Mechanism of sugar transport through the sugar-specific LamB channel ofEscherichia coli outer membrane

Summary Lipid bilayer experiments were performed with the sugar-specific LamB (maltoporin) channel ofEscherichia coli outer membrane. Single-channel analysis of the conductance steps caused by LamB showed that there was a linear relationship between the salt concentration in the aqueous phase and the channel conductance, indicating only small or no binding between the ions and the channel interior. The total or the partial blockage of the ion movement through the LamB channel was not dependent on the ion concentration in the aqueous phase. Both results allowed the investigation of the sugar binding in more detail, and the stability constants of the binding of a large variety of sugars to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the sugars. The channel was highly cation selective, both in the presence and absence of sugars, which may be explained by the existence of carbonyl groups inside the channel. These carbonyl groups may also be involved in the sugar binding via hydrogen bonds. The kinetics of the sugar transport through the LamB channel were estimated relative to maltose by assuming a simple one-site, two-barrier model from the relative rates of permeation taken from M. Luckey and H. Nikaido (Proc. Natl. Acad. Sci. USA 77:165–171 (1980a)) and the stability constants for the sugar binding given in this study.     

Heptose-deficient mutants ofEscherichia coli K12 deficient in up to three major outer membrane proteins

Molecular Genetics and Genomics - Mutants ofEscherichia coli K12, deficient in up to three major outer membrane proteinsb,c andd have been constructed. Mutants that lack the lipopolysaccharide...     

Expression of foreign antigens on the surface ofEscherichia coli by fusion to the outer membrane protein TraT

ThetraT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between anEscherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane ofE. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence oftraT, was amplified and obtained by PCR. This sequence was then subcloned downstream of thetac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane ofE. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as anE. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

Role of the Escherichia coli Tat pathway in outer membrane integrity

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.     

Role of a major outer membrane protein in Escherichia coli.

Mutants of Escherichia coli B/r lacking a major outer membrane protein, protein b, were obtained by selecting for resistance to copper. These mutants showed a decreased ability to utilize a variety of metabolites when the metabolites were present at low concentrations. Also, mutants of E. coli K-12 lacking proteins b and c from the outer membrane were shown to have an identical defect in the uptake of various metabolites. These results are discussed with regard to their implications as to the role of these proteins in permeability of the outer membrane,     

Sorting of lipoproteins to the outer membrane in E. coli

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane. The structures of LolA and LolB are remarkably similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta-barrel and an alpha-helical lid. Structural differences between the two proteins reveal the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB. Strong inner membrane retention of lipoproteins occurs with Asp at position 2 and a few limited residues at position 3. The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing their retention in the inner membrane. The positive charge of phosphatidylethanolamine and the negative charge of Asp at position 2 are essential for Lol avoidance. The Lol avoidance signal is speculated to cause the formation of a tight lipoprotein-phosphatidylethanolamine complex that has five acyl chains and therefore cannot be recognized by LolCDE.     

Sorting of lipoproteins to the outer membrane in E. coli

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane. The structures of LolA and LolB are remarkably similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta-barrel and an alpha-helical lid. Structural differences between the two proteins reveal the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB. Strong inner membrane retention of lipoproteins occurs with Asp at position 2 and a few limited residues at position 3. The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing their retention in the inner membrane. The positive charge of phosphatidylethanolamine and the negative charge of Asp at position 2 are essential for Lol avoidance. The Lol avoidance signal is speculated to cause the formation of a tight lipoprotein-phosphatidylethanolamine complex that has five acyl chains and therefore cannot be recognized by LolCDE.     

Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli.

Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented.     

Influence of membrane components in the binding of proteins to membrane surfaces

We have quantified the enhancement of membrane binding of activated and deactivated Galpha(s) and Galpha(q) subunits, Gbetagamma subunits, and phospholipase Cbeta(2) by lipid rafts and by the presence of membrane-associated protein partners. Membrane binding studies show that lipid rafts do not affect the intrinsic membrane affinity of Galpha(q)(GDP) and Galpha(s)(GDP), supporting the idea that these proteins partition evenly between the domains. Visualization of lipid rafts on monolayers by use of a probe that does not enter raft domains shows that neither activated nor deactivated Galpha(q)(GDP) subunits distribute evenly between the raft and nonraft domains, contrary to previous suggestions. Membrane binding of deactivated Galpha(q) and Galpha(s)(GDP) became weaker when Gbetagamma subunits were present, in contrast with the behavior predicted by thermodynamics. However, activated Galpha subunits and phospholipase Cbeta(2) were recruited to membrane surfaces by protein partners by predicted amounts. Our studies suggest that the anomalous behavior seen for deactivated Galpha subunits in the presence of Gbetagamma subunits may be due to conformational changes in the N-terminus and/or occlusion of a portion of its membrane interaction region by Gbetagamma. Even though membrane recruitment was clearly observed for one protein partner, the presence of a second partner of lower affinity did not further promote membrane binding. For these proteins, the formation of larger protein complexes with very high membrane affinities is unlikely.     

In vitro reassembly of the membranous vesicle from Escherichia coli outer membrane components: Role of individual compnents and magnesium ions in reassembly

A method was developed for the reassembly of membranous vesicle from the sodium dcoxycholate-dissociated outer membrane components of Escherichia coli. The removal of the detergent by dialysis and the presence of Mg²⁺ were essential for the reassembly.Membrane protein alone did not form any membranous structure. Closed membranous vesicles similar to the native outer membrane were reassembled only when protein was mixed with both lipopolysaccharide and phospholipid in deoxycholate solution and subsequently dialyzed. The membrane showed a distinct trilaminar structure with a center-to-center distance between two dark lines of 53 Å, which is a characteristic of the native outer membrane. This characteristic trilaminar structure was shown to be due to the presence of lipopolysaccharide. Phospholipd was required for the vesicularization of membrane. Lipopolysaccharide and/or phospholipid formed a membranous structure in the absence of protein, while the morphology of their negatively stained sample was quite different from that of the native outer membrane unless the outer membrane protein was added to the reassembly mixture.The protein from the cytoplasmic membrane was unable to reform membranous vesicle with lipopolysaccharide and phospholipid, indicating that the reassembly system discriminated outer membrane proteins from cytoplasmic membrane proteins.     

Selectivity in lipid binding to the bacterial outer membrane protein OmpF

The outer membrane porin OmpF from Escherichia coli has been reconstituted into lipid bilayers of defined composition, and fluorescence spectroscopy is used to characterize its interaction with the surrounding lipid. OmpF is a trimer within the membrane. It contains two Trp residues per monomer, Trp(214) at the lipid-protein interface and Trp(61) at the trimer interface. The fluorescence of Trp-214 in the mutant W61F is quenched by dibromostearoylphosphatidylcholine (di(Br(2)C18:0)PC), whereas the fluorescence of Trp(61) in the mutant W214F is not quenched by di(Br(2)C18:0)PC when fluorescence is excited directly through the Trp rather than through the Tyr residues. Measurements of relative fluorescence quenching for OmpF reconstituted into mixtures of lipid X and di(Br(2)C18:0)PC have been analyzed to give the binding constant of lipid X for OmpF, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). The phosphatidylcholine showing the strongest binding to OmpF is dimyristoyloleoylphosphatidylcholine (di(C14:1)PC) with binding constants decreasing with either increasing or decreasing fatty acyl chain length. Comparison with various theories for hydrophobic matching between lipids and proteins suggests that in the chain length range from C14 to C20, hydrophobic matching is achieved largely by distortion of the lipid bilayer around the OmpF, whereas for chains longer than C20, distortion of both the lipid bilayer and of the protein is required to achieve hydrophobic matching. Phosphatidylcholine and phosphatidylethanolamine bind with equal affinity to OmpF, but the affinity for phosphatidylglycerol is about half that for phosphatidylcholine.     

Deoxyribonucleic acid and outer membrane: binding to outer membrane involves a specific protein.

The binding of deoxyribonucleic acid (DNA) to the outer membrane of Escherichia coli was examined. The amount of DNA found to be bound to outer membrane was low and was estimated to be about 0.4% of the total DNA. Treatment of cells with chloramphenicol or rifampin caused a disassociation of the apparent DNA-outer membrane complex. The results presented here suggest that the binding between membrane and DNA is specific and involves a membrane protein having a molecular weight of 13,000.     

Mutant ofEscherichia coli sensitive to proteinase

A mutant ofEscherichia coli sensitive to proteinase (MP2) has been isolated and characterized briefly. MP2 cells were impaired by Pronase, showing a decrease of the cell turbidity both in growing cell and in cell suspension, while the parental cells remained unimpaired. Pronase conceivably digested cellular protein, but DNA remained with cells. This Pronase action was prevented by the inhibitor of protein synthesis, while it was stimulated by bacitracin. The mutant was susceptible to an externally added repressor. A transient repression of arginine enzyme synthesis was observed only when the cell extract of a repressible strain was added, not by the extract of a nonrepressible strain. It was concluded that the proteinase-sensitive mutant has a lower permeability barrier to macromolecules.     

Phage receptors of Escherichia coli. Basic components of the outer membrane of E. coli as phage receptors

Major outer membrane components which determine the structure and the barrier function of membrane Gram-negative bacteria are receptors for many bacteriophages. LPS--the major component of the outer membrane of Enterobacteria can be used by some phages with wide host range specificity. The other component of the outer membrane frequently include phage receptor component is OmpA protein. OmpA protein different areas can be used as receptors for different phages T--even group. A large group of phage receptors compose porin proteins, which are discovered in 32 species of bacteria. The synthesis of major porin proteins, which a receptor for several phages, are regulated by sufficiently complex system of some genes. These genes are sensitive to the changes of environment.     

Binding of metallic ions to the outer membrane of Escherichia coli

The binding of metal ions by the outer membrane of Escherichia coli K-12 strain AB264 was investigated by using outer membrane obtained after Triton X-100 extraction of purified cell envelopes. Binding studies, conducted under saturating conditions, indicated a selective trapping of certain metallic ions. Low-dose electron microscopy of metal-loaded samples revealed an aggregative deposition of lead on one surface of the membrane which suggests that at least one distinctive binding site is asymmetrically arranged in these outer membrane vesicles.