Clark S R, Oresnik I J, Hynes M F
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Mol Gen Genet. 2001 Jan;264(5):623-33. doi: 10.1007/s004380000348.
The rpoN gene, which codes for the alternative transcription factor sigma54, was cloned and sequenced from Rhizobium leguminosarum strain VF39SM. Construction of a rpoN mutant allowed analysis of the role of RpoN as a transcriptional regulator of genes carrying lacZ reporter fusions. Analysis of a rpoN::lacZ transcriptional fusion in the rpoN background revealed that this gene was negatively autoregulated. Site-directed mutagenesis was used to demonstrate that this autoregulation was dependent on a reverse complement RpoN binding site located upstream of the rpoN gene. rpoN was shown to be required for full microaerobic expression of both copies of fixGHIS, as well as of fixNOQP, despite the absence of apparent rpoN binding sites upstream of fixG. Moreover, rpoN was found to be required for full microaerobic expression of fnrN, which in turn is absolutely required for microaerobic induction of fixGHIS. This suggests that the reduced fixG::lacZ expression seen in the rpoN background is due to the dependence of fnrN expression on RpoN.
编码替代转录因子sigma54的rpoN基因，是从豆科根瘤菌VF39SM菌株中克隆并测序的。构建rpoN突变体有助于分析RpoN作为携带lacZ报告基因融合体的基因转录调节因子的作用。对rpoN背景下的rpoN::lacZ转录融合体进行分析发现，该基因存在负向自调控。利用定点诱变证明，这种自调控依赖于位于rpoN基因上游的反向互补RpoN结合位点。结果表明，尽管在fixG上游没有明显的rpoN结合位点，但rpoN对于fixGHIS两个拷贝以及fixNOQP的完全微氧表达是必需的。此外，发现rpoN对于fnrN的完全微氧表达是必需的，而fnrN又是fixGHIS微氧诱导所绝对必需的。这表明在rpoN背景下观察到的fixG::lacZ表达降低是由于fnrN表达对RpoN的依赖性。
