The orphan response regulator EpsW is a substrate of the DifE kinase and it regulates exopolysaccharide in Myxococcus xanthus

Here we attempted to identify the downstream target of the DifE histidine kinase in the regulation of exopolysaccharide (EPS) production in the Gram-negative bacterium Myxococcus xanthus. This bacterium is an important model system for the studies of Type IV pilus (T4P) because it is motile by social (S) motility which is powered by T4P retraction. EPS is critical for S motility because it is the preferred anchor for T4P retraction in this bacterium. Previous studies identified the Dif chemosensory pathway as crucial for the regulation of EPS production. However, the downstream target of the DifE kinase in this pathway was unknown. In this study, EpsW, an orphan and single-domain response regulator (RR), was identified as a potential DifE target first by bioinformatics. Subsequent experiments demonstrated that epsW is essential for EPS biosynthesis in vivo and that EpsW is directly phosphorylated by DifE in vitro. Targted mutagenesis of epsW suggests that EpsW is unlikely the terminal RR of the Dif pathway. We propose instead that EpsW is an intermediary in a multistep phosphorelay that regulates EPS in M. xanthus.