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-氨基酸对细菌的新陈代谢和可能的毒性还不是很清楚。我们将研究一系列对不同D-氨基酸的潜在可直接选择的抗性标记，用于三种NIAID A类制剂的选择试剂豁免菌株的遗传操作，即鼠疫耶尔森氏菌、图拉氏方济氏菌和炭疽芽孢杆菌。为了同时解毒和利用D-丝氨酸作为尿路中的碳源和氮源，致尿路病原性大肠埃希氏菌具有D-丝氨酸脱氨酶基因座dsdCXA。我们对这一系统进行了深入的研究，并建议将dsdXA基因或同源物的重组版本引入不同的A类生物防御剂中，以评估D-丝氨酸脱氨酶(DsdA)作为非抗生素、直接选择标记的用途。我们的初步结果表明，鼠疫耶尔森氏菌在复杂的介质中生长时对D-丝氨酸敏感。 我们还将评估D-酪氨酸、D-半胱氨酸、D-蛋氨酸、D-苯丙氨酸和D-色氨酸是否会抑制不同试剂在复杂介质和各自定义的介质的修改版本中的生长。在大肠杆菌和枯草芽孢杆菌中，对这些特定的抑制性氨基酸有各种已知的异化机制。我们将调查已知的异化基因提供可直接选择的表型的能力。作为另一种方法，我们将评估真菌或哺乳动物来源的D-氨基酸氧化酶基因的重组版本是否可以用于直接选择对单一D-氨基酸或D-氨基酸的鸡尾酒的抗性。 我们对可直接选择标记的寻找也导致了对碲酸盐抗性的检测。至少有五种不同的细菌基因系统被鉴定出具有这种抗性。我们的初步结果表明，炭疽杆菌和鼠疫杆菌在复杂的介质中对亚碲酸钾敏感。我们将尝试将亚碲酸盐抗性标记改造到这三个物种中。 我们对D-氨基酸和亚碲酸盐的敏感性和抗性机制的探讨 R21计划有望为在这三个菌剂中产生新的选择标记提供多种途径。我们的工作很有可能为如何在更多具有生物防御兴趣的细菌病原体中设计类似的特征提供见解。

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.