Identification and analysis of Escherichia coli proteins that interact with the histidine kinase NtrB in a yeast two-hybrid system.

In this work we used the yeast two-hybrid (Y2H) system to deepen our understanding of protein-protein interactions that are involved in the nitrogen regulatory network in Escherichia coli. Three different genes, encoding GlnB, GlnK and AspA, respectively, were found among 64 positive clones identified from E. coli Sau3AI Y2H libraries using the nitrogen regulator NtrB as bait. Structural and functional analysis of the prey clones provided information on library features and the degree of saturation achieved in the screens. Further analysis revealed that the C-terminal kinase domain of NtrB is required for the interaction with GlnK, while AspA(91-312) interacts specifically with the conserved histidine phosphotransfer domain of NtrB, thus providing additional evidence for the involvement of the conserved transmitter module of the histidine kinase NtrB in input sensory functions.