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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）：