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Isolation of transposon Tn5 insertion mutants of Rhodobacter capsulatus unable to reduce trimethylamine-N-oxide and dimethylsulphoxide

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Abstract

1) Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) strain 37b4 was subjected to transposon Tn5 mutagenesis. 2) Kanamycin-resistant transconjugants were screened for their inability to reduce trimethylamine-N-oxide (TMAO) as judged by the lack of alkali production during anaerobic growth on plates containing glucose as carbon source and cresol red as pH indicator. 3) Of 6 mutants examined, all were found to have considerably decreased levels of methylviologen-dependent TMAO reductase activity and dimethylsulphoxide (DMSO) reductase activity. 4) Periplasmic fractions of one of these mutants (DK9) and of the parent strain were subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis. The gels were stained for TMAO-reductase and DMSO-reductase. With the wild-type strain, only a single polypeptide band, Mr=46,000, stained for TMAO and DMSO reductase activity. In mutant DK9 this band was not detectable. 5) In contrast to the parent strain, harvested washed cells of mutant DK9 were unable to generate a cytoplasmic membrane potential in the presence of TMAO or DMSO under dark anaerobic conditions. 6) In contrast to the parent strain, DK9 was unable to grow in dark anaerobic culture with fructose as the carbon source and TMAO as oxidant.

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Abbreviations

TMAO:

trimethylamine-N-oxide

DMSO:

dimethylsulphoxide

PMS:

phenazine methosulphate

Δψ:

cytoplasmic membrane potential

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Author notes

  1. D. J. Kelly

    Present address: Department of Microbiology, University of Sheffield, Western Bank, S10 2TN, Sheffield, UK

Authors and Affiliations

  1. Department of Biochemistry, University of Birmingham, P.O. Box 363, B15 2TT, Birmingham, UK

    D. J. Kelly, D. J. Richardson & J. B. Jackson

  2. Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, Oxford, UK

    S. J. Ferguson

Authors
  1. D. J. Kelly
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  2. D. J. Richardson
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  3. S. J. Ferguson
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  4. J. B. Jackson
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Kelly, D.J., Richardson, D.J., Ferguson, S.J. et al. Isolation of transposon Tn5 insertion mutants of Rhodobacter capsulatus unable to reduce trimethylamine-N-oxide and dimethylsulphoxide. Arch. Microbiol. 150, 138–144 (1988). https://doi.org/10.1007/BF00425153

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  • DOI: https://doi.org/10.1007/BF00425153

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