Recombineering in Mycobacterium tubercolosis

描述（由申请人提供）：结核分枝杆菌比任何其他单一感染因子杀死更多的人。尽管结核病持续威胁全球健康，并且耐药性结核病在美国流行，但是用于诊断、预防和治愈结核病的新策略一直缓慢出现。近年来，分枝杆菌遗传学研究取得了相当大的进展，转化、突变和重组菌株构建方法的发展促进了更复杂的遗传分析。然而，在M.结核病仍然是个问题，需要多步质粒或噬菌体构建来产生所需的突变体。此外，这些多步骤过程在M中通常需要数周或数月。结核病，由于其极其缓慢的增长与24小时的双重时间。重组工程的遗传学方法已经被开发用于E.大肠杆菌及其遗传元件，利用高频率噬菌体介导的重组系统，使用PCR产物或寡核苷酸在一个步骤中构建突变体。这些E.大肠杆菌系统的分枝杆菌尚未成功，这表明更富有成效的方法可能是开发基于分枝杆菌噬菌体编码的重组系统的重组工程系统。分枝杆菌重组工程系统的发展将能够构建一组定义的遗传破坏，覆盖整个分枝杆菌。结核病基因组，这将提供一个有用的资源，整个社区的分枝杆菌遗传学家。初步数据支持分枝杆菌噬菌体TM 4编码其自身重组功能的假设，因为当共电穿孔到M.恶臭此外，这些重组体的形成不依赖于宿主RecA或RecB活性。由于TM4不编码任何可识别的重组基因，因此TM4编码可用于重组工程目的的新型重组系统是合理的。此外，分枝杆菌噬菌体Che9c和Halo都含有编码RecE或RecT样蛋白的基因，这些蛋白被预测为介导噬菌体重组系统。这些推定的重组蛋白将在功能和生化特性，并用于开发重组系统在快速和缓慢生长的分枝杆菌。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis kills more people than any other single infectious agent. In spite of the continuing global health threat of tuberculosis and the prevalence of drug-resistance tuberculosis in the United States, new strategies for the diagnosis, prevention and cure of tuberculosis have been slow to emerge. Considerable advances have been made in mycobacterial genetics in recent years, and the development of methods for transformation, mutation, and construction of recombinant strains has facilitated more sophisticated genetic analyses. However, the construction of defined gene replacements and null mutants in M. tuberculosis continues to be problematic, requiring multi-step plasmid or phage constructions to make the desired mutants. Moreover, these multi-step processes often take many weeks or months in M. tuberculosis due to its extremely slow-growth with a 24 hours double-time. Genetic methods for recombineering have been developed for E. coli and its genetic elements that take advantage of high-frequency phage-mediated recombination systems to construct mutants in a single step, using either PCR products or oligonucleotides. Adaptation of the these E. coli systems to the mycobacteria has yet to be successful, suggesting that a more fruitful approach may be to develop recombineering systems based on mycobacteriophage-encoded recombination systems. The development of a mycobacterial recombineering system would enable the construction of a defined set of genetic disruptions covering the entire M. tuberculosis genome that would provide a useful recourse to the entire community of mycobacterial geneticists. Preliminary data support the hypothesis that mycobacteriophage TM4 encodes its own recombination functions, since combinations of TM4 cosmids give rise to wild-type TM4 plaques when co-electroporated into M. smegmatis. Moreover, the formation of these recombinants is independent on either the host RecA or RecB activities. Since TM4 does not encode any recognizable recombination genes, it is plausible that TM4 encodes a novel recombination system that can be exploited for recombineering purposes. Moreover, both mycobacteriophage Che9c and Halo contain genes that do encode either RecE- or RecT-like proteins that are predicted to mediate phage recombination systems. These putative recombination proteins will be functionally and biochemically characterized and used to develop recombineering systems in both fast- and slow-growing mycobacteria.