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。

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.