In low GC content Gram-positive bacteria, the HPr protein is the master regulator of carbon metabolism. HPr is a key component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system that interacts with and/or phosphorylates proteins relevant to carbon catabolite repression. HPr can be phosphorylated by two distinct kinases as follows: the bifunctional enzyme HPr kinase/Ser(P)-HPr phosphorylase (HprK/P) phosphorylating the serine 46 residue (Ser(P)-HPr) and acting as a phosphorylase on Ser(P)-HPr; and the PEP-requiring enzyme I (EI) generating histidine 15-phosphorylated HPr (His(P)-HPr). The various HPr forms interact with numerous enzymes and modulate their activity. By carrying out a genome-wide yeast two-hybrid screen of a Bacillus subtilis library, we identified a novel HPr-interacting protein, the transcriptional activator YesS, which regulates the expression of pectin/rhamnogalacturonan utilization genes. Remarkably, yeast tri-hybrid assays involving the ATP-dependent HprK/P and the PEP-dependent EI suggested that YesS interacts with HPr and His(P)-HPr but not with Ser(P)-HPr. These findings were confirmed by in vitro interaction assays using the purified HPr-binding domain of the YesS protein. Furthermore, pectin utilization and in vivo YesS-mediated transcriptional activation depended upon the presence of His(P)-HPr, indicating that HPr-mediated YesS regulation serves as a novel type of carbon catabolite repression. In the yeast two-hybrid assays, B. subtilis HprK/P and EI were active and specifically recognized their substrates. Both kinases formed long lived complexes only with the corresponding nonphosphorylatable mutant HPr. These findings suggest that two-hybrid assays can be used for the identification of unknown kinases of phosphorylated bacterial proteins detected in phosphoproteome analyses.