Department of Biological Sciences, Myongji University, Yongin, Gyeonggido, Republic of Korea.
The Natural Science Research Institute, Myongji University, Yongin, Gyeonggido, Republic of Korea.
Microbiol Spectr. 2023 Jun 15;11(3):e0001423. doi: 10.1128/spectrum.00014-23. Epub 2023 Apr 26.
Peptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are tightly regulated under harsh environmental stresses, few related mechanisms have been investigated. In this study, we aimed to investigate the coordinated and distinct roles of the PG dd-carboxypeptidases (DD-CPases) DacC and DacA in cell growth under alkaline and salt stresses and shape maintenance in Escherichia coli. We found that DacC is an alkaline DD-CPase, the enzyme activity and protein stability of which are significantly enhanced under alkaline stress. Both DacC and DacA were required for bacterial growth under alkaline stress, whereas only DacA was required for growth under salt stress. Under normal growth conditions, only DacA was necessary for cell shape maintenance, while under alkaline stress conditions, both DacA and DacC were necessary for cell shape maintenance, but their roles were distinct. Notably, all of these roles of DacC and DacA were independent of ld-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and the outer membrane lipoprotein Lpp. Instead, DacC and DacA interacted with penicillin-binding proteins (PBPs)-dd-transpeptidases-mostly in a C-terminal domain-dependent manner, and these interactions were necessary for most of their roles. Collectively, our results demonstrate the coordinated and distinct novel roles of DD-CPases in bacterial growth and shape maintenance under stress conditions and provide novel insights into the cellular functions of DD-CPases associated with PBPs. Most bacteria have a peptidoglycan architecture for cell shape maintenance and protection against osmotic challenges. Peptidoglycan dd-carboxypeptidases control the amount of pentapeptide substrates, which are used in the formation of 4-3 cross-links by the peptidoglycan synthetic dd-transpeptidases, penicillin-binding proteins (PBPs). Seven dd-carboxypeptidases exist in Escherichia coli, but the physiological significance of their redundancy and their roles in peptidoglycan synthesis are poorly understood. Here, we showed that DacC is an alkaline dd-carboxypeptidase for which both protein stability and enzyme activity are significantly enhanced at high pH. Strikingly, dd-carboxypeptidases DacC and DacA physically interacted with PBPs, and these interactions were necessary for cell shape maintenance as well as growth under alkaline and salt stresses. Thus, cooperation between dd-carboxypeptidases and PBPs may allow E. coli to overcome various stresses and to maintain cell shape.
肽聚糖（PG）是维持细菌形状和适应渗透压的关键结构，它是细菌的必需架构。尽管在恶劣的环境压力下，PG 的合成和修饰受到严格调控，但相关机制研究甚少。在这项研究中，我们旨在研究 PG 双羧肽酶（DD-CPases）DacC 和 DacA 在大肠杆菌中碱性和盐胁迫下细胞生长和形状维持中的协调和独特作用。我们发现 DacC 是一种碱性 DD-CPase，其酶活性和蛋白稳定性在碱性胁迫下显著增强。DacC 和 DacA 都需要在碱性胁迫下生长，而只有 DacA 需要在盐胁迫下生长。在正常生长条件下，只有 DacA 对细胞形状维持是必需的，而在碱性胁迫条件下，DacA 和 DacC 都对细胞形状维持是必需的，但它们的作用是不同的。值得注意的是，DacC 和 DacA 的所有这些作用都不依赖于转肽酶，转肽酶是 PG 3-3 交联和 PG 与外膜脂蛋白 Lpp 之间形成共价键所必需的。相反，DacC 和 DacA 与青霉素结合蛋白（PBPs）-转肽酶相互作用，主要是通过 C 端结构域依赖的方式，这些相互作用是它们大多数作用所必需的。总之，我们的研究结果表明，DD-CPases 在应激条件下的细菌生长和形状维持中具有协调和独特的新作用，并为与 PBPs 相关的 DD-CPases 的细胞功能提供了新的见解。大多数细菌都有肽聚糖结构来维持细胞形状和抵抗渗透压的挑战。肽聚糖双羧肽酶控制五肽底物的数量，这些底物被肽聚糖合成的双转肽酶青霉素结合蛋白（PBPs）用于形成 4-3 交联。在大肠杆菌中存在七种双羧肽酶，但它们冗余的生理意义及其在肽聚糖合成中的作用知之甚少。在这里，我们表明 DacC 是一种碱性双羧肽酶，其蛋白稳定性和酶活性在高 pH 值下显著增强。值得注意的是，双羧肽酶 DacC 和 DacA 与 PBPs 物理相互作用，这些相互作用对于细胞形状的维持以及在碱性和盐胁迫下的生长都是必需的。因此，双羧肽酶与 PBPs 之间的合作可能使大肠杆菌能够克服各种应激并维持细胞形状。
