Conjugation and recombination in mycobacteria

描述(申请人提供)：结核分枝杆菌每年造成200多万人死亡。此外，结核病的全球负担因其与艾滋病病毒的致命联系以及多药耐药菌株的出现而加剧，这增加了对遏制结核病/艾滋病流行的新疗法的需求。新药和疫苗的设计需要了解分枝杆菌的生物学，并开发基因工具来操纵它们的基因组，以确定发病和耐药性的分子基础。虽然转化和转导在分枝杆菌遗传学的发展中都起到了重要的作用，但对配偶DNA转移的了解却很少。接合作用为分枝杆菌之间的DNA转移提供了一种重要的替代方法，特别是作为一种基因传递系统，用于在菌株之间移动标记并产生靶向突变。在上一个授权期，我们描述了污垢分枝杆菌的DNA转移过程，这一过程不同于迄今所描述的任何连接系统。 这项建议旨在通过定义和鉴定介导DNA转移的顺式作用DNA序列和反式作用蛋白来表征耻垢分枝杆菌的接合系统。这样的分析将提供有关DNA转移过程的重要机制信息，并使供体细胞和受体细胞之间的差异得以确定。此外，通过修改可转移的质粒，将建立一个新的等位基因交换系统，使之能够捕获染色体DNA片段，并通过转移介导的重组产生靶向突变。将该体系应用于生长缓慢的分枝杆菌病原体，将是当前分子方法的一个有价值的新补充。此外，已从禽分枝杆菌中分离出编码DNA松弛酶的质粒，这些松弛酶与革兰氏阴性细菌中经典的配偶转移所需的DNA松弛酶有关。将研究这些质粒在生长缓慢的分枝杆菌之间转移的能力，以了解接合在分枝杆菌种群之间横向转移中的作用及其在耐药性传播中的可能作用。其目的是：1.鉴定DNA转移所需的顺式作用序列，并开发转移作为研究分枝杆菌的分子遗传学工具。 2.鉴定和鉴定供体细胞和受体细胞中的反式作用转移功能。 3.检测快生和慢生分枝杆菌之间染色体和质粒DNA的转移。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis accounts for over 2 million deaths per year. Furthermore, the global burden of tuberculosis has been compounded by its deadly association with the AIDS virus and by the emergence of multi-drug resistant strains, which have increased the demand for new treatments to stem the tuberculosis/AIDS epidemic. The design of new drugs and vaccines requires an understanding of the biology of mycobacteria and the development of genetic tools to manipulate their genomes in order to determine the molecular basis of pathogenesis and drug resistance. Although both transformation and transduction have had important roles in the development of mycobacterial genetics, little is known about conjugal DNA transfer. Conjugation offers an important alternative for transferring DNA between mycobacteria and, in particular, as a gene delivery system for moving markers between strains and generating targeted mutations. During the last grant-period, we described a DNA transfer process in Mycobacterium smegmatis that is different from any conjugation system described to-date. This proposal is designed to characterize the M. smegmatis conjugation system by defining and identifying both cis-acting DNA sequences and trans-acting proteins that mediate DNA transfer. Such analyses will provide important mechanistic information about the process of DNA transfer and allow differences between donor and recipient cells to be determined. Moreover, by modifying transferable plasmids, a new allele-exchange system will be established, enabling the capture of segments of chromosomal DNA and the generation of targeted mutations by transfer-mediated recombination. The application of this system to the slow-growing mycobacterial pathogens will be a valuable new addition to current molecular approaches. In addition, plasmids have been isolated from Mycobacterium avium that encode DNA relaxases related to those required for classical conjugal transfer in gram-negative bacteria. The ability of these plasmids to transfer among slow-growing mycobacteria will be investigated to understand the role of conjugation in lateral transfer among mycobacterial populations and its possible role in the spread of drug resistance. The aims are: 1. To characterize cis-acting sequences required for DNA transfer and to develop transfer as a molecular genetic tool for the study of mycobacteria. 2. To identify and characterize trans-acting transfer functions in both donor and recipient cells. 3. To examine transfer of chromosomal and plasmid DNA among fast- and slow-growing mycobacteria.