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：
7442165
负责人：
Randall K Holmes
金额：
$ 22.66万
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 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7442165
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新型的遗传工具和选择方法，这些方法不需要使用抗生素来研究细菌选择剂和生物粘液剂。我们将使用丙氨酸种族酶（ALR）基因作为此处提出的研究的可选遗传标记。我们选择丙氨酸种族的基本原理如下：1）丙氨酸种族酶合成D-丙氨酸是必需的，D-丙氨酸是合成细胞壁肽聚糖的必不可少的前体。完全缺乏丙氨酸的种族友活性的细菌突变体会产生有缺陷的，无连接的肽聚糖。在没有D-丙氨酸的情况下，它们非常容易受到渗透裂解的杀伤，通常表现出有条件的致命性，也表现出对外源性D-丙氨酸的生长和生存能力的要求。我们的策略将是利用丙氨酸种族隔酶突变体的有条件致命表型作为一种稳定，易于使用的，非抗生素的基于基于抗生素的，可挑选的表型，用于在细菌选择剂的遗传研究中使用。为此，我们将构建和表征麦芽芽孢杆菌的丙氨酸结果缺失（DALR）在随后的遗传研究中使用，我们将使用由B. pseudomallei的BPSL2179基因编码的克隆的丙氨酸丙氨酸种族酶来补充的标记，以辅助对状态的b. pseymallei的标准型，并构造了对状态的a antry and？没有补充D-丙氨酸的培养基。我们将将ALR纳入一组新型且高度柔韧的分子遗传工具集中，包括可选的质粒载体和可选的转座子，用于用于B. mallei。我们还将执行并行实验，以开发这些工具，以与B. pseudomallei一起使用，B. pseudomallei（B类选择的剂，会导致人类中的黑细胞病症。在未来的研究中，我们将使用新的遗传工具在此提案中开发出来，用于在主要研究者的实验室开始始于主要研究者的Mallei和B. pseudomallei的致病机制的分子研究。最后，我们期望我们和其他研究人员都可以对本提案中开发的新型遗传策略和工具进行调整，以与Burkholderia物种以外的细菌精选药物一起使用，这对BiodeFense很重要。