Rajagopalan Ramya, Kroos Lee
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA.
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA
J Bacteriol. 2017 Apr 25;199(10). doi: 10.1128/JB.00788-16. Print 2017 May 15.
undergoes multicellular development when starved. Thousands of rod-shaped cells coordinate their movements and aggregate into mounds in which cells differentiate into spores. Mutations in the operon impair development. The operon encompasses a clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) system. Null mutations in , a small gene at the beginning of the operon, suppress the developmental defects caused by null mutations in the downstream and genes but failed to suppress defects caused by a small in-frame deletion in We provide evidence that the original mutant has a second-site mutation. We show that null mutants exhibit developmental defects indistinguishable from and null mutants, and a null mutation in suppresses the defects of a null mutation. The similarity of DevTRS proteins to components of the CRISPR-associated complex for antiviral defense (Cascade), together with our molecular characterization of mutants, support a model in which DevTRS form a Cascade-like subcomplex that negatively autoregulates transcript accumulation and prevents DevI overproduction that would strongly inhibit sporulation. Our results also suggest that DevI transiently inhibits sporulation when regulated normally. The mechanism of transient inhibition may involve MrpC, a key transcription factor, whose translation appears to be weakly inhibited by DevI. Finally, our characterization of a mutant indicates that very little transcript is required for sporulation, which is surprising since Exo proteins help form the polysaccharide spore coat. CRISPR-Cas systems typically function as adaptive immune systems in bacteria. The CRISPR-Cas system of has been proposed to prevent bacteriophage infection during development, but how controls sporulation has been elusive. Recent evidence supported a model in which DevR and DevS prevent overproduction of DevI, a predicted 40-residue inhibitor of sporulation. We provide genetic evidence that DevT functions together with DevR and DevS to prevent DevI overproduction. We also show that spores form about 6 h earlier in mutants lacking than in the wild type. Only a minority of natural isolates appear to have a functional promoter and , suggesting that a functional CRISPR-Cas system evolved recently in niches where delayed sporulation and/or protection from bacteriophage infection proved advantageous.
饥饿时会经历多细胞发育。数千个杆状细胞协调它们的运动并聚集形成丘，其中细胞分化为孢子。操纵子中的突变会损害发育。该操纵子包含一个成簇的规律间隔短回文重复序列相关（CRISPR-Cas）系统。操纵子起始处的一个小基因发生无效突变，可抑制下游基因和基因无效突变导致的发育缺陷，但无法抑制基因中一个小的框内缺失所引起的缺陷。我们提供证据表明原始突变体存在第二位点突变。我们表明基因无效突变体表现出与基因和基因无效突变体无法区分的发育缺陷，并且基因的无效突变可抑制基因无效突变的缺陷。DevTRS蛋白与抗病毒防御的CRISPR相关复合物（Cascade）的组分相似，以及我们对突变体的分子特征分析，支持了一个模型，即DevTRS形成类似Cascade的亚复合物，该亚复合物负向自动调节转录物积累并防止DevI过量产生，而DevI过量产生会强烈抑制孢子形成。我们的结果还表明，正常调节时DevI会短暂抑制孢子形成。短暂抑制的机制可能涉及关键转录因子MrpC，其翻译似乎受到DevI的微弱抑制。最后，我们对突变体的特征分析表明，孢子形成所需的转录物非常少，这令人惊讶，因为Exo蛋白有助于形成多糖孢子壁。CRISPR-Cas系统通常在细菌中作为适应性免疫系统发挥作用。已提出的CRISPR-Cas系统可在发育过程中防止噬菌体感染，但它如何控制孢子形成一直不清楚。最近的证据支持一个模型，即DevR和DevS可防止DevI过量产生，DevI是一种预测的40个残基的孢子形成抑制剂。我们提供遗传证据表明DevT与DevR和DevS共同作用以防止DevI过量产生。我们还表明，缺乏基因的突变体中孢子形成比野生型早约6小时。只有少数自然分离株似乎具有功能性的启动子和，这表明功能性的CRISPR-Cas系统最近在延迟孢子形成和/或免受噬菌体感染被证明有利的生态位中进化而来。
