces griseus by ADP-ribosylation and adenylylation. Biochem Biophys Res Commun. 1994 Oct 28;204(2):598–605. doi: 10.1006/bbrc.1994.2501. [DOI] [PubMed] [Google Scholar]
  21. Post L. E., Nomura M. DNA sequences from the str operon of Escherichia coli. J Biol Chem. 1980 May 25;255(10):4660–4666. [PubMed] [Google Scholar]
  22. Russell C. B., Dahlquist F. W. Exchange of chromosomal and plasmid alleles in Escherichia coli by selection for loss of a dominant antibiotic sensitivity marker. J Bacteriol. 1989 May;171(5):2614–2618. doi: 10.1128/jb.171.5.2614-2618.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Salles C., Créancier L., Claverys J. P., Méjean V. The high level streptomycin resistance gene from Streptococcus pneumoniae is a homologue of the ribosomal protein S12 gene from Escherichia coli. Nucleic Acids Res. 1992 Nov 25;20(22):6103–6103. doi: 10.1093/nar/20.22.6103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sander P., Meier A., Böttger E. C. rpsL+: a dominant selectable marker for gene replacement in mycobacteria. Mol Microbiol. 1995 Jun;16(5):991–1000. doi: 10.1111/j.1365-2958.1995.tb02324.x. [DOI] [PubMed] [Google Scholar]
  25. Shapiro M. DNAdraw: a program for preparing DNA and protein data for publication. Biotechniques. 1995 Jun;18(6):1064–1067. [PubMed] [Google Scholar]
  26. Skorupski K., Taylor R. K. Positive selection vectors for allelic exchange. Gene. 1996 Feb 22;169(1):47–52. doi: 10.1016/0378-1119(95)00793-8. [DOI] [PubMed] [Google Scholar]
  27. Skrzypek E., Haddix P. L., Plano G. V., Straley S. C. New suicide vector for gene replacement in yersiniae and other gram-negative bacteria. Plasmid. 1993 Mar;29(2):160–163. doi: 10.1006/plas.1993.1019. [DOI] [PubMed] [Google Scholar]
  28. Solenberg P. J., Baltz R. H. Hypertransposing derivatives of the streptomycete insertion sequence IS493. Gene. 1994 Sep 15;147(1):47–54. doi: 10.1016/0378-1119(94)90037-x. [DOI] [PubMed] [Google Scholar]
  29. Solenberg P. J., Baltz R. H. Transposition of Tn5096 and other IS493 derivatives in Streptomyces griseofuscus. J Bacteriol. 1991 Feb;173(3):1096–1104. doi: 10.1128/jb.173.3.1096-1104.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Speyer J. F. A simple and effective electroporation apparatus. Biotechniques. 1990 Jan;8(1):28–30. [PubMed] [Google Scholar]
  31. 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]
  32. Stuy J. H., Walter R. B. Cloning, characterization, and DNA base sequence of the high-level streptomycin resistance gene strA1 of Haemophilus influenzae Rd. J Bacteriol. 1992 Sep;174(17):5604–5608. doi: 10.1128/jb.174.17.5604-5608.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES