No abstract

The membrane-bound protein EIICB Glc encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucosephosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-␣-D-glucopyranoside, a substrate of EIICB Glc , reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB Glc . In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.In Escherichia coli K-12, as in many other gram-positive and gram-negative bacteria, the phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems (PTSs) are the major transport and sensor systems for carbohydrates. All of the PTSs except the mannose-specific PTS consist of five conserved functional domains, designated enzyme I (EI) (gene, ptsI), the histidine-containing phosphoryl carrier protein HPr (gene, ptsH), enzyme IIA (EIIA), enzyme IIB (EIIB), and enzyme IIC (EIIC) (for reviews see references 30 and 31). Depending on the organism or system, these functional PTS domains exist as single or multidomain proteins. The two cytoplasmic proteins, EI and HPr, are the general components of all PTS, whereas the EII complexes are carbohydrate specific. The protein kinase EI uses phosphoenolpyruvate in an autophosphorylation reaction, and the phosphoryl group is subsequently transferred to HPr, EIIA, and EIIB. Finally, the carbohydrate substrate, which is bound by the integral membrane domain of EIIC, is phosphorylated and concomitantly translocated across the membrane. The preferred carbon source of E. coli, D-glucose, is taken up by two different EIIs, the highaffinity glucose-specific molecule EII Glc (Glc-PTS) and the low-affinity mannose-specific molecule EII Man (Man-PTS).The Glc-PTS consists of the cytoplasmic protein EIIA Glc , encoded by the crr g...

The glucose-phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory and regulatory system. In addition to its central role in glucose uptake, it informs other global regulatory networks about carbohydrate availability and the physiological status of the cell. The expression of the ptsG gene encoding the glucose-PTS transporter EIICB Glc is primarily regulated via the repressor Mlc, whose inactivation is glucose dependent. During transport of glucose and dephosphorylation of EIICB Glc , Mlc binds to the B domain of the transporter, resulting in derepression of several Mlc-regulated genes. In addition, Mlc can also be inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. In this study, we identified the binding site for Mlc in the carboxy-terminal region of MtfA by measuring the effect of mutated MtfAs on ptsG expression. In addition, we demonstrated the ability of MtfA to inactivate an Mlc super-repressor, which cannot be inactivated by EIICB Glc , by using in vivo titration and gel shift assays. Finally, we characterized the proteolytic activity of purified MtfA by monitoring cleavage of amino 4-nitroanilide substrates and show Mlc's ability to enhance this activity. Based on our findings, we propose a model of MtfA as a glucose-regulated peptidase activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as they enter the stationary phase. This proteolytic activity of MtfA modulated by Mlc constitutes a newly identified PTS output signal that responds to changes in environmental conditions. D espite the fact that Escherichia coli K-12 has been an important model organism for many decades, the function of Ͼ20% of all putative open reading frames in its genome remains unknown. Moreover, in many cases, no obvious sequence similarities with previously characterized proteins exist. The mtfA gene (formerly named yeeI), a monocistronic gene located at 44.1 min in the E. coli chromosome, has clearly belonged to this group. Intensive sequence similarity searches revealed that orthologs of MtfA (mnemonic for Mlc titration factor A) exist in more than 100 proteobacteria of the alpha, beta, and gamma subdivisions. In a previous report (2), we were able to demonstrate that E. coli MtfA is a cytoplasmic protein that binds to the carboxy-terminal part of the Mlc repressor protein (mnemonic for "making large colonies" [gene, dgsA]) with a very high affinity. Mlc is one of the global regulators of carbohydrate metabolism in E. coli and is especially involved in the regulation of the ptsG gene expression, which encodes the glucose transporter EIICB Glc . The EIICB Glc together with the cytoplasmic protein EIIA Glc , encoded by the crr gene (as part of the ptsHI-crr operon), forms the glucosespecific phosphoenolpyruvate (PEP)-dependent carbohydratephosphotransferase system (glucose-PTS). Both proteins take part in a phosphorylation cascade, which begins with an autophosphorylation reaction of the so-called enzyme I (EI; gene, ptsI) at the expense...