Antonie van Leeuwenhoek

, Volume 60, Issue 1, pp 43–48 | Cite as

Characterization of new plasmids from methylotrophic bacteria

  • V. Brenner
  • I. Holubová
  • O. Benada
  • J. Hubáček


Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotrophMethylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genusMethylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and theE. coli plasmid pK19 Kmr, which were checked for conjugative transfer fromE. coli into the methylotrophic host.

Key words

methylotroph plasmids conjugation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Allen LN & Hanson RS (1985) Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth ofMethylobacterium organophilum on methanol. J. Bacteriol. 161: 955–962Google Scholar
  2. 2.
    Nunn D & Lidstrom M (1986) Phenotypic characterization of 10 methanol oxidation mutant classes ofMethylobacterium sp. strain AM1. J. Bacteriol. 166: 592–598Google Scholar
  3. 3.
    Anderson D & Lidstrom M (1988) Themox FG region encodes four polypeptides in the methanol-oxidizing bacteriumMethylobacterium sp. strain AM1. J. Bacteriol. 170: 2254–2262Google Scholar
  4. 4.
    Machlin S, Tam P, Bastien C & Hanson R (1987) Genetic and physical analysis ofMethylobacterium organophilum. XX genes encoding methanol oxidation. J. Bacteriol. 170: 141–148Google Scholar
  5. 5.
    Tsygankov YD & Chistoserdov AY (1985) Specific-purpose broad-host-range vectors. Plasmid 14: 118–125Google Scholar
  6. 6.
    Vries JE, Harms N, Hoogendijk J & Stouthamer AH (1989) Isolation and characterization ofParacoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property. Arch. Microbiol. 152: 52–57Google Scholar
  7. 7.
    Vries GE, Kües U & Stahl U (1990) Physiology and genetics of methylotrophic bacteria. FEMS Microbiol. Reviews 75: 57–102Google Scholar
  8. 8.
    Ueda S, Kitamoto N, Tamura Y, Sakakibara Y & Shimizu S (1987) Isolation and characterization of a plasmid in a methylotrophic bacterium. J. Ferment. Technol. 65(5): 589–591Google Scholar
  9. 9.
    Nöling J, Frylink MJ, & de Vos WM (1989) Isolation and characterization of plasmid DNA from the methanogenMethanobacterium thermoformicicum. In 6th International symposium on microbial growth on C1 compounds, Abstr. Book P331, GöttingenGoogle Scholar
  10. 10.
    Maniatis T, Fritsch E & Sambrook J (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  11. 11.
    Serebrijski IG, Kazakova SM & Tsygankov YD (1989) Construction of Hfr-like donors of the obligate methanoloxidizing bacteriumMethylobacillus flagellatum KT. FEMS Microbiol. Lett. 59: 203–206Google Scholar
  12. 12.
    Birnboim HC & Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7: 1513–1523Google Scholar
  13. 13.
    Hogrefe C & Friedrich B (1984) Isolation and characterization of megaplasmid DNA lithoautotrophic bacteria. Plasmid 12: 161–169Google Scholar
  14. 14.
    Brenner V, Holubová I & Hubáček J (1990) An efficient method for isolation of plasmid DNA from methylotrophic bacteria. Folia Microbiol. 35: 454–455Google Scholar
  15. 15.
    Davis RW, Simon M & Davidson N (1971) Electron microscopic heteroduplex methods for mapping regions of base sequence homology in nucleic acid. In: Grossmann L & Moldave K (Eds) Methods in Enzymology 21 (pp 413–428). Academic Press, New York, LondonGoogle Scholar
  16. 16.
    Kado CI & Liu ST (1981) Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145: 1365–1373Google Scholar
  17. 17.
    Lyon B, Kearney P, Sinclair M & Holloway B (1988) Comparative mapping ofMethylophilus spp. using cosmid gene libraries and prime plasmids. J. Gen. Microbiol. 134: 123–132Google Scholar
  18. 18.
    Tatra P & Goodwin D (1985) Mapping of some genes involved in C-1 metabolism in the facultative methylotrophMethylobacterium sp. strain AM1 (Pseudomonas AM1). Arch. Microbiol. 143(2): 169–178Google Scholar
  19. 19.
    Kim YM & Lidstrom ME (1989) Plasmid analysis in pink facultative methylotrophic bacteria using a modified acetone-alkaline hydrolysis method. FEMS Microbiol. Lett. 60: 125–130Google Scholar
  20. 20.
    Green P, Bousfield I & Hood D (1988) Three new methylobacterium species:N. rhodesianum, sp. nov.,N. zatmanii sp. nov., andM. fujisawaense sp. nov. Int. J. Syst. Bact. 38: 124–127Google Scholar
  21. 21.
    Sharpe GS (1984) Broad host range cloning vectors for Gram-negative bacteria. Gene 29: 93–102Google Scholar
  22. 22.
    Meyer R, Laux R, Boch G, Hinds M, Bayly R & Shapiro JA (1982) Broad-host-range IncP-4 plasmid R1162: Effects of deletions and insertions on plasmid maintenance and host range. J. Bacteriol. 152: 140–150Google Scholar
  23. 23.
    Gautier F & Bonewald R (1980) The use of plasmid R1162 and derivatives for gene cloning in the methanol-utilizingPseudomonas AM1. Mol. Gen. Genet. 178: 375–380Google Scholar
  24. 24.
    Kües U & Stahl U (1989) Replication of plasmids in gramnegative bacteria. Microbiol. Rev. 53: 451–516Google Scholar
  25. 25.
    Appleyard RK (1954) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived fromEscherichia coli K-12. Genetics 39: 440–452Google Scholar
  26. 26.
    Pridmore RD (1987) New and versatile cloning vectors with kanamycin resistance marker. Gene 56: 309–312Google Scholar

Copyright information

© Kluwer Academic Publishers 1991

Authors and Affiliations

  • V. Brenner
    • 1
  • I. Holubová
    • 1
  • O. Benada
    • 1
  • J. Hubáček
    • 1
  1. 1.Institute of MicrobiologyCzechoslovak Academy of SciencesPrague 4Czechoslovakia

Personalised recommendations