Defining the Bacillus subtilis σW regulon:: A comparative analysis of promoter consensus search, run-off transcription/macroarray analysis (ROMA), and transcriptional profiling approaches
Defining the Bacillus subtilis σW regulon:: A comparative analysis of promoter consensus search, run-off transcription/macroarray analysis (ROMA), and transcriptional profiling approaches
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DOI：
10.1006/jmbi.2001.5372
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发表时间：
2002-02-22
影响因子：
5.6
通讯作者：
Helmann, JD
中科院分区：
文献类型：
--
作者：
Cao, M;Kobel, PA;Helmann, JD
枯草芽孢杆菌细胞质外功能（ECF）σ因子σ（W）控制由碱休克强烈诱导的大调节子。为了确定sigma（W）的生理作用，我们试图确定sigma（W）控制下的完整基因集。以前，我们描述了一个启动子共识搜索程序，以确定西格玛（W）控制的基因。在此，我们介绍了一种新的方法来确定额外的目标启动子：径流转录，然后通过宏阵列分析（ROMA）。我们比较了所得到的目标与那些在传统的转录谱研究和使用共识搜索方法确定的列表。虽然转录谱鉴定出强烈依赖于σ（W）的基因用于体内表达，但由于其他启动子元件的掩蔽效应、与其他ECF σ因子的重叠识别或两者，一些σ（W）依赖性启动子未被检测到。总之，共有搜索、ROMA和转录谱分析方法建立了至少30个启动子位点（控制类似于60个基因）作为sigma（W）激活的直接靶点。值得注意的是，没有一种方法能识别出超过80%的相似调节子。因此，我们建议在研究中采用两种或更多种互补方法的组合，以寻求在定义细菌调节子时实现最大覆盖。我们的研究结果表明，sigma（W）控制基因，保护细胞免受损害细胞壁生物合成的试剂，但未能揭示任何连接到操纵子可能在适应碱性生长条件的功能。这与sigW突变体在碱休克中存活的能力不受影响的观察结果一致。我们得出结论，在B。枯草芽孢杆菌突然施加碱胁迫可能通过损害细胞壁生物合成机制发挥功能的能力来激活σ（W）胁迫响应。(C)2002年爱思唯尔科学有限公司
The Bacillus subtilis extracytoplasmic function (ECF) sigma factor sigma(W) controls a large regulon that is strongly induced by alkali shock. To define the physiological role of sigma(W) we have sought to identify the complete set of genes under sigma(W) control. Previously, we described a promoter consensus search procedure to identify sigma(W) controlled genes. Herein, we introduce a novel method to identify additional target promoters: run-off transcription followed by macroarray analysis (ROMA). We compare the resulting list of targets with those identified in conventional transcriptional profiling studies and using the consensus search approach. While transcriptional profiling identifies genes that are strongly dependent on sigma(W) for in vivo expression, some sigma(W)-dependent promoters are not detected due to the masking effects of other promoter elements, overlapping recognition with other ECF sigma factors, or both. Taken together, the consensus search, ROMA, and transcriptional profiling approaches establish a minimum of 30 promoter sites (controlling similar to60 genes) as direct targets for activation by sigma(W). Significantly, no single approach identifies more than similar to80% of the regulon so defined. We therefore suggest that a combination of two or more complementary approaches be employed in studies seeking to achieve maximal coverage when defining bacterial regulons. Our results indicate that sigma(W) controls genes that protect the cell against agents that impair cell wall biosynthesis but fail to reveal any connection to operons likely to function in adaptation to alkaline growth conditions. This is consistent with the observation that a sigW mutant is unaffected in its ability to survive alkali shock. We conclude that in B. subtilis sudden imposition of alkali stress activates the sigma(W) stress response, perhaps by impairing the ability of the cell wall biosynthetic machinery to function. (C) 2002 Elsevier Science Ltd.