Developments in Defining dif.

The Gram-negative soil bacterium Myxococcus xanthus has a complex life cycle that involves vegetative growth and, when nutrients are limiting, formation of spore-containing fruiting bodies. The progression of the developmental cycle depends in part upon the ability of M. xanthus cells to sense and respond to their environment and to coordinate two different motility systems to achieve directed movements. The dif genes were originally isolated by Yang et al. (13) as part of a search for mutants defective in fruiting body formation. It was later determined that they encode a chemosensory pathway that regulates the production of extracellular polysaccharides (EPS), which are essential for M. xanthus social (S) motility and fruiting body formation (9, 14). Social motility is powered by the extension and retraction of type IV pili (TFP) (8, 11); when pili bind to EPS found on the surface of cells or on slime trails, they trigger retraction, pulling cells forward (10). Single and combinatorial dif mutants show that, despite the strong homology of dif-encoded proteins to core components of bacterial chemosensory systems, the Dif pathway does not regulate EPS production directly through a phosphorylated response regulator protein but rather through unidentified intermediates (1, 5, 14). To clarify Dif signaling, Black et al. (2) have now studied in vitro phosphorylation and dephosphorylation of purified Dif proteins to establish the relationships between these proteins and their respective biochemical properties. These results provide a solid foundation upon which to base future work to untangle the various and varied aspects of Dif regulation. As shown in Fig. 1, five of the six dif genes show good homology to characterized chemosensory proteins: DifA is a membranebound receptor similar to other methyl-accepting chemotaxis proteins (MCPs), DifC is a CheW-like coupling protein, DifE contains a CheA-like autokinase domain, DifD is a CheY-like response regulator protein, and DifG is a homolog of the Bacillus subtilis CheY phosphatase, CheC. Yeast twoand three-hybrid experiments indicate that DifA, DifC, and DifE form a ternary complex similar to those found in other chemosensory systems (15). Yeast two-hybrid studies also revealed binding affinities between DifE and DifD and between DifD and DifG (15). Mutational inactivation of difA, difC, or difE caused the loss of EPS production by M. xanthus (1, 14). However, inactivation of DifD or DifG caused EPS overproduction (5). To explain their data, Black and colleagues proposed that the receiver domain protein DifD serves as a phosphate sink, with DifG acting as a phosphatase to regenerate the population of phosphate-accepting DifD (Fig. 1). Thus, the DifA-DifC-DifE ternary complex positively regulates EPS production, while DifD and DifG act in concert as negative regulators. The biochemical studies now published by Black et al. (2) provide clear confirmation that DifE is indeed an autokinase that can efficiently autophosphorylate in vitro. Furthermore, the incubation of phosphorylated DifE (DifE P) with DifD results in the rapid transfer of phosphate to DifD, as expected. However, DifD P proved unusually stable with a half-life of 30 min; dephosphorylation of DifD was greatly accelerated by relatively low concentrations of DifG, showing that DifG is an efficient phosphatase. DifD and DifG are likely to serve as a phosphate sink for DifE. However, the biochemical activities established here do not account for the phenotype of a difD difG double mutant, which produces more EPS than do difD and difG single mutants (4). DifG may influence the Dif pathway downstream of DifE phosphorylation since DifG does not affect DifE autokinase activity in vitro. Interestingly, DifE autokinase activity was inhibited by the addition of DifA and DifC. In other systems, including the M. xanthus Frz chemosensory system, the MCP and coupling protein strongly enhance the level of autophosphorylation of the CheA histidine kinase homolog (7). The authors suggest that this modification may reflect the diversity of inputs for the Dif system. For example, the Dif pathway regulates the ability of M. xanthus to respond to phosphatidylethanolamine (PE) (6), in addition to regulating EPS production. EPS production is subject to positive stimulation of the Dif system by type IV pili (TFP), which are responsible for powering S motility (4). Conversely, the Dif pathway inhibits cell reversals when cells respond to PE (6). The authors propose that one way that Dif could accommodate these distinct inputs is by maintaining populations of DifA in different conformations. Further work is needed to determine if and how DifE autokinase activity might be altered by its association with DifA and DifC based on the nature of the Dif stimulus. Understanding Dif function will require the assimilation of observations concerning both EPS production and PE responses. The various dif mutants produce differing effects on PE-based behaviors. For example, wild-type cells show a reduction in cell reversal frequency when exposed to PE; mutants lacking DifA, DifC, or DifE do not. This phenotype might at first be attributed to the failure of these mutant strains to produce EPS; however, the DifD mutant, which produces an * Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720. Phone: (510) 642-2293. Fax: (510) 643-6334. E-mail: [email protected]. Published ahead of print on 2 July 2010.