Kashyap Des R, Botero Lina M, Franck William L, Hassett Daniel J, McDermott Timothy R
Dept. of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT 59717, USA.
J Bacteriol. 2006 Feb;188(3):1081-8. doi: 10.1128/JB.188.3.1081-1088.2006.
Seminal regulatory controls of microbial arsenite [As(III)] oxidation are described in this study. Transposon mutagenesis of Agrobacterium tumefaciens identified genes essential for As(III) oxidation, including those coding for a two-component signal transduction pair. The transposon interrupted a response regulator gene (referred to as aoxR), which encodes an ntrC-like protein and is immediately downstream of a gene (aoxS) encoding a protein with primary structural features found in sensor histidine kinases. The structural genes for As(III) oxidase (aoxAB), a c-type cytochrome (cytc2and molybdopterin biosynthesis (chlE) were downstream of aoxR. The mutant could not be complemented by aoxSR in trans but was complemented by a clone containing aoxS-aoxR-aoxA-aoxB-cytc2 and consistent with reverse transcriptase (RT) PCR experiments, which demonstrated these genes are cotranscribed as an operon. Expression of aoxAB was monitored by RT-PCR and found to be up-regulated by the addition of As(III) to cell cultures. Expression of aoxAB was also controlled in a fashion consistent with quorum sensing in that (i) expression of aoxAB was absent in As(III)-unexposed early-log-phase cells but was observed in As(III)-unexposed, late-log-phase cells and (ii) treating As(III)-unexposed, early-log-phase cells with ethyl acetate extracts of As(III)-unexposed, late-log-phase culture supernatants also resulted in aoxAB induction. Under inducing conditions, aoxS expression was readily observed in the wild-type strain but significantly reduced in the mutant, indicating that AoxR is autoregulatory and at least partially controls the expression of the aox operon. In summary, regulation of A. tumefaciens As(III) oxidation is complex, apparently being controlled by As(III) exposure, a two-component signal transduction system, and quorum sensing.
本研究描述了微生物亚砷酸盐[As(III)]氧化的重要调控机制。根癌土壤杆菌的转座子诱变鉴定出了As(III)氧化所必需的基因，包括编码双组分信号转导对的那些基因。转座子中断了一个响应调节基因（称为aoxR），该基因编码一种ntrC样蛋白，且紧挨着一个编码具有传感器组氨酸激酶中发现的一级结构特征的蛋白的基因（aoxS）下游。As(III)氧化酶（aoxAB）、一种c型细胞色素（cytc2）和钼蝶呤生物合成（chlE）的结构基因位于aoxR的下游。该突变体不能被反式的aoxSR互补，但能被包含aoxS - aoxR - aoxA - aoxB - cytc2的克隆互补，这与逆转录酶（RT）PCR实验一致，该实验表明这些基因作为一个操纵子共同转录。通过RT - PCR监测aoxAB的表达，发现向细胞培养物中添加As(III)可使其上调。aoxAB的表达也以与群体感应一致的方式受到控制，即：(i)在未接触As(III)的对数早期细胞中aoxAB不表达，但在未接触As(III)的对数后期细胞中可观察到表达；(ii)用未接触As(III)的对数后期培养上清液的乙酸乙酯提取物处理未接触As(III)的对数早期细胞也会导致aoxAB的诱导。在诱导条件下，野生型菌株中很容易观察到aoxS的表达，但在突变体中显著降低，表明AoxR是自我调节的，并且至少部分控制aox操纵子的表达。总之，根癌土壤杆菌As(III)氧化的调控是复杂的，显然受As(III)暴露、双组分信号转导系统和群体感应的控制。
