Regulation of the carnitine pathway in Escherichia coli:: Investigation of the cai-fix divergent promoter region
Regulation of the carnitine pathway in Escherichia coli:: Investigation of the cai-fix divergent promoter region
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DOI：
10.1128/jb.180.10.2599-2608.1998
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发表时间：
1998-05-01
影响因子：
3.2
通讯作者：
Mandrand-Berthelot, MA
中科院分区：
文献类型：
--
作者：
Buchet, A;Eichler, K;Mandrand-Berthelot, MA
厌氧性肉碱代谢需要发散的结构操纵子caitabcde和fixabcx。转录单镜LACZ融合研究表明，在存在肉碱的存在下，两个操纵子在厌氧生长过程中均均可表达，对常见的环境刺激反应（例如葡萄糖和硝酸盐），并且由同一通用调节剂（CRP）（CRP和FNR）阳性地调节，以及H -ns，介导肉碱信号的CAIF特异性调节蛋白过量生产恢复了FNR中的诱导突变体，对应于其作为主要靶标的远无生物病的作用。成绩单分析确定了两个不同的转录起点，启动了289 bp。 DNASE I足迹揭示了三个具有不同亲和力的地点，以结合该调节区域内的CRP-CRP复合物。定点诱变实验表明，以前报道的以CAI转录起始位点为中心的完美CRP图案1在唯一CAI激活中起着直接作用。相反，位于固定启动子的-69.5的CRP位点2中的突变仅导致固定表达降低三倍。因此，位于固定的-126.5处的第三个CRP位点的作用可能是为了加强位点2的作用，通过删除分析，发现了固定mRNA启动位点的关键50 bp顺式作用序列与固定mRNA启动位点重叠，以删除分析， CAI转录是必需的。该区域被认为与CAIF调节剂介导的信号的转导有关。
The divergent structural operons caiTABCDE and fixABCX of Escherichia coli are required for anaerobic carnitine metabolism. Transcriptional monocopy lacZ fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS, Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in an fnr mutant, corresponding to its role as the primary target far anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at -41.5 of the cai transcriptional start site, plays a direct role in the sole cai activation. In contrast, mutation in CRP site 2, positioned at -69.5 of the fix promoter, caused only a threefold reduction in fix expression. Thus, the role of the third CRP site, located at -126.5 of fix, might be to reinforce the action of site 2, A critical 50-bp cis-acting sequence overlapping the fix mRNA start site was found, by deletion analysis, to be necessary for cai transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.