Determining mechanisms underlying bacteriophage dynamics during abiotic perturbation.

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The long-term goal of my research program is to determine the mechanisms by which microbial communities remain resilient during disturbances (perturbations) in their physical (abiotic) environment. Abiotic environmental factors such as temperature, pH (acidity or alkalinity), and osmolality (the concentration of solute particles in a solution) strongly impact bacterial growth, yet we do not understand how they affect microbial communities. One of the ways that bacterial community dynamics are affected during abiotic perturbations, is through changes in the infection ability and reproduction of viruses that infect bacteria, known as bacteriophages or phages. Although phages are the most abundant organisms on Earth, we know very little about the complexities of the relationship between them and their bacterial hosts, particularly in the context of abiotic perturbations. Understanding the molecular mechanisms underlying this interaction is essential to understanding microbial communities associated with various hosts and environments. This research proposal focuses on unraveling the bacterial as well as phage-dependent mechanisms by which phage infection is resilient to environmental perturbations, specifically osmolality. We have previously shown that shifts in environmental parameters such as external osmolality lead to irreversible changes in both bacterial and phage populations. Our preliminary experimental and computational data indicate that phages that interact with bacterial osmolality sensing proteins may take advantage of a disrupted environment to increase infection. We will now determine the molecular links between bacterial and phage responses to abiotic perturbation: 1) We will investigate the bacterial genes involved in the response to osmotic stress and phage infection in E. coli by applying single-cell imaging and microfluidics approaches in combination with bulk assays. We will test the resilience of the system to osmolality perturbations in a mouse model to determine the in vivo relevance of these genes and their behavior in the context of a complex bacterial community. The combination of experimental and computational analyses will allow us to converge on general mechanisms that in the future we will test in other bacteria-phage pairs and perturbations. 2) We will identify phage genes involved in resilience to environmental stress by combining isolation approaches with in vitro assays and computational analyses. We will create a library of phages against hundreds of bacterial species to identify genetic features predictive of resilience to environmental perturbations. Finally, we will evolve phages to further identify genes involved in resilience to abiotic insults. The completion of this research will not only allow for assignment of novel features in currently largely under-characterized phage genomes, but it will also provide crucial missing knowledge to enable the prediction of microbial community dynamics.