Mechanisms of gene regulation in bacterial defense islands

PROJECT SUMMARY Mechanisms of gene regulation in bacterial defense islands Advancements in bioinformatics and the availability of tens of thousands of bacterial genome sequences have enabled the discovery of diverse pathways in bacteria that defend against environmental stress and bacteriophage (phage) infection. Over 100 different antiphage immune systems have been identified, and each bacterial genome encodes a different combination of ~5-20 such systems. Over the past five years, the molecular mechanisms of many antiphage immune systems have been deciphered, but because immune systems are typically studied one-by-one in model organisms, how these systems are regulated in their native hosts is not well understood. Our lab previously identified the transcriptional regulators CapH+CapP and CapW, which are each associated with diverse antiphage immune systems and control their expression in response to phage infection and/or DNA damage. Our fortuitous discovery of CapH+CapP and CapW implies that many similar regulators remain to be discovered. The aim of my research is to identify and characterize novel transcriptional regulators associated with antiphage immune systems in clinically-important bacterial pathogens. While studying ubiquitination-related BilABCD and BubABCD systems, I identified the novel transcriptional regulators CapK and CapS, and further found that these proteins co-occur with a variety of antiphage immune systems. I will define the upstream signal(s) and molecular mechanisms of CapK+CapS, thereby expanding our knowledge of how bacteria respond to phage infection. Building off my work on CapK+CapS, I will perform comprehensive bioinformatics searches of bacterial genomes to identify new immune system-associated transcriptional regulators, then use my established experimental workflow to validate their activity and define their molecular mechanisms. Complementing this work, I will perform gene expression profiling (RNA-Seq) on a large panel of clinical Enterobacter isolates with distinct sets of antiphage immune systems, to determine whether and how these systems are regulated in response to phage infection, DNA damage, and other stress. This work will define the global transcriptional control networks that regulate expression of antiphage immune systems and coordinate the bacterial response to infection. Together, the work proposed here will comprehensively define the mechanisms of transcriptional regulation in antiphage immune systems, and establish gene expression regulation as a widespread means of bacterial immune system control.