Proteins of two-component systems (TCS) have essential functions in the sensing of external and selfgenerated signals in bacteria and in the generation of appropriate output responses. Accordingly, in Myxococcus xanthus, TCS are important for normal motility and fruiting body formation and sporulation. Here we analyzed the M. xanthus genome for the presence and genetic organization of genes encoding TCS. Two hundred seventy-two TCS genes were identified, 251 of which are not part of che gene clusters. We report that the TCS genes are unusually organized, with 55% being orphan and 16% in complex gene clusters whereas only 29% display the standard paired gene organization. Hybrid histidine protein kinases and histidine protein kinases predicted to be localized to the cytoplasm are overrepresented among proteins encoded by orphan genes or in complex gene clusters. Similarly, response regulators without output domains are overrepresented among proteins encoded by orphan genes or in complex gene clusters. The most frequently occurring output domains in response regulators are involved in DNA binding and cyclic-di-GMP metabolism. Our analyses suggest that TCS encoded by orphan genes and complex gene clusters are functionally distinct from TCS encoded by paired genes and that the connectivity of the pathways made up of TCS encoded by orphan genes and complex gene clusters is different from that of pathways involving TCS encoded by paired genes. Experimentally, we observed that orphan TCS genes are overrepresented among genes that display altered transcription during fruiting body formation. The systematic analysis of the 25 orphan genes encoding histidine protein kinases that are transcriptionally up-regulated during development showed that 2 such genes are likely essential for viability and identified 7 histidine protein kinases, including 4 not previously characterized that have important function in fruiting body formation or spore germination.A fundamental property of cells is their ability to sense and respond to external stimuli and self-generated signals. In the case of bacteria, this ability maximizes their chances of survival. Signal transduction proteins have essential functions in stimulus sensing, information processing, and the generation of output responses. Despite the multitude of cues that bacteria need to monitor, the signal transduction schemes involved center on a few types (11, 65): ligand-regulated one-component systems, which consist of single protein molecules containing both a sensing domain and an output domain; cyclic-di-GMP synthetases, phosphodiesterases, adenylate and guanylate cyclases, which act by modifying the level of secondary messenger molecules; methyl-accepting chemotaxis proteins that modulate the activity of chemosensory systems; and systems in which information transfer depends on covalent modification by phosphorylation/dephosphorylation by means of either Ser/ Thr/Tyr protein kinases or histidine protein kinases (HPKs). HPKs are the more common type of protein kinase in bacte...

Genetic programs underlying multicellular morphogenesis and cellular differentiation are most often associated with eukaryotic organisms, but examples also exist in bacteria such as the formation of multicellular, spore-filled fruiting bodies in the order Myxococcales. Most members of the Myxococcales undergo a multicellular developmental program culminating in the formation of spore-filled fruiting bodies in response to starvation. To gain insight into the evolutionary history of fruiting body formation in Myxococcales, we performed a comparative analysis of the genomes and transcriptomes of five Myxococcales species, four of these undergo fruiting body formation (Myxococcus xanthus, Stigmatella aurantiaca, Sorangium cellulosum, and Haliangium ochraceum) and one does not (Anaeromyxobacter dehalogenans). Our analyses show that a set of 95 known M. xanthus development-specific genes--although suffering from a sampling bias--are overrepresented and occur more frequently than an average M. xanthus gene in S. aurantiaca, whereas they occur at the same frequency as an average M. xanthus gene in S. cellulosum and in H. ochraceum and are underrepresented in A. dehalogenans. Moreover, genes for entire signal transduction pathways important for fruiting body formation in M. xanthus are conserved in S. aurantiaca, whereas only a minority of these genes are conserved in A. dehalogenans, S. cellulosum, and H. ochraceum. Likewise, global gene expression profiling of developmentally regulated genes showed that genes that upregulated during development in M. xanthus are overrepresented in S. aurantiaca and slightly underrepresented in A. dehalogenans, S. cellulosum, and H. ochraceum. These comparative analyses strongly indicate that the genetic programs for fruiting body formation in M. xanthus and S. aurantiaca are highly similar and significantly different from the genetic program directing fruiting body formation in S. cellulosum and H. ochraceum. Thus, our analyses reveal an unexpected level of plasticity in the genetic programs for fruiting body formation in the Myxococcales and strongly suggest that the genetic program underlying fruiting body formation in different Myxococcales is not conserved. The evolutionary implications of this finding are discussed.

In Myxococcus xanthus, two-component systems have crucial roles in regulating motility behavior and development. Here we describe an orphan response regulator, consisting of an N-terminal receiver domain and a C-terminal DNA binding domain, which is required for A and type IV pilus-dependent gliding motility. Genetic evidence suggests that phosphorylation of the conserved, phosphorylatable aspartate residue in the receiver domain is required for DigR activity. Consistent with the defect in type IV pilus-dependent motility, a digR mutant is slightly reduced in type IV pilus biosynthesis, and the composition of the extracellular matrix fibrils is abnormal, with an increased content of polysaccharides and decreased accumulation of the FibA metalloprotease. By using genome-wide transcriptional profiling, 118 genes were identified that are directly or indirectly regulated by DigR. These 118 genes include only 2, agmQ and cheY4, previously implicated in A and type IV pilus-dependent motility, respectively. In silico analyses showed that 36% of the differentially expressed genes are likely to encode exported proteins. Moreover, four genes encoding homologs of extracytoplasmic function (ECF) sigma factors, which typically control aspects of cell envelope homeostasis, are differentially expressed in a digR mutant. We suggest that the DigR response regulator has an important function in cell envelope homeostasis and that the motility defects in a digR mutant are instigated by the abnormal cell envelope and abnormal expression of agmQ and cheY4.

SummaryIn prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus , aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.

SummaryStarvation-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 Aand 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 Aand 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.