Structural and ligand binding analyses of the periplasmic sensor domain of RsbU in Chlamydia trachomatis supports role in TCA cycle regulation.

Chlamydia trachomatis are obligate intracellular bacteria that undergo dynamic morphologic and physiologic conversions upon gaining access to a eukaryotic cell. These conversions likely require the detection of key environmental conditions and regulation of metabolic activity. Chlamydia encodes homologs to proteins in the Rsb phosphoregulatory partner-switch pathway, best described in Bacillus subtilis. ORF CT588 has strong sequence similarity to RsbU cytoplasmic phosphatase domain but also contains a unique periplasmic sensor domain that is expected to control phosphatase activity. A 1.7 Å crystal structure of the periplasmic domain of the RsbU protein from C. trachomatis (PDB 6MAB) displays close structural similarity to DctB from Vibrio and Sinorhizobium. DctB has been shown both structurally and functionally to specifically bind to the TCA cycle intermediate succinate. Surface plasmon resonance and differential scanning fluorimetry of TCA intermediates and potential metabolites from a virtual screen of RsbU revealed that alpha-ketoglutarate, malate, and oxaloacetate bound to the RsbU periplasmic domain. Substitutions in the putative binding site resulted in reduced binding capabilities. A RsbU-null mutant showed severe growth defects which could be restored through genetic complementation. Chemical inhibition of ATP synthesis by oxidative phosphorylation phenocopied the growth defect observed in the RsbU null strain. Altogether, these data support a model with the Rsb system responding differentially to TCA cycle intermediates to regulate metabolism and key differentiation processes.