Two intercellular signals required for fruiting body formation in Myxococcus xanthus act sequentially but non-hierarchically.

Starvation-induced fruiting body formation in Myxococcus xanthus depends on intercellular signalling. A-signal functions after 2 h of starvation and its synthesis depends on the asg genes. C-signal functions after 6 h of starvation and is generated by proteolytic cleavage of a precursor by the protease PopC. Previous gene expression studies suggested that the A- and C-signal lie on a hierarchical pathway. Here we explored the causal relationship between the A- and C-signal. The asgA and asgB mutants have reduced popC expression, PopC accumulation and C-signal accumulation. popC expression was shown not to depend on A-signal but on the AsgA and AsgB proteins. Restored popC expression in the two mutants rescued PopC and C-signal accumulation as well as C-signalling and the developmental defects of the two mutants without restoring A-signalling. Based on these results we suggest that A- and C-signal do not lie on a hierarchical, dependent pathway. Instead the A- and C-signal act sequentially and without a causal relationship suggesting that they are linked by a shared timing mechanism, which ensures the early and late onset of A-signalling and C-signalling, respectively, during starvation. This pathway topology represents a novel architecture for bacterial intercellular signalling systems involving more than one signal.