Doctoral Dissertations

Date of Award

8-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Gladys Alexandre

Committee Members

Francisco Barrera, Elizabeth Fozo, Barry Bruce, Todd Reynolds

Abstract

Bacterial membranes act as barriers between a cell and its external environment. The membrane insulates and maintains charge gradients, including the proton (ion) gradient (∆pH) and the transmembrane electrical potential (∆ψ) which comprise the proton (ion) motive force (PMF or IMF). These concentration and electrical gradients (or membrane energetics) power diverse physiologies including cell growth, division, motility, ATP synthesis, pH homeostasis, and molecular transport. Abiotic factors like temperature, osmolarity, or pH alter membrane physicochemical properties and perturb membrane energetics. Cells must sense changes in their environments and membrane properties to modify cellular components for survival. The Progestin and AdipoQ Receptor (PAQR) family, a group of poorly characterized membrane proteins that share a core seven transmembrane structure, is conserved in multiple phyla of Bacteria and Eukarya. Eukaryotic PAQRs sense changes in membrane fluidity and modify fatty acid and/or lipid metabolism to fluidize the membrane. The molecular mechanisms of PAQRs are not well understood, and the physiological functions of bacterial PAQRs are unknown. This dissertation characterizes bacterial P AQR homologs, which we refer to as TrhA, for ‘Transmembrane Homeostasis protein A’. We show that bacterial TrhAs in Escherichia coli (gene yqfA) and Bacillus subtilis (gene yplQ) maintain membrane energetics homeostasis. In both species, trhA mutants had depolarized membrane potentials, low ATP levels, motility defects, perturbed PMFs, and pH or osmotic stress imbalances. Membrane fatty acid and fluidity analyses revealed that lack of TrhA provokes subtle defects in unsaturated fatty acid production that do not translate to consistent fluidity defects. Bacterial TrhAs have pleiotropic effects on cellular physiology, including motility. Genetic motility suppressors and E. coli TrhA site-specific mutants, used to identify essential or auxiliary sequence motifs for membrane energetics homeostasis, suggest that energetics and motility are uncoupled in trhA mutants: some mutants rescue energetics but not motility, while others rescue motility but not energetics. Together, bacterial TrhAs universally modulate membrane energetics, likely through their subtle, unknown roles in unsaturated fatty acid metabolism. A role in membrane energetics has not been described for eukaryotic homologs, suggesting functional divergence of eukaryotic PAQRs from bacterial PAQRs.

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