Novel genetic tools for Burkholderia mallei and other bacterial Select Agents

针对鼻疽伯克霍尔德氏菌和其他细菌选择剂的新型遗传工具

基本信息

批准号：
7287118
负责人：
Randall K Holmes
金额：
$ 19.25万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7287118
关键词：
Aftercare Alanine Alanine Racemase Alleles Amino Acids Antibiotics Bacteria Burkholderia Burkholderia mallei Burkholderia pseudomallei Carbon Catabolism Categories Cell Wall Complement Condition Cytolysis DNA DNA Transposable Elements Defect Equus caballus Escherichia coli Eubacterium Exhibits Future Genes Genetic Genetic Markers Genetic Recombination Genome Glanders Goals Growth Human Insertional Mutagenesis Isomerism Laboratories Link Malleus Melioidosis Methods Molecular Molecular Genetics Muramic Acid Mus Nitrogen Pathway interactions Penicillins Peptidoglycan Phenotype Plasmid Cloning Vector Plasmids Principal Investigator Reporter Research Research Personnel Site Source Standards of Weights and Measures System Variant base biodefense concept crosslink design diaminopyrimidine gene replacement genetic manipulation improved interest killings macrophage mutant novel promoter research study site-specific integration tool

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to develop novel genetic tools and selection methods that do not require the use of antibiotics for research on bacterial select agents and biodefense. We will use the alanine racemase (alr) gene as a selectable genetic marker for the studies proposed here. Our rationale for choosing alanine racemase is as follows: 1) Alanine racemase is required for synthesis of D-alanine, which is an essential precursor for synthesis of cell wall peptidoglycan. Bacterial mutants that are completely deficient in alanine racemase activity produce defective, un-cross-linked peptidoglycan. In the absence of D-alanine they are highly susceptible to killing by osmotic lysis and typically exhibit both conditional lethality and a requirement for exogenous D-alanine for growth and viability. Our strategy will be to exploit the conditional lethal phenotype of alanine racemase mutants as a stable, easy-to-use, non-antibiotic-based, counterselectable phenotype for use in genetic studies of bacterial select agents. Toward that end, we will construct and characterize an alanine-racemase deletion (Dalr) variant of B. mallei for use in subsequent genetic studies, and we will use the cloned alanine racemase encoded by the BPSL2179 gene of B. pseudomallei as the selectable marker to complement the conditional-lethal phenotype of the ?alr variant and enable it to grow on standard bacteriologic medium without supplemental D-alanine. We will incorporate alr into a novel and highly flexible set of molecular genetic tools, including selectable plasmid vectors and selectable transposons, for use in B. mallei. We will also perform parallel experiments to develop these tools for use with B. pseudomallei, the category B select agent that causes melioidosis in humans. In future studies, we will use the novel genetic tools to be developed in this proposal for molecular studies on pathogenic mechanisms in B. mallei and B. pseudomallei that were recently begun in the principal investigator's laboratory. Finally, we expect that the novel genetic strategies and tools developed in this proposal can be adapted, both by us and by other investigators, for use with bacterial select agents other than Burkholderia species that are important for Biodefense.

描述（由申请人提供）：该提案的长期目标是开发不需要使用抗生素的新型遗传工具和选择方法，用于细菌选择剂和生物防御的研究。我们将使用丙氨酸消旋酶 (alr) 基因作为本文提出的研究的可选择遗传标记。我们选择丙氨酸消旋酶的理由如下： 1）丙氨酸消旋酶是合成D-丙氨酸所必需的，D-丙氨酸是合成细胞壁肽聚糖的重要前体。完全缺乏丙氨酸消旋酶活性的细菌突变体会产生有缺陷的、未交联的肽聚糖。在缺乏 D-丙氨酸的情况下，它们非常容易被渗透裂解杀死，并且通常表现出条件致死性，并且需要外源 D-丙氨酸才能生长和生存。我们的策略是利用丙氨酸消旋酶突变体的条件致死表型作为稳定、易于使用、非抗生素、可反选择的表型，用于细菌选择剂的遗传研究。为此，我们将构建并表征鼻疽伯克霍尔德氏菌的丙氨酸消旋酶缺失（Dalr）变体，以用于后续的遗传学研究，并且我们将使用伪鼻疽伯克氏菌的 BPSL2179 基因编码的克隆丙氨酸消旋酶作为选择标记补充 ?alr 变体的条件致死表型，使其能够在不添加 D-丙氨酸的标准细菌学培养基上生长。我们将把 ar 纳入一套新颖且高度灵活的分子遗传工具中，包括可选择的质粒载体和可选择的转座子，用于鼻疽。我们还将进行平行实验来开发这些工具，用于类鼻疽杆菌（引起人类类鼻疽的 B 类选择因子）。在未来的研究中，我们将使用本提案中开发的新型遗传工具，对鼻疽伯克氏菌和类鼻疽伯克氏菌致病机制进行分子研究，这些研究最近在主要研究者的实验室中开始。最后，我们期望我们和其他研究人员都可以调整本提案中开发的新遗传策略和工具，以与对生物防御很重要的伯克霍尔德氏菌以外的细菌选择剂一起使用。