Unraveling how N. meningitidis distinguish self from non-self DNA during DNA transformation

自然转化是指被定义为具有遗传能力的细菌从细胞外环境中获取裸露的DNA，并通过重组将其整合到基因组中的过程。至少有60种细菌是可以自然转化的，这个数字被认为被大大低估了。自然转化是一种极其宝贵的研究工具，有助于生物学中最重要的发现之一(即遗传物质是DNA)。此外，现在清楚的是，通过自然转化进行的水平基因转移是产生遗传多样性的强大机制，也是细菌进化的主导力量。这可能会产生毁灭性的后果，例如通过促进毒力或抗生素耐药基因的传播。IV型菌毛(TFP)或相关细胞器在DNA转化中发挥关键作用。此外，它们也是细菌中最普遍的定植因素之一。因此，TFP一直并将继续被深入研究，因为它们可能成为开发针对细菌病原体的新疗法的主要靶点。细菌病原体通过感染人类、牲畜和农作物而给人类的健康和经济带来沉重的负担。在现有的模型中，裸露的DNA需要与TFP结合，然后才能被细菌回缩，但这一点仍有待正式证明。在一些物种中，只有自己的DNA被摄取，因为它包含一个被称为摄取序列的签名序列基序，这一事实加强了TFP中DNA受体的存在。在奈瑟氏菌中，这个DNA摄取序列(DUS)基序长10个碱基对，大约发现。每个基因组2000个拷贝。有能力的细菌如何结合DNA，并在摄取过程中区分自己和非自己的DNA仍然是一个谜。DUS受体的鉴定和分析将是一项重大突破，将提高我们对DNA转化和TFP生物学的理解。这一建议最初源于我们最近对参与TFP生物学的脑膜炎奈瑟菌蛋白的表征。其中三种蛋白质(comp、PilV和PilX)是TFP的次要成分(低丰度)。COMP在竞争能力中起着关键作用。这促使我们纯化CoMP蛋白并测试其DNA结合活性，这导致了一个重大发现：CoMP具有DNA结合活性，并显示出对DUS的偏好(未发表的数据)。在这个项目中，我们计划使用生化和细胞分析以及高分辨率结构技术来彻底表征COMP的DNA结合活性。

Natural transformation is the process by which bacteria, defined as genetically competent, take up naked DNA from the extracellular environment and incorporate it into their genomes by recombination. At least 60 bacterial species are naturally transformable, a number that is thought to be considerably underestimated. Natural transformation is an extremely valuable tool for research and was instrumental to one of the most important discoveries in biology (i.e. that the genetic material is DNA). Moreover, it is now clear that horizontal gene transfer by natural transformation is a powerful mechanism for generating genetic diversity and a dominant force in bacterial evolution. This can have devastating consequences, e.g. by promoting spread of virulence or antibiotic resistance genes.Type IV pili (Tfp), or related organelles, play a key role in DNA transformation. In addition, they are one of the most widespread colonization factors in bacteria. Therefore, Tfp have been and continue to be intensively studied as they might be primary targets for the development of new therapies against bacterial pathogens that place a heavy burden on human health and economy by infecting mankind, livestock and crops.In current models, naked DNA needs to be bound by Tfp before it is taken up by the bacteria upon pilus retraction, but this remains to be formally demonstrated. The existence of a DNA receptor in Tfp is strengthened by the fact that in some species only self DNA is taken up because it contains a signature sequence motif known as the Uptake Sequence. In Neisseria, this DNA Uptake Sequence (DUS) motif is 10 bp long and is found in approx. 2,000 copies per genome. How competent bacteria bind DNA and are able to discriminate self from non-self DNA during uptake remains a mystery. The identification and analysis of a DUS receptor would represent a major breakthrough and would improve both our understanding of DNA transformation and Tfp biology in general.This proposal originally derives from our recent characterization of N. meningitidis proteins involved in Tfp biology. Three of these proteins (ComP, PilV and PilX) are minor (low abundance) components of the Tfp. ComP plays a key role in competence. This prompted us to purify the ComP protein and test its DNA-binding activity, which led to a major finding: ComP has DNA-binding activity and shows a preference for the DUS (unpublished data). During this project, we plan to thoroughly characterize ComP's DNA-binding activity using biochemical and cellular assays, together with high resolution structural techniques.