Novel genetic tools for Burkholderia mallei and other bacterial Select Agents

描述(由申请人提供)： 这项提议的长期目标是开发新的基因工具和选择方法，这些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)基因作为可选择的遗传标记，用于这里提出的研究。我们选择丙氨酸消旋酶的理由如下：1)D-丙氨酸的合成需要丙氨酸消旋酶，D-丙氨酸是合成细胞壁肽聚糖的重要前体。完全缺乏丙氨酸外消旋酶活性的细菌突变株会产生有缺陷的、未交联肽聚糖。在没有D-丙氨酸的情况下，它们非常容易被渗透裂解杀死，通常表现出条件致命性和生长和生存所需的外源D-丙氨酸。我们的战略将是利用丙氨酸外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、非基于抗生素的、可逆选择的表型，用于细菌选择剂的遗传学研究。为此，我们将构建和鉴定马来芽孢杆菌的丙氨酸消旋酶缺失(DALR)变体，用于后续的遗传学研究，我们将使用假鼻疽杆菌BPSL2179基因编码的克隆的丙氨酸外消旋酶作为选择标记来补充？ALR变体的条件致死表型，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整合到一套新颖和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B.Mallei。我们还将进行平行实验，以开发这些工具用于假鼻疽杆菌，这是一种导致人类类鼻疽病的B类选择剂。在未来的研究中，我们将使用本提案中开发的新的遗传工具来研究最近在首席研究员实验室开始的马利氏杆菌和假鼻疽杆菌的致病机制的分子研究。最后，我们预计，在这项建议中开发的新的遗传策略和工具可以被我们和其他研究人员采用，用于除伯克霍尔德氏菌以外的对生物防御重要的细菌选择剂。

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.