Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli

The global regulator Mlc controls several genes implicated in sugar utilization systems, notably the phosphotransferase system (PTS) genes, ptsG, manXYZ and ptsHI, as well as the malT activator. No specific low molecular weight inducer has been identified that can inactivate Mlc, but its activity appeared to be modulated by transport of glucose via Enzyme IICB(Glc) (PtsG). Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICB(Glc). We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICB(Glc) phosphorylation by the general PTS proteins and also Enzyme IICB(Glc)-mediated phosphorylation of alpha-methylglucoside. Binding of Mlc to Enzyme IICB(Glc) in vitro required the IIB domain and the IIC-B junction region. Moreover, we show that these same regions are sufficient for Mlc regulation in vivo, via cross-dephosphorylation of IIB(Glc) during transport of other PTS sugars. The control of Mlc activity by sequestration to a transport protein represents a novel form of signal transduction in gene regulation.     

YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12

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 glucose-phosphotransferase 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-alpha-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.     

Correlation between growth rates, EIIACrr phosphorylation, and intracellular cyclic AMP levels in Escherichia coli K-12

In Escherichia coli K-12, components of the phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) represent a signal transduction system involved in the global control of carbon catabolism through inducer exclusion mediated by phosphoenolpyruvate-dependent protein kinase enzyme IIA(Crr) (EIIA(Crr)) (= EIIA(Glc)) and catabolite repression mediated by the global regulator cyclic AMP (cAMP)-cAMP receptor protein (CRP). We measured in a systematic way the relation between cellular growth rates and the key parameters of catabolite repression, i.e., the phosphorylated EIIA(Crr) (EIIA(Crr) approximately P) level and the cAMP level, using in vitro and in vivo assays. Different growth rates were obtained by using either various carbon sources or by growing the cells with limited concentrations of glucose, sucrose, and mannitol in continuous bioreactor experiments. The ratio of EIIA(Crr) to EIIA(Crr) approximately P and the intracellular cAMP concentrations, deduced from the activity of a cAMP-CRP-dependent promoter, correlated well with specific growth rates between 0.3 h(-1) and 0.7 h(-1), corresponding to generation times of about 138 and 60 min, respectively. Below and above this range, these parameters were increasingly uncoupled from the growth rate, which perhaps indicates an increasing role executed by other global control systems, in particular the stringent-relaxed response system.