Wisconsin Energy Institute, Great Lakes Bioenergy Research Center, University of Wisconsin-Madison , Madison, Wisconsin, USA.
Laboratory of Genetics, University of Wisconsin-Madison , Madison, Wisconsin, USA.
mBio. 2023 Aug 31;14(4):e0063123. doi: 10.1128/mbio.00631-23. Epub 2023 Jun 7.
Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling. IMPORTANCE By coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.
细胞的伸长和分裂是细菌生命周期的重要方面，对于生存和复制必须进行协调。这些过程的调节失常的影响尚不清楚，因为这些系统通常不易进行传统的遗传操作。最近，我们报道了革兰氏阴性菌中的 CenKR 双组分系统 (TCS)，该系统在 α-变形菌中具有遗传可操作性、广泛保守，并且直接调节细胞伸长和分裂的关键成分的表达，包括编码 Tol-Pal 复合物亚基的基因。在这项工作中，我们表明过表达 导致细胞丝状化和链状化。使用低温电子显微镜 (cryo-EM) 和低温电子断层扫描 (cryo-ET)，我们生成了野生型细胞和 过表达菌株的细胞 envelope 和分裂隔膜的高分辨率二维 (2D) 图像和三维 (3D) 体积，发现这些形态变化源于外膜 (OM) 和肽聚糖 (PG) 收缩的缺陷。通过监测 Pal 的定位、PG 生物合成以及细菌细胞骨架蛋白 MreB 和 FtsZ，我们开发了一个模型，说明 CenKR 活性增加如何导致细胞伸长和分裂的变化。该模型预测，增加 CenKR 活性会降低 Pal 的迁移率，延迟 OM 收缩，并最终破坏 MreB 和 FtsZ 的中隔定位，并干扰 PG 合成和重塑的空间调节。重要性通过协调细胞的伸长和分裂，细菌保持其形状，支持关键的 envelope 功能，并协调分裂。在一些研究充分的革兰氏阴性菌中，调节和组装系统已被牵连到这些过程中。然而，我们缺乏关于这些过程及其在细菌系统发育中的保守性的信息。在 和其他 α-变形菌中，CenKR 是一种必需的双组分系统 (TCS)，它调节已知或预测在细胞 envelope 生物合成、伸长和/或分裂中发挥作用的基因的表达。在这里，我们利用 CenKR 的独特特征来了解增加其活性如何影响细胞伸长/分裂，并使用抗生素来确定调节这个 TCS 的活性如何导致细胞形态的变化。我们的研究结果为 CenKR 活性如何控制细菌 envelope 的结构和功能、细胞伸长和分裂机械的定位以及在健康、宿主-微生物相互作用和生物技术中具有重要意义的生物体中的细胞过程提供了新的见解。
