Adenylate Charge Regulates Sensor Kinase CheS 3 To Control Cyst Formation in Rhodospirillum centenum

Abstract

ABSTRACT Rhodospirillum centenum forms metabolically dormant cysts under unfavorable growth conditions such as desiccation or nutrient starvation. The development of cysts is tightly regulated and involves a cyst-repressing chemotaxis-like signal transduction pathway called the Che 3 signaling cascade. The Che 3 cascade is comprised of a methyl chemoreceptor (MCP 3 ), receptor-methylating/demethylating proteins CheB 3 and CheR 3 , two CheW 3 linker proteins, a CheA 3 -CheY hybrid histidine kinase, and a single-domain response regulator, CheY 3 . In addition to Che-like components, the Che 3 cascade also contains a second hybrid histidine kinase, CheS 3 . Recent biochemical and genetic studies show that CheA 3 does not serve as a phosphor donor for CheY 3 ; instead, CheA 3 inhibits a CheS 3 →CheY 3 two-component system by phosphorylating an inhibitory receiver domain of CheS 3 . In this study, we show that in addition to phosphorylation by CheA 3 , the phosphorylation state of CheS 3 is also regulated by the cellular energy level as quantified by the molar ratio of ATP/(ATP + ADP). A 35% decrease in cellular energy is shown to occur in vivo upon a nutrient downshift that gives rise to cyst formation. When this energy decline is replicated in vitro , the phosphorylation level of CheS 3 is reduced by ~75%. Finally, we also show that ADP-mediated reduction of CheS 3 phosphorylation is a consequence of ADP enhancing autodephosphorylation of CheS 3 . IMPORTANCE Upon starvation, Rhodospirillum centenum undergoes a developmental process that forms metabolically dormant cysts, which withstand desiccation and nutritional limitation. This study explores the role of the cellular energy state as measured by the ratio of ATP to ADP as an important regulator of cyst formation in Rhodospirillum centenum . We show that R. centenum cells experience a significant reduction in ATP during cyst formation using ATP/(ATP + ADP) as a measurement. When this in vivo level of energy starvation is simulated in vitro , CheS 3 phosphorylation is reduced by 75%. This profound reduction in CheS 3 autophosphorylation is contrasted with a much lower 25% decrease in CheA 3 phosphorylation in response to a similar downward shift in ATP/(ATP + ADP). We argue that even though adenylate energy affects all ATP-dependent enzymes to an extent, the enhanced inhibition of CheS 3 activity in response to a reduction in the ATP/(ATP + ADP) ratio likely functions as an important input signal to regulate cyst development.