Mechanisms for sensing and responding to interbacterial antagonism

项目概要 越来越明显的是，种间对抗是细菌界生命所固有的。并且 然而，尽管我们对细菌在与其同胞发生冲突时所采用的抗菌机制的理解 最近，我们对细菌感知和响应方式的了解呈指数级增长。 对抗性威胁仍然有限。在这个提案中，我们测试了细菌对存在做出反应的假设 通过激活多方面的防御程序来对抗拮抗细菌竞争者。这个 假设源于我们的发现，即铜绿假单胞菌激活广泛的转录后 响应细菌间拮抗作用的调节程序。该途径，我们称之为 PARA （铜绿假单胞菌对拮抗作用的反应），当铜绿假单胞菌细胞亚群时触发 由于拮抗攻击而导致裂解。检测细胞中尚未鉴定的分子 裂解物导致小RNA介导的Gac/Rsm全局转录后调节的激活 程序。这种反应的激活对于铜绿假单胞菌在攻击期间的生存至关重要，因为突变体无法 在与拮抗生物的竞争过程中，安装反应会遭受严重的适应性缺陷。最后，我们 证明防御反应需要多种同时作用的机制，包括 未知功能的途径。在本提案的目标 1 中，我们将描述这样一条途径，我们将其 命名为 ARC1（拮抗反应复合物 1）。我们的初步数据表明 ARC1 是一种大膜 相关蛋白复合物，为铜绿假单胞菌提供保护，抵抗毒素介导的拮抗作用 由竞争物种的 VI 型分泌系统提供。我们对 ARC1 的研究将阐明其范围 它提供保护的威胁，并提供对其防御功能的机械洞察。在 目标 2，我们将寻求互补的遗传和生化方法来表征信号 存在于铜绿假单胞菌细胞裂解物中，负责触发 PARA。最后，在目标 3 中，我们超越了 P。 铜绿假单胞菌并询问 PARA 背后的调节成分（Gac/Rsm 途径）在多大程度上 - 通常具有防御其他假单胞菌属细菌间拮抗作用的功能。我们也 检查以下假设：Gac/Rsm 调节子的变异反映了对特定细菌的适应 不同物种遇到的威胁。通过这项工作，我们将回答长期存在的问题 领域，包括定义重要的全球监管计划的进化相关功能，以及 提供长而难以捉摸的信号分子的分子表征。此外，通过 ARC1 的表征，我们的工作将定义参与保守膜的机制基础 细菌间防御功能未知的复合物。总体而言，拟议的工作将拓宽我们的范围 了解细菌间拮抗作用塑造细菌进化过程的方式。

Project Summary It is increasingly evident that interspecies antagonism is intrinsic to life in the bacterial kingdom. And yet, while our understanding of the antibacterial mechanisms bacteria employ in conflicts with their brethren has recently grown exponentially, our knowledge of the means by which bacteria sense and respond to antagonistic threats remains limited. In this proposal, we test the hypothesis that bacteria react to the presence of an antagonistic bacterial competitor through the activation of a multifaceted defensive program. This hypothesis grew from our discovery that Pseudomonas aeruginosa activates an extensive posttranscriptional regulatory program in response to interbacterial antagonism. The pathway, which we term PARA (Pseudomonas aeruginosa response to antagonism), is triggered when a subpopulation of P. aeruginosa cells succumb to lysis as a result of an antagonistic attack. Detection of as yet unidentified molecule(s) in cellular lysate leads to the activation of the small RNA-mediated Gac/Rsm global posttranscriptional regulatory program. Activation of this response is crucial for P. aeruginosa survival during attack, as a mutant unable to mount the response suffers a severe fitness defect during competition with antagonistic organisms. Finally, we demonstrate that the defensive response requires multiple, simultaneously acting mechanisms, including pathways of unknown function. In Aim 1 of this proposal, we will characterize one such pathway, which we name ARC1 (antagonism response complex 1). Our preliminary data indicate that ARC1 is a large membrane- associated protein complex that provides P. aeruginosa protection against antagonism mediated by toxins delivered by the type VI secretion system of a competitor species. Our studies of ARC1 will elucidate the range of threats towards which it provides protection and provide mechanistic insight into its defensive functions. In Aim 2, we will pursue complementary genetic and biochemical approaches directed at characterizing the signal present in P. aeruginosa cellular lysate responsible for triggering PARA. Finally, in Aim 3, we move beyond P. aeruginosa and ask to what extent the regulatory components behind PARA – the Gac/Rsm pathway – function generally to defend against interbacterial antagonism in other Pseudomonas species. We also examine the hypothesis that variability in the Gac/Rsm regulon reflects adaptation to the specific bacterial threats encountered by different species. Through this work, we stand to answer longstanding questions in the field, including defining the evolutionarily relevant function of an important global regulatory program and providing a molecular characterization of a long elusive signaling molecule. Additionally, through the characterization of ARC1, our work will define the mechanistic basis for participation of a conserved membrane complex of unknown function in interbacterial defense. Overall, the proposed work stands to broaden our understanding of the ways in which interbacterial antagonism shapes the course of bacterial evolution.