Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA.
Department of Biology, Indiana University, Bloomington, Indiana, USA.
J Bacteriol. 2024 Jul 25;206(7):e0041323. doi: 10.1128/jb.00413-23. Epub 2024 Jun 14.
Many bacteria build elaborate molecular machines to import DNA via natural competence, yet this activity is often not identified until strains have been handled and domesticated in laboratory settings. For example, one of the best studied Gram-positive model organisms, has a poorly transformable ancestor. Transformation in the ancestral strain is inhibited by a transmembrane peptide, ComI, which is encoded on an extrachromosomal plasmid. Although ComI was shown to be necessary and sufficient to inhibit transformation when produced at high levels under an inducible promoter, the mechanism by which ComI inhibits transformation is unknown. Here, we examine the native regulation and mechanism of transformation inhibition by ComI. We find that under native regulation, ComI expression is restricted in the absence of the plasmid. In the presence of the plasmid, we find that ComI is expressed at higher levels in cells that are differentiating into a competent state. The subcellular localization of ComI, however, does not depend on any other competence proteins, and permeabilization activity is concentration-dependent. Time-lapse microscopy reveals that competent cells producing ComI are first permeabilized and then die. Based on these observations, we propose a new model for the mechanism of ComI in which response to competence activation leads to selective elimination of the competent subpopulation.
Natural transformation mechanisms have been studied across several bacterial systems, but few examples of inhibition exist. This work investigates the mechanism of action of a plasmid-encoded transmembrane inhibitor of natural transformation. The data reveal that the peptide can cause cell permeabilization. Permeabilization is synergistic with entry of into the "competent" state, such that cells with the ability to be transformed are preferentially killed. These findings reveal a self-preservation mechanism coupled to the physiological state of the cells that ensures that the population can maintain an unaltered plasmid and its predicted prophage.
许多细菌构建精心设计的分子机器，通过自然感受态来导入 DNA，但这种活性通常只有在菌株在实验室环境中经过处理和驯化后才会被识别。例如，一种研究得最好的革兰氏阳性模式生物，有一个转化效率很低的祖先。在祖先菌株中，转化受到跨膜肽 ComI 的抑制，ComI 编码在一个染色体外质粒上。尽管当 ComI 在诱导型启动子下高水平产生时，已显示出 ComI 是抑制转化所必需且充分的，但 ComI 抑制转化的机制尚不清楚。在这里，我们研究了 ComI 对天然转化的调控和抑制机制。我们发现，在天然调节下，当不存在质粒时，ComI 的表达受到限制。在存在质粒的情况下，我们发现 ComI 在分化为感受态的细胞中表达水平更高。然而，ComI 的亚细胞定位并不依赖于任何其他感受态蛋白，并且通透活性是浓度依赖性的。延时显微镜观察显示，产生 ComI 的感受态细胞首先被通透，然后死亡。基于这些观察结果，我们提出了一个新的 ComI 作用机制模型，该模型认为对感受态激活的反应导致对感受态亚群的选择性消除。
自然转化机制已在几个细菌系统中进行了研究，但抑制作用的例子很少。这项工作研究了一种质粒编码的跨膜天然转化抑制剂的作用机制。数据显示该肽可引起细胞通透。通透与进入“感受态”状态的协同作用，使得具有转化能力的细胞优先死亡。这些发现揭示了一种与细胞生理状态相关的自我保护机制，确保了群体能够维持未改变的质粒及其预测的噬菌体。
