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: 10179834
• 负责人: Matthew B Neiditch
• 金额: $ 59.37万
• 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/10179834
• 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/antagonist; 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.

项目概述：目前和第7次霍乱弧菌大流行引起的埃尔托生物型编码两个 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 这些岛的获得被认为是引发第七次大流行的关键，这些基因的功能 仍然是个未知数本建议的主要目的是了解其功能和调节 我们发现一种新的细菌胞苷脱氨酶（CDA）调节系统编码在VSP-1上 和许多其他变形菌。这种新的CDA调节系统由多结构域蛋白组成， DcdV（脱氧胞苷脱氨酶弧菌）及其抑制剂DifV（DcdV抑制因子弧菌） 编码在dcdV的5'端的222 NT区。我们的生物信息学分析表明， 它们与VSP-1编码的DncV/CapV环状GMP-AMP噬菌体在细菌基因组中显著共存 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difV不存在的情况下表达DcdV导致细胞表达和V中dNTP池的破坏。 霍乱弧菌和大肠杆菌。脱氧胞苷脱氨酶（DCD）酶在维持 核苷酸稳态，超突变和病毒防御在细菌和真核生物，但在许多 在某些方面，DcdV及其直系同源物与任何其他先前研究的DCD都有很大不同。例如所有 先前描述的DCD是单结构域蛋白，而DcdV具有相关的N-末端核苷酸 我们遗传学研究表明，NK结构域对于DcdV活性是必需的。此外，其他DCD DcdV受dTTP的变构结合负调控，而DcdV则受DifV调控。出于一系列的原因 根据提案中描述的初步研究，我们假设通过抑制DcdV来激活DcdV。 DifV使细胞核苷酸库偏斜。更具体地，DcdV驱动dUTP浓度的增加， dCTP和dTTP浓度的降低作为双重噬菌体防御机制，即，防止 积累用于噬菌体基因组复制的dNTP底物并促进dUMP掺入噬菌体 基因组DcdV究竟是如何发挥作用的，DifV是如何抑制这种功能的， 例如，作为噬菌体防御机制的一部分，该系统对细菌存活的影响仍有待阐明。 我们建议研究DcdV功能的机制基础，DifV对其的调节，以及DcdV的生物学贡献。 这种新发现的调节系统对霍乱弧菌和其他细菌的生理学的影响。这些目标 将在细胞和原子水平上使用细胞生物学，遗传学，生物化学，显微镜， 和结构生物学。通过定义这种新的CDA调节系统的机制和功能，我们期望 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的影响。