Aktivierung eines kontakt-abhängigen Signalsystems durch regulierte Proteolyse in Myxococcus xanthus

In response to starvation M. xanthus initiates a complex developmental program that culminates in the formation of spore-filled fruiting bodies. The C-signal induces multiple responses including aggregation of cells into fruiting bodies, sporulation and expression of developmentally regulated genes. The C-signal is a 17 kDa cell surface-associated protein (p17) that is synthesized by proteolytic cleavage of the 25 kDa full-length CsgA protein (p25). We have previously shown that the protease (PopC) involved in cleavage of p25 is a serine protease, developmentally regulated and likely to be secreted. Based on the this information, we developed a three-tiered strategy to identify popC candidates. First, 146 genes likely to encode proteases in the M. xanthus genome were identified. Second, among these genes 32 likely to encode secreted serine proteases (including 10 trypsin-like proteases, 10 subtilisin-like proteases, eight HtrA homologs and four homologs of Rhomboid proteases) were identified. Based on the biochemical characteristics of these four protease families, the HtrA and Rhomboid homologs were unlikely to be identical to PopC. Finally, DNA microarray expression data from developing cells showed that four of the subtilisin-like proteases are up-regulated during development. To test whether any of these four proteases could be identical to PopC we inactivated the four corresponding genes by insertion mutagenesis. Here we show that MXAN0206 encodes PopC. Inactivation of popC results in a mutant that aggregates to construct abnormal fruiting bodies and has a severe sporulation defect. popC is likely in an operon with a downstream gene. Complementation of the popC mutant with a wild type copy of the popC gene restored the developmental defects, thus, providing evidence that PopC is important for development. Interestingly, accumulation of p17 cannot be detected in the popC mutant. popC encodes a 51 kDa subtilase-like protease containing the characteristic catalytic triad of subtilases. An active site mutant of popC displays the same developmental defects as a mutant with an insertion in popC and is unable to synthesize p17. PopC accumulates in vegetative and starving cells; however, PopC is selectively secreted in starving cells. Additionally we could show in an in vitro protease assay, that purified PopC cleaves p25 directly to generate p17. We propose that regulated MXAN0206 secretion guarantees that p25 and PopC are only present in the same cell compartment during starvation, thus, restricting p17 synthesis to starving cells.