Archives of Microbiology

, Volume 152, Issue 1, pp 52–57 | Cite as

Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property

  • Gert E. de Vries
  • Nellie Harms
  • Jasper Hoogendijk
  • Adriaan H. Stouthamer
Original Papers


A selection scheme was devised to isolate Paracoccus denitrificans mutants with increased recipient qualities in transfer experiments, using broad host range plasmids. In some of the mutants obtained, a DNA modifying activity that prevents the activity of the restriction endonucleases BamHI and BglII on isolated P. denitrificans DNA had simultaneously been lost. From a detailed analysis of the restriction properties of the enzymes SAU3 AI, MboI and DpnI, it was concluded that a subset of GATC sequences in P. denitrificans DNA may be methylated at an unusual position. It was concluded that P. denitrificans possesses at least one potent host-dependent restriction/modification system which affects conjugation. In addition to the class of restriction-defective mutants, at least one other class of enhanced transfer mutants with unknown defect(s) was isolated. Strains, in which the two mutant classes were combined, exhibited transfer frequencies which were significantly higher than strains containing either mutation alone. Such double mutant strains appeared to be well suited for future experiments like complementation analysis, transposon mutagenesis and gene replacement by homologous recombination.

Key words

Conjugation Restriction Modification Mutants Plasmids Paracoccus 


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  1. Allen LN, Hanson RS (1985) Construction of broad host range cosmid cloning vectors: Identification of genes necessary for growth of Methylobacterium organophilum on methanol. J Bacteriol 161:955–962Google Scholar
  2. Bagdasarian MM, Amann E, Lurz R, Rückert R, Bagdasarian M (1983) Activity of the hybrid trp-lac(tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broadhost range, controlled-expression vectors. Gene 26:273–282Google Scholar
  3. Datta N, Hedges RW (1972) Host range of R-factors. J Gen Microbiol 70:453–460Google Scholar
  4. Ehrlich M, Gama-Sosa MA, Carreira LH, Ljungdahl LG, Kuo KC, Gehrke CW (1985) DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine. Nucl Acids Res 13:1399–1412Google Scholar
  5. Frey J, Bagdasarian M, Feiss D, Franklink FCH, Deshusses J (1983) Stable cosmid vectors that enable the introduction of cloned fragments into a wide range of Gram-negative bacteria. Gene 24:299–308Google Scholar
  6. Gautier F, Bonewald R (1980) The use of plasmid R1162 and derivatives for gene cloning in the methanol-utilizing Pseudomonas AM1. Mol Gen Genet 178:375–380Google Scholar
  7. Harms N, Vries GE de, Maurer K, Hoogendijk J, Stouthamer AH (1987) Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans. J Bacteriol 169:3969–3975Google Scholar
  8. Janulaitis A, Klimasauskas S, Petrusyte M, Butkus V (1983) Cytosine modification in DNA by BcnI methylase yields N4-methylcytosine. FEBS Lett 161:131–134Google Scholar
  9. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  10. 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
  11. Nunn DN, Lidstrom ME (1986) Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1. J Bacteriol 166:581–590Google Scholar
  12. Paraskeva C (1979) Transfer of kanamycin resistance mediated by plasmid R 68.45 in Paracoccus denitrificans. J Bacteriol 139:1062–1064Google Scholar
  13. Ruvkun GB, Ausubel F (1981) A general method for site-directed mutagenesis in prokaryotes. Nature (Lond) 289:85–88Google Scholar
  14. Schmidhauser TJ, Helinski DR (1985) Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of Gram-negative bacteria. J Bacteriol 164:446–455Google Scholar
  15. Simon R, Priefer U, Pühler A (1983) Vector plasmids for in vivo and in vitro manipulations of Gram-negative bacteria. In: Pühler A (ed) Molecular genetics of the bacteria-plant interactions. Springer, Berlin Heidelberg New York, pp 98–106Google Scholar
  16. Spence DW, Barr GC (1981) A method for transformation of Paracoccus denitrificans. FEMS Microbiol Lett 12:159–161Google Scholar
  17. Verseveld HW van, Boon JP, Stouthamer AH (1979) Growth yields of oxidative phosphorylation of Paracoccus denitrificans during two-(carbon) substrate-limited growth. Arch Microbiol 122: 213–223Google Scholar
  18. Vries GE de (1986) Molecular biology of bacterial methanol oxidation. FEMS Microbiol Rev 39:235–258Google Scholar
  19. Vries GE de, Harms N, Maurer K, Papendrecht A, Stouthamer AH (1988) Physiological regulation of Paracoccus denitrificans methanol dehydrogenase synthesis and activity. J Bacteriol 170:3731–3737Google Scholar
  20. Willets NS, Crowther C (1981) Mobilization of the non-conjugative incQ plasmid RSF1010. Gen Res 37:311–316Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Gert E. de Vries
    • 1
  • Nellie Harms
    • 1
  • Jasper Hoogendijk
    • 1
  • Adriaan H. Stouthamer
    • 1
  1. 1.Department of MicrobiologyFree University AmsterdamAmsterdamThe Netherlands

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