Systems approach to study sRNAs in Escherichia coli

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The overall objective of my NSERC research program is to understand the role of small non-coding RNAs (sRNA) to cell stress in bacteria. I propose to combine high-throughput and molecular biology approaches to study how sRNA-mediated gene regulation contributes to the growth and stress responses of the model bacterium, Escherichia coli. E. coli expresses some 200 sRNAs, most of which are encoded in intergenic regions of the genome or embedded within untranslated portions of protein-coding genes. sRNA genes are typically induced by changes in the growth environment due, for example, to nutrient stress or chemical perturbants. Once expressed, sRNAs regulate protein synthesis by base-pairing to partially complementary mRNAs and altering translation and/or RNA stability. Gene-regulation by sRNAs is thought to be vital to bacterial stress responses. Paradoxically, there are few examples where disruption of sRNA-mediated gene regulation results in a discernable phenotype in bacterial growth or survival. Indeed, much of our knowledge of sRNA function comes from in-depth studies on a small handful of sRNAs, leaving the function of most unknown. In recent years, my group has pioneered the development of tools for large-scale investigations of gene function in E. coli. We developed a suite of instrumentation and software for measuring bacterial growth rates in high-density colony arrays. We have also created an imaging device, comprised of a 3D-printed housing, single board computer, camera and software, for studying fluorescence in colony arrays. These tools have us uniquely positioned for success in systems approaches to understanding gene function where we typically use chemical or genetic perturbation. To date, our efforts have focused exclusively on protein-coding genes that contribute to bacterial fitness. In the proposed research we will work to understand the importance of sRNAs to bacterial growth and cell stress responses. Currently the dark matter of bacterial genomes, we aim to describe a genome-wide functional network for sRNAs in E. coli. Short-term objectives include: 1. Probe the functional network of sRNAs in bacterial cell stress 2. Test hypotheses posed by the sRNA functional network 3. Measure sRNA expression dynamics in different growth conditions While approaches to date aimed at understanding sRNA function have largely been reductionist, the proposed approach will allow us to interrogate a large number of perturbations and combinations thereof. These will include genetic mutations, through deletion of one or more sRNA genes at a time, and diverse environmental stresses. In addition we will profile transcription of sRNA promoters across hundreds of conditions with high temporal resolution. This systems approach will provide new hypotheses for detailed study using conventional biochemical and molecular genetic methods. In all we will address a major knowledge gap in understanding the importance of bacterial sRNAs to cell stress.