Collaborative Research: Experimental Evolution of Peptidoglycan in the Bacterial Symbiont Vibrio Fischeri

细菌细胞壁通常由由肽聚糖(PG)组成的相互连接的网状物组成。PG是这些微生物所特有的。它对细菌的生存是必不可少的，并且在不同的细菌群中具有非常保守的结构。PG在植物和动物中不存在，鉴于其在细菌中的重要性和保守性，它是抗生素和先天免疫的极好靶标。因此，PG是细菌检测和控制策略的重点。阻断细菌形成正常PG结构的能力通常是致命的，但在极少数情况下，具有新PG的细菌是自然进化的。该项目使用与动物相关的模型细菌费氏弧菌来探索PG在实验上被迫进化时会发生什么。这些结果将揭示肽聚糖的自然进化，揭示其在细菌中的功能限制，以及将PG作为一种控制细菌的手段的局限性。此外，费氏弧菌是一种在夏威夷鱿鱼上定居的天然共生细菌，PG是这种共生关系中的关键信号分子。因此，它提供了研究肽聚糖结构如何影响宿主动物组织检测和响应其驻留细菌(即其微生物群)的能力的机会。这项工作对社会的更广泛影响包括跨学科的研究生和本科生培训，对社区K-12学校的推广，以及为一本适合课堂使用的关于细菌共生体的书做出贡献。该项目将阐明细菌细胞壁肽聚糖(PG)结构的异常变化是如何进化的，并将有助于确定PG进化的限制因素。该项目将研究易驯化的费氏弧菌如何使用迭代阻止PG生物合成的正常和替代途径的策略来进化新的PG结构(S)。产生新型PG的可行突变体将被挑选出来。这样的突变体很可能具有明显的非野生类型的生长、细胞形状、运动性等特性；然而，在培养中的几轮生长将导致进化出适应新PG的菌株。表型测试和全基因组重测序的结合将为这些进化菌株的系统类型分析奠定基础。对遗传和表型数据的综合分析将提高我们对细胞壁如何与其他细胞成分和过程协调的理解，并将导致发现细菌细胞中新的表型重要的相互连接。这些结果将使我们了解PG的自然进化，并导致对PG未来如何进化的更具预测性的理解，例如，在抗生素使用的选择压力下。鱼腥藻是鱿鱼共生模型中的共生体，共生体PG触发宿主的发育变化。这项研究将深入了解PG结构与其在这种共生中的功能之间的关系，对理解高等生物的天然免疫具有更广泛的意义。

Bacterial cell walls are usually composed of an interconnected mesh made of peptidoglycan (PG). PG is unique to these microorganisms. It is essential for bacterial survival, and has a remarkably conserved structure throughout diverse bacterial groups. PG is not found in plants and animals, and given its essentialness and conservation in bacteria, it is an excellent target for antibiotics and innate immunity. Thus, PG is a focus of strategies for bacterial detection and control. Blocking the ability of bacteria to make a normal PG structure is generally lethal, yet in rare instances bacteria with novel PG have evolved naturally. This project uses the model animal-associated bacterium Vibrio fischeri to explore what happens when PG is experimentally forced to evolve. The results will shed light on the natural evolution of peptidoglycan, on the constraints of its function in bacteria, and on the limits of targeting PG as a means of controlling bacteria. Moreover, V. fischeri is a natural symbiotic bacterium that colonizes a Hawaiian squid, and PG is a key signaling molecule in this symbiosis. Thus, it offers the opportunity to examine how peptidoglycan structure affects the ability of host animal tissue to detect and respond to its resident bacteria (i.e., its microbiome). The broader impacts of this work for society include interdisciplinary graduate and undergraduate student training, outreach to community K-12 schools, and contributions to a book on bacterial symbionts geared for use in the classroom. This project will elucidate how unusual changes to bacterial cell-wall peptidoglycan (PG) structure can evolve, and it will help define constraints on PG evolution. This project will examine how the tractable bacterium Vibrio fischeri can evolve new PG structure(s) using a strategy of iteratively blocking both normal and alternative pathways to PG biosynthesis. Viable mutants generating novel PG will be selected. Such mutants are likely to have distinctly non-wild-type properties of growth, cell shape, motility, etc.; however, rounds of growth in culture will lead to evolved strains that have accommodated the new PG. A combination of phenotypic testing and whole-genome resequencing will underpin a systems-type analysis of these evolved strains. The integrated analysis of genetic and phenotypic data will improve our understanding of how the cell wall is coordinated with other cellular components and processes, and it will lead to the discovery of new phenotypically important interconnections in bacterial cells. The results will inform our understanding of the natural evolution of PG and lead to a more predictive understanding of how PG might evolve in the future, e.g. under selective pressure from antibiotic use. V. fischeri is a symbiont in a model squid symbiosis, in which symbiont PG triggers developmental changes in the host. This study will provide insight into the relationship between PG structure and its function in this symbiosis, with broader implications for understanding innate immunity in higher organisms.