Genetic analysis of beneficial bacterial colonization

项目总结 我的实验室的目标是描述细菌和它们之间的分子通讯 动物寄主导致了特定的和可复制的殖民。为了实现这一目标，实验室研究了 鱼弧菌-鱿鱼系统，在该系统中，动物的光器官仅由一种细菌定植 物种。这个系统是有利的，因为细菌通过自然的感染途径定居，所有的 动物在三小时内被细菌接种到海水中，细菌可能会受到 对于详细的基因操作，感染的准确位置可以直接在活体动物宿主中成像，并且 对动物宿主的化学分析使详细的分子研究成为可能。关注鱿鱼是怎么回事 被特定的共生体繁殖，从而排除了数百万竞争的细菌 海水，揭示了细菌聚集和生物膜形成在促进特定宿主- 微生物相互作用。我们小组提出的问题包括：(1)共生体如何调节 有益的生物膜？生物膜为微生物提供了一个受保护的环境，在这个环境中，它们可以共同行动 并抵抗先天免疫侮辱和抗菌化合物。V.FISCHERI对共生生物膜的阐述是 进入宿主所需的，提供了在自然宿主的背景下研究这一过程的机会 殖民模式。我们过去的工作确定Bink是生物膜形成的关键负面调节因子，并且 宿主中浮游植物向生物膜的转变。在这项研究中，我们研究了Bink如何解释来自主机的信号 以及这些信息是如何传递给V.FISCHERI的。我们研究了信号转导和寻找的机制 鉴定和鉴定调节Bink活性的配体。(2)哪些新的细菌因素起关键作用？ 在殖民进程中的作用？我们已经成功地将全球遗传方法应用于 确定费氏弧菌的细菌定植因子。重点放在新的和未被研究的细菌基因 费氏弧菌-鱿鱼系统具有阐明蛋白质功能的潜力，我们鉴定了一种蛋白质 对生物膜的形成和鱿鱼的定居有很大的影响。该蛋白质被注释为推定的 RNA结合蛋白，我们将描述这种蛋白的作用和分子机制。 确定它如何影响共生生物膜的形成。(3)小分子对微生物组有何影响 专一性和殖民性？我们已经开始鉴定存在于宿主中的化合物和 与共生行为共同规范。我们将整合遗传学方法来阐明信号通路 寄主殖民的背景。费斯切里-乌贼系统的一个主要优点是能够审问 细菌在完好无损的动物宿主中的行为，以及这些项目的完成将使 了解有益微生物对动物定居的潜在机制。

PROJECT SUMMARY The objective of my laboratory is to characterize how molecular communication between bacteria and their animal hosts leads to specific and reproducible colonization. To accomplish this goal, the laboratory studies the Vibrio fischeri-squid system, in which the animal’s “light organ” is colonized exclusively by one bacterial species. This system is advantageous because bacteria colonize through the natural route of infection, all animals are colonized within three hours of bacterial inoculation into the seawater, the bacteria can be subject to detailed genetic manipulation, the precise site of infection can be imaged directly in the live animal host, and chemical analysis of the animal host enables detailed molecular investigations. Focusing on how squid are reproducibly colonized by the specific symbiont, to the exclusion of the millions of competing bacteria in seawater, has revealed key roles for bacterial aggregation and biofilm formation in promoting specific host- microbe interactions. Questions that our group is asking include: (1) How does a symbiont regulate a beneficial biofilm? Biofilms provide microbes with a protected environment in which they can act collectively and resist innate immune insults and antimicrobial compounds. V. fischeri elaboration of a symbiotic biofilm is required for entry into the host, providing an opportunity to study this process in the context of a natural host colonization model. Our past work identified BinK as a key negative regulator of biofilm formation and the planktonic-to-biofilm transition in the host. In this study, we examine how BinK interprets signals from the host and how that information is transmitted to V. fischeri. We examine mechanisms of signal transduction and seek to identify and characterize a ligand that regulates BinK activity. (2) What novel bacterial factors play critical functions in colonization processes? We have had success in applying global genetic approaches to identify bacterial colonization factors in V. fischeri. With a focus on novel and understudied bacterial genes for which the V. fischeri-squid system has the potential to elucidate protein functions, we identified a protein that has a substantial impact on biofilm formation and squid colonization. The protein is annotated as a putative RNA-binding protein, and we will characterize the molecular mechanisms by which this protein acts and determine how it impacts symbiotic biofilm formation. (3) How do small molecules influence microbiome specificity and colonization? We have begun to identify compounds that are present in the host and that are co-regulated with symbiotic behaviors. We will integrate genetic approaches to elucidate signaling pathways in the context of host colonization. A major strength of the V. fischeri-squid system is the ability to interrogate bacterial behavior in the intact animal host, and completion of these projects will enable a deeper understanding of the mechanisms underlying animal colonization by beneficial microbes.