Regulation of secretion of the signalling protease PopC in Myxococcus xanthus
In response to starvation Myxococcus xanthus initiates a developmental program that culminates in fruiting body formation. Completion of this developmental program depends on cell-cellcommunication involving at least two intercellular signals, the A-signal and the C-signal. The contact-dependent int...
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|Summary:||In response to starvation Myxococcus xanthus initiates a developmental program that culminates in fruiting body formation. Completion of this developmental program depends on cell-cellcommunication involving at least two intercellular signals, the A-signal and the C-signal. The contact-dependent intercellular C-signal function to induce and coordinate the two morphogenetic events in fruiting body formation, aggregation and sporulation, temporally and spatially coordinated. The intercellular C-signal is a 17 kDa protein (p17), which is generated by proteolytic cleavage of the full-length 25 kDa csgA protein (p25), and is essential for fruiting body formation. p25 and PopC, the protease that cleaves p25, accumulate in the outer membrane and cytoplasm, respectively in vegetative cells. PopC is specifically secreted during starvation. Therefore, restriction of p25 cleavage to starving cells depends on a compartmentalization mechanism that involves the regulated secretion of PopC in response to starvation. In this report, the main focus is on understanding the mechanism underlying regulated secretion of the PopC protease.
We first focused on the identification of proteins required for PopC secretion. PopC lacks a signal peptide and is secreted in an unprocessed form. We report that two incomplete type III secretion systems, a type VI secretion system and type I secretion systems are not involved in PopC secretion. From a collection of mutants generated by random transposon mutagenesis and unable to complete fruiting body formation, we identified seven mutants unable to secrete PopC. None of the insertions were in genes coding for known secretion systems. The mutations were divided into three classes based on the insertion sites. The class I mutation was in a gene cluster largely encoding proteins of unknown function, predicted to localize to the cell envelope, and with a narrow phylogenetic distribution except for a D,D-carboxypeptidase and two Ser/Thr kinases. The class II mutations were in two clusters encoding paralogous proteins of unknown function predicted to localize to the cytoplasm. Several of the class II genes are phylogenetically widely distributed and frequently present in gene clusters linked to genes encoding secretion systems. We speculate that the class I mutation affect a novel type of secretion system involved in PopC secretion and that the class II mutations either affect proteins with accessory or regulatory functions in PopC secretion.
Next, we focused on elucidating the molecular mechanism underlying the activation of PopC secretion in response to starvation. Our data demonstrate that PopC secretion is controlled at the post-translational level by a regulatory cascade involving the RelA and PopD proteins. Specifically, RelA is required for activation of PopC secretion in response to starvation and PopD, which is encoded in an operon with PopC, interacts directly with PopC and acts as an inhibitor of PopC secretion. On the basis of genetic and biochemical data we suggest that PopC and PopD form a cytoplasmic complex that blocks PopC secretion in the presence of nutrients. In response to starvation, RelA is activated resulting in induction of the stringent response. Activated RelA by an unknown mechanism induces the proteolytic degradation of PopD in the PopC/PopD complex in that way releasing PopC for secretion. On the basis of these data, we suggest that the generation of p17 depends on a two-step proteolytic cascade involving degradation of PopD and, subsequently, the specific cleavage of p25 by PopC.
The current model for intercellular A-signaling in M. xanthus proposes that starvation induces the release of extracellular A-signal proteases. These proteases are thought to cleave surface-exposed proteins and extracellular proteins thereby generating the A-signal amino acids and peptides, which serve to measure the density of starving cells early during development. DNA microarray analyses (S. Wegener-Feldbrügge, unpubl.) previously suggested that the primary defect in the asgA and asgB mutants, which are unable to generate the A-signal, is not a reduced capacity in protein secretion but a reduced expression of genes encoding secreted proteases including popC. Here, genetic analyses demonstrated that restored expression of popCD rescues development of asgA and asgB mutants without restoring A-signaling. Thus, ectopic expression of popCD leads to a bypass of the requirement for the A-signal during development. We suggest that the inability of asgA and asgB mutants to undergo development is the result of at least two defects: (i) reduced expression of the genes encoding the A-signal proteases; and, (ii) reduced expression of the popC gene.|