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Evaluation and characterization of catabolite-responsive elements (cre) of Bacillus subtilis

机译:枯草芽孢杆菌分解代谢物响应元件(cre)的评估和表征

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摘要

A global mechanism of catabolite repression of the genus Bacillus comprises negative regulation exerted through the binding of the CcpA protein to the catabolite-responsive elements (cres) of the target genes. We searched for cre sequences in the Bacillus subtilis genome using a query sequence, WTGNAANCGNWNNCW (N and W stand for any base and A or T, respectively), picking out 126 putative and known cre sequences. To examine their cre function, we integrated spac promoter (Pspac)-cre-lacZ fusions into the amyE locus. Examination of catabolite repression of β-galactosidase synthesis in the integrants led us to the following conclusions: (i) lower mismatching of cre sequences to the query sequence is required for their function; (ii) although cre sequences are partially palindromic, low mismatching in the same direction as that of transcription of the target genes is more critical for their function than that in the inverse direction; and (iii) yet, a more palindromic nature of cre sequences is desirable for a better function. Furthermore, the alignment of 22 cres that function in vivo implicated a consensus sequence, WWTGNAARCGNWWWCAWW (R stands for G or A). Interestingly, in the case where cre sequences are located in the protein-coding regions of the target genes, their conserved bases are preferentially the third bases of codons where base degeneracy is allowed.
机译:芽孢杆菌属的分解代谢物阻遏的整体机制包括通过CcpA蛋白与靶基因的分解代谢物响应元件(cres)结合而施加的负调控。我们使用查询序列WTGNAANCGNWNNCW在枯草芽孢杆菌基因组中搜索cre序列(N和W分别代表任何碱基和A或T),挑选出126个推定的已知cre序列。为了检查其cre功能,我们将spac启动子(Pspac)-cre-lacZ融合体整合到amyE基因座中。对整合子中β-半乳糖苷酶合成的分解代谢物阻遏作用的研究得出以下结论:(i)cre序列与查询序列的错配率较低,这是其功能所必需的; (ii)尽管cre序列是部分回文的,但与靶基因转录方向相同的低错配对其功能比与反方向的错配更为关键; (iii)然而,cre序列具有更多回文性质对于更好的功能是理想的。此外,在体内起作用的22 cres的比对涉及一个共有序列WWTGNAARCGNWWWCAWW(R代表G或A)。有趣的是,在cre序列位于靶基因的蛋白质编码区中的情况下,其保守碱基优选是允许碱基简并的密码子的第三个碱基。

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