Molecular Characterization of New Bacterial Factors in Phospholipid Transport and Genome Integrity

尽管是研究最深入的微生物之一，也是开发抗微生物药物的主力，但只有69%的大肠杆菌基因被赋予了功能，而31%(4,247个基因中的1,336个)没有实验数据。这些未定性(定义不明确或孤立的)基因的生物学作用可以根据它们与注释基因产品的关联来预测，这是通过我们产生的大规模蛋白质或遗传相互作用网络中的所谓“关联内疚”原理来实现的。然而，这种研究补充而不是取代了直接机械调查的需要。在这个研究计划中，我提出了两个目标，这两个目标建立在我长期的研究计划和坚实的初步数据基础上。我的细菌研究计划的长期目标是：(1)从我们的相互作用图中表征与保守过程有关的孤儿基因在大肠杆菌中的生物学作用；以及(2)增进我们对可能对耐药细菌产生影响的重要细菌机制的理解。为了实现这些目标，我之前的NSERC发现基金揭示了一些大肠杆菌孤儿基因功能预测的机制。更具体地说，我首次提出了一些方法来理解3个孤儿大肠杆菌基因之间的联系的生物学意义，以及我们在之前的相互作用筛选中确定的被广泛研究的磷脂运输和基因组完整性过程。我们发现与这些特定过程相关的孤儿现在在研究界得到了越来越多的关注，因此，我优先考虑这3个孤儿候选对象的详细机械特征。越来越多且仍在发展的证据表明，脂多糖是如何合成、运输和插入外膜的，但磷脂通过辅助蛋白在细菌内膜和外膜之间运输的机制尚不清楚。此外，新出现的研究表明，聚合酶PHP(聚合酶和组氨酸磷酸酶)结构域能够在染色体复制过程中协调校对和聚合酶活动。因此，我利用了一种与PHP域具有高度序列相似性的孤儿蛋白的结构，以了解它作为核酸酶在基因组完整性中的作用。因此，我假设两个靶孤儿参与维持外膜脂质不对称，第三个孤儿在基因组完整性过程中作为校对核酸酶发挥作用。我将使用生化、遗传、结构和分子方法来检验这些假说，我预计这将为革兰氏阴性菌如何在磷脂运输和基因组完整性的复杂细菌过程中组织目标孤儿提供新的知识，并为研究微生物生理学的研究人员产生有价值的机械见解。

Despite being one of the best-studied microbes and a workhorse for the development of antimicrobials, only 69% of Escherichia coli genes have been assigned function, while 31% (1,336 of 4,247) have no experimental data. The biological roles of these uncharacterized (ill-defined or orphan) genes can be predicted based on their association with annotated gene products through the so-called "guilt-by-association" principle from large-scale protein or genetic interaction networks we have generated. However, such studies complement rather than replace the need for direct mechanistic investigations. In this research program, I propose two objectives that are built on my long-term research program and solid preliminary data. The long-term goals of my bacterial research program are to: (1) characterize the biological roles of orphan genes in E. coli that are connected with conserved processes from our interaction maps; and (2) advance our understanding of vital bacterial machineries that could have consequences to drug-resistant bacteria. Towards achieving these goals, my previous NSERC discovery grant revealed mechanisms for some of the E. coli orphan gene function predictions. More specifically, I propose for the first time, methods to understand the biological significance of connections between 3 orphan E. coli genes and the widely-studied phospholipid trafficking and genome integrity processes that we identified in previous interaction screens. Orphans we found to associate with these particular processes are now receiving increased attention in the research community, and as such I have prioritized these 3 orphan candidates for detailed mechanistic characterization. Accumulating and still evolving evidence has shown how lipopolysaccharide is synthesized, trafficked and inserted into the outer membrane, yet the mechanisms by which phospholipids are transported via auxiliary proteins between the bacterial inner and outer membrane is not known. As well, emerging studies suggest that the polymerase PHP (Polymerase and Histidinol Phosphatase) domain enables coordination of proofreading and polymerase activities during chromosomal replication. I thus exploited the structure of an orphan protein that has high sequence similarity to the PHP domain to understand its role as a nuclease in genome integrity. Therefore, I hypothesize that two target orphans are involved in the maintenance of outer membrane lipid asymmetry, and a third orphan to function as a proofreading nuclease in the genome integrity process. I will test these hypotheses using biochemical, genetic, structural and molecular approaches, which I anticipate will provide new knowledge of how a Gram-negative bacteria organizes targeted orphans in the intricate bacterial processes of phospholipid trafficking and genome integrity, and generate valuable mechanistic insights for researchers studying the physiology of microbes.