Constitutive activation of a hybrid two-component regulator reveals cross-regulation of polysaccharide utilization...

Human gut microbes, such as Bacteroides, rely on specialized gene clusters known as polysaccharide utilization loci (PULs) to metabolize diverse dietary and host-derived glycans. A major class of transcription regulators of these PULs is the hybrid two-component system (HTCS) containing a histidine sensor kinase and a response regulator within a single transmembrane polypeptide chain. Characterizing HTCS-mediated PUL regulation is often challenging, because the specific glycan signals required to activate most HTCSs remain unknown. Here, we characterized structural details of a highly conserved HTCS activation mechanism and developed a universal activation strategy by mutating the interdomain latch motif that inhibits the DNA-binding activities. Using the response regulator portion of BT4124 from Bacteroides thetaiotaomicron as a model system, crystallographic analyses reveal a "closed" inactive conformation anchored by a hydrogen-bond network formed by the conserved latch residues between the receiver and DNA-binding domains. Molecular dynamic simulation with the deep-learning BioEmu shows that the "AD" mutation of the latch residues destabilizes the inhibitory interface, shifting the conformation equilibrium predominantly to an active, "open" conformation. This constitutively active variant, BT4124R^AD, allows us to map specific DNA-binding sites within the potential regulated promoters in vitro and characterize transcription regulation in cells. Induced expression of BT4124R^AD not only down-regulates local homogalacturonan (HG) utilization genes, but also cross-represses multiple PULs associated with other homogalacturonan-related pectic glycans. These findings highlight a complex cross-regulatory network governing pectin degradation and establish the targeted latch mutation as a potential broadly applicable tool for deciphering the regulatory networks of HTCSs in Bacteroides.