Demonstration of interactions among Myxococcus xanthus Dif chemotaxis-like proteins by the yeast two-hybrid system

The Myxococcus xanthus dif locus encodes several bacterial chemotaxis homologues that are crucial for fibril exopolysaccharide (EPS) production, social gliding motility, and fruiting body development. In primary sequence, DifA is homologous to methyl-accepting chemotaxis protein, DifC to CheW, DifD to CheY, DifE to CheA, and DifG to CheC. In this study, the interactions among the Dif chemotaxis-like proteins were investigated using the yeast two-hybrid (Y2H) system. DifC was found to interact with both DifA and DifE. Using a modified Y2H or a "three-hybrid" system, it was demonstrated that DifC is capable of mediating the formation of DifA, DifC, and DifE ternary protein complexes. The conserved domains of DifE, based on sequence analysis, likely reflect functional conservations of CheA-type kinases, because its P2 domain interacts with DifD, P5 with DifC, and the P3 domain appears to dimerize. Similarly, C-terminal regions of DifA appear to dimerize as well. In addition, DifG was found to interact with DifD, which is consistent with the hypothesis that DifG is a phosphatase of DifD-phosphate. These findings support the models in which Dif proteins constitute a unique chemotaxis-like signal transduction pathway with central functions in regulating EPS production in M. xanthus.