Transcriptional analysis of the promoter region of the Pseudomonas putida branched-chain keto acid dehydrogenase operon
Transcriptional analysis of the promoter region of the Pseudomonas putida branched-chain keto acid dehydrogenase operon
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DOI：
10.1128/jb.172.10.5655-5663.1990
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发表时间：
1990-10
影响因子：
3.2
通讯作者：
K. T. Madhusudhan;G. Huang;G. Burns;J. R. Sokatch
中科院分区：
文献类型：
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作者：
K. T. Madhusudhan;G. Huang;G. Burns;J. R. Sokatch
支链酮酸脱氢酶是恶臭假单胞菌在含有支链氨基酸的基本培养基中生长时产生的多酶复合物。克隆并测序了一个1.87 kb的DNA片段，其中含有0.24 kb的E1 α结构基因和1.6 kb的上游DNA。bkd操纵子的第一个基因bkdA1上游的非编码DNA有854个碱基对（bp），转录和翻译起始之间有592个碱基对。非编码区的G + C含量为56.7%，而操纵子的所有结构基因的G + C含量为65.2%。在与bkd操纵子相反的链上发现了从碱基774开始的部分开放阅读框。当bkd启动子克隆到启动子探针载体pKT 240中时，在恶臭假单胞菌中获得了链霉素抗性，而在两个方向上的启动子的大肠杆菌中没有获得链霉素抗性，这表明bkd启动子是双向的或者在该区域中存在两个启动子。一系列的有序删除两侧的建议的转录开始的网站显示，几乎700 bp的翻译开始上游需要表达。在含有具有完整bkd启动子的构建体的恶臭假单胞菌KT 2440的rpoN突变体中也获得了链霉素抗性，表明bkd操纵子不需要rpoN σ因子来表达。在pKT 240中含有bkd启动子、bkdA1和bkdA2的另一构建体用于转化恶臭假单胞菌JS 113，其是不能产生支链酮酸脱氢酶的E1亚基的突变体。在这种情况下，获得了bkd操纵子的非常高的诱导型表达。
Branched-chain keto acid dehydrogenase is a multienzyme complex produced by Pseudomonas putida when it is grown in a minimal medium containing branched-chain amino acids. A 1.87-kilobase (kb) DNA fragment was cloned and sequenced which contained 0.24 kb of the E1 alpha structural gene and 1.6 kb of upstream DNA. There were 854 base pairs (bp) of noncoding DNA upstream of bkdA1, the first gene of the bkd operon, and 592 bp between the transcriptional and translational starts. The G + C content of the noncoding region was 56.7% compared with 65.2% for all the structural genes of the operon. A partial open reading frame was found on the strand opposite that of the bkd operon beginning at base 774. When the bkd promoter was cloned into the promoter probe vector pKT240, streptomycin resistance was obtained in P. putida but not Escherichia coli with the promoter in both orientations, which indicates either that the bkd promoter is bidirectional or that there are two promoters in this region. A series of ordered deletions on both sides of the proposed site of the start of transcription revealed that almost 700 bp upstream of the start of translation were required for expression. Streptomycin resistance was also obtained in an rpoN mutant of P. putida KT2440 containing constructs with the intact bkd promoter, indicating that the bkd operon does not require the rpoN sigma factor for expression. Another construct containing the bkd promoter, bkdA1, and bkdA2 in pKT240 was used to transform P. putida JS113, a mutant which was unable to produce the E1 subunits of the branched-chain keto acid dehydrogenase. In this case, very high inducible expression of the bkd operon was obtained.