The contribution of novel cytidine deaminase regulatory systems to bacterial evolution

新型胞苷脱氨酶调节系统对细菌进化的贡献

• 批准号: 10339467
• 负责人: Matthew B Neiditch
• 金额: $ 57.87万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-05 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10339467
• 关键词: Address; Bacteria; Bacterial Genome; Bacterial Physiology; Bacteriophages; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Process; Biology; Cells; Cellular biology; Cessation of life; Collaborations; Cyclic GMP; Cytidine Deaminase; Defense Mechanisms; Discipline; Dissection; Enterobacter cloacae; Enzymes; Escherichia coli; Eukaryota; Evolution; Filament; Gene Expression; Genes; Genetic; Genetic study; Genome; Genomic Islands; Homeostasis; In Vitro; Infection; Island; Life; Microscopy; Mutation; N-terminal; Names; Nucleic Acids; Nucleotides; Organism; Orthologous Gene; Peptides; Phosphotransferases; Physiology; Play; Protein Biochemistry; Proteins; Proteobacteria; Publications; Recording of previous events; Regulation; Research; Role; Signal Transduction; Structural Protein; Structure; System; Tertiary Protein Structure; Testing; Toxic effect; Trans-Activators; Trees; Untranslated RNA; Ursidae Family; Vibrio; Vibrio cholerae; Viral; Virus Diseases; Water; base; clinically relevant; deoxycytidine deaminase; experience; experimental study; gene function; inhibitor; mutant; novel; overexpression; pandemic disease; pathogenic bacteria; prevent; protein complex; small molecule; structural biology; tool; virtual

Project Summary: The current and 7th pandemic of Vibrio cholerae caused by the El Tor biotype encodes two novel genetic islands called the Vibrio Seventh Pandemic Islands 1 and 2 (VSP-1 and VSP-2). Although acquisition of these islands is proposed to be key to initiation of the 7th pandemic, the function of these genes remains virtually unknown. The over-arching purpose of this proposal is to understand the function and regulation of a novel bacterial cytidine deaminase (CDA) regulatory system that we have discovered is encoded on VSP-1 and in many other Proteobacteria. This new CDA regulatory system consists of the multi-domain protein we named DcdV (deoxycytidine deaminase Vibrio) and its inhibitor named DifV (DcdV inhibitory factor Vibrio) encoded in a 222 NT region 5’ of dcdV. These genes were first identified as our bioinformatic analysis indicated that they significantly cooccur in bacterial genomes with the VSP-1 encoded DncV/CapV cyclic GMP-AMP phage defense system that we previously discovered. Consistent with a potential role of DcdV-DifV to regulate phage defense, expressing DcdV in the absence of difV causes cell filamentation and disruption of dNTP pools in V. cholerae and Escherichia coli. Deoxycytidine deaminases (DCD) enzymes play critical roles in maintaining nucleotide homeostasis, hypermutation, and viral defense in both bacteria and eukaryotes, but in numerous respects, DcdV and its orthologs are quite different from any other previously studied DCDs. For example, all previously described DCDs are single domain proteins, while DcdV has an associated N-terminal nucleotide kinase (NK) domain that our genetic studies show is essential for DcdV activity. Furthermore, other DCDs are negatively regulated by allosteric binding of dTTP, while DcdV is instead regulated by DifV. For a litany of reasons based on preliminary studies described in the proposal, we hypothesize that activation of DcdV via inhibition of DifV skews the cellular nucleotide pool. More specifically, DcdV drives an increase in dUTP concentration and decrease in dCTP and dTTP concentrations as a two-fold phage defense mechanism, i.e., preventing accumulation of dNTP substrates for phage genome replication and promoting dUMP incorporation into phage genomes. Exactly how DcdV functions mechanistically, how this function is inhibited by DifV, and the contribution of this system to bacterial survival, for example, as part of a phage defense mechanism remains to be elucidated. We propose to study the mechanistic basis of DcdV function, its regulation by DifV, and the biological contribution of this newly discovered regulatory system to bacterial physiology in V. cholerae and other bacteria. These aims will be pursued at the cellular and atomic level using the tools of cell biology, genetics, biochemistry, microscopy, and structural biology. By defining the mechanism and function of this novel CDA regulatory system we expect that our research will have a broad impact in multiple disciplines across both prokaryotic and eukaryotic fields.

项目摘要：目前和第七次由El Tor生物型引起的霍乱弧菌大流行编码两种 新的遗传岛称为弧菌第七大流行岛1和2(VSP-1和VSP-2)。虽然 这些岛屿的获取被认为是启动第7次大流行的关键，这些基因的功能 几乎仍然是未知的。这项建议的总体目的是了解其功能和规则。 我们发现的一种新的细菌胞苷脱氨酶(CDA)调控系统编码在VSP-1上 在许多其他变形杆菌中。这个新的CDA调控系统由多结构域蛋白WE组成 命名为DcdV(脱氧胞苷脱氨酶弧菌)及其抑制物命名为DifV(DcdV抑制因子弧菌) 编码在DCDV的222个核苷酸的5‘区中。这些基因首次被确认为我们的生物信息学分析表明 它们显著地与VSP-1编码的DncV/CapV环状GMP-AMP噬菌体共存于细菌基因组中 我们之前发现的防御系统。与DcdV-DifV调节噬菌体的潜在作用一致 防御，在没有difV的情况下表达DcdV会导致细胞丝状化和dNTP池的破坏。 霍乱和大肠埃希氏菌。脱氧胞苷脱氨酶(DCD)在维持 在细菌和真核生物中，核苷酸动态平衡、超突变和病毒防御，但在许多 方面，DcdV及其同源基因与以前研究的任何DCDs都有很大的不同。例如，所有 以前描述的DCDs是单域蛋白，而DcdV有一个相关的N-末端核苷酸 我们的遗传学研究表明，激酶(NK)结构域对DcdV的活性是必不可少的。此外，其他DCD是 DTTP受变构结合负性调节，而DcdV受DifV调节。出于一系列原因 根据提案中描述的初步研究，我们假设DcdV通过抑制 DifV扭曲了细胞的核苷酸池。更具体地说，DcdV推动dUTP浓度的增加和 降低dCTP和dTTP浓度作为一种双重的噬菌体防御机制，即防止 噬菌体基因组复制dNTP底物的积累和促进Dump掺入噬菌体的研究 基因组。DcdV到底是如何发挥作用的，DifV是如何抑制这一功能的，以及DifV的贡献 以细菌存活为例，该系统作为噬菌体防御机制的一部分还有待阐明。 我们建议研究DcdV功能的机制基础，DifV对DcdV的调节以及生物贡献 这一新发现的霍乱弧菌和其他细菌的细菌生理调控系统。这些目标 将利用细胞生物学、遗传学、生物化学、显微镜、 和结构生物学。通过界定这一新型CDA监管体系的机制和功能，我们期待 我们的研究将在原核生物和真核生物领域的多个学科中产生广泛影响。