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

细菌细胞壁通常由肽聚糖（PG）组成的相互连接的网状结构组成。PG是这些微生物所特有的。它对细菌的生存至关重要，并且在不同的细菌群中具有非常保守的结构。PG不存在于植物和动物中，鉴于其在细菌中的重要性和保存性，它是抗生素和先天免疫的一个很好的靶点。因此，PG是细菌检测和控制策略的重点。阻断细菌生成正常PG结构的能力通常是致命的，但在极少数情况下，具有新型PG的细菌已经自然进化。该项目使用模型动物相关细菌费氏弧菌来探索PG在实验中被迫进化时会发生什么。该结果将揭示肽聚糖的自然进化，其在细菌中的功能限制，以及靶向PG作为控制细菌手段的局限性。此外，V. fischeri是一种天然的共生细菌，在夏威夷鱿鱼中定植，PG是这种共生关系中的关键信号分子。因此，它提供了一个机会来研究肽聚糖结构如何影响宿主动物组织检测其常驻细菌（即微生物组）和对其作出反应的能力。这项工作对社会的广泛影响包括跨学科的研究生和本科生培训，社区K-12学校的推广，以及为一本针对课堂使用的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进化的限制条件。该项目将研究易处理的细菌费氏弧菌如何通过迭代阻断PG生物合成的正常和替代途径来进化新的PG结构。将选择产生新型PG的可行突变体。这些突变体可能在生长、细胞形状、运动性等方面具有明显的非野生型特性；然而，在培养物中的多轮生长将导致进化菌株适应新的PG。表型测试和全基因组重测序的结合将支持这些进化菌株的系统型分析。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重要表型相互联系。这些结果将有助于我们了解PG的自然进化，并对PG在未来的进化方式（例如在抗生素使用的选择性压力下）有更预测性的理解。V. fischeri是一种模式乌贼共生中的共生体，共生体PG触发宿主的发育变化。这项研究将为这种共生关系中PG结构及其功能之间的关系提供深入的见解，对理解高等生物的先天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.