Mohammad Aboulwafa

The Ascorbate Transporter of Escherichia coli

The sgaTBA genes of Escherichia coli encode a putative 12-transmembrane alpha-helical segment (12 TMS) transporter, an enzyme IIB-like protein and an enzyme IIA-like protein of the phosphotransferase system (PTS), respectively. We show that all three proteins as well as the energy-coupling PTS proteins, enzyme I and HPr, are required for the anaerobic utilization and uptake of L-ascorbate in vivo and its phosphoenolpyruvate-dependent phosphorylation in vitro. The transporter exhibits an apparent K(m) for L-ascorbate of 9 micro M and is highly specific. The sgaTBA genes are regulated at the transcriptional level by the yjfQ gene product, as well as by Crp and Fnr. The yjfR gene product is essential for L-ascorbate utilization and probably encodes a cytoplasmic L-ascorbate 6-phosphate lactonase. We conclude that SgaT represents a novel prototypical enzyme IIC that functions with SgaA and SgaB to allow phosphoryl transfer from HPr(his-P) to L-ascorbate via the phosphoryl transfer pathway: [pathway: see text].

Dependency of sugar transport and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system on membranous phosphatidyl glycerol in Escherichia coli: Studies with a pgsA mutant lacking phosphatidyl glycerophosphate synthase

It has been reported that phosphatidyl glycerol (PG) is specifically required for the in vitro activities of the hexose-phosphorylating Enzymes II of the Escherichia coli phosphoenolpyruvate-dependent sugar transporting phosphotransferase system (PTS). We have examined this possibility by measuring the properties of a null pgsA mutant that lacks detectable PG. The mutant showed lower in vitro phosphorylation activities towards several sugars when both PEP-dependent and sugar-phosphate-dependent [14C]sugar phosphorylation reactions were measured. The order of dependency on PG for the different enzymes II was: IIMannose > IIGlucose > IIFructose > IIMannitol. Nonsedimentable (40000 rpm for 2 h) Enzymes II exhibited a greater dependency on PG than pelletable Enzymes II. Western blot analyses showed that the glucose Enzyme II is present in normal amounts. Transport and fermentation measurements revealed diminished activities for all Enzymes II. Thermal stability of all of these enzymes except the mannitol-specific Enzyme II was significantly decreased by the pgsA mutation, and sensitivity to detergent treatments was enhanced. Sugar transport proved to be the most sensitive indicator of proper Enzyme II-phospholipid association. Our results show that PG stimulates but is not required for Enzyme II function in E. coli.

Regulation of crp Gene Expression by the Catabolite Repressor/Activator, Cra, in Escherichia coli

Growth of E. coli on several carbon sources is dependent on the catabolite repressor/activator (Cra) protein although a Cra consensus DNA-binding site is not present in the control regions of the relevant catabolic operons. We show that Cra regulates growth by activating expression of the crp gene. It thereby mediates catabolite repression of catabolic operons by an indirect mechanism.

Dependency of sugar transport and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system on membranous phosphatidylethanolamine in Escherichia coli : studies with a pssA mutant lacking phosphatidylserine synthase

An isogenic pair of Escherichia coli strains lacking (pssA) and possessing (wild-type) the enzyme phosphatidylserine synthase was used to estimate the effects of the total lack of phosphatidylethanolamine (PE), the major phospholipid in E. coli membranes, on the activities of several sugar permeases (enzymes II) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The mutant exhibits greatly elevated levels of phosphatidylglycerol (PG), a lipid that has been reported to stimulate the in vitro activities of several PTS permeases. The activities, thermal stabilities, and detergent sensitivities of three PTS permeases, the glucose enzyme II (IIGlc), the mannose enzyme II (IIMan) and the mannitol enzyme II (IIMtl), were characterized. Western blot analyses revealed that the protein levels of IIGlc were not appreciably altered by the loss of PE. In the pssA mutant, IIGlc and IIMan activities were depressed both in vivo and in vitro, with the in vivo transport activities being depressed much more than the in vitro phosphorylation activities. IIMtl also exhibited depressed transport activity in vivo but showed normal phosphorylation activities in vitro. IIMan and IIGlc exhibited greater thermal lability in the pssA mutant membranes than in the wild-type membranes, but IIMtl showed enhanced thermal stability. All three enzymes were activated by exposure to TritonX100 (0.4%) or deoxycholate (0.2%) and inhibited by SDS (0.1%), but IIMtl was the least affected. IIMan and, to a lesser degree, IIGlc were more sensitive to detergent treatments in the pssA mutant membranes than in the wild-type membranes while IIMtl showed no differential effect. The results suggest that all three PTS permeases exhibit strong phospholipid dependencies for transport activity in vivo but much weaker and differential dependencies for phosphorylation activities in vitro, with IIMan exhibiting the greatest and IIMtl the least dependency. The effects of lipid composition on thermal sensitivities and detergent activation responses paralleled the effects on in vitro phosphorylation activities. These results together with those previously published suggest that, while the in vivo transport activities of all PTS enzymes II require an appropriate anionic to zwitterionic phospholipid balance, the in vitro phosphorylation activities of these same enzymes show much weaker and differential dependencies. Alteration of the phospholipid composition of the membrane thus allows functional dissection of transport from the phosphorylation activities of PTS enzyme complexes.

Characterization of Soluble Enzyme II Complexes of the Escherichia coli Phosphotransferase System

Plasmid-encoded His-tagged glucose permease of Escherichia coli, the enzyme IIBCGlc (IIGlc), exists in two physical forms, a membrane-integrated oligomeric form and a soluble monomeric form, which separate from each other on a gel filtration column (peaks 1 and 2, respectively). Western blot analyses using anti-His tag monoclonal antibodies revealed that although IIGlc from the two fractions migrated similarly in sodium dodecyl sulfate gels, the two fractions migrated differently on native gels both before and after Triton X-100 treatment. Peak 1 IIGlc migrated much more slowly than peak 2 IIGlc. Both preparations exhibited both phosphoenolpyruvate-dependent sugar phosphorylation activity and sugar phosphate-dependent sugar transphosphorylation activity. The kinetics of the transphosphorylation reaction catalyzed by the two IIGlc fractions were different: peak 1 activity was subject to substrate inhibition, while peak 2 activity was not. Moreover, the pH optima for the phosphoenolpyruvate-dependent activities differed for the two fractions. The results provide direct evidence that the two forms of IIGlc differ with respect to their physical states and their catalytic activities. These general conclusions appear to be applicable to the His-tagged mannose permease of E. coli. Thus, both phosphoenolpyruvate-dependent phosphotransferase system enzymes exist in soluble and membrane-integrated forms that exhibit dissimilar physical and kinetic properties.

Soluble sugar permeases of the phosphotransferase system in Escherichia coli: evidence for two physically distinct forms of the proteins in vivo

The bacterial phosphoenolpyruvate‐dependent phosphotransferase system (PTS) consists of a set of cytoplasmic energy‐coupling proteins and various integral membrane permeases/sugar phosphotransferases, each specific for a different sugar. We have conducted biochemical analyses of three PTS permeases (enzymes II), the glucose permease (IIGlc), the mannitol permease (IIMtl) and the mannose permease (IIMan). These enzymes each catalyse two vectorial/chemical reactions, sugar phosphorylation using phosphoenolpyruvate (PEP) as the phosphoryl donor, dependent on enzyme I, HPr and IIA as well as IIBC (the PEP reaction), and transphosphorylation using a sugar phosphate (glucose‐6‐P for IIGlc and IIMan; mannitol‐1‐P for IIMtl) as the phosphoryl donor, dependent only on IIBC (the TP reaction). When crude extracts of French‐pressed or osmotically shocked Escherichia coli cells are centrifuged in an ultracentrifuge at high speed, 5–20% of the enzyme II activity remains in the high‐speed supernatant, and passage through a gel filtration column gives two activity peaks, one in the void volume exhibiting high PEP‐dependent and TP activities, and a second included peak with high PEP‐dependent activity and high (IIMan), moderate (IIGlc) or negligible (IIMtl) TP activities. Both log and stationary phase cells exhibit comparable relative amounts of pelletable and soluble enzyme II activities, but long‐term exposure of cells to chloramphenicol results in selective loss of the soluble fraction with retention of much of the pelleted activity concomitant with extensive protein degradation. Short‐term exposure of cells to chloramphenicol results in increased activities in both fractions, possibly because of increased lipid association, with more activation in the soluble fraction than in the pelleted fraction. Western blot analyses show that the soluble IIGlc exhibits a subunit size of about 45 kDa, and all three soluble enzymes II elute from the gel filtration column with apparent molecular weights of 40–50 kDa. We propose that enzymes II of the PTS exist in two physically distinct forms in the E. coli cell, one tightly integrated into the membrane and one either soluble or loosely associated with the membrane. We also propose that the membrane‐integrated enzymes II are largely dimeric, whereas the soluble enzymes II, retarded during passage through a gel filtration column, are largely monomeric.

Characterization of the E. coli glucose permease fused to the maltose-binding protein.

The ptsG gene that encodes the major glucose transporter of Escherichia coli, IIGlc, was inserted into a pMALE‐ampr expression vector down‐stream of the malE gene which encodes the E. coli maltose‐binding protein (MBP). IIGlc‐MBP in the 2 h high speed supernatant of cell lysates eluted from a gel filtration column showing two activity peaks. The glucose‐6‐phosphate‐dependent transphosphorylation (TP) activity of the membrane bound oligomeric peak 1 showed substrate inhibition while that of the soluble monomeric peak 2 did not. Purification of peak 2 yielded activity with weak substrate inhibition, and further gel filtration analyses showed that upon purification, some of the monomeric IIGlc‐MBP associated to higher molecular size forms. Assays of the phosphoenolpyruvate‐dependent and transphosphorylation reactions showed that the specific activity of the purified enzyme from peak 1 was approximately double that from peak 2. The results show that the monomeric IIGlc‐MBP exhibits no substrate inhibition although the oligomeric form does. Purification promotes subunit association, an increase in catalytic activity, and restoration of substrate inhibition. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Biophysical Studies of the Membrane-Embedded and Cytoplasmic Forms of the Glucose-Specific Enzyme II of the E. coli Phosphotransferase System (PTS)

The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-IIGlc-YFP and MBP-IIGlc-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET-) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET+). The monomeric species could form a heterodimeric species (FRET+) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-IIGlc activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-IIGlc retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-IIGlc indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-IIGlc.

In vitro Interconversion of the Soluble and Membrane- Integrated Forms of the Escherichia coli Glucose Enzyme II of the Phosphoenolpyruvate-Dependent Sugar-Transporting Phosphotransferase System

In previous publications, we have shown that integral membrane sugar permeases of the bacterial phosphotransferase system can exist in a ‘soluble’ (probably micellar) monomeric form (SII) as well as a membrane-integrated dimeric form (MII). We here show that the two forms of the his-tagged glucose permease of Escherichia coli can be interconverted in vitro. Conversion of MII to SII is promoted by (1) low protein concentration, (2) detergent, (3) high pH, and (4) phospholipase A2 treatment. Conversion of SII to MII is promoted by: (1) high protein concentration, (2) adherence to and elution from an Ni2+ column, (3) neutral pH, and (4) incorporation into phospholipid liposomes.

Lipid dependencies, biogenesis and cytoplasmic micellar forms of integral membrane sugar transport proteins of the bacterial phosphotransferase system

Permeases of the prokaryotic phosphoenolpyruvate-sugar phosphotransferase system (PTS) catalyse sugar transport coupled to sugar phosphorylation. The lipid composition of a membrane determines the activities of these enzyme/transporters as well as the degree of coupling of phosphorylation to transport. We have investigated mechanisms of PTS permease biogenesis and identified cytoplasmic (soluble) forms of these integral membrane proteins. We found that the catalytic activities of the soluble forms differ from those of the membrane-embedded forms. Transport via the latter is much more sensitive to lipid composition than to phosphorylation, and some of these enzymes are much more sensitive to the lipid environment than others. While the membrane-embedded PTS permeases are always dimeric, the cytoplasmic forms are micellar, either monomeric or dimeric. Scattered published evidence suggests that other integral membrane proteins also exist in cytoplasmic micellar forms. The possible functions of cytoplasmic PTS permeases in biogenesis, intracellular sugar phosphorylation and permease storage are discussed.