A single-domain response regulator activates exopolysaccharide biosynthesis by interaction with the initiating phosphoglycosyl transferase

Exported polysaccharides play crucial functions in bacteria. Polysaccharide biosynthesis in the ubiquitous Wzx/Wzy- and ABC-transporter-dependent pathways starts with the transfer of a sugar-1-phosphate from a nucleotide-sugar donor to undecaprenyl phosphate, a reaction catalyzed by a phosphoglycosyl transferase (PGT). Both reaction substrates are limited and shared among multiple glycoconjugate pathways, raising the question of how bacteria regulate these pathways. In <i>Myxococcus xanthu</i>s, EpsZ, which belongs to the family of large monotopic PGTs (monoPGTs), starts the Wzx/Wzy-dependent exopolysaccharide (EPS) biosynthesis. The Dif chemosensory system regulates EPS biosynthesis by an unknown mechanism via the phosphorylated single-domain response regulator EpsW (EpsW~P). Here, we show that EpsW~P stimulates EPS biosynthesis at the post-translational level. Moreover, MiniTurbo-based proximity labeling experiments suggest that EpsW~P interacts directly with EpsZ. Additionally, heterologous expression of these two proteins in <i>Salmonella enterica</i> demonstrates that EpsW stimulates EpsZ enzymatic activity. <i>S. enterica</i> WbaP, the prototype large monoPGT, forms a functional homodimer, with dimerization involving a distinct cytoplasmic β-hairpin. However, AlphaFold-based structural modeling shows that EpsZ lacks this β-hairpin, suggesting an alternative mechanism for dimerization. Structural modeling of an EpsZ-EpsW heterocomplex suggests that EpsW~P, by direct interaction, promotes the formation of the stable EpsZ dimer. These findings suggest a new model for the regulation of polysaccharide biosynthesis in which EpsW~P allosterically facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating EPS biosynthesis at its initial step. Genomics and structural modeling suggest that the regulation of large monoPGTs by a single-domain response regulator is widespread in myxobacteria and potentially beyond.IMPORTANCEBacteria produce various polysaccharides with important biological functions and biotechnological applications. Polysaccharide synthesis is energy-costly and requires substrates that are in limited supply, raising the question of how bacteria regulate these pathways. Here, we explored the regulation of exopolysaccharide biosynthesis in <i>Myxococcus xanthus</i>. We demonstrate that the phosphorylated single-domain response regulator EpsW activates exopolysaccharide biosynthesis at the post-translational level by stimulating the activity of the phosphoglycosyl transferase EpsZ. By interacting with EpsZ, phosphorylated EpsW facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating exopolysaccharide biosynthesis at its initial step. We propose that this previously unrecognized regulatory mechanism is broadly conserved, not only in myxobacteria but also beyond.