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

弧菌物种海洋几丁质降解涉及的遗传网络的功能表征

• 批准号: RGPIN-2019-05807
• 负责人: Thomas, Nikhil
• 金额: $ 2.33万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761046
• 关键词: Functional; characterization; genetic; networks; involved

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.

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。海洋细菌弧菌物种通过自然降解地球上第二丰富的碳生物聚合物甲壳素而成为碳动员的核心。弧菌的基因调控回路和细胞生物学 我之前获得的关于弧菌遗传学的 NSERC 资助使我们能够鉴定 ExsA 和 HlyU 蛋白作为参与细胞的基因调控因子。我们现在建议研究弧菌几丁质代谢的全基因组方面，以更好地了解弧菌在海洋几丁质和碳循环中的作用。然而，对于弧菌如何代谢几丁质的详细了解，碳同化、修饰酶、信号通路和遗传调控因素方面的知识仍然不完整。该研究计划的主要目标是发现弧菌几丁质利用中涉及的全基因组调控机制以及质谱分析。技术，将用于研究海洋微生物副溶血弧菌的几丁质降解。我们的研究计划包括 4 个主要目标： 目标 1：创建高密度转座子插入库。将在胶体几丁质生长培养基上筛选副溶血弧菌突变体，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。 目标 2：鉴定副溶血弧菌基因和其他遗传调控元件（例如非编码 RNA）。使用基于群体的 Tn-seq 方法结合下一代测序 (NGS) 和生物信息学，有助于几丁质的利用。使用代谢组学和定量酶报告分析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 CFIA 合作，我们将研究与来自牡蛎养殖场和加拿大沿海水域的新分离的环境弧菌中的几丁质代谢相关的遗传多样性。我们将获得与几丁质酶、多糖转运、信号通路以及未知调控机制（例如非编码 RNA 和翻译后）相关的全基因组知识。该研究计划与达尔豪斯大学在 i) 海洋研究和 ii) 基因组学应用方面的战略举措相一致。该项目将支持 HQP 的发展和“组学”研究领域的培训，该领域正在迅速扩大，需要专门的生物信息学技能。