Determination of glucose preference over mannitol by a general PTS component HPr

Abstract

Bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) plays a major role in the transport of a variety of sugar substrates and phosphorylates them at the same time. It consists of two general cytoplasmic proteins, enzyme I (EI) and the histidine phosphocarrier protein, HPr, which are used for all PTS sugars, and a variable number of sugar-specific transport complexes collectively known as enzyme II (EII). EII complexes usually consist of three domains (A, B and C). They contain a membrane-bound C domain that takes charge of transporting sugar into the cytoplasm. These PTS proteins are known to participate in intracellular signal transduction and regulation of metabolic enzymes as well as transport function. Carbon catabolite repression (CCR) is the regulatory phenomenon to ensure sequential utilization of carbon sources in microorganisms. Its molecular mechanism has been most intensively studied in the model organisms Escherichia coli and Bacillus subtilis. Glucose is the most preferred carbon source in these organisms, being transported via the PTS. Previous studies on CCR suggested a model based on inducer exclusion that includes inhibition of transport of several non-PTS sugars and induction prevention that includes lowering of adenylate cyclase activity. Both pathways are regulated mainly by the phosphorylation state of enzyme IIAGlc (EIIAGlc) encoded by the crr (catabolite repression resistant) gene. In E. coli, the preferential utilization of glucose over non-PTS carbon sources such as lactose is known to be strictly dependent on the phosphorylation state of the EIIAGlc. On the contrary, the mechanism of the preference among PTS sugars has never been addressed. The regulatory mechanism of glucose-mannitol diauxie is thought to be far different from that of glucose-lactose diauxie. In this study, the molecular mechanism of the glucose preference over another PTS sugar, mannitol in E. coli was investigated. Surprisingly, the crr mutant retained the glucose preference over mannitol, whereas this selectivity disappeared in a mutant devoid of MtlR that negatively regulates regulating the expression of the mtlADR operon required for the transport and metabolism of mannitol. MtlR showed a high affinity interaction with the dephosphorylated form of the general PTS component HPr, which increased during glucose uptake. This interaction decreased the expression of the mtlADR operon by enhancing the repressor activity of MtlR and resulted in the inhibition of mannitol utilization. The regulatory machinery of the mtl operon mediated by MtlR is still unclear since MtlR cannot directly interact with the promoter region of the mtl operon. To gain further insight, the crystal structure of MtlR-HPr complex was solved but no DNA binding domain was identified. Based on various experiments performed in this study, an unknown DNA-binding protein seems to be required to mediate the DNA binding of MtlR. Taken together, the results reveal HPr as the key player in the sugar selectivity between glucose and mannitol.