Heinzen R A, Mo Y Y, Robertson S J, Mallavia L P
Department of Microbiology, Washington State University, Pullman 99164-4233, USA.
Gene. 1995 Mar 21;155(1):27-34. doi: 10.1016/0378-1119(94)00888-y.
We have identified and sequenced four genes that encode the protein subunits comprising the succinate dehydrogenase enzyme complex (Sdh) of the rickettsia Coxiella burnetii. The Sdh-encoding gene cluster (sdhCDAB) begins 3326 bp upstream from the citrate synthase-encoding gene (gltA) start codon and is read with opposite polarity. An open reading frame encoding the N-terminal 280 amino acids (aa) of 2-oxoglutarate dehydrogenase (SucA) begins 24 bp downstream from the stop codon of the gene specifying the iron-sulfur subunit (sdhB) of Sdh. The deduced aa sequence of Sdh subunits and the N-terminal portion of SucA revealed significant aa identity with the Esherichia coli homologues ranging from a low of 36.6% for SdhD to a high of 61.2% for SdhA and SdhB. Primer extension identified transcription start points (tsp) for sdh and sucA. The region upstream from the sdh tsp, but not the sucA tsp, displayed homology to promoter consensus sequences of E. coli. Further evidence that sucA transcription can occur independent of sdh transcription was provided by demonstrating that a TnphoA insertion disrupting sdhB had no effect on the production of SucA by an E. coli cell-extract-directed in vitro transcription/translation system. The plasmid clone pLPM60, which carries the C. burnetii sdhCDAB coding and upstream regulatory regions, rescued an E. coli sdhA mutant (MOB252), indicating functional expression of the rickettsial locus. A cell extract of MOB252 transformed with pLPM60 showed a sixfold greater level of Sdh enzyme activity over the E. coli wild type. A plasmid clone lacking the sdh upstream regulatory region did not complement nor produce sdh mRNA by dot blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
我们已鉴定并测序了四个基因，它们编码构成立克次氏体伯氏考克斯氏体琥珀酸脱氢酶复合物（Sdh）的蛋白质亚基。编码Sdh的基因簇（sdhCDAB）起始于柠檬酸合酶编码基因（gltA）起始密码子上游3326 bp处，并以相反的极性进行转录。一个编码2-氧代戊二酸脱氢酶（SucA）N端280个氨基酸（aa）的开放阅读框起始于指定Sdh铁硫亚基（sdhB）的基因终止密码子下游24 bp处。Sdh亚基和SucA N端部分的推导氨基酸序列与大肠杆菌同源物显示出显著的氨基酸同一性，范围从SdhD的低36.6%到SdhA和SdhB的高61.2%。引物延伸确定了sdh和sucA的转录起始点（tsp）。sdh tsp上游区域，但不是sucA tsp上游区域，与大肠杆菌启动子共有序列显示出同源性。通过证明破坏sdhB的TnphoA插入对大肠杆菌细胞提取物指导的体外转录/翻译系统产生SucA没有影响，进一步证明了sucA转录可以独立于sdh转录发生。携带伯氏考克斯氏体sdhCDAB编码和上游调控区域的质粒克隆pLPM60挽救了大肠杆菌sdhA突变体（MOB252），表明立克次氏体基因座的功能表达。用pLPM60转化的MOB252细胞提取物显示，Sdh酶活性水平比大肠杆菌野生型高六倍。通过斑点印迹分析，缺乏sdh上游调控区域的质粒克隆既不能互补也不产生sdh mRNA。（摘要截断于250字）
