Regulation of the glucose-specific phosphotransferase system (PTS) of Staphylococcus carnosus by the antiterminator protein GlcT.
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The ptsG operon of Staphylococcus carnosus consists of two adjacent genes, glcA and glcB, encoding glucose- and glucoside-specific enzymes II, respectively, the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The expression of the ptsG operon is glucose-inducible. Putative RAT (ribonucleic antiterminator) and terminator sequences localized in the promoter region of glcA suggest regulation via antitermination. The glcT gene was cloned and the putative antiterminator protein GlcT was purified. Activity of this protein was demonstrated in vivo in Escherichia coli and Bacillus subtilis. In vitro studies led to the assumption that phosphoenolpyruvate-dependent phosphorylation of residue His105 via the general PTS components enzyme I and HPr facilitates dimerization of GlcT and consequently activation. Because of the high similarity of the two ptsG-RAT sequences of B. subtilis and S. carnosus, in vivo studies were performed in B. subtilis. These indicated that GlcT of S. carnosus is able to recognize ptsG-RAT sequences of B. subtilis and to cause antitermination. The specific interaction between B. subtilis ptsG-RAT and S. carnosus GlcT demonstrated by surface plasmon resonance suggests that only the dimer of GlcT binds to the RAT sequence. HPr-dependent phosphorylation of GlcT facilitates dimer formation and may be a control device for the proper function of the general PTS components enzyme I and HPr necessary for glucose uptake and phosphorylation by the corresponding enzyme II. (+info)
Molecular analysis of sucrose metabolism of Erwinia amylovora and influence on bacterial virulence.
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Sucrose is an important storage and transport sugar of plants and an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight pathogen Erwinia amylovora, a chromosomal fragment which enabled Escherichia coli to utilize sucrose as sole carbon source was cloned. By transposon mutagenesis, the scr regulon of E. amylovora was tagged, and its nucleotide sequence was determined. Five open reading frames, with the genes scrK, scrY, scrA, scrB, and scrR, had high homology to genes of the scr regulons from Klebsiella pneumoniae and plasmid pUR400. scrB and scrR of E. amylovora were fused to a histidine tag and to the maltose-binding protein (MalE) of E. coli, respectively. ScrB (53 kDa) catalyzed the hydrolysis of sucrose with a K(m) of 125 mM. Binding of a MalE-ScrR fusion protein to an scrYAB promoter fragment was shown by gel mobility shifts. This complex dissociated in the presence of fructose but not after addition of sucrose. Expression of the scr regulon was studied with an scrYAB promoter-green fluorescent protein gene fusion and measured by flow cytometry and spectrofluorometry. The operon was affected by catabolite repression and induced by sucrose or fructose. The level of gene induction correlated to the sucrose concentration in plant tissue, as shown by flow cytometry. Sucrose mutants created by site-directed mutagenesis did not produce significant fire blight symptoms on apple seedlings, indicating the importance of sucrose metabolism for colonization of host plants by E. amylovora. (+info)
Characterization of the ccpA gene of Enterococcus faecalis: identification of starvation-inducible proteins regulated by ccpA.
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Inactivation of ccpA in Enterococcus faecalis leads to reduction of the growth rate, derepression of the galKETR operon in the presence of a mixture of glucose and galactose, and reduction of transcription of ldh in the presence of glucose. Moreover, the E. faecalis ccpA gene fully complements a Bacillus subtilis ccpA mutant, arguing for similar functions of these two homologous proteins. Protein comparison on two-dimensional gels from the wild-type cells and the ccpA mutant cells revealed a pleiotropic effect of the mutation on gene expression. The HPr protein of the carbohydrate-phosphotransferase system was identified by microsequencing, and a modification of its phosphorylation state was observed between the wild-type and the mutant strains. Moreover, at least 16 polypeptides are overexpressed in the mutant, and 6 are repressed. Interestingly, 13 of the 16 polypeptides whose synthesis is enhanced in the mutant were also identified as glucose starvation proteins. The N-terminal amino acid sequences of four of them match sequences deduced from genes coding for L-serine dehydratase, dihydroxyacetone kinase (two genes), and a protein of unknown function from Deinococcus radiodurans. (+info)
A mutation which affects both the specificity of PtsG sugar transport and the regulation of ptsG expression by Mlc in Escherichia coli.
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Normally glucosamine (GlcN) is not a substrate for EIICB(Glc) of the glucose phosphotransferase system (PTS), encoded by ptsG, but it is transported by the mannose (Man) PTS, encoded by manXYZ. A mutation, umgC, has been described in Escherichia coli which allows a strain mutated in the Man PTS to grow on GlcN. The umgC mutation was mapped to the ptsG region and was proposed to make ptsG expression constitutive. Transcription of ptsG is regulated by the repressor Mlc so that mutations in mlc enhance the expression of ptsG. An mlc mutation, however, is not sufficient to allow good growth on GlcN, unlike the umgC mutation. The umgC mutation is shown to enhance expression of ptsG even in the absence of any PTS sugar transport, but the increase is greater in the presence of GlcN or Man. The umgC mutation also increases expression of the ptsHI and manXYZ operons, which are both regulated by Mlc. The umgC mutation was sequenced and two mutations were found: one, G176D, within the IIC membrane domain and the second, E472K, within the soluble IIB domain of PtsG. The cloned UmgC allele shows the enhanced transport and regulatory characteristics of the chromosomal mutation. Analysis of the two mutations present individually on plasmids shows that the IIC mutation is responsible for both the effect on sugar specificity and regulation. (+info)
Catabolite repression and induction of the Mg(2+)-citrate transporter CitM of Bacillus subtilis.
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In Bacillus subtilis the citM gene encodes the Mg(2+)-citrate transporter. A target site for carbon catabolite repression (cre site) is located upstream of citM. Fusions of the citM promoter region, including the cre sequence, to the beta-galactosidase reporter gene were constructed and integrated into the amyE site of B. subtilis to study catabolic effects on citM expression. In parallel with beta-galactosidase activity, the uptake of Ni(2+)-citrate in whole cells was measured to correlate citM promoter activity with the enzymatic activity of the CitM protein. In minimal media, CitM was only expressed when citrate was present. The presence of glucose in the medium completely repressed citM expression; repression was also observed in media containing glycerol, inositol, or succinate-glutamate. Studies with B. subtilis mutants defective in the catabolite repression components HPr, Crh, and CcpA showed that the repression exerted by all these medium components was mediated via the carbon catabolite repression system. During growth on inositol and succinate, the presence of glutamate strongly potentiated the repression of citM expression by glucose. A reasonable correlation between citM promoter activity and CitM transport activity was observed in this study, indicating that the Mg(2+)-citrate uptake activity of B. subtilis is mainly regulated at the transcriptional level. (+info)
A novel regulatory role of glucose transporter of Escherichia coli: membrane sequestration of a global repressor Mlc.
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External glucose stimulates transcription of several genes including ptsG encoding IICB(Glc), a membrane component of the phosphotransferase system (PTS), by relieving the negative regulation of a global repressor Mlc in Escherichia coli. We investigate here how glucose modulates Mlc action. The Mlc-mediated repression is eliminated by a ptsI mutation, while Mlc is constitutively active in a ptsG mutant. We show that IICB(Glc)-FLAG interacts physically with Mlc in crude extracts prepared from cells in which IICB(Glc) is supposed to exist as the non-phosphorylated form. The IICB(Glc)-Mlc interaction is no longer observed when IICB(Glc) is phosphorylated. Exogenously added purified Mlc binds to purified IICB(Glc)-FLAG. We also demonstrate that Mlc is associated with membrane when IICB(Glc) is dephosphorylated while it is in the cytoplasm when IICB(Glc) is phosphorylated or absent. We conclude that IICB(Glc) regulates the cellular localization of Mlc, depending on its phosphorylation state, which is determined by the availability of external glucose. Thus, glucose induces the transcription of Mlc-regulated promoters by sequestering Mlc to the membrane through dephosphorylation of IICB(Glc). (+info)
Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli.
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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. (+info)
A novel membrane anchor function for the N-terminal amphipathic sequence of the signal-transducing protein IIAGlucose of the Escherichia coli phosphotransferase system.
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Enzyme IIA(Glucose) (IIA(Glc)) is a signal-transducing protein in the phosphotransferase system of Escherichia coli. Structural studies of free IIA(Glc) and the HPr-IIA(Glc) complex have shown that IIA(Glc) comprises a globular beta-sheet sandwich core (residues 19-168) and a disordered N-terminal tail (residues 1-18). Although the presence of the N-terminal tail is not required for IIA(Glc) to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIA(Glc) to the membrane-bound IIBC(Glc). The sequence of the N-terminal tail suggests that it has the potential to form an amphipathic helix. Using CD, we demonstrate that a peptide, corresponding to the N-terminal 18 residues of IIA(Glc), adopts a helical conformation in the presence of either the anionic lipid phosphatidylglycerol or a mixture of anionic E. coli lipids phosphatidylglycerol (25%) and phosphatidylethanolamine (75%). The peptide, however, is in a random coil state in the presence of the zwitterionic lipid phosphatidylcholine, indicating that electrostatic interactions play a role in the binding of the lipid to the peptide. In addition, we show that intact IIA(Glc) also interacts with anionic lipids, resulting in an increase in helicity, which can be directly attributed to the N-terminal segment. From these data we propose that IIA(Glc) comprises two functional domains: a folded domain containing the active site and capable of weakly interacting with the peripheral IIB domain of the membrane protein IIBC(Glc); and the N-terminal tail, which interacts with the negatively charged E. coli membrane, thereby stabilizing the complex of IIA(Glc) with IIBC(Glc). This stabilization is essential for the final step of the phosphoryl transfer cascade in the glucose transport pathway. (+info)