Generation of Bradyrhizobium japonicum mutants with increased N 2O reductase activity by selection after introduction of a mutated dnaQ gene

Manabu Itakura, Kazufumi Tabata, Shima Eda, Hisayuki Mitsui, Kiriko Murakami, Junichi Yasuda, Kiwamu Minamisawa

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22 Citations (Scopus)

Abstract

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N2O) reductase (N2OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N2O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N2O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N2OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N2OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.

Original languageEnglish
Pages (from-to)7258-7264
Number of pages7
JournalApplied and environmental microbiology
Volume74
Issue number23
DOIs
Publication statusPublished - 2008 Dec

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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