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：由 El Tor 生物型引起的当前第七次霍乱弧菌大流行编码两种 称为弧菌第七大流行岛 1 和 2（VSP-1 和 VSP-2）的新型基因岛。 据称，占领这些岛屿是引发第七次大流行的关键，这些基因的功能 该提案的首要目的仍然是了解其功能和监管。 我们发现一种新型细菌胞苷脱氨酶 (CDA) 调节系统由 VSP-1 编码 在许多其他变形菌中，这种新的 CDA 调节系统由我们的多结构域蛋白组成。 命名为DcdV（脱氧胞苷脱氨酶弧菌）及其抑制剂命名为DifV（DcdV抑制因子弧菌） 我们的生物信息学分析表明，这些基因首先被鉴定为 dcdV 的 222 NT 区域 5’。 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着共存 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。 在缺乏 difV 的情况下表达 DCV 会导致 V 中的细胞丝状化和 dNTP 池的破坏。 霍乱和大肠杆菌脱氧胞苷脱氨酶 (DCD) 在维持体内发挥着关键作用。 细菌和真核生物中的核苷酸稳态、超突变和病毒防御，但在许多 就各方面而言，DcdV 及其直系同源物与之前研究的任何其他 DCD 都有很大不同。 之前描述的 DCD 是单结构域蛋白，而 DCV 具有相关的 N 端核苷酸 我们的遗传研究表明激酶 (NK) 结构域对于 DCdV 活性至关重要。此外，其他 DCD 也是如此。 由于多种原因，DcdV 受到 dTTP 变构结合的负向调节，而 DcdV 则受到 DifV 的调节。 根据提案中描述的初步研究，我们通过抑制 DifV 会扭曲细胞核苷酸池。更具体地说，DcdV 会导致 dUTP 浓度增加， dCTP 和 dTTP 浓度的降低作为双重噬菌体防御机制，即防止 噬菌体基因组复制的 dNTP 底物积累并促进 dUMP 掺入噬菌体 DcdV 的具体功能、DifV 如何抑制该功能以及其贡献 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 对其的调节以及生物学贡献 这一新发现的调节系统对霍乱弧菌和其他细菌的细菌生理学的影响。 将利用细胞生物学、遗传学、生物化学、显微镜等工具在细胞和原子水平上进行研究， 通过定义这种新型 CDA 调控系统的机制和功能，我们期望 我们的研究将对原核和真核领域的多个学科产生广泛的影响。