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，对地球的许多生态系统产生重大影响。海洋细菌弧菌是碳动员的核心，通过自然降解地球上第二丰富的碳生物聚合物，几丁质。我的NSERC提案的重点是了解弧菌sp.的遗传调控回路和细胞生物学。我之前的NSERC对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控因子。为了更好地了解弧菌在海洋几丁质和碳循环中的作用，我们现在建议研究弧菌在海洋几丁质代谢的全基因组方面。研究人员已经确定了一组有助于弧菌物种利用几丁质的基因，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。关于弧菌如何代谢几丁质的详细知识尚不完整。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。本研究计划的主要目标是发现涉及几丁质弧菌利用的全基因组调控机制。创新的基因组学和代谢组学方法以及质谱技术将用于研究海洋微生物副溶血性弧菌对几丁质的降解。我们的研究计划包括4个主要目标：目标1：建立一个高密度的副溶血性弧菌突变体转座子插入文库，该文库将在胶体几丁质生长培养基上筛选，作为唯一的碳源，以有条件地选择具有几丁质利用缺陷的突变体。目的2：利用基于群体的n-seq方法，结合下一代测序（NGS）和生物信息学，鉴定副溶血性弧菌基因和其他有助于几丁质利用的遗传调控元件（如非编码rna或遗传位点）。目的3：利用代谢组学和定量酶报告基因检测对副溶血性弧菌几丁质降解基因调控网络进行功能表征。目标4：与加拿大食品检验局合作，我们将调查从牡蛎养殖场和加拿大沿海水域新分离的环境弧菌中与几丁质代谢相关的遗传多样性。我们将获得有关几丁质酶、多糖转运、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。该研究项目与达尔豪斯大学在1)海洋研究和2)基因组学应用方面的战略计划保持一致。该项目将支持“基因组学”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.