Light is an essential environmental cue for diverse organisms. Many prokaryotic blue light photoreceptors use Light, Oxygen, Voltage (LOV) sensory domains to control the activities of diverse output domains, including histidine kinases (HK). Upon activation, these proteins autophosphorylate a histidine residue before subsequently transferring the phosphate to an aspartate residue in the receiver domain of a cognate response regulator (RR). Such phosphorylation, activates the output domain of the RR, leading to changes in gene expression, protein-protein interactions or enzymatic activities. Here we focus on one such light sensing LOV-HK from the marine bacterium Erythrobacter litoralis HTCC2594 (EL368), seeking to understand how kinase activity and subsequent downstream effects are regulated by light. We found that photoactivation of EL368 led to a significant enhancement in the incorporation of phosphate within the HK domain. Further enzymatic studies showed that the LOV domain affected both the LOV-HK turnover rate (kcat) and Km in a light-dependent manner. Using in vitro phosphotransfer profiling, we identified two target RRs for EL368 and two additional LOV-HKs (EL346 and EL362) encoded within the host genome. The two RRs include a PhyR-type transcriptional regulator (EL_PhyR) and a receiver-only protein (EL_LovR), reminiscent of stress-triggered systems in other bacteria. Taken together, our data provide a biochemical foundation for this light-regulated signaling module of sensors, effectors and regulators that control bacterial responses to environmental conditions.

To survive and adapt to environmental changes, bacteria commonly use two component signaling systems. Minimally, these pathways use histidine kinases (HKs) to detect environmental signals, harnessing these to control phosphorylation levels of receiver (REC) domains of downstream response regulators that convert this signal into physiological responses. Studies of several prototypical REC domains suggest that phosphorylation shifts these proteins between inactive and active structures that are globally similar and well-folded. However, it is unclear how globally these findings hold within REC domains in general, particularly when considered within full-length proteins. Here we present EL_LovR, a full-length REC-only protein that is phosphorylated in response to blue light in the marine α-proteobacterium Erythrobacter litoralis HTCC2594. Notably, EL_LovR is similar to comparable REC-only proteins used in bacterial general stress responses, where genetic evidence suggests that their potent phosphatase activity is important to shut off such systems. Size exclusion chromatography, light scattering and solution NMR experiments show that EL_LovR is monomeric and unfolded in solution under conditions routinely used for other REC structure determinations. Addition of Mg2+ and phosphorylation induce progressively greater degrees of tertiary structure stabilization, with the solution structure of the fully-activated EL_LovR adopting the canonical receiver domain fold. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink that depletes the HK phosphoryl group, promoting the phosphatase activity of this enzyme. Our findings demonstrate that EL_LovR undergoes substantial ligand-dependent conformational changes that have not been reported for other RRs, expanding the scope of conformational changes and regulation used by REC domains, critical components of bacterial signaling systems.

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