Cellular homeostasis pathways in bacteria

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The overarching goal of my research is to uncover the fundamental principles that drive bacterial success, enabling them to colonize and proliferate in every corner of this planet. To accomplish this goal, I have developed a unified bacterial-centric research vision cutting across classically defined subfields. I meld my long history of unraveling the intricacies of bacterial control mechanisms with an ability to develop and implement novel global technologies to open up understudied areas and computational approaches to extend findings beyond model organisms. The current grant explores three important and related areas. First, fueled by two novel CRISPRi strategies that we developed, we continue our quest to identify cellular construction principles by exploring three understudied sets of genes: cell envelope genes, essential genes, and genes that accelerate growth transitions. We tackle the redundancy of function that has prevented genetic analysis of the envelope with double CRISPRi, a technology that allows simultaneous knockdown of two genes via adjacently encoded sgRNAs. We unravel the tradeoffs underlying the expression of essential genes with mismatched CRISPRi, which uses single mismatches in the base pairing region of sgRNAs to predictably titrate their efficacy. By measuring the fitness impact of graded knockdown, we determine the expression-fitness relationships of essential genes and how they are affected by environmental and genetic changes. Finally, we identify essential and non-essential genes that accelerate growth transitions. Second, we continue our studies of the general principles controlling translational output both by exploring the extent to which ribosomes themselves influence the upstream process of transcription (transcription/translation coupling) and the downstream process of mRNA degradation, and by determining whether alternative ribosomal proteins produced under stress conditions result in new translational properties. These studies are enabled by new technologies we developed for genome-wide measurement of ribosome spacing and mRNA degradation. Third, we have begun an exciting new study of gene regulatory networks throughout the bacterial kingdom. This effort is fueled by our new statistically rigorous, phylogenetic foot-printing approach, which we have validated to have a low false positive rate coupled with high recall and precision. We plan to leverage the vast existing database of bacterial genomes to examine evolution of gene regulatory networks across bacteria. Our studies also address an overwhelming current challenge: to develop experimental and computational approaches that enable researchers to comprehensively explore the regulatory wiring and functional diversity of bacteria that thrive in a wide variety of rapidly changing environments. Such approaches can synergize with and exploit metagenomic data to empower mechanistic interrogation of gene function in understudied organisms.