Dissecting the role of YceG in E. coli cell wall biogenesis

描述（由申请人提供）：大肠杆菌的细胞包膜是一个由三层组成的复杂结构-内膜和外膜，中间夹着一层薄薄的肽聚糖（PG）。细菌必须完成扩展、重塑和降解PG的精细任务才能生长和分裂，同时保持这一重要的承受应力结构的完整性(1)。对细胞壁生长过程的任何扰动都可能导致细胞壁的灾难性破坏，并最终导致细胞裂解。因此，许多临床上有价值的抗生素，包括青霉素和其他ss-内酰胺类，通过抑制PG生物发生所需的蛋白质来靶向这种独特的分子。尽管在过去的几十年里我们对细胞壁有了相当多的了解，但其生物发生的许多方面仍有待阐明。大肠杆菌中大约三分之一的蛋白质与细胞包膜有关，但其中一半的蛋白质功能未知，这一事实突出了这一点(2)。为了进一步扩大我们对细胞壁的认识，并充分探索未来抗生素的潜在靶点，将重点放在与细胞包膜相关的未表征基因的进一步研究上是至关重要的。通过对大肠杆菌中新的细胞形态发生因子的基因筛选，我发现功能未知的基因yceG可能是一种潜在的细胞壁重塑因子。我的初步生化实验表明，YceG确实是一种新的细胞壁水解酶。生物信息学分析表明，yceG在许多其他革兰氏阴性和革兰氏阳性细菌中具有进化保守性。本实验旨在1)确定YceG在体外的酶促特异性，2)确定YceG在体内的生理作用，3)探索YceG的调控机制。了解这种独特的细胞壁水解酶的功能和调控将为发现细胞壁组装过程中的新漏洞提供必要的见解，这些漏洞可以通过急需的新型抗菌疗法来靶向。

DESCRIPTION (provided by applicant): The cell envelope of E. coli is a complex structure composed of three layers - an inner and outer membrane with a thin layer of peptidoglycan (PG) sandwiched between. Bacteria must carry out the delicate task of expanding, remodeling, and degrading PG to grow and divide, all while maintaining the integrity of this essential stress-bearing structure (1). Any perturbations to the process of cell wall growth can lead to a catastrophic breach in the cell wall and ultimately cause cell lysis. As such, many clinically valuable antibiotics, including penicillin and other ss-lactams, target this unique molecule by inhibiting proteins required for PG biogenesis. Despite the considerable knowledge we have acquired about the cell wall over the past several decades, many aspects of its biogenesis remain to be elucidated. This is highlighted by the fact that approximately one- third of proteins in E. coli are associated with the cell envelope, but half of those proteins have unknown functions (2). To further expand our knowledge of the cell wall and fully explore the potential array of targets for future antibiotics, it is of utmost importance to focus additional studies on he uncharacterized genes connected to the cell envelope. Using a genetic screen for the identification of new cell morphogenesis factors in E. coli, I implicated the gene of unknown function, yceG, as a potential cell wall remodeling factor. My preliminary biochemical experiments show that YceG is indeed a novel cell wall hydrolase. Bioinformatic analysis indicates that yceG is evolutionarily conserved across many other gram-negative and gram-positive bacteria. The proposed experiments aim to 1) determine the enzymatic specificity of YceG in vitro, 2) to define the physiological role of YceG in vivo, and 3) to explore how YceG is regulated. Knowledge of the function and regulation of this unique cell wall hydrolase will provide insight necessary to uncover new vulnerabilities in the cell wall assembly process that can be targeted by much needed novel antibacterial therapies.