Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp. Strain HW13 Isolated from a Pristine Environment

The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R. eutropha JMP134. The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134. Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not. Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC.真养产碱菌（Ralstonia eutropha）JMP134的tfd基因，是目前2,4-二氯苯氧乙酸（2,4-dichlorophenoxyacetic acid, 2,4-D）降解菌中唯一得到充分表征的2,4-D降解基因簇。本研究从从未接触过人为化学品的夏威夷埋藏土壤中分离得到的慢生根瘤菌（Bradyrhizobium）菌株HW13中，克隆并表征了一类全新的2,4-D降解基因家族cadRABKC。根据推导得到的氨基酸序列，可推断cadR基因编码一种AraC/XylS型转录调控因子。cadABC基因经预测编码2,4-D加氧酶亚基，其推导氨基酸序列分别与洋葱伯克霍尔德菌（Burkholderia cepacia）AC1100的2,4,5-三氯苯氧乙酸（2,4,5-trichlorophenoxyacetic acid, 2,4,5-T）加氧酶亚基TftA、TftB，以及红球菌（Rhodococcus erythropolis）NI86/21的推定铁氧还蛋白ThcC具有46%、44%和37%的序列同一性。该基因簇与真养产碱菌JMP134的2,4-D双加氧酶基因tfdA存在显著差异。根据推导氨基酸序列，cadK基因被推定编码2,4-D转运蛋白，其序列与菌株JMP134的2,4-D转运蛋白TfdK具有60%的序列同一性。携带cadRABKC基因簇的苜蓿中华根瘤菌（Sinorhizobium meliloti）Rm1021，可将包括2,4-D和2,4,5-T在内的多种苯氧乙酸类化合物转化为对应的苯酚衍生物。移码突变实验表明，在菌株Rm1021中，cadRABC基因均对2,4-D转化至关重要，而cadK并非必需基因。本研究共发现5株2,4-D降解菌，包括慢生根瘤菌和鞘氨醇单胞菌（Sphingomonas）菌株，均含有cadA基因同源物，提示这些2,4-D降解菌拥有与HW13菌株cadABC相似的2,4-D降解基因。