Demonstration of Interactions Among Dif Proteins and the Identification of Kapb as a Regulator of Exopolysaccharide in Myxococcus Xanthus

Myxococcus xanthus Dif proteins are chemotaxis homologues that regulate exopolysaccharide (EPS) biogenesis. Previous genetic studies suggested that Dif protein might interact with one another as do the chemotaxis proteins in enterics. The interactions among Dif proteins were since investigated with the yeast two-hybrid (Y2H) system. The results indicate that DifC interacts with both DifA and DifE. Using a modified Y2H system, DifC was shown to be able to bring DifA and DifE into a protein complex. Further Y2H experiments demonstrated that the different conserved domains of DifE likely function as their counterparts of CheA-type kinases because the putative P2 domain of DifE interacts with DifD, P5 with DifC and the dimerization domain P3 with itself. Similarly, DifA can interact with itself through its C-terminal region. In addition, DifG was found to interact with the CheY homologue DifD. These findings support the notion that Dif proteins constitute a unique chemotaxis-like signal transduction pathway in M. xanthus.

In addition, KapB, a TPR (Tetratricopeptide repeats) protein, was identified as an interacting partner of DifE byY2H library screening. Further analysis demonstrated that the N-terminal half of KapB interacted with the putative P2 domain of DifE. KapB had been previously reported to interact with several Serine/Threonine (Ser/Thr) kinase pathways including the Pkn4-Pfk pathway. This pathway is implicated in glycogen metabolism in M. xanthus by a previous report. In this study, kapB as well as pfkn deletion mutants were found to overproduce EPS. It was also found that the Dif pathway is involved in glycogen metabolism because the glycogen level is altered in dif mutants. These results indicate EPS biogenesis and glycogen metabolism may be coordinately regulated. This coordination of the Dif-regulated EPS production and the Pkn4-regulated glycogen metabolism appears to involve KapB. This is the first example of a TPR protein mediating the interplays of a histidine kinase pathway and a Ser/Thr kinase pathway.