Two-component systems (TCSs) are virtually ubiquitous signaling pathways, which typically comprise a histidine kinase (HK) that monitors an external signal and a response regulator (RR) that controls cellular responses, communicating via kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute entire TCS signaling pathways in single proteins, with a relaxed specificity of intramolecular phosphotransfer compared to the extreme intermolecular specificity typical of classical HK-RR TCSs.

We investigated the specificity of phosphotransfer within hybrid HKs, focusing on the GacS-GacA system of Pseudomonas brassicacearum. To explore potential phosphotransfer interactions between the different hybrid HKs of the GacS kinase cluster, we focused experimentally on the receiver domains of the hybrid HKs, creating chimeric proteins by replacing the GacS native receiver domain with receiver domains from each of the GacS receiver cluster hybrid HKs. Our results demonstrate a link between the evolutionary history of domains in hybrid HKs and their ability to interact to form functional signaling pathways (‘phylogenetic promiscuity’), and support the use of phylogenetic arguments to infer possible functional interactions between proteins.