Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA.
Graduate Program in Genetics, University of Iowa, Iowa City, IA, 52242, USA.
Mol Microbiol. 2018 Nov;110(4):533-549. doi: 10.1111/mmi.14107. Epub 2018 Oct 14.
Clostridioides (formerly Clostridium) difficile produces two major toxins, TcdA and TcdB, upon entry into stationary phase. Transcription of tcdA and tcdB requires the specialized sigma factor, σ , which also directs RNA Polymerase to transcribe tcdR itself. We fused a gene for a red fluorescent protein to the tcdA promoter to study toxin gene expression at the level of individual C. difficile cells. Surprisingly, only a subset of cells became red fluorescent upon entry into stationary phase. Breaking the positive feedback loop that controls σ production by engineering cells to express tcdR from a tetracycline-inducible promoter resulted in uniform fluorescence across the population. Experiments with two regulators of tcdR expression, σ and CodY, revealed neither is required for bimodal toxin gene expression. However, σ biased cells toward the Toxin-ON state, while CodY biased cells toward the Toxin-OFF state. Finally, toxin gene expression was observed in sporulating cells. We conclude that (i) toxin production is regulated by a bistable switch governed by σ , which only accumulates to high enough levels to trigger toxin gene expression in a subset of cells, and (ii) toxin production and sporulation are not mutually exclusive developmental programs.
艰难梭菌（以前称为梭状芽孢杆菌）在进入静止期时会产生两种主要毒素，TcdA 和 TcdB。tcdA 和 tcdB 的转录需要专门的 σ 因子，该因子还指导 RNA 聚合酶转录 tcdR 本身。我们将红色荧光蛋白的基因融合到 tcdA 启动子上，以研究单个艰难梭菌细胞中毒素基因的表达。令人惊讶的是，只有一部分细胞在进入静止期时会变成红色荧光。通过工程细胞以四环素诱导型启动子表达 tcdR，打破控制 σ 产生的正反馈环，导致整个群体的荧光均匀。对 tcdR 表达的两个调节剂 σ 和 CodY 的实验表明，两者都不是二相毒素基因表达所必需的。然而，σ 使细胞偏向于 Toxin-ON 状态，而 CodY 使细胞偏向于 Toxin-OFF 状态。最后，在产孢细胞中观察到毒素基因的表达。我们得出结论：（i）毒素的产生受到由 σ 控制的双稳态开关调节，只有在足够高的水平下，σ 才会触发一部分细胞中的毒素基因表达；（ii）毒素的产生和产孢不是相互排斥的发育程序。
