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Research in the Stock laboratory is broadly focused on bacterial signal transduction, molecular information processing pathways that enable bacteria to sense changes in their environments and elicit appropriate adaptive responses. Such processes are important for host-microbe interactions, and thus are relevant to therapeutic strategies to promote beneficial host-microbe interactions or to develop new antibiotics to combat infectious disease.
The current project is focused on characterizing regulatory pathways that allow bacteria in the human gut to metabolize dietary fiber.
Dietary fibers are mainly plant-derived complex carbohydrates (polysaccharides) that are undigestible by animals but represent a rich and abundant carbon source for gut microbiota. Fiber impacts the composition of bacteria species in gut microbiota, which is often associated with human health. To utilize polysaccharides, bacteria such as Bacteroidetes, one of the two predominant phyla in the human gut, rely on comprehensive metabolic systems for breaking down polysaccharides in the extracellular environment, transporting intermediates into the bacterial cells, and further transforming them into simple components that can be used as carbon sources for growth. The many proteins involved in these systems are produced from gene clusters known as polysaccharide utilization loci, or PULs. Bacteria contain numerous PULs to metabolize different polysaccharides. In Bacteroides thetaiotaomicron, PULs comprise almost one fifth of the genome. Regulation is important! PULs for a specific polysaccharide are transcriptionally expressed only when breakdown intermediates indicative of that specific polysaccharide are present.
Many PULs are regulated by Two-Component Systems. Two-Component Systems, which have long been a focus of research in the Stock laboratory, are the predominant multi-component regulatory scheme in bacteria. Canonical systems involve a central phosphotransfer reaction between two conserved proteins: a sensor histidine kinase, which detects specific signals and a response regulator, typically a transcription factor, which, when phosphorylated, binds to DNA to regulate transcription of specific genes. The Two-Component Systems that regulate PULs have a novel twist. The histidine kinase and response regulator transcription factor are fused together into a single polypeptide chain, creating a Hybrid Two-Component System (HTCS). Little is known about these unusual hybrid signaling proteins. Our goal is to characterize them at functional and structural levels.
The specific project will focus on one Hybrid Two-Component System from Bacteroides thetaiotaomicron. Expression plasmids for the intact protein and domain constructs will be created, proteins will be purified, activities will be assessed, and structures will be determined by X-ray crystallography and/or cryo-electron microscopy (cryo-EM). Functional characterization will include phosphorylation and DNA-binding assays in the presence and absence of activating ligands. These unusual proteins, in which signaling occurs within a single polypeptide chain, provide a unique opportunity to visualize how ligand binding to extracellular domains is propagated across a membrane to control activities of intracellular domains. The signaling mechanisms will be further studied in Bacteroides cells by fluorescence microscopy and other cellular assays to examine how protein localization and interactions impact the signaling.
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