Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Jun;173(12):3807–3813. doi: 10.1128/jb.173.12.3807-3813.1991

Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase.

M L Holmes 1, S D Nuttall 1, M L Dyall-Smith 1
PMCID: PMC208012  PMID: 1711028

Abstract

We report here on advances made in the construction of plasmid shuttle vectors suitable for genetic manipulations in both Escherichia coli and halobacteria. Starting with a 20.4-kb construct, pMDS1, new vectors were engineered which were considerably smaller yet retained several alternative cloning sites. A restriction barrier observed when plasmid DNA was transferred into Haloferax volcanii cells was found to operate via adenine methylation, resulting in a 10(3) drop in transformation efficiency and the loss of most constructs by incorporation of the resistance marker into the chromosome. Passing shuttle vectors through E. coli dam mutants effectively avoided this barrier. Deletion analysis revealed that the gene(s) for autonomous replication of pHK2 (the plasmid endogenous to Haloferax strain Aa2.2 and used in the construction of pMDS1) was located within a 4.2-kb SmaI-KpnI fragment. Convenient restriction sites were identified near the termini of the novobiocin resistance determinant (gyrB), allowing the removal of flanking sequences (including gyrA). These deletions did not appear to significantly affect transformation efficiencies or the novobiocin resistance phenotype of halobacterial transformants. Northern blot hybridization with strand- and gene-specific probes identified a single gyrB-gyrA transcript of 4.7 kb. This is the first demonstration in prokaryotes that the two subunits of DNA gyrase may be cotranscribed.

Full text

PDF
3809

Images in this article

3810Image
on p.3810
3810Image
on p.3810

Selected References

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

  1. Blaseio U., Pfeifer F. Transformation of Halobacterium halobium: development of vectors and investigation of gas vesicle synthesis. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6772–6776. doi: 10.1073/pnas.87.17.6772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Charlebois R. L., Lam W. L., Cline S. W., Doolittle W. F. Characterization of pHV2 from Halobacterium volcanii and its use in demonstrating transformation of an archaebacterium. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8530–8534. doi: 10.1073/pnas.84.23.8530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cline S. W., Doolittle W. F. Efficient transfection of the archaebacterium Halobacterium halobium. J Bacteriol. 1987 Mar;169(3):1341–1344. doi: 10.1128/jb.169.3.1341-1344.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colman S. D., Hu P. C., Bott K. F. Mycoplasma pneumoniae DNA gyrase genes. Mol Microbiol. 1990 Jul;4(7):1129–1134. doi: 10.1111/j.1365-2958.1990.tb00687.x. [DOI] [PubMed] [Google Scholar]
  5. Dimri G. P., Das H. K. Cloning and sequence analysis of gyrA gene of Klebsiella pneumoniae. Nucleic Acids Res. 1990 Jan 11;18(1):151–156. doi: 10.1093/nar/18.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dundas I. E. Physiology of halobacteriaceae. Adv Microb Physiol. 1977;15:85–120. doi: 10.1016/s0065-2911(08)60315-x. [DOI] [PubMed] [Google Scholar]
  7. Gellert M., Mizuuchi K., O'Dea M. H., Itoh T., Tomizawa J. I. Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4772–4776. doi: 10.1073/pnas.74.11.4772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hane M. W., Wood T. H. Escherichia coli K-12 mutants resistant to nalidixic acid: genetic mapping and dominance studies. J Bacteriol. 1969 Jul;99(1):238–241. doi: 10.1128/jb.99.1.238-241.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Heitman J., Model P. Site-specific methylases induce the SOS DNA repair response in Escherichia coli. J Bacteriol. 1987 Jul;169(7):3243–3250. doi: 10.1128/jb.169.7.3243-3250.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Higgins N. P., Peebles C. L., Sugino A., Cozzarelli N. R. Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1773–1777. doi: 10.1073/pnas.75.4.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holmes M. L., Dyall-Smith M. L. A plasmid vector with a selectable marker for halophilic archaebacteria. J Bacteriol. 1990 Feb;172(2):756–761. doi: 10.1128/jb.172.2.756-761.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Holmes M. L., Dyall-Smith M. L. Mutations in DNA gyrase result in novobiocin resistance in halophilic archaebacteria. J Bacteriol. 1991 Jan;173(2):642–648. doi: 10.1128/jb.173.2.642-648.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hopewell R., Oram M., Briesewitz R., Fisher L. M. DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology among gyrase proteins and implications for 4-quinolone action and resistance. J Bacteriol. 1990 Jun;172(6):3481–3484. doi: 10.1128/jb.172.6.3481-3484.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Inoue S., Ohue T., Yamagishi J., Nakamura S., Shimizu M. Mode of incomplete cross-resistance among pipemidic, piromidic, and nalidixic acids. Antimicrob Agents Chemother. 1978 Aug;14(2):240–245. doi: 10.1128/aac.14.2.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lam W. L., Doolittle W. F. Shuttle vectors for the archaebacterium Halobacterium volcanii. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5478–5482. doi: 10.1073/pnas.86.14.5478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lodwick D., Ross H. N., Harris J. E., Almond J. W., Grant W. D. dam methylation in the archaebacteria. J Gen Microbiol. 1986 Nov;132(11):3055–3059. doi: 10.1099/00221287-132-11-3055. [DOI] [PubMed] [Google Scholar]
  17. Menzel R., Gellert M. Modulation of transcription by DNA supercoiling: a deletion analysis of the Escherichia coli gyrA and gyrB promoters. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4185–4189. doi: 10.1073/pnas.84.12.4185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Menzel R., Gellert M. Regulation of the genes for E. coli DNA gyrase: homeostatic control of DNA supercoiling. Cell. 1983 Aug;34(1):105–113. doi: 10.1016/0092-8674(83)90140-x. [DOI] [PubMed] [Google Scholar]
  19. Shimmin L. C., Dennis P. P. Characterization of the L11, L1, L10 and L12 equivalent ribosomal protein gene cluster of the halophilic archaebacterium Halobacterium cutirubrum. EMBO J. 1989 Apr;8(4):1225–1235. doi: 10.1002/j.1460-2075.1989.tb03496.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Timmers K., Sternglanz R. Ionization and divalent cation dissociation constants of nalidixic and oxolinic acids. Bioinorg Chem. 1978 Aug;9(2):145–155. doi: 10.1016/s0006-3061(00)80286-0. [DOI] [PubMed] [Google Scholar]
  21. Yamagishi J., Yoshida H., Yamayoshi M., Nakamura S. Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli. Mol Gen Genet. 1986 Sep;204(3):367–373. doi: 10.1007/BF00331012. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Yoshida H., Kojima T., Yamagishi J., Nakamura S. Quinolone-resistant mutations of the gyrA gene of Escherichia coli. Mol Gen Genet. 1988 Jan;211(1):1–7. doi: 10.1007/BF00338386. [DOI] [PubMed] [Google Scholar]

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

RESOURCES