Coli Phosphotransferase System Research Articles

The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS): an interface between energy and signal transduction

The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates, and is involved in signal transduction. It comprises two general phosphotransferase proteins (EI and HPr) and a—species dependent—variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC). EI and HPr transfer phosphoryl groups from PEP to the IIA units. IIA and IIB sequentially transfer phosphates to the sugar, which is translocated by the IIC unit. The ratio of phosphorylated to non-phosphorylated IIA and IIB varies with transport activity, and the phosphorylation state of some of the IIA and IIB serves as signal input for regulation of catabolite repression, intermediate metabolism, gene expression and chemotaxis in response to the availability of carbohydrates and PEP (glycolytic activity). PTS occur in about one-third of all eubacteria and in a few archaebacteria but not in animals and plants. Uniqueness and pleiotropic function make the PTS a potential target for anti-infectives. The PTS transporter for mannose is utilized as a gate for the penetration of bacteriophage lambda DNA across, and insertion of certain bacteriocins (small antimicrobial peptides) into the inner membrane. The PTS of Escherichia coli is in the focus of this review, but occasionally comparisons with other species are made. The topics are: History; Modular design of the E. coli PTS; Structure function and catalytic mechanism of the protein modules; Regulation of and by the PTS; The PTS in pathogenicity and virulence; Computational models; Metabolic engineering.

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.

EI of the Phosphotransferase System ofEscherichia coli: Mathematical Modeling Approach to Analysis of Its Kinetic Properties

The mathematical model of the operation of the first enzyme of the Escherichia coli phosphotransferase system, EI, is proposed. Parameters of the kinetic model describing the operation of EI under different conditions are identified on the basis of a large amount of known experimental data. The verified model is employed to predict modes of operation of EI under both in vivo physiological conditions and in vitro nonphysiological conditions. The model predicts that under in vivo physiological conditions, the rate of phosphotransfer from EI to the second protein of the phosphotransferase system HPr by the dimer is much higher than by the monomer. A hypothesis is proposed on the basis of calculations that the transfer by a monomer plays a role in the regulation of chemotaxis. At submicromolar pyruvate concentration, the model predicts nonmonotonic dependence of the phosphotransfer rate on the substrate (PEP) concentration.

Spatial and temporal organization of the E. coli PTS components

The phosphotransferase system (PTS) controls preferential use of sugars in bacteria. It comprises of two general proteins, enzyme I (EI) and HPr, and various sugar-specific permeases. Using fluorescence microscopy, we show here that EI and HPr localize near the Escherichia coli cell poles. Polar localization of each protein occurs independently, but HPr is released from the poles in an EI- and sugar-dependent manner. Conversely, the β-glucoside-specific permease, BglF, localizes to the cell membrane. EI, HPr and BglF control the β-glucoside utilization (bgl) operon by modulating the activity of the BglG transcription factor; BglF inactivates BglG by membrane sequestration and phosphorylation, whereas EI and HPr activate it by an unknown mechanism in response to β-glucosides availability. Using biochemical, genetic and imaging methodologies, we show that EI and HPr interact with BglG and affect its subcellular localization in a phosphorylation-independent manner. Upon sugar stimulation, BglG migrates from the cell periphery to the cytoplasm through the poles. Hence, the PTS components appear to control bgl operon expression by ushering BglG between the cellular compartments. Our results reinforce the notion that signal transduction in bacteria involves dynamic localization of proteins.

Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products.

In Escherichia coli, the uptake and phosphorylation of glucose is carried out mainly by the phosphotransferase system (PTS). Despite the efficiency of glucose transport by PTS, the required consumption of 1 mol of phosphoenolpyruvate (PEP) for each mol of internalized glucose represents a drawback for some biotechnological applications where PEP is a precursor of the desired product. For this reason, there is considerable interest in the generation of strains that can transport glucose efficiently by a non-PTS mechanism. The purpose of this work was to study the effect of different gene expression levels, of galactose permease (GalP) and glucokinase (Glk), on glucose internalization and phosphorylation in a E. coli PTS(-) strain. The W3110 PTS(-), designated VH32, showed limited growth on glucose with a specific growth rate (mu) of 0.03 h(-1). A low copy plasmid family was constructed containing E. coli galP and glk genes, individually or combined, under the control of a trc-derived promoter set. This plasmid family was used to transform the VH32 strain, each plasmid having different levels of expression of galP and glk. Experiments in minimal medium with glucose showed that expression of only galP under the control of a wild-type trc promoter resulted in a mu of 0.55 h(-1), corresponding to 89% of the mu measured for W3110 (0.62 h(-1)). In contrast, no increase in specific growth rate (mu) was observed in VH32 with a plasmid expressing only glk from the same promoter. Strains transformed with part of the plasmid family, containing both galP and glk genes, showed a mu value similar to that of W3110. Fermentor experiments with the VH32 strain harboring plasmids pv1Glk1GalP, pv4Glk5GalP, and pv5Glk5GalP showed that specific acetate productivity was twofold higher than in W3110. Introduction of plasmid pLOI1594, coding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, to strain VH32 carrying one of the plasmids with galP and glk caused a twofold increase in ethanol productivity over strain W3110, also containing pLOI1594.

Photolysis of 2-azi-2-deoxy- d- arabino-hexitol and analogous hexitols; the ineffectiveness of certain carbohydrate diazirines as photoaffinity labels

Three diazirines 2-azi-2-deoxy- d- arabino-hexitol (4), its 1-deoxy analogue (5), and 3-azi-3,6-dideoxy- l- xylo-hexitol (9) were synthesised and their products of photolysis analysed by TLC. Diazirine 4 gave exclusively 2-deoxy- d- arabino-hexose (12), 5 gave predominantly 1,2-dideoxy- d- erythro-3-hexulose (10) and 1-deoxy- d-glucitol (11), and 9 did not yield any main product. Carrying out the irradiation of 4 in D 2O gave selectively (2 S)-2-deoxy- d- arabino-(2- 2H)hexose (12a). The results indicate that photolysis of a diazirine flanked by a hydroxymethyl group, as in compound 4, leads to a rapid and stereoselective intramolecular reaction of the intermediate. This may be an explanation of why compound 4 is ineffective as a photoaffinity reagent for mannitol permease ( d-mannitol-specific enzyme II) of the E. coli phosphotransferase system for which it is a substrate. A secondary hydroxymethylene group has a less pronounced effect and still allows some reaction with the medium.

Effect of Bdellovibrio bacteriovirus infection on the phosphoenolpyruvate: Sugar phosphotransferase system in Escherichia coli: Evidence for activation of cytoplasmic proteolysis

Intact cells of Bdellovibrio bacteriovorus strain 109J were found to be incapable of taking up 14C-methyl α-glucoside, mannitol or fructose, and extracts derived from these cells exhibited negligible activities of the protein components of the phosphoenolypyruvate: sugar phosphotransferase system (PTS). Escherichia coli strain ML35 cells exhibited high in vivo sugar uptake activities that were progressively lost over a period of 2 h at 30°C following the entry of B. bacteriovorus into the periplasm of E. coli. It vitro complementation assays revealed that the E. coli PTS enzymes, enzyme 1. HPr, and the glucose- and mannitoi-specific enzymes II, were all lost almost in parallel with the disappearance of uptake activity. Thus, loss of activity in vivo was not due to membrane leakiness, energy depletion, or preferential inhibition or inactivation of any one protein component of the PTS. Instead, loss of PTS activity was attributed to digestion of the protein constituents of the system by proteases present in the cytoplasm of the host cell after bdeliovibrio entry. Both ethylenediaminetetraacetate and phenylmethylsulphonyl fluoride partially protected against inactivation in vitro, and the two inhibitors together gave full protection, suggesting that both metallo-and seryl-proteases were responsible for the inactivation. Protease activity increased progressively with time following bdellovibrio entry and appeared to degrade the E. coli PTS enzymes in vivo. Preliminary evidence suggested that the proteases responsible for PTS enzyme degradation may be encoded by the B. bacteriovorus chromosome.

Structure and evolution of a multidomain multiphosphoryl transfer protein: Nucleotide sequence of the fruB(HI) gene in Rhodobacter capsulatus and comparisons with homologous genes from other organisms

The gene order of the fructose (fru) operon and nucleotide sequence of the first gene (fruB(HI) of Rhodobacter capsulatus are reported, analyzed and compared with homologous genes from other bacteria, and the gene products are identified. Included within the region reported is a gene encoding a multiphosphoryl transfer protein (MTP) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). MTP consists of three moieties: a fructose-specific enzyme III (IIIfru)-like N-terminal moiety (residues 1 to 143) followed by an FPr(HPr)-like moiety (residues 157 to 245) and an enzyme I-like moiety (residues 273 to 827). The enzyme III-like moiety closely resembles the N-terminal 143 residues of the IIIfru-FPR fusion protein from Salmonella typhimurium (40.6% identity throughout its length) and the C-terminal 145 residues of the mannitol-specific enzyme II (IImtl) (37.8% identity throughout its length with the IIImtl moiety of IImtl). The FPr-like domain of MTP resembles the S. typhimurium FPr (42.4% identity) and the Escherichia coli or S. typhimurium HPr (38.8% identity). The enzyme I-like moiety resembles the E. coli enzyme I (38.9% identity). Predicted phosphorylation sites within the three functional units of MTP (His62 in the IIIfru-like moiety; His171 in the FPr-like moiety and His457 in the enzyme I-like moiety) were identified on the basis of sequence comparisons with the homologous proteins from enteric bacteria. The three functional domains of MTP are joined by two flexible "linkage" regions, rich in alanine, glycine and proline, which show 47% sequence identity with each other. They also exhibit a high degree of sequence identity with the linkage region of the mannose-specific enzyme III (IIIman) of the E. coli PTS as well as several other proteins of bacterial, eukaryotic and viral origin. At the RNA level, these linker regions formed hairpin structures with high (90%) G + C content. Analyses of the IIIfru-FPr fusion protein of S. typhimurium revealed that between the IIIfru and FPr moieties of this protein is a stretch of 142 amino acids that do not show homology to known PTS proteins. This region and the adjacent FPr-like region contain a sequence of 110 residues exhibiting 59% similarity to the receiver consensus motif defined by Kofoid and Parkinson. Because the Salmonella IIIfru-FPr fusion protein has been implicated in transcriptional regulation, this region of the Salmonella protein may prove to have regulatory significance.(ABSTRACT TRUNCATED AT 400 WORDS)

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate:glucose phosphotransferase system.

The overall stereochemical course of the reactions leading to the phosphorylation of methyl alpha-D-glucopyranoside by the glucose-specific enzyme II (enzyme IIGlc) of the Escherichia coli phosphotransferase system has been investigated. With [(R)-16O,17O,18O]phosphoenolpyruvate as the phosphoryl donor and in the presence of enzyme I, HPr, and enzyme IIIGlc of the phosphotransferase system, membranes from E. coli containing enzyme IIGlc catalyzed the formation of methyl alpha-D-glucopyranoside 6-phosphate with overall inversion of the configuration at phosphorus (with respect to phosphoenolpyruvate). It has previously been shown that sequential covalent transfer of the phosphoryl group of phosphoenolpyruvate to enzyme I, to HPr, and to enzyme IIIGlc occurs before the final transfer from phospho-enzyme IIIGlc to the sugar, catalyzed by enzyme IIGlc. Because overall inversion of the configuration of the chiral phospho group of phosphoenolpyruvate implies an odd number of transfer steps, the phospho group has been transferred at least five times, and transfer from phospho-enzyme IIIGlc to the sugar must occur in two steps (or a multiple thereof). On the basis that no membrane protein other than enzyme IIGlc is directly involved in the final phospho transfer steps, our results imply that a covalent phospho-enzyme IIGlc is an intermediate during transport and phosphorylation of glucose by the E. coli phosphotransferase system.