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

Abstract

ABSTRACT 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.