Functional characterization of genetic networks involved in marine chitin degradation by Vibrio species

海洋和其他海洋环境中的碳动员是一种生物地球化学活动，对地球上的许多生态系统产生重大影响。海洋细菌弧菌通过自然降解地球上第二丰富的碳生物聚合物甲壳素，对碳动员起着核心作用。我的NSERC建议的重点是了解弧菌的遗传调控电路和细胞生物学。我之前的NSERC关于弧菌遗传学的资助使我们能够确定ExsA和HlyU蛋白是参与细胞表面结构和分泌系统的遗传调节蛋白。我们现在建议研究弧菌甲壳素代谢的全基因组方面。为了更好地了解弧菌在海洋甲壳素和碳循环中的作用，研究人员已经确定了一组有助于弧菌利用甲壳素的基因，然而，碳同化、修饰酶、信号通路和遗传调控因素的方面仍然不清楚。弧菌如何代谢甲壳素的详细知识还不完整。我们相信，解决这一知识差距的遗传方法将提供对海洋和海洋环境中这些微生物如何动员甲壳素和碳的理解。*这项研究提案的主要目标是发现弧菌利用甲壳素所涉及的全基因组调控机制。创新的基因组和代谢方法，以及质谱学技术，将用于研究海洋微生物副溶血性弧菌对甲壳素的降解。我们的研究计划包括4个主要目标：*目标1：建立副溶血弧菌高密度转座子插入文库，该文库将在胶体几丁质生长介质上筛选，作为有条件地筛选具有甲壳素利用缺陷的突变株的唯一碳源。*目标2：利用基于群体的TN-seq方法，结合下一代测序(NGS)和生物信息学，鉴定副溶血性弧菌的基因和其他影响甲壳素利用的遗传调控元件(如非编码RNA或遗传位点)。*目标3：利用代谢组学和定量酶报告分析从功能上表征副溶血性弧菌几丁质降解基因调控网络。*目标4：与CFIA合作，我们将研究新分离的环境弧菌中与甲壳素代谢相关的遗传多样性。我们将从牡蛎养殖场和加拿大沿海水域获得有关几丁质酶、多糖运输、信号通路和未知调控机制(如非编码RNA和翻译后修饰)的全基因组知识。这项研究计划与Dalhousie在i)海洋研究和ii)基因组应用方面的战略倡议相一致。该项目将支持基因组研究部门的HQP开发和培训，该部门正在迅速扩大，需要一套专门的生物信息学技能。

Carbon mobilization in oceans and other marine environments is a biogeochemical activity that significantly impacts many of Earth's ecosystems. Marine bacterial Vibrio species are central to carbon mobilization by naturally degrading the second most abundant carbon biopolymer on earth, chitin. My NSERC proposal is focused on understanding the genetic regulatory circuits and cell biology of Vibrio sp. My previous NSERC grant on Vibrio genetics allowed us to identify ExsA and HlyU proteins as genetic regulators involved in cell surface structures and secretion systems. We now propose to investigate genome-wide aspects of chitin metabolism by Vibrio sp. to better understand the role of Vibrios in ocean chitin and carbon cycling.******Researchers have identified a set of genes that contribute to chitin utilization by Vibrio species, however, aspects of carbon assimilation, modifying enzymes, signalling pathways, and genetic regulatory factors remain obscure. Detailed knowledge of how Vibrio bacteria metabolize chitin is incomplete. We believe that genetic approaches to address this knowledge gap will provide an understanding of how chitin and carbon are mobilized by these microbes in ocean and marine environments.******The main goal of this research proposal is to discover genome-wide regulatory mechanisms involved in Vibrio chitin utilization. Innovative genomic and metabolomic approaches, alongside mass spectrometry technology, will be used to study chitin degradation by the marine microbe Vibrio parahaemolyticus. Our research proposal consists of 4 main objectives: ***Objective 1: To create a high-density transposon insertion library of V. parahaemolyticus mutants that will be screened on a colloidal chitin growth medium as the only carbon source to conditionally select against mutants with chitin utilization defects. ***Objective 2: To identify V. parahaemolyticus genes, and other genetic regulatory elements (e.g. non-coding RNAs or genetic loci) that contribute to chitin utilization using a population-based Tn-seq approach combined with next generation sequencing (NGS) and bioinformatics.***Objective 3: To functionally characterize V. parahaemolyticus chitin degradation gene regulatory networks using metabolomics and quantitative enzymatic reporter assays.***Objective 4: In collaboration with the CFIA, we will investigate genetic diversities linked to chitin metabolism within newly isolated environmental Vibrio sp. from oyster farms and Canadian coastal waters.******We will gain genome-wide knowledge relating to chitinase enzymes, polysaccharide transport, signalling pathways, and unknown regulatory mechanisms such as non-coding RNAs and post-translational modifications. This research program aligns with Dalhousie's Strategic Initiatives in i) Oceans Research, and ii) Genomics Applications. The project will support HQP development and training in the omic' research sector which is rapidly expanding and requires a specialized bioinformatic skill set.*****