Use of an EZ-Tn5-based random mutagenesis system to identify a novel toxin regulatory locus in Clostridium perfringens strain 13.
Use of an EZ-Tn5-based random mutagenesis system to identify a novel toxin regulatory locus in Clostridium perfringens strain 13.
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DOI：
10.1371/journal.pone.0006232
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发表时间：
2009-07-14
期刊：
影响因子：
3.7
通讯作者：
McClane BA
中科院分区：
文献类型：
--
作者：
Vidal JE;Chen J;Li J;McClane BA
尽管对于探索细菌发病机制和生理学有用，但当前的随机诱变系统在研究产毒素细菌产气荚膜梭菌方面受到限制。开发了一种基于 EZ-Tn5 的随机诱变方法用于产气荚膜梭菌。该诱变系统鉴定了一个控制菌株 13（产气荚膜梭菌 A 型菌株）产生毒素的新调控位点。该新基因座编码与金黄色葡萄球菌 Agr 群体感应系统的 AgrB 和 AgrD 成分同源的蛋白质以及两种假设的蛋白质，被发现可调节菌株 13 的 α 毒素和产气荚膜溶血素 O (PFO) 的早期产生。菌株 13 ΔagrB 突变体的 PFO 产生可以通过遗传互补或物理互补（即通过菌株的共培养）来恢复13 ΔagrB 突变体，具有菌株 13 或产气荚膜梭菌 C 型 CN3685 的 pfoA 突变体。在所有已测序的产气荚膜梭菌菌株（包括 B、C、D 和 E 型分离株）中都可以识别出类似的 AgrB 和 AgrD 编码位点，表明该调节位点有助于大多数产气荚膜梭菌菌株的毒素调节。在菌株 13 中，发现 agrB 和 agrD 基因在操纵子中与编码假设蛋白质的两个上游基因共转录。本研究中开发的新的基于 Tn5 的随机诱变系统比以前报道的产气荚膜梭菌随机诱变方法更有效和随机。它允许鉴定出与金黄色葡萄球菌 Agr 系统同源的新型产气荚膜梭菌毒素调节位点，并且其功能符合类 Agr 群体感应系统的预期。由于之前的研究表明α毒素和perfringolysin O是导致小鼠模型中菌株13诱导的梭菌性肌坏死的原因，因此新的agr调节位点可能对菌株13的毒力具有重要意义。
Although useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens. An EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO) by strain 13. PFO production by the strain 13 ΔagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 ΔagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins. The new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.
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影响因子：
6.7
作者：
Li J;McClane BA
通讯作者：
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影响因子：
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作者：
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