Generation of Bradyrhizobium japonicum Mutants with Increased N 2 O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N 2 O) reductase (N 2 OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N 2 O respiration. Mutati...

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Veröffentlicht in:Applied and environmental microbiology 2008-12, Vol.74 (23), p.7258-7264
Hauptverfasser: Itakura, Manabu, Tabata, Kazufumi, Eda, Shima, Mitsui, Hisayuki, Murakami, Kiriko, Yasuda, Junichi, Minamisawa, Kiwamu
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container_end_page 7264
container_issue 23
container_start_page 7258
container_title Applied and environmental microbiology
container_volume 74
creator Itakura, Manabu
Tabata, Kazufumi
Eda, Shima
Mitsui, Hisayuki
Murakami, Kiriko
Yasuda, Junichi
Minamisawa, Kiwamu
description We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N 2 O) reductase (N 2 OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N 2 O 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) N 2 O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N 2 OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N 2 OR 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.
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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) N 2 O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N 2 OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N 2 OR activity of the wild-type USDA110. 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title Generation of Bradyrhizobium japonicum Mutants with Increased N 2 O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene
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