Phosphate flow between hybrid histidine kinases CheA₃ and CheS₃ controls Rhodospirillum centenum cyst formation

Abstract

Genomic and genetic analyses have demonstrated that many species contain multiple chemotaxis-like signal transduction cascades that likely control processes other than chemotaxis. The Che₃ signal transduction cascade from Rhodospirillum centenum is one such example that regulates development of dormant cysts. This Che-like cascade contains two hybrid response regulator-histidine kinases, CheA₃ and CheS₃, and a single-domain response regulator CheY₃. We demonstrate that cheS₃ is epistatic to cheA₃ and that only CheS₃∼P can phosphorylate CheY₃. We further show that CheA₃ derepresses cyst formation by phosphorylating a CheS₃ receiver domain. These results demonstrate that the flow of phosphate as defined by the paradigm E. coli chemotaxis cascade does not necessarily hold true for non-chemotactic Che-like signal transduction cascades.

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 .

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.

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.