Two-Component System Design Principles

This project will characterize a large family of transcription factors that regulate utilization of complex dietary and host glycans in Bacteroides, a major component of the human gut microbiota. Bacteroides specialize in utilization of complex carbohydrates found in dietary fiber and the mucous that lines the gut, converting the glycans into nutrients used by themselves, other bacteria, and humans. Bacteroides devote ~20% of their genomes to genes encoding proteins necessary for capturing, processing, importing and metabolizing glycans, with genes for utilization of specific glycans clustered into dozens of different polysaccharide utilization loci (PULs). Regulation of PULs determines which glycans are utilized, shaping the composition of the gut microbiota and the balance between diet and host glycan utilization, with impacts on human health including immunomodulation, neurotransmitter release and bowel inflammation. Hybrid Two-Component System (HTCS) transcription factors are a prevalent strategy used for regulating PULs. HTCSs are an unusual variation of Two-Component System (TCS) signaling in which the transmembrane sensor histidine kinase and response regulator transcription factor are combined into a single protein chain. Despite the critical role these regulators play in a central genus of the human gut microbiota, remarkably little is known about their function. Specifically, the genes regulated by each HTCS have not been identified, the regulatory network that produces prioritized utilization of different complex glycans has not been defined, and the mechanism of coordination of HTCSs that enables regulation of multiple PULs necessary for utilization of a single complex dietary fiber is unknown. Several obstacles have hindered investigations to date. The specific activating ligands of many HTCSs are unknown; DNA-binding sites for HTCS transcription factors are poorly defined; and direct vs. indirect effects on gene expression in response to specific glycans cannot be distinguished. Our recent studies have revealed a conserved feature of HTCSs that can be exploited to activate DNA binding independent of an activating ligand. This strategy will be employed to define the regulons of each HTCS using CHiP-seq and RNA-seq to identify DNA-binding motifs and establish regulatory sites for activation or repression. These data will enable identification of cross-regulation of multiple PULs by individual HTCSs, thus establishing a regulatory network that will provide insights into prioritization of glycan utilization. Mechanisms of coordination of HTCSs involved in pectin utilization will be explored using regulon analyses, HTCS interaction assays, and HTCS localization studies. Structural characterization of HTCSs by x-ray crystallography and cryoEM will be pursued to provide a comprehensive structural description of phosphotransfer signaling from ligand-binding input to DNA-binding output, furthering understanding of TCS signaling and elucidating features specific to HTCSs adapted for regulation of polysaccharide utilization. These studies will provide foundational knowledge about complex carbohydrate utilization by Bacteroides as a step toward developing dietary strategies to improve gut health.