In this study we have identified an additional signaling protein in the Che3 pathway and further defined a complex signal transduction mechanism involving the histidine kinase CrdS, the transcription factor CrdA, and CheA3, which together regulate entry into development in M. xanthus
. Our in vitro
biochemical and in vivo
phenotypic data allow us to propose a model whereby both CrdS and CheA3 cooperatively regulate the phosphorylation state of CrdA (). CrdA~P thereby alters transcription, affecting developmental gene expression (18
). In our model, CrdS is able to act as a dual kinase/phosphatase, directly regulating the phosphorylation state of CrdA. When CrdS senses the appropriate signal, it undergoes autophosphorylation at histidine 371 and subsequently phosphorylates the response regulator CrdA on conserved aspartate residue 53. CrdS is then able to act directly on CrdA~P, leading to a dramatic change in the stability of the phosphoryl group, resulting in rapid dephosphorylation of CrdA. CrdS-mediated dephosphorylation of CrdA is predicted to utilize a mechanism similar to that proposed for NarX-mediated dephosphorylation of NarL (8
). Additionally, we have shown that purified CheA3 is able to dephosphorylate CrdA, consistent with our in vivo
analysis and previous results suggesting that Che3 negatively regulates CrdA during development (18
). Although we have not observed CheA3 kinase activity in our assays, we have demonstrated that CheA3 acts to alter CrdA~P stability. This does not exclude the possibility that CheA3 may also act as a kinase under conditions not yet identified. For instance, M. xanthus
FrzE (CheA1) is active only when both CheW and MCP proteins are present in vitro
). If CheA3 can also function as a kinase under some conditions, this would lead to a more complex regulatory mechanism by which CheA3, like CrdS, could act both as a kinase and as a phosphatase.
FIG 5 Model for regulation of CrdA by both CrdS and CheA3. CrdS serves as the primary histidine kinase regulating the phosphorylation of CrdA. CheA3 can act as a phosphatase on CrdA~P to keep CrdA~P levels low. Activation of CrdA by phosphorylation allows CrdA (more ...)
Identification of CrdS as a putative kinase for CrdA was accomplished by comparing the genomes of M. xanthus and other members in the Myxococcales order. Because crdS, crdA, and crdB cooccur with similar gene neighborhoods, we hypothesized that CrdS and CrdA comprised a cognate histidine kinase-response regulator pair. Thus, the presence of the che3 gene cluster appears to be a recent addition for M. xanthus and its close relative, Stigmatella aurantiaca. Phenotypic analysis of crdS, crdA, and cheA3 mutants provided in vivo evidence that both CrdS and CheA3 regulate CrdA. Mutations in crdS are delayed in aggregation, displaying a phenotype similar to that observed for mutations in crdA. In contrast, overproduction of CrdS in the otherwise wild-type parent background led to an opposing phenotype in which cells were observed to aggregate prematurely, similar to the cheA3 mutant. Lastly, the crdA mutation was found to be epistatic to enhanced production of CrdS (by the PpilA-crdS expression construct) indicating that both CrdS and CheA3 signal transduction are dependent on the presence of CrdA. Thus, CrdS and CrdA comprise a prototypical TCS that is regulated in parallel by CheA3, the central processor within the Che3 chemosensory system.
In M. xanthus, there is relatively little biochemical data detailing HK and RR specificity. We have provided kinetic data for CrdS autophosphorylation, phosphotransfer to CrdA, and dephosphorylation of CrdA by both CrdS and CheA3. The soluble cytoplasmic portion of CrdS, CrdSsoluble, was maximally phosphorylated within 30 minutes, with a corresponding Km of 25 µM and a Vmax of 0.73 µM min−1 using ATP as a substrate. Phosphorylated CrdS and CrdA are relatively stable, exhibiting half-lives of ~120 h for CrdS~P and 54 minutes for CrdA~P. Additionally, phosphotransfer from CrdS~P to CrdA is highly specific, with complete loss of CrdS~P occurring in less than 5 s in the presence of CrdA, while CrdS~P displayed a very low capacity for transfer to both of the alternative targets provided, NtrC_1189 and NtrC_4261. The results indicate high fidelity for the CrdS-CrdA phosphotransfer reaction.
Perhaps our most important observation is that CheA3 can act as a CrdA phosphatase, as indicated by the significant decrease in the half-life for CrdA~P from 54 minutes to 9 minutes when incubated with CheA3. No such difference was observed when an alternative CheA homolog, DifE (or CheA2), was provided in vitro
. Thus, it appears that CheA3 in M. xanthus
may serve a role similar to that of CheA3 in Rhodobacter sphaeroides
. In R. sphaeroides
, CheA3 acts as a phosphatase capable of affecting CheY6~P stability (37
). Interestingly, both CheA3 in M. xanthus
and CheA3 in R. sphaeroides
decrease the half-life of the phosphorylated RR by approximately 4- to 5-fold (37
). While the overall effect of RR dephosphorylation appears to be similar, the underlying mechanism of CheA3-dependent CrdA dephosphorylation is not understood and is currently being investigated.
Many organisms contain complex signaling cascades to control critical, energy-intensive processes such as development. Thus, it is not surprising that M. xanthus
possesses a complicated mechanism to regulate CrdA phosphorylation. However, it is not known how CrdA fits into the overall developmental program. Recent results illustrate that several NtrC-like activators participate within a complex cascade to regulate development for M. xanthus
). No interaction between those NLAs and CrdA has been demonstrated. Additionally, it is not known how CrdS and CheA3 cooperate to regulate CrdA activity. One possibility is that CrdS and components upstream of CheA3 detect similar or related stimuli. CrdB contains a peptidoglycan-binding OmpA domain and requires CheA3 to process signals (18; S. Müller and J. Kirby, unpublished data). Similarly, the crdS
gene cluster encodes a putative Pbp1a peptidoglycan-binding protein. Thus, it is possible that CrdB and CrdS respond to envelope stress to regulate the overall status of CrdA phosphorylation within the cell to affect development.