Determinants of gene targeting and regulation by response regulators in Clostridioides difficile

Bacteria use two-component signal transduction systems to sense the conditions of the environment and adapt their behavior to ensure survival. The separate roles of signal perception and response output are carried out by histidine kinase and response regulator proteins, respectively. Most response regulators alter gene transcription, but it is not yet possible to predict which genes they regulate. The precise prediction of these gene regulatory outputs could enable researchers to predict, and doctors and patients to mitigate, various bacterial behaviors. This dissertation presents the elucidation of the genes and biological functions regulated by two response regulators, RR_1586 and RR_1677, from the hypervirulent human pathogen Clostridioides difficile R20291. The data presented herein supports the conclusion that RR_1586 regulates genes involved in phosphate transport, and further characterization of its activity suggests several mechanisms it uses to regulate those genes. The bacterial one-hybrid assay used in this study to elucidate the gene regulatory targets of RR_1586 could potentially be used to find a generalizable solution to predicting gene targets of all response regulators from genome sequences alone. In the process of optimizing the bacterial one-hybrid assay for such a broadly impactful endeavor, it was found that RR_1677 appears to regulate the processes of protein synthesis and cell wall synthesis. A bioinformatics pipeline was also constructed by combining several existing utilities to analyze and interpret the experimental results. Two additional lines of research are also presented. The first is the adaptation of Fourier-transform infrared spectroscopy to observe response regulator phosphorylation. This method offers an alternative from the more labor-intensive methods currently available. The second is an analysis of the biophysical interactions between a response regulator from Saccharomyces cerevisiae and its binding partner, a histidine-containing phosphotransfer protein. Characterization of this interaction elucidates the physical impetus behind the evolutionary conservation of a specific glycine residue near the active site whose role was previously unknown

Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins

Abstract Background Many bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners. Results We report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1. Conclusions Phosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator