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

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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.