Generation of Bradyrhizobium japonicum Mutants with Increased N2O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene
Generation of Bradyrhizobium japonicum Mutants with Increased N2O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene
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DOI：
10.1128/aem.01850-08
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发表时间：
2008-12-01
影响因子：
4.4
通讯作者：
Minamisawa, Kiwamu
中科院分区：
文献类型：
--
作者：
Itakura, Manabu;Tabata, Kazufumi;Minamisawa, Kiwamu
通过引入含有突变B的质粒，获得了两个日本慢生根瘤菌USDA 110的一氧化二氮（N2 O）还原酶（N2 OR）活性增加的有益突变体。日本血吸虫dnaQ基因（pKQ2），在N2O呼吸选择压力下富集培养。dnaQ编码DNA聚合酶III的α亚基，其突变在大肠杆菌中产生强的突变子表型。pKQ2导入B。Japonicum USDA 110增加了对卡那霉素自发抗性的菌落的出现频率。在厌氧条件下，在5%（vol/vol）或20%（vol/vol）N2O气氛下，在有和没有pKQ2的情况下进行一系列USDA 110的重复培养。在第10个培养周期，USDA 110（pKQ2）的细胞群显示出比野生型菌株更高的N2OR活性。通过植物传代获得的四种缺乏pKQ2的细菌突变体显示出野生型USDA 110的7至12倍的N2OR活性。虽然两个突变体具有弱的或无效的共生固氮表型，其余两个（5M09和5M14）具有相同的共生固氮能力和异养生长的野生型USDA 110在培养物中。
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.