D-Amino acid and tellurite resistance in NIAID Category A bacterial pathogens

描述（由申请人提供）： 除了少数例外，D-氨基酸对细菌的代谢和可能的毒性还没有得到很好的理解。我们将研究一系列潜在的直接选择性抗性标记，以不同的D-氨基酸用于选择代理豁免菌株的三个NIAID A类代理，鼠疫耶尔森氏菌，土拉弗朗西斯菌和炭疽芽孢杆菌的遗传操作。为了同时解毒和使用D-丝氨酸作为泌尿道中的碳源和氮源，致尿路疾病的大肠杆菌菌株具有D-丝氨酸脱氨酶基因座dsdCXA。我们已经深入研究了这个系统，并建议将重组版本的dsdXA基因或同源物引入不同的A类生物防御剂，以评估D-丝氨酸脱氨酶（DsdA）作为非抗生素，直接选择标记的效用。初步结果表明，Y.鼠疫杆菌在复杂培养基中生长时对D-丝氨酸敏感。 我们还将评估D-酪氨酸、D-半胱氨酸、D-甲硫氨酸、D-苯丙氨酸和D-色氨酸是否会抑制复合培养基及其各自限定培养基的改良版本中不同试剂的生长。E.大肠杆菌和枯草芽孢杆菌对这些特定的抑制性氨基酸。我们将研究已知的异化基因提供直接选择表型的能力。作为一种替代方法，我们将评估真菌或哺乳动物衍生的D-氨基酸氧化酶基因的重组版本是否可用于直接选择对单一D-氨基酸或D-氨基酸混合物的抗性。 我们对直接选择标记的研究也导致了对亚碲酸盐抗性的研究。至少有五种不同的细菌基因系统被鉴定出赋予这种抗性。初步结果表明，B. anthracis和Y.鼠疫菌在复杂介质中对亚碲酸钾敏感。我们将尝试将亚碲酸盐抗性标记工程化到这三个物种中。 我们的探索敏感性和耐药机制的D-氨基酸和亚碲酸盐在这一点上， R21项目有望为在这三种药物中产生新的选择性标记提供多种途径。我们的工作很可能为如何在其他生物防御感兴趣的细菌病原体中设计类似性状提供见解。

DESCRIPTION (provided by applicant): With a few exceptions, the metabolism and possible toxicity of D-amino acids for bacteria is not well understood. We will investigate an array of potentially directly selectable resistance markers to different D-amino acids for use in genetic manipulation of select agent exempt strains of the three NIAID class A agents, Yersinia pestis, Francisella tularensis and Bacillus anthracis. In order to simultaneously detoxify and use D-serine as a carbon and nitrogen source in the urinary tract, uropathogenic strains of Escherichia coli possess the D-serine deaminase locus, dsdCXA. We have intensively studied this system and propose to introduce recombinant versions of the dsdXA genes or homologs into the different class A biodefense agents in order to assess the utility of D-serine deaminase (DsdA) as a non-antibiotic, directly selectable marker. Our preliminary results indicate that Y. pestis is sensitive to D-serine when grown in complex media. We will also assess if D-tyrosine, D-cysteine, D-methionine, D-phenylalanine and D-tryptophan will be inhibit growth of the different agents in complex media and modified versions of their respective defined media. There are a variety of known dissimilatory mechanisms in E. coli and Bacillus subtilus for these particular inhibitory amino acids. We will investigate the ability of known dissimilatory genes to provide a directly selectable phenotype. As an alternative approach, we will assess if recombinant versions of fungal or mammalian derived D-amino acid oxidase genes can be used for direct selection of resistance to single D-amino acids or cocktails of D-amino acids. Our search for directly selectable markers has also led to an examination of tellurite resistance. There are at least five different bacterial gene systems identified that confer such resistance. Our preliminary results indicate that B. anthracis and Y. pestis are sensitive to potassium tellurite in complex media. We will attempt to engineer tellurite resistance markers into the three species. Our exploration of sensitivity and resistance mechanisms for D-amino acids and tellurite in this R21 project is expected to provide multiple avenues for generation of new selective markers in these three agents. Our work is very likely to provide insights into how to engineer similar traits in additional bacterial pathogens of biodefense interest.