Revealing Stochastic Switches in Bacteria

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PROJECT SUMMARY Stochastic switches are a broad class of genetic mechanisms that enable single cells to switch certain genes on and off randomly, without responding to their environment. Such switches are prevalent in pathogenic bacteria, where they are often involved in generating diverse surface protein repertoires across the bacterial population, which enables a subset of cells to avoid detection by the immune system. In general, stochastic switches provide a strategy for survival in fluctuating environments, by maintaining subpopulations of cells in pre-adapted states that are prepared for future, possibly unpredictable, environmental stresses. In particular, these strategies are known to be important in antibiotic persistence, a non-genetic, reversible, physiological state with enhanced tolerance for antibiotics that occurs in a subpopulation of bacterial cells. This grant applies novel microfluidic devices that enable single cell observations persister cell lineages, with transcriptomics, and bioinformatics to study three major facets of stochastic switching. We use microscopy and synthetic biology to understand why bacterial aggregation, a behavior that enhances survival under antimicrobial treatment, is regulated by stochastic switching, and how to reverse aggregate states using small molecule inhibitors of key genetic pathways. We use a novel custom microfluidics setup that enables single- cell lineage tracking on hundreds of thousands of cells to observe antibiotic persister states that could not previously be observed, and apply transcriptomics to reveal molecular mechanisms of persistence. We use a new population genetic approach to modeling bacterial recombination, which can be flexibly applied to infer recombination parameters from large-scale genomic and metagenomic sequencing datasets. We apply this method to study how stochastic switching is influenced by recombination in the context of the human gut microbiome. The proposed research will substantially advance understanding of the role of stochastic switches, aggregation, and recombination in bacterial adaptation. Through its emphasis on precise quantification using powerful single cell microfluidics and microscopy, the research will yield new avenues to address antibiotic persistence of bacteria, to perturb bacterial aggregated states, and to understand how the human gut environment selects for and maintains antibiotic resistance and surface antigen genes.