EVALUATING ELONGASOME TUG-OF-WAR AS A KEY REGULATOR OF BACTERIAL CELL WALL SYNTHESIS

评估延长体拔河作用作为细菌细胞壁合成的关键调节因子

• 批准号: BB/X001482/1
• 负责人: Seamus Holden
• 金额: $ 47.88万
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX001482%2F1
• 关键词: EVALUATING; ELONGASOME; TUG; WAR; KEY

PURPOSE OF RESEARCHAlmost all bacteria are surrounded by a mesh-like peptidoglycan cell wall that is essential for their survival. Due to the cell's high internal osmotic pressure (turgor), defects in cell wall structure cause bacteria to die by cell lysis. How bacteria regulate cell wall synthesis to build a strong, precisely shaped and structured cell wall remains a major puzzle bridging physics and bacteriology. Many rod-shaped bacteria, including major antibiotic resistant pathogens, grow by adding new material to the cylindrical portion of their cell wall, thus leading to cell elongation. This is achieved by an essential multi-protein synthesis machinery called the elongasome, which inserts glycan strands around the circumference of the cell thereby elongating and reinforcing the cell wall and giving cells their rod shape. It is likely that the length of new elongasome-synthesized circumferential glycan strands has substantial effect on key cell wall properties including vulnerability to lysis upon antibiotic treatment or changing environmental conditions. However, how cells regulate the length of circumferential glycan strands is unknown.This proposal focusses on two current knowledge gaps: how do rod-shaped bacteria regulate the length of circumferential glycan strands, and how does this affect bacterial cell wall properties and cell fitness?We will address these questions by testing two central hypotheses:(1) Molecular motor tug-of-war, where multiple synthesis complexes pull individual elongasomes in opposite directions, is a key regulator of elongasome processivity, ie the length of elongasome synthesis events, which, in turn, determines the length of new circumferential glycan strands.(2) Elongasome processivity and associated glycan strand length are key determinants of cell wall material properties and cell fitness in rod-shaped bacteriaWe will test these hypotheses in key Gram-positive and -negative model organisms Bacillus subtilis and Escherichia coli. TIMELINESS-During a successful BBSRC DTP PhD studentship supervised by the co-PIs, we developed a single molecule tracking method that allows us to accurately determine elongasome processivity for the first time. -We found strong evidence that B. subtilis elongasome processivity is regulated by synthase tug-of-war, and that elongasome processivity has substantial effect on cell shape. -This makes us uniquely placed to determine (i) the molecular mechanism of elongasome tug-of-war in B. subtilis, (ii) whether tug-of-war is a conserved cell wall regulatory mechanism active in Gram-positive and -negative rod-shaped model bacteria B. subtilis and E. coli, and (iii) how it affects the cell wall properties and cell fitness of both organisms.VALUE FOR MONEYThis project leverages established specialist instrumentation: a unique Holden lab custom single molecule microscope, a BBSRC 19ALERT funded microscope with integrated microfluidics, a small scale bacterial fermentation setup, and extensive apparatus for cell wall biochemical analyses. The project thus requires no investment in equipment except ongoing maintenance. Furthermore we have a large number of relevant and readily constructed bacterial strains from ongoing BBSRC DTP PhD and responsive mode grants and highly relevant interdisciplinary expertise from all PIs. As one PI has an ongoing Wellcome fellowship, first 11 months of his PI time are of no cost to UKRI.OUTCOMES-Fundamental knowledge gain: The proposal will substantially advance our understanding of bacterial envelope biophysics, bacterial molecular machines, and bacterial cell biology. -Potential applications in biomedicine and biotechnology: The pilot studies carried out in WP4 will evaluate the utility of our fundamental research in biomedicine (to identify novel synergistic antibiotic combinations) and in biotechnology (to reduce cell lysis during fermentation condition used in heterologous protein production).

研究目的：几乎所有的细菌都被网状的肽聚糖细胞壁所包围，这对细菌的生存至关重要。由于细胞内部的高渗透压（膨胀），细胞壁结构的缺陷导致细菌因细胞裂解而死亡。细菌如何调节细胞壁合成以构建坚固、精确形状和结构的细胞壁仍然是连接物理学和细菌学的主要难题。许多杆状细菌，包括主要的抗生素耐药病原体，通过向其细胞壁的圆柱形部分添加新材料来生长，从而导致细胞伸长。这是通过一种重要的多蛋白质合成机制来实现的，这种机制被称为长体，它在细胞周围插入聚糖链，从而延长和加强细胞壁，使细胞呈棒状。很可能新的细长体合成的环状聚糖链的长度对细胞壁的关键特性有实质性的影响，包括在抗生素治疗或改变环境条件下的裂解脆弱性。然而，细胞如何调节周聚糖链的长度是未知的。这个建议集中在两个目前的知识空白：棒状细菌如何调节周聚糖链的长度，以及这如何影响细菌细胞壁特性和细胞适应性？我们将通过测试两个中心假设来解决这些问题：(1)分子马达拉锯战，多个合成配合物将单个长粒体拉向相反的方向，是长粒体进程的关键调节器，即长粒体合成事件的长度，这反过来决定了新的环状聚糖链的长度。(2)在杆状细菌中，长体加工和相关的聚糖链长度是细胞壁材料特性和细胞适应性的关键决定因素。我们将在革兰氏阳性和阴性模式生物枯草芽孢杆菌和大肠杆菌中验证这些假设。及时性-在合作pi的指导下，我们成功地开发了一种单分子跟踪方法，使我们能够第一次准确地确定长粒体的加工能力。-我们发现了强有力的证据，证明枯草芽孢杆菌的长体进程是由合酶拔河调节的，并且长体进程对细胞形状有实质性的影响。这使得我们能够确定(i)枯草芽孢杆菌中长体拔河的分子机制，（ii）拔河是否是一种在革兰氏阳性和阴性杆状模型细菌枯草芽孢杆菌和大肠杆菌中活跃的保守细胞壁调节机制，以及（iii）它如何影响这两种生物的细胞壁特性和细胞适应性。该项目利用已建立的专业仪器：独特的霍尔顿实验室定制单分子显微镜，BBSRC 19ALERT资助的集成微流体显微镜，小型细菌发酵装置，以及用于细胞壁生化分析的广泛仪器。因此，除了持续的维护外，该项目不需要对设备进行投资。此外，我们从正在进行的BBSRC DTP博士和响应模式资助中获得了大量相关且易于构建的细菌菌株，以及来自所有pi的高度相关的跨学科专业知识。因为一个PI有一个正在进行的惠康奖学金，他的PI时间的前11个月是没有成本的UKRI。结果：获得基础知识：该提案将大大提高我们对细菌包膜生物物理学，细菌分子机器和细菌细胞生物学的理解。-在生物医学和生物技术方面的潜在应用：在WP4进行的试点研究将评估我们在生物医学（确定新的增效抗生素组合）和生物技术（减少异种蛋白质生产中发酵条件下的细胞裂解）方面的基础研究的效用。

项目成果

Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis

分子运动拔河比赛调节枯草芽孢杆菌细胞壁合成动力学

• DOI: 10.1101/2023.05.10.540107
• 发表时间: 2023
• 作者: Middlemiss S