Figure 1.
Gene arrangement of the R. centenum che3 cluster and domain organizations of CheA3, CheS3, and CheY3.
Arrow length is proportional to gene length. Abbreviations: REC, receiver domain; PAS, Per, Arnt, Sim domains; HWE_HK, HWE superfamily of histidine kinases; Hpt, histidine phosphotransfer domain; CA, catalytic and ATP-binding domain. Conserved histidine and aspartate residues as putative phosphorylation sites are denoted for each protein. The start and end amino acid positions of the receiver domains as well as those of the full proteins are also labeled according to the prediction by SMART [48].
Figure 2.
Characterization of cheS3, cheA3 and cheY3 mutants.
The encystment phenotypes of 11 strains including wild type and single, double, and point mutants of cheS3, cheA3 and cheY3 were measured qualitatively by phase contrast microscopy and quantitatively by flow cytometry. (A) Growth on nutrient-rich CENS medium reveals hyper-cyst strains that overproduce cysts relative to wild type. (B) Growth on nutrient-limiting CENBA medium identifies hypo-cyst strains that under-produce cyst cells relative to wild type. Error bars in the bar graphs represent standard deviation obtained from two biological replicates. * (p<0.05), ** (p<0.01) when compared to the wild type (wt) strain in an unpaired t-test.
Figure 3.
Metal ion dependent phosphorylation of CheA3 and CheS3.
(A) Four possible phosphorylation states of phosphorylated hybrid histidine kinases (HHKs). (B) Metal cation dependencies of phosphorylation of isolated HHKs CheS3 and CheA3 and their receiver domain mutants.
Figure 4.
Identification of intramolecular phosphoryl transfer within CheA3 and CheS3.
(A) CheA3∼P is acid- and alkaline-labile, whereas the REC mutant CheA3:D663A∼P is acid-labile and base-resistant. (B) Both CheS3∼P and its REC mutant CheS3:D54A are acid-labile and alkaline-stable. (C) CheA3:D663A∼P phosphorylates CheA3-REC truncation protein in Buffer 15 containing K+ and 18 mM Mg2+. (D) Phosphoryl transfer from CheS3:D54A∼P to CheS3-REC1 truncation protein was not observed in Buffer 15.
Table 1.
Half-lives of phospho-HHKs.
Figure 5.
Intermolecular phosphoryl transfer events assayed among CheS3, CheA3, and CheY3.
2–5 µM CheS3, CheA3, or their REC mutant forms were autophosphorylated in 200 µM ATP for 30 min before 1/10 volumes of 65 mM CheY3 or REC domain truncations were added. (A, B) Neither CheA3∼P nor CheA3:D663A∼P are able to phosphorylate CheY3 in Buffer 9 containing K+ and 6 mM Ca2+. (C, D) CheS3∼P and CheS3:D54A∼P phosphorylates CheY3 within 15 sec of CheY3 addition in Buffer 5 containing Na+, 3 mM Ca2+, and 3 mM Mg2+. (E) Intermolecular phosphoryl transfer analyses of CheA3 and CheS3. (A) CheA3:D663A∼P phosphorylates CheS3-REC1 in Buffer 15 containing K+ and 18 mM Mg2+. (F) CheS3 is unable to phosphorylate CheA3-REC in Buffer 15.
Figure 6.
Model for regulation of Che3 signal transduction pathway.
(A) In the absence of unknown signals, CheA3 is deactivated; CheS3 autophosphorylates and transfers phosphates to its cognate response regulator CheY3; activated CheY3 then interacts with downstream components to repress cyst formation. (B) In the presence of an unknown signal (denoted by a red star), CheA3 autophosphorylation is activated; His-phosphorylated CheA3 constantly transfers the phosphates to its C-terminal REC domain, which serves as a phosphate sink. CheA3∼P also phosphorylates the REC1 domain of CheS3, inhibiting CheS3 kinase activity and CheY3 remains unphosphorylated. Cyst formation is therefore derepressed without activated CheY3. The thickness of the arrows represents the level of phosphate flow.