Masepohl B, Krey R, Klipp W
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Germany.
J Gen Microbiol. 1993 Nov;139(11):2667-75. doi: 10.1099/00221287-139-11-2667.
Synthetic oligonucleotides, which were designed according to amino acid sequences conserved between Rhodospirillum rubrum and Azospirillum brasilense DraT and DraG, respectively, were used to identify the corresponding genes of Rhodobacter capsulatus. Sequence analysis of a 1904 bp DNA fragment proved the existence of R. capsulatus draT and draG. These two genes were separated by 11 bp only, suggesting that R. capsulatus draT and draG were part of one transcriptional unit. In contrast to R. rubrum, A. brasilense and Azospirillum lipoferum, the R. capsulatus draTG genes were not located upstream of the structural genes of nitrogenase nifHDK but close to the dctP gene at a distance of about 1000 kb from the nifHDK genes. Deletion mutations in the draTG gene region were constructed and introduced into R. capsulatus wild-type and a nifHDK deletion strain. The resulting mutant strains were examined for post-translational regulation of the molybdenum and the alternative nitrogenase in response to ammonia and darkness. Under 'switch-off' conditions the modified (ADP-ribosylated) and the non-modified forms of component II of both the molybdenum and the alternative nitrogenase were detected in a draTG wild-type background by immunoblot analysis, whereas only the non-modified forms were present in the draTG deletion strains. Nitrogenase activity in these strains was followed by the acetylene reduction assay. In contrast to the wild-type, draTG mutants were not affected in nitrogenase activity in response to ammonia or darkness. These results demonstrated that the draTG genes are required for post-translational regulation of both the molybdenum and the heterometal-free nitrogenase in R. capsulatus.
根据红螺菌（Rhodospirillum rubrum）与巴西固氮螺菌（Azospirillum brasilense）DraT和DraG之间保守的氨基酸序列分别设计的合成寡核苷酸，用于鉴定荚膜红细菌（Rhodobacter capsulatus）的相应基因。对一个1904 bp的DNA片段进行序列分析，证实了荚膜红细菌draT和draG的存在。这两个基因仅相隔11 bp，表明荚膜红细菌draT和draG是一个转录单元的一部分。与红螺菌、巴西固氮螺菌和脂环固氮螺菌（Azospirillum lipoferum）不同，荚膜红细菌draTG基因并不位于固氮酶nifHDK结构基因的上游，而是靠近dctP基因，与nifHDK基因相距约1000 kb。构建了draTG基因区域的缺失突变体，并将其导入荚膜红细菌野生型和nifHDK缺失菌株中。对所得突变菌株进行检测，以研究钼和替代固氮酶在响应氨和黑暗时的翻译后调控。在“关闭”条件下，通过免疫印迹分析在draTG野生型背景中检测到钼和替代固氮酶的组分II的修饰（ADP-核糖基化）形式和未修饰形式，而在draTG缺失菌株中仅存在未修饰形式。通过乙炔还原测定法跟踪这些菌株中的固氮酶活性。与野生型不同，draTG突变体对氨或黑暗响应时的固氮酶活性不受影响。这些结果表明，draTG基因是荚膜红细菌中钼和无杂金属固氮酶翻译后调控所必需的。
