Molecular and General Genetics MGG

, Volume 246, Issue 4, pp 411–418 | Cite as

Isolation of cryptic plasmids from moderately halophilic eubacteria of the genus Halomonas. Characterization of a small plasmid from H. elongata and its use for shuttle vector construction

  • Carmen Vargas
  • Rosario Fernández-Castillo
  • David Cánovas
  • Antonio Ventosa
  • Joaquin J. Nieto
Original Paper


Three cryptic plasmids have been isolated from moderately halophilic eubacteria belonging to three species of the genus Halomonas. These three plasmids were designated pHE1 (4.2 kb, isolated from H. elongata ATCC 33174), pHI1 (48 kb, isolated from “H. israelensis” ATCC 43985), and pHS1 (ca. 70 kb, isolated from H. subglaciescola UQM 2927). Because of its small size, the plasmid pHE1 was selected for further characterization and construction of a shuttle vector for Halomonas strains. pHE1 was cloned into pBluescript KS and a detailed restriction map was constructed. Hybridization experiments excluded the existence of sequences homologous to pHE1 in total DNA from other strains of the genus Halomonas. Moreover, no DNA homology with pMH1, the only plasmid described so far from moderate halophiles, was found. Since pHE1 appeared to be unable to replicate in Escherichia coli cells, a number of mobilizable pHE1-derived hybrid plasmids were constructed that could be selected and maintained both in E. coli and in H. elongata. Finally, an improved shuttle vector, pHS15, was generated. The vector pHS15 contains an origin of replication from E. coli as well as one from H. elongata, a streptomycin resistance gene for positive selection in moderate halophiles, a number of unique restriction sites commonly used for cloning, and the mobilization functions of the broad host range IncP plasmid RK2. The vector pHS15 was readily mobilized by the RK2 derivative pRK2013 to all Halomonas strains tested so far. This is the first report on the development of a cloning vector useful for moderately halophilic eubacteria.

Key words

Moderate halophiles Halomonas elongata Plasmids Cloning vectors 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1989) Current protocols in molecular biology. Greene Publishing Associates, John Wiley and Sons, New YorkGoogle Scholar
  2. Csonka LN, Hanson AD (1991) Prokaryotic osmoregulation: genetics and physiology. Annu Rev Microbiol 45:569–606Google Scholar
  3. Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351Google Scholar
  4. Eckardt T (1978) A rapid method for the identification of plasmid DNA in bacteria. Plasmid 1:584–588Google Scholar
  5. Fernández-Castillo R, Vargas C, Nieto JJ, Ventosa A, Ruiz-Berraquero F (1992) Characterization of a plasmid from moderately halophilic eubacteria. J Gen Microbiol 138:1133–1137Google Scholar
  6. Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652Google Scholar
  7. Franzmann PD, Wehmeyer U, Stackebrandt E (1988) Halomonadaceae fam. nov., a new family of the class Proteobacteria to accommodate the genera Halomonas and Deleya. Syst Appl Microbiol 11:16–19Google Scholar
  8. Galinski EA (1989) The potential use of halophilic eubacteria for the production of organic chemicals and enzyme protective agents. In: Da Costa MS, Duarte JC, Williams RAD (eds) Microbiology of extreme environments and its potential for biotechnology. Elsevier Applied Science, London, pp 375–379Google Scholar
  9. Galinski EA, Tindall BJ (1992) Biotechnological prospects for halophiles and halo-tolerant microorganisms. In: Herber RA, Sharp RJ (eds) Molecular biology and biotechnology of extremophiles. Blackie and Son, London, pp 76–114Google Scholar
  10. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–561Google Scholar
  11. Heitman J, Model P (1987) Site-specific methylases induce the SOS DNA repair response in Escherichia coli. J Bacteriol 169:3243–3250Google Scholar
  12. Hengen PN, Iyer VN (1992) DNA cassettes containing the origin of transfer (oriT) of two broad-host-range transfer systems. Biotechniques 13:57–62Google Scholar
  13. Holmes ML, Nuttall SD, Dyall-Smith ML (1991) Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. J Bacteriol 173:3807–3813Google Scholar
  14. Kamekura M (1986) Production and function of enzymes of eubacterial halophiles. FEMS Microbiol Rev 39:145–150Google Scholar
  15. Kogut M, Mason JR, Russell NJ (1992) Isolation of salt-sensitive mutants of the moderately halophilic eubacterium Vibrio costicola. Curr Microbiol 24:325–328Google Scholar
  16. Kushner DJ (1978) Life in high salt and solute concentrations: halophilic bacteria. In: Kushner DJ (ed) Microbial life in extreme environments. Academic Press, London, pp 317–368Google Scholar
  17. Kushner DJ, Kamekura M (1988) Physiology of halophilic eubacteria. In: Rodriguez-Valera F (ed) Halophilic bacteria, vol I. CRC Press, Boca Raton, Florida, pp 109–140Google Scholar
  18. Morelle G (1989) A plasmid extraction procedure on a miniprep scale. BRL Focus 11:7–8Google Scholar
  19. Nieto JJ, Fernández-Castillo R, Márquez MC, Ventosa A, Ruiz-Berraquero F (1989) A survey of metal tolerance in moderately halophilic eubacteria. Appl Environ Microbiol 55:2385–2390Google Scholar
  20. Nieto JJ, Fernández-Castillo R, García MT, Mellado E, Ventosa A (1993) Survey of antimicrobial susceptibility of moderately halophilic eubacteria and extremely halophilic aerobic archaeobacteria: utilization of antimicrobial resistance as a genetic marker. Syst Appl Microbiol 16:352–360Google Scholar
  21. Plazinski J, Cen YH, Rolfe BG (1985) General method for the identification of plasmid species in fast-growing soil microorganisms. Appl Environ Microbiol 48:1001–1003Google Scholar
  22. Prentki P, Krisch HM (1984) In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313Google Scholar
  23. Priefer UB, Simon R, Puhler A (1985) Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions. J Bacteriol 163:324–330Google Scholar
  24. Rodríguez-Valera F (1986) The ecology and taxonomy of aerobic chemoorganotrophic halophilic eubacteria. FEMS Microbiol Rev 39:17–22Google Scholar
  25. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  26. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  27. Ventosa A (1988) Taxonomy of moderately halophilic heterotrophic eubacteria. In: Rodríguez-Valera F (ed) Halophilic bacteria, vol 1. CRC Press, Boca Raton, Florida, pp 71–84Google Scholar
  28. Vreeland RH (1992) The family Halomonadaceae. In: Balows A, Truper HG, Dworkin M, Harder W, Schleifer KH (eds) The prokaryotes, 2nd edn. Springer-Verlag, New York, pp 3181–3188Google Scholar
  29. Wood WB (1969) Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol 16:118–133Google Scholar
  30. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Carmen Vargas
    • 1
  • Rosario Fernández-Castillo
    • 1
  • David Cánovas
    • 1
  • Antonio Ventosa
    • 1
  • Joaquin J. Nieto
    • 1
  1. 1.Department of Microbiology and Parasitology, Faculty of PharmacyUniversity of SevillaSevillaSpain

Personalised recommendations