Identification and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli
Identification and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli
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DOI：
10.1128/jb.178.5.1248-1257.1996
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发表时间：
1996-03-01
影响因子：
3.2
通讯作者：
MandrandBerthelot, MA
中科院分区：
文献类型：
--
作者：
Eichler, K;Buchet, A;MandrandBerthelot, MA
参与肉毒碱代谢的大肠杆菌caiTABCDE和fixABCX操纵子的表达由肉毒碱和厌氧诱导。当克隆到多拷贝质粒中时，发现与caiTABCDE操纵子相邻的3'区域增加由染色体caiB基因合成的肉毒碱脱氢酶活性水平。测定了核苷酸序列，显示它含有一个393 bp的开放阅读框，命名为cai F，其转录方向与cai操纵子相反。该开放阅读框编码131个氨基酸的蛋白质，预测分子量为15，438 Da，其与数据库中可获得的蛋白质没有任何显著同源性。在体内过表达一贯导致16 kDa的蛋白质的合成。CaiF基因在厌氧条件下转录为单顺反子mRNA，与肉毒碱的存在无关。引物延伸分析将转录起始位点定位到caiF起始密码子上游的位置82。其前面是以位置-41.5为中心的环AMP受体蛋白基序。CaiF的过量产生导致在肉毒碱的存在下刺激发散的cai和fix操纵子的转录。这表明CaiF通过与肉毒碱相互作用起激活剂的作用，从而介导肉毒碱代谢的诱导。此外，CaiF可以反式补充在肉毒碱途径中受损的实验室菌株MC 4100的调节缺陷。CaiF-lacZ操纵子融合体的表达受到FNR调节剂介导的厌氧诱导和环AMP受体蛋白激活。组蛋白样蛋白H-NS和NarL（加硝酸盐）调节剂作为阻遏物。由于caiF基因受到多重控制，它似乎是肉毒碱代谢调节的关键因素。
Expression of the Escherichia coli caiTABCDE and fixABCX operons involved in carnitine metabolism is induced by both carnitine and anaerobiosis. When cloned into a multicopy plasmid, the 3' region adjacent to the caiTABCDE operon was found to increase levels of carnitine dehydratase activity synthesized from the chromosomal caiB gene. The nucleotide sequence was determined, and it was shown to contain an open reading frame of 393 bp named caiF which is transcribed in the direction opposite that of the cai operon. This open reading frame encodes a protein of 131 amino acids with a predicted molecular mass of 15,438 Da which does not have any significant homology with proteins available in data libraries. In vivo overexpression consistently led to the synthesis of a 16-kDa protein. The caiF gene was transcribed as a monocistronic mRNA under anaerobiosis independently of the presence of carnitine. Primer extension analysis located the start site of transcription to position 82 upstream of the caiF initiation codon. It was preceded by a cyclic AMP receptor protein motif centered at position -41.5. Overproduction of CaiF resulted in the stimulation of transcription of the divergent cai and fix operons in the presence of carnitine. This suggested that CaiF by interacting with carnitine plays the role of an activator, thereby mediating induction of carnitine metabolism. Moreover, CaiF could complement in trans the regulatory defect of laboratory strain MC4100 impaired in the carnitine pathway. Expression of a caiF-lacZ operon fusion was subject to FNR regulator-mediated anaerobic induction and cyclic AMP receptor protein activation. The histone-like protein H-NS and the NarL (plus nitrate) regulator acted as repressors. Because of the multiple controls to which the caiF gene is subjected, it appears to be a key element in the regulation of carnitine metabolism.