Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation inMyxococcus xanthus

Abstract

Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifEϳP), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifDϳP). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifDϳP has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifDϳP as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifEϳP.

CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate

“…DifD is involved in both polysaccharide biosynthesis associated with social motility and chemotaxis. This stability has been related to the slow gliding movement of M. xanthus and its prolonged adaptation time for chemotaxis (5). At this time, we do not know why CheY3-P has such a prolonged half-life; however, it points to CheX, besides CheA2, playing a critical role in modulating CheY-P concentration.…”

PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide

“…In M. xanthus, phosphorylation and dephosphorylation among Dif chemosensory proteins are essential for the regulation of EPS production (Black et al, 2010). Also, many other proteins are likely to play minor regulatory roles in M. xanthus EPS production (Berleman et al, 2011;Black et al, 2006;Dana & Shimkets, 1993;Kimura et al, 2004;Petters et al, 2012;Weimer et al, 1998).…”

Chemosensory signaling systems that control bacterial survival

“…DifG subsequently regenerates unphosphorylated DifD by functioning as a DifD~P phosphatase. This model is supported by in vivo experiments which shows an interaction between DifD and DifG in two hybrid experiments and in vitro where biochemically isolated DifG dephosphorylates DifD~P [22]. Furthermore, since the CheY-like DifD functions as a negative regulator of EPS biosynthesis, it has been hypothesized that unidentified mediators exist that relay a signal transduced from DifE to produce an uncharacterized output [21] (Figure 2A).…”