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传染性病原体杀死的人都多。尽管结核病继续对全球健康构成威胁，而且耐药结核病在美国流行，但诊断、预防和治疗结核病的新战略迟迟没有出现。近年来，分枝杆菌遗传学取得了相当大的进展，重组菌株的转化、突变和构建方法的发展促进了更复杂的遗传分析。然而，在结核分枝杆菌中构建确定的基因替代和零突变体仍然存在问题，需要多步骤的质粒或噬菌体构建来制造所需的突变体。此外，由于结核分枝杆菌生长极其缓慢，24小时两倍时间，这些多步骤过程通常需要数周或数月。大肠杆菌及其遗传元件的基因重组方法已经开发出来，利用高频噬菌体介导的重组系统，利用PCR产物或寡核苷酸在一步中构建突变体。这些大肠杆菌系统对分枝杆菌的适应尚未成功，这表明更有效的方法可能是开发基于分枝噬菌体编码重组系统的重组系统。分枝杆菌重组系统的发展将能够构建覆盖整个结核分枝杆菌基因组的一套明确的遗传破坏，这将为整个分枝杆菌遗传学家社区提供有用的资源。初步数据支持这样的假设，即分枝噬菌体TM4编码其自身的重组功能，因为TM4小体的组合在共电穿孔到耻毛分枝杆菌时产生野生型TM4斑块。此外，这些重组体的形成不依赖于宿主RecA或RecB的活性。由于TM4不编码任何可识别的重组基因，因此有可能TM4编码了一种新的重组系统，可以用于重组目的。此外，分枝噬菌体Che9c和Halo都含有编码RecE或rect样蛋白的基因，这些蛋白被预测会介导噬菌体重组系统。这些假定的重组蛋白将被功能和生化特征，并用于在快速和缓慢生长的分枝杆菌中开发重组系统。

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.