Investigating the contextual essentiality of the Escherichia coli efflux system

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Efflux pumps are membrane-spanning proteins that actively transport molecules across membranes to the extracellular environment. Bacterial efflux pumps have received significant attention due to their involvement in antibiotic resistance. Despite this association, a growing body of evidence suggests that these proteins also play important roles in bacterial physiology. During metabolism, organisms produce waste metabolites that can be toxic if they reach high intracellular levels. It has been hypothesized that efflux pumps are responsible for the removal of toxic waste metabolites. Furthermore, there is also evidence that efflux pumps are involved in pH homeostasis. Using Escherichia coli as a model organism, the proposed research program is designed to provide a holistic understanding of the physiological roles of efflux-pumps in different environmental and genetic contexts, with a particular focus on their connection to metabolism and cellular homeostasis.******Aim 1 of this proposal is the characterization of a conditional synthetic-sick interaction that we have observed between two efflux pumps. Deletion of two major facilitator efflux pumps causes severe growth defects when E. coli is grown in a nutrient-limited medium. We hypothesize that the loss of these efflux pumps leads to the accumulation of toxic metabolites and/or an inability of the cell to regulate cytoplasmic pH. To investigate this hypothesis we will integrate transcriptomic-metabolomic analyses and we will assess the intracellular pH of the strain. By investigating the basis of this conditional synthetic-sick interaction, we will gain insight into the essentiality of efflux pumps in bacterial physiology. ******Aim 2 is the first systems-level study of E. coli efflux pump essentiality in different environmental and genetic conditions. By profiling a wide variety of growth conditions in combination with extensive genetic manipulation, we will establish the contextual essentiality of the E. coli efflux family. We will characterize any conditionally essential genes and genetic interactions using the transcriptomic/metabolomic approach described in Aim 1, combined with an assessment of changes in pH tolerance and phenotypic analysis. Such studies will provide a repertoire of efflux metabolic waste substrates, bacterial stress responses induced by the loss of efflux, and a systems-level understanding of the efflux pumps involved in cellular homeostasis. ******Despite E. coli being a well-characterised organism, fundamental questions remain regarding the physiological roles of efflux pumps. This study is designed to address these questions and will provide the first systems-level investigation into the contextual essentiality of the E. coli efflux family. Such information will contribute to our broader understanding of bacterial physiology and will offer important insight into the origins of these proteins, which could be applicable to other bacterial species and organisms. **