Kashyap Des R, Botero Lina M, Lehr Corinne, Hassett Daniel J, McDermott Timothy R
Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana 59717, USA.
J Bacteriol. 2006 Feb;188(4):1577-84. doi: 10.1128/JB.188.4.1577-1584.2006.
Transposon Tn5-B22 mutagenesis was used to identify genetic determinants required for arsenite [As(III)] oxidation in an Agrobacterium tumefaciens soil isolate, strain 5A. In one mutant, the transposon interrupted modB, which codes for the permease component of a high-affinity molybdate transporter. In a second mutant, the transposon insertion occurred in mrpB, which is part of a seven-gene operon encoding an Mrp-type Na+:H+ antiporter complex. Complementation experiments with mod and mrp operons PCR cloned from the genome-sequenced A. tumefaciens strain C58 resulted in complementation back to an As(III)-oxidizing phenotype, confirming that these genes encode activities essential for As(III) oxidation in this strain of A. tumefaciens. As expected, the mrp mutant was extremely sensitive to NaCl and LiCl, indicating that the Mrp complex in A. tumefaciens is involved in Na+ circulation across the membrane. Gene expression studies (lacZ reporter and reverse transcriptase PCR experiments) failed to show evidence of transcriptional regulation of the mrp operon in response to As(III) exposure, whereas expression of the mod operon was found to be up-regulated by As(III) exposure. In each mutant, the loss of As(III)-oxidizing capacity resulted in conversion to an arsenate [As(V)]-reducing phenotype. Neither mutant was more sensitive to As(III) than the parental strain.
转座子Tn5-B22诱变被用于鉴定土壤根癌农杆菌菌株5A中砷酸盐[As(III)]氧化所需的遗传决定因素。在一个突变体中，转座子中断了modB，该基因编码一种高亲和力钼酸盐转运体的通透酶成分。在第二个突变体中，转座子插入发生在mrpB中，mrpB是一个七基因操纵子的一部分，该操纵子编码一种Mrp型Na+:H+反向转运体复合物。用从基因组测序的根癌农杆菌菌株C58中PCR克隆的mod和mrp操纵子进行互补实验，结果互补回As(III)氧化表型，证实这些基因编码该根癌农杆菌菌株中As(III)氧化所必需的活性。正如预期的那样，mrp突变体对NaCl和LiCl极其敏感，表明根癌农杆菌中的Mrp复合物参与跨膜Na+循环。基因表达研究（lacZ报告基因和逆转录酶PCR实验）未能显示出mrp操纵子对As(III)暴露有转录调控的证据，而mod操纵子的表达被发现受As(III)暴露上调。在每个突变体中，As(III)氧化能力的丧失导致转变为砷酸盐[As(V)]还原表型。两个突变体对As(III)的敏感性均不高于亲本菌株。
