Rajagopalan Ramya, Wielgoss Sébastien, Lippert Gerardo, Velicer Gregory J, Kroos Lee
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA.
Institute of Integrative Biology (IBZ), ETH Zurich, Zurich, Switzerland.
J Bacteriol. 2015 Apr;197(7):1249-62. doi: 10.1128/JB.02542-14. Epub 2015 Feb 2.
During starvation-induced development of Myxococcus xanthus, thousands of rod-shaped cells form mounds in which they differentiate into spores. The dev locus includes eight genes followed by clustered regularly interspaced short palindromic repeats (CRISPRs), comprising a CRISPR-Cas system (Cas stands for CRISPR associated) typically involved in RNA interference. Mutations in devS or devR of a lab reference strain permit mound formation but impair sporulation. We report that natural isolates of M. xanthus capable of normal development are highly polymorphic in the promoter region of the dev operon. We show that the dev promoter is predicted to be nonfunctional in most natural isolates and is dispensable for development of a laboratory reference strain. Moreover, deletion of the dev promoter or the small gene immediately downstream of it, here designated devI (development inhibitor), suppressed the sporulation defect of devS or devR mutants in the lab strain. Complementation experiments and the result of introducing a premature stop codon in devI support a model in which DevRS proteins negatively autoregulate expression of devI, whose 40-residue protein product DevI inhibits sporulation if overexpressed. DevI appears to act in a cell-autonomous manner since experiments with conditioned medium and with cell mixtures gave no indication of extracellular effects. Strikingly, we report that devI is entirely absent from most M. xanthus natural isolates and was only recently integrated into the developmental programs of some lineages. These results provide important new insights into both the evolutionary history of the dev operon and its mechanistic role in M. xanthus sporulation.
Certain mutations in the dev CRISPR-Cas (clustered regularly interspaced short palindromic repeat-associated) system of Myxococcus xanthus impair sporulation. The link between development and a CRISPR-Cas system has been a mystery. Surprisingly, DNA sequencing of natural isolates revealed that many appear to lack a functional dev promoter, yet these strains sporulate normally. Deletion of the dev promoter or the small gene downstream of it suppressed the sporulation defect of a lab strain with mutations in dev genes encoding Cas proteins. The results support a model in which the Cas proteins DevRS prevent overexpression of the small gene devI, which codes for an inhibitor of sporulation. Phylogenetic analysis of natural isolates suggests that devI and the dev promoter were only recently acquired in some lineages.
在饥饿诱导的黄色粘球菌发育过程中，数千个杆状细胞形成土堆，它们在其中分化为孢子。dev基因座包括八个基因，后面跟着成簇的规律间隔短回文重复序列（CRISPRs），构成一个CRISPR - Cas系统（Cas代表与CRISPR相关），通常参与RNA干扰。实验室参考菌株的devS或devR基因突变允许土堆形成，但会损害孢子形成。我们报告说，能够正常发育的黄色粘球菌自然分离株在dev操纵子的启动子区域高度多态。我们表明，dev启动子在大多数自然分离株中预计无功能，并且对于实验室参考菌株的发育是可有可无的。此外，删除dev启动子或其紧下游的小基因（此处命名为devI，发育抑制剂）可抑制实验室菌株中devS或devR突变体的孢子形成缺陷。互补实验以及在devI中引入提前终止密码子的结果支持了一个模型，即DevRS蛋白负向自动调节devI的表达，如果devI过度表达，其40个氨基酸的蛋白产物DevI会抑制孢子形成。DevI似乎以细胞自主的方式起作用，因为条件培养基和细胞混合物实验没有显示出细胞外效应。引人注目的是，我们报告说大多数黄色粘球菌自然分离株完全没有devI，并且它只是最近才整合到一些谱系的发育程序中。这些结果为dev操纵子的进化历史及其在黄色粘球菌孢子形成中的机制作用提供了重要的新见解。
黄色粘球菌dev CRISPR - Cas（成簇的规律间隔短回文重复序列相关）系统中的某些突变会损害孢子形成。发育与CRISPR - Cas系统之间的联系一直是个谜。令人惊讶的是，自然分离株的DNA测序显示，许多似乎缺乏功能性的dev启动子，但这些菌株仍能正常形成孢子。删除dev启动子或其下游的小基因可抑制编码Cas蛋白的dev基因突变的实验室菌株的孢子形成缺陷。结果支持了一个模型，即Cas蛋白DevRS可防止小基因devI的过度表达，devI编码一种孢子形成抑制剂。对自然分离株的系统发育分析表明，devI和dev启动子只是最近才被一些谱系获得。
