Transcriptome analysis was used to investigate the global stress response of the gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ alpha-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon, which responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example, citM, ylxF, yloA, ykoJ, and several genes of the flgB operon. However, high-affinity CssR binding was observed only for htrA, htrB, and, possibly, citM. In addition, the DNA macroarray approach revealed that several genes of the sporulation pathway are downregulated by AmyQ overexpression and that a group of motility-specific (sigmaD-dependent) transcripts were clearly upregulated. Subsequent flow-cytometric analyses demonstrate that, upon overproduction of AmyQ as well as of a nonsecretable variant of the alpha-amylase, the process of sporulation is severely inhibited. Similar experiments were performed to investigate the expression levels of the hag promoter, a well-established reporter for sigmaD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of alpha-amylase overproduction.

译文

转录组分析用于研究由解淀粉芽孢杆菌分泌充分的AmyQ α-淀粉酶过量产生引起的革兰氏阳性细菌枯草芽孢杆菌的整体应激反应。当枯草芽孢杆菌的蛋白质分泌最佳时,在指数生长结束时和固定相进行对照和过量生产菌株的分析。表达增加的基因包括htrA和htrB,它们是CssRS调节子的一部分,它们对高水平的蛋白质分泌和热应激做出反应。在相同的分泌胁迫条件下,与野生型相比,对cssS突变体的转录组谱进行分析,揭示了几个以CssRS依赖性方式改变转录的基因,例如citM,ylxF,yloA,ykoJ和flgB操纵子的几个基因。然而,仅在htrA,htrB和可能的citM中观察到高亲和力的CssR结合。此外,DNA macroarray方法揭示了孢子形成途径的几个基因被AmyQ过表达下调,并且一组运动性特异性 (sigmaD依赖性) 转录本被明显上调。随后的流式细胞仪分析表明,在过量生产AmyQ以及不可分泌的 α-淀粉酶变体后,孢子形成过程受到严重抑制。进行了类似的实验以研究hag启动子的表达水平,hag启动子是sigmaD依赖性基因表达的公认报告基因。这种方法证实了基于我们的DNA宏观阵列分析的观察结果,并使我们得出结论,在所有 α-淀粉酶过度生产的条件下,与运动有关的几个基因的表达水平都保持在高水平。

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