Baek Seung-Hun, Hartsock Angela, Shapleigh James P
Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA.
J Bacteriol. 2008 Jan;190(1):78-86. doi: 10.1128/JB.00792-07. Epub 2007 Nov 2.
Agrobacterium tumefaciens can grow anaerobically via denitrification. To learn more about how cells regulate production of nitrite and nitric oxide, experiments were carried out to identify proteins involved in regulating expression and activity of nitrite and nitric oxide reductase. Transcription of NnrR, required for expression of these two reductases, was found to be under control of FnrN. Insertional inactivation of the response regulator actR significantly reduced nirK expression and Nir activity but not nnrR expression. Purified ActR bound to the nirK promoter but not the nor or nnrR promoter. A putative ActR binding site was identified in the nirK promoter region using mutational analysis and an in vitro binding assay. A nirK promoter containing mutations preventing the binding of ActR showed delayed expression but eventually reached about 65% of the activity of an equivalent wild-type promoter lacZ fusion. Truncation of the nirK promoter revealed that truncation up to and within the ActR binding site reduced expression, but fragments lacking the ActR binding site and retaining the NnrR binding site showed expression as high as or higher than the full-length fragment. Additional experiments revealed that expression of paz, encoding the copper protein pseudoazurin, was highly reduced in the actR or fnrN mutants and that ActR binds to the paz promoter. Inactivation of paz reduced Nir activity by 55%. These results help explain why Nir activity is very low in the actR mutant even though a nirK promoter with mutations in the ActR binding site showed significant expression.
根癌土壤杆菌可通过反硝化作用进行厌氧生长。为了更深入了解细胞如何调节亚硝酸盐和一氧化氮的产生，开展了实验以鉴定参与调节亚硝酸盐和一氧化氮还原酶表达及活性的蛋白质。发现这两种还原酶表达所需的NnrR转录受FnrN控制。反应调节因子ActR的插入失活显著降低了nirK表达和Nir活性，但未降低nnrR表达。纯化的ActR与nirK启动子结合，但不与nor或nnrR启动子结合。使用突变分析和体外结合试验在nirK启动子区域鉴定出一个假定的ActR结合位点。含有阻止ActR结合的突变的nirK启动子显示表达延迟，但最终达到等效野生型启动子lacZ融合活性的约65%。nirK启动子的截短显示，截短至ActR结合位点及其内部会降低表达，但缺乏ActR结合位点且保留NnrR结合位点的片段显示出与全长片段一样高或更高的表达。进一步的实验表明，编码铜蛋白假天青蛋白的paz在actR或fnrN突变体中的表达大幅降低，且ActR与paz启动子结合。paz的失活使Nir活性降低了55%。这些结果有助于解释为什么在actR突变体中Nir活性非常低，尽管在ActR结合位点有突变的nirK启动子显示出显著表达。
