The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK(2)) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK(2) complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V(max) of 1.25 micromol P(i)/min/mg and a K(m) of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA(Glc), a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.

The signal-transducing protein EIIA Glc , a component of the phosphoenolpyruvate-glucose phosphotransferase system, plays a key role in carbon regulation in enteric bacteria, such as Escherichia coli and Salmonella typhimurium. The phosphorylation state of EIIA Glc governs transport and metabolism of a number of carbohydrates. When glucose as preferred carbon source is transported, EIIA Glc becomes predominantly unphosphorylated and allosterically inhibits several permeases, including the maltose ATP-binding cassette transport system (MalFGK 2 ) in a process termed "inducer exclusion." We have mapped the binding surface of EIIA Glc that interacts with the MalK subunits by using synthetic cellulose-bound peptide arrays like pep scan-and substitutional analyses. Three regions constituting two binding sites were identified encompassing residues 69 -79 (I), 87-91 (II), and 118 -127 (III). Region III is MalK-specific, whereas residues from regions I and II partly overlap but are not identical to the binding interfaces for interaction with glycerol kinase and lactose permease. These results were fully verified by studying the inhibitory effect of purified EIIA Glc variants carrying mutations at positions representative of each of the three regions on the ATPase activity of the purified maltose transport complex reconstituted into proteoliposomes. Moreover, a synthetic peptide encompassing residues 69 -91 was demonstrated to partially inhibit ATPase activity. We also show for the first time that the N-terminal domain of EIIA Glc is essential for inducer exclusion.Complex bacterial activities, like those involved in the quest of food, require the coordination of entire metabolic networks. In enteric bacteria, such as Escherichia coli and Salmonella typhimurium, the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) 2 plays a key role in this process as a signal transduction system (1). The PTS comprises a cascade of protein kinases and phosphocarriers that constitute a series of transport systems, which couple transport and phosphorylation of numerous sugars. The pathway requires the sequential transfer of a phosphoryl group from phosphoenolpyruvate via EI, HPr to the sugar-specific EII components. "Catabolite repression" allows for the preferential utilization of so-called Class A sugars mostly transported by the PTS, when cells are cultured in the presence of other PTS or non-PTS sugars (Class B). Consequently, the PTS regulates the uptake of Class B-sugars by both transcriptional and post-transcriptional mechanisms. The phosphorylation state of the glucose-specific EIIA Glc component plays a key role in this process. Phosphorylated

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