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

研究目的几乎所有的细菌都被网状肽聚糖细胞壁所包围，这是它们生存所必需的。由于细胞的高内部渗透压（膨压），细胞壁结构的缺陷导致细菌通过细胞溶解而死亡。细菌如何调节细胞壁的合成，以建立一个强大的，精确的形状和结构的细胞壁仍然是一个主要的难题桥梁物理学和细菌学。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。这是通过一种称为延长体的基本多蛋白质合成机制实现的，该机制将聚糖链插入细胞周围，从而延长和加强细胞壁，并使细胞呈杆状。新的延长酶体合成的圆周聚糖链的长度可能对关键细胞壁性质具有实质性影响，包括在抗生素处理或改变环境条件时对裂解的脆弱性。然而，细胞如何调节环聚糖链的长度是未知的。这一建议集中在两个目前的知识差距：杆状细菌如何调节环聚糖链的长度，以及这如何影响细菌细胞壁的性质和细胞健身？我们将通过测试两个中心假设来解决这些问题：（1）分子马达拔河，其中多个合成复合物将单个延长体拉向相反的方向，是延长体持续合成能力的关键调节因子，即延长体合成事件的长度，这反过来又决定了新的圆周聚糖链的长度。(2)在杆状细菌中，伸长体的持续合成能力和相关的聚糖链长度是决定细胞壁材料特性和细胞适应性的关键因素。我们将在关键的革兰氏阳性和阴性模式生物枯草芽孢杆菌和大肠杆菌中检验这些假设。时间表-在一个成功的BBSRC DTP博士研究生在co-PI的监督下，我们开发了一种单分子跟踪方法，使我们能够首次准确地确定延长体的持续合成能力。- 我们找到了有力的证据证明B.枯草杆菌延长体的持续合成能力受合酶拔河的调节，并且延长体的持续合成能力对细胞形状具有实质性的影响。- 这使得我们独特地处于确定（i）B中延长体拔河的分子机制的位置。（ii）拔河是否是革兰氏阳性和阴性杆状模型细菌B中活跃的保守的细胞壁调节机制。subtilis和E.大肠杆菌，以及（iii）它如何影响细胞壁的性质和细胞健身的两种生物体。价值为moneyThis项目利用既定的专业仪器：一个独特的霍尔顿实验室定制单分子显微镜，BBSRC 19 ALERT资助的显微镜与集成微流体，小规模的细菌发酵设置，和广泛的细胞壁生化分析仪器。因此，该项目不需要对设备进行投资，但需要进行持续的维护。此外，我们拥有大量相关且易于构建的细菌菌株，这些菌株来自正在进行的BBSRC DTP博士学位和响应模式赠款以及来自所有PI的高度相关的跨学科专业知识。作为一个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UKRI.OUTCOMES-Fundamental知识增益：该提案将大大提高我们的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中的潜在应用：在WP 4中进行的试点研究将评估我们在生物医学（以确定新的协同抗生素组合）和生物技术（以减少异源蛋白生产中使用的发酵条件下的细胞裂解）中的基础研究的效用。

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).