Novel genetic tools for Burkholderia mallei and other bacterial Select Agents

描述（由申请人提供）： 该提案的长期目标是开发新的遗传工具和选择方法，不需要使用抗生素来研究细菌选择剂和生物防御。我们将使用丙氨酸消旋酶（alr）基因作为本文提出的研究的选择性遗传标记。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必需前体。丙氨酸消旋酶活性完全缺陷的细菌突变体产生缺陷的、未交联的肽聚糖。在不存在D-丙氨酸的情况下，它们对通过渗透裂解的杀伤高度敏感，并且通常表现出条件致死性和生长和存活所需的外源D-丙氨酸。我们的策略将是利用丙氨酸消旋酶突变体的条件致死表型作为一种稳定的，易于使用的，非抗生素为基础的，用于细菌选择剂的遗传研究中的反选择表型。为此，我们将构建并表征B的丙氨酸消旋酶缺失（Dalr）变体。我们将使用克隆的由B的BPSL 2179基因编码的丙氨酸消旋酶。类鼻疽作为选择性标记，以补充条件致死表型的？alr变体，并使其能够在没有补充D-丙氨酸的标准细菌培养基上生长。我们将把alr纳入一套新的和高度灵活的分子遗传学工具，包括可选择的质粒载体和可选择的转座子，用于B。鼻疽我们还将进行并行实验，以开发这些工具用于B。类鼻疽，导致人类类鼻疽病的B类选择因子。在未来的研究中，我们将使用新的遗传工具来开发这个建议的致病机制的分子研究在B。鼻疽和B.最近在首席研究员的实验室开始的假鼻疽。最后，我们希望在这项建议中开发的新的遗传策略和工具可以适应，无论是由我们和其他研究人员，用于与细菌选择剂以外的伯克霍尔德氏菌物种是重要的生物防御。

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.