In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σ^B). In C. difficile, σ^B has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σ^B antibody to demonstrate time-limited overproduction of σ^B in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σ^B gene expression. Inducible σ^B overproduction uncouples σ^B expression from its native regulatory network and allows for the refinement of the previously proposed σ^B regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σ^B was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σ^B-dependent gene activation.

中文翻译：

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重新定义艰难梭菌 σB 调节子：σB 激活参与解毒自由基的基因，这些自由基可能因暴露于抗微生物剂和过氧化氢而产生。

在许多革兰氏阳性细菌中，一般的应激反应在转录水平上由替代 sigma 因子 sigma B (σ ^B )调节。在艰难梭菌中，σ ^B参与保护免受压力源，如活性氧 (ROS) 和抗菌化合物。在这里，我们使用了一种抗 σ ^B抗体来证明 σ ^B在艰难梭菌中的时间限制过度产生，尽管它在较高的细胞浓度下具有毒性。这种毒性最终导致用于脱水四环素诱导的 σ ^B基因表达的质粒丢失。诱导型 σ ^B生产过剩解除 σ ^B表达来自其本地调节网络，并允许改进先前提出的 σ ^B调节子。发现至少 32% 的调节子由参与反应自由基的基因组成。直接基因激活由艰难梭菌σ^乙通过证实在体外的特异性靶基因（转录的径流cd0350，cd3614，cd3605，和cd2963）。最后，我们证明了不同的抗菌剂和过氧化氢以依赖于该西格玛因子的方式诱导这些基因，使用基于板的荧光素酶报告基因测定。总之，我们的工作表明，对抗菌素的致命暴露可能导致有毒自由基的形成，从而导致 σ ^B依赖性基因激活。