Generation of Bradyrhizobium japonicum mutants with increased N2O 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. 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.
Itakura
Manabu
M
Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8577, Japan.
Tabata
Kazufumi
K
Eda
Shima
S
Mitsui
Hisayuki
H
Murakami
Kiriko
K
Yasuda
Junichi
J
Minamisawa
Kiwamu
K
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D020369:Bradyrhizobium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004258:DNA Polymerase III / Q000235:genetics*; D050505:Mutant Proteins / Q000096:biosynthesis* / Q000235:genetics; D009609:Nitrous Oxide / Q000378:metabolism; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids
D001426:Bacterial Proteins; D050505:Mutant Proteins; D010088:Oxidoreductases; C022763:nitrous oxide reductase; D004258:DNA Polymerase III; D009609:Nitrous Oxide