Research On An Interaction Between PTS Component-EI And A Histidine Kinase-response Regulator Hybrid Protein In Gluconobacter Oxydans

While Gluconobacter oxydans may contain an incomplete phosphoenolpyruvate sugar phosphotransferase system (PTS), which is composed of EI, HPr and EIIA, the function of each component remains unknown. We screened a histidine kinase-response regulator hybrid protein (HK) from G. oxydans which interacts with El by yeast two-hybrid assay, and the specificity of the interaction was verified by GST pull down assay in vitro and bimolecular fluorescence complementary assay in vivo, respectively. Furthermore, the N-terminal domains of El and HK were preliminarily identified to involve the interaction.Sequence analysis revealed that HK may be a member of two-component system, which can transmit the signal through phosphate transferring. To analyze the role of HK in bacteria, its ATPase activity was firstly determined in vitro. We further analyzed the autophosphorylation activity of HK and discovered that HK is capable of sensing the concentration of an important second messenger in cells - cAMP. A low concentration of cAMP can enhance its autophosphorylation activity significantly. All the properties identified indicated that HK is indeed a member of two-component system in bacteria.As PTS and two-component system shared a similar phosphate transfer mechanism, the interaction between El and HK indicated that functional association may exist between the two systems, a proposal important for exploring the metabolism of carbon and nitrogen of G. oxydans under stress.