Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli.

Supplementation of the growth medium with high concentrations of NaCl, KCl, or sucrose caused a drastic change in the ratio of the two peptidoglycan-associated major outer membrane proteins of Escherichia coli K-12 in that the amounts of proteins b and c present in cell envelope preparations decreased and increased, respectively. Kinetic studies showed that, after the osmolarity of the medium was changed, one protein was hardly incorporated into the membrane, whereas the other was incorporated with an increased rate. After about 1.5 to 2 generations, the cell envelopes obtained the b/c ratio characteristic for the new medium, and both proteins were subsequently incorporated in the cell ensured this new ratio. Once proteins b and c were incorporated in the cell envelope, they were not converted into each other by changes in osmolarity of the growth medium.     

Effect of outer membrane permeability on chemotaxis in Escherichia coli.

The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.     

Effect of ethylenediaminetetraacetate on phospholipids and outer membrane function in Escherichia coli.

Treatment of Escherichia coli K-12 strain S15, containing a normal amount of phospholipase A, with ethylenediaminetetraacetate (EDTA) resulted in an increase in sensitivity of the organism to actinomycin D. Strain S17, a mutant deficient in both detergent-resistant phospholipase A and detergent-sensitive phospholipase A, was considerably less sensitive to the antibiotic after the treatment. Both strains released lipopolysaccharide after EDTA treatment, indicating that this outer membrane component alone is not the barrier to actinomycin in these organisms. The phospholipase A-deficient strain released less alkaline phosphatase, a periplasmic enzyme. EDTA treatment of S15 resulted in the accumulation of free fatty acids, indicative of phospholipase A activation. Cells briefly treated with EDTA regained the barrier to actinomycin when incubated in growth media, and the cessation of the accumulation of free fatty acids was in approximate temporal agreement with restoration of the barrier. Cells in which phospholipase A was activated by brief exposure to EDTA synthesized relatively more phosphatidylethanolamine than did untreated cells in the initial period after dilution into growth media. These experiments suggest that the EDTA-induced loss of outer membrane barrier function of E. coli K-12 is mediated through the activation of phospholipase A.     

Comparison of the polypeptide composition of Escherichia coli outer membranes prepared by two methods.

Escherichia coli outer membranes were prepared by centrifugation to equilibrium in sucrose gradients and then treated with Sarkosyl in the presence of ethylenediaminetetraacetate. The polypeptide profiles of the two outer membrane preparations were compared by two-dimensional polyacrylamide gel electrophoresis. The patterns obtained were not identical, and Sarkosyl removed several minor proteins from the outer membrane.     

Neisseria meningitidis: heterogenicity in the outer membrane proteins released into the growth medium

Eight strains of Neisseria meningitidis belonging to different serogroups were analysed for their virulence in mice and their release of outer membrane proteins into the medium during growth. All strains released proteins. No detectable lipopolysaccharide was observed. However, SDS-PAGE showed a heterogenicity in the protein number and profile among the different strains of N. meningitidis tested.     

Proteomic analysis of the Escherichia coli outer membrane.

Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Traditional biochemical and genetic approaches have yielded a wealth of knowledge relating to the function of OMPs. Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understanding of the organization, expression and function of the OMPs in this Gram-negative bacterium. In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs. We propose a rapid method for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two-dimensional electrophoresis, followed by protein identification using mass spectrometry. Applying this method to examine E. coli K-12 cells grown in minimal media we identified 21 out of 26 (80%) of the predicted integral OMPs that are annotated in SWISS-PROT release 37 and predicted to separate within the range of pH 4-7 and molecular mass 10-80 kDa. Five outer membrane lipoproteins were also identified and only minor contamination by nonmembrane proteins was observed. Importantly, this research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two-dimensional electrophoresis. Two of the identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and translated. Furthermore, we show that like the E. coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, suggesting a putative role for this protein in iron transport. In an additional demonstration we show the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.     

Protein interactions in the outer membrane of Escherichia coli.

Specific protein interactions in Escherichia coli outer membrane were analyzed using chemical cross-linking with truly cleavable reagents and symmetrical two-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The major outer membrane proteins were shown to form cross-linked complexes. These include multimers of lambda receptor, protein I, II, III and the free form of lipoprotein. Lipoprotein was also found to be cross-linked to proteins II and III. The identity of many of these complexes was verified using appropriate mutants missing the proteins in question. No new protein interactions were detected in the mutants even when three of the major proteins were missing. Proteins II, III and the free form of lipoprotein could also be cross-linked to the peptidoglycan layer of the cell wall.     

Effect of variations in lipopolysaccharide on the fluidity of the outer membrane of Escherichia coli.

The lipid hydrocarbon chains in the outer membrane of gram-negative bacteria appear from previous experiments to be less mobile than in the cytoplasmic membrane. To determine whether lipopolysaccharide, a unique outer membrane component, is a cause of this restricted mobility, outer membranes differing in the amount of lipopolysaccharide, and the length of the polysaccharide side chain, were prepared from Escherichia coli J5. Cytoplasmic membranes were prepared for comparison. The probes, 5- and 12-doxylstearate, were introduced into these membranes, electron spin resonance spectra were analyzed, and the order parameter (S) and empirical motion parameter (tau0) were calculated. Outer membrane preparations containing long chain lipopolysaccharide were much less fluid by these criteria than were preparations containing short chain lipopolysaccharide. Removing about 40% of the lipopolysaccharide from the former preparations greatly increased their fluidity. The lipid in the cytoplasmic membrane preparations was more fluid than in the outer membrane and cytoplasmic membranes were similar to each other regardless of the composition of the outer membrane. These results indicate that lipopolysaccharide, and especially the polysaccharide portion, directly or indirectly causes the restricted mobility of the lipid hydrocarbon chains observed in the outer membrane.     

Ion channel activities in the Escherichia coli outer membrane.

The electrical properties of Escherichia coli cells were examined by the patch-clamp technique. Giant cells or giant spheroplasts were generated by five different methods. By electron micrographic and other criteria we determined that the patches are most likely from the outer membrane. We regularly observed currents through at least two types of channels in this membrane. The first current is mechanosensitive and voltage-dependent, and can be observed in single gene mutants of the known major porins (ompF, ompC, phoE, lamB); this channel may represent a minor porin or a new class of outer membrane protein. The possible identity of the second, voltage-sensitive channel with one of the known outer membrane proteins is being explored. The high-resistance seals consistently formed on these patches and the presence of gated ion channels suggest that most of the pores of the outer membrane are not statically open, as commonly held, but are closed at rest and may be openable by physiological stimuli.     

Escherichia coli K-12 tolF mutants: alterations in protein composition of the outer membrane.

Outer membrane materials prepared from three independently isolated spontaneous Escherichia coli tolF mutants contained no detectable protein Ia. The loss of this protein was nearly completely compensated for by an increase in other major outer membrane proteins, Ib and II. Thus, the major outer membrane proteins accounted for 40% of the total cell envelope protein in both tol+ and tolF strains. No changes were found in the levels of inner membrane proteins prepared from tolF strains when compared with similar preparations from the tol+ strain. Phage-resistant mutants were selected starting with a tolF strain by using either phage TuIb or phage PA2. These phage-resistant tolF strains contained neither protein Ia nor protein Ib. The mutation leading to the loss of protein Ib in these strains is independent of the tolF mutation and is located near malP on the E. coli genetic map.     

Polyamines decrease Escherichia coli outer membrane permeability.

The permeability of the outer membranes of gram-negative bacteria to hydrophilic compounds is mostly due to the presence of porin channels. We tested the effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on two processes known to depend on intact porin function: fluxes of beta-lactam antibiotics in live cells and chemotaxis. In both cases, inhibition was observed. Measurements of the rate of permeation of cephaloridine and of chemotaxis in swarm plates and capillary assays were used to determine the concentration dependence of this modulation. The effective concentration ranges depended on the nature of the polyamine and varied from submillimolar for spermine to tens of millimolar for cadaverine. Both OmpC and OmpF porins were inhibited, although the effects on OmpC appeared to be milder. These results are in agreement with our observations that polyamines inhibit porin-mediated ion fluxes in electrophysiological experiments, and they suggest that a low-affinity polyamine binding site might exist in these porins. These results reveal the potential use of porins as targets for blocking agents and suggest that polyamines may act as endogenous modulators of outer membrane permeability.     

Strain and temperature-dependent variation in the amount of BtuB polypeptide in the Escherichia coli K-12 outer membrane

Abstract The outer membrane protein BtuB of Escherichia coli K-12 is the receptor for vitamin B₁₂; it is normally present in approx. 200 copies per cell. We describe here the conditions by which BtuB was readily observed in electropherograms of outer membrane preparations. These conditions are as follows. (1) Incorporation of 8 M urea in sodium dodecyl sulfate-polyacrylamide gel systems improved detection of the polypeptide. (2) In most E. coli K-12 strains examined, BtuB was most abundant when cells were grown at 27°C. This thermoregulation of BtuB was independent of envZ and envY , two regulatory genes for outer membrane proteins.     

Strain specificity of outer membrane proteins in Escherichia coli.

Outer membrane proteins of various strains of Escherichia coli were compared using three different systems of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The outer membranes of E. coli K-12, E. coli B, and E. coli J-5 had distinctive protein compositions. As regards proteins which interact with peptidoglycan, E. coli K-12 contained O-8 and O-9, while E. coli B possessed one protein which migrated to the position of O-9. Although E. coli J-5 possessed two such proteins, O-8' and O-9', their positions on polyacrylamide gel were different from those of O-8 and O-9. Protein O-7, which migrates slightly more slowly than O-8, was found specifically in E. coli K-12. Proteins O-10 and O-11 were found in all strains tested, although the relative amounts were different depending on the strain. Strains of E. coli K-12 and E. coli J-5 gave three major bands, O-2a, O-2b, and O-3, in the region of high molecular weight. These proteins were repressed by iron in the cultivation media. Strains of E. coli B, on the other hand, gave only O-2b and O-3. E. coli J-5 gave two other major bands in this region, but the amounts were not controlled by iron in the cultivation media.     

Effect of growth rate and cell shape on the peptidoglycan composition in Escherichia coli.

The muropeptide composition of peptidoglycan from Escherichia coli W7 cultivated at different growth rates in chemostat cultures was compared by using high-pressure liquid chromatography. At a low growth rate (D = 0.1 h-1), about 40% more covalently bound lipoprotein and at least twofold more diaminopimelyl-diaminopimelic acid cross-bridges were found than at a high growth rate (D = 0.8 h-1). The total degree of cross-linkage was only slightly increased, and the fraction of trimeric muropeptides and the average length of the glycan chains were not changed significantly. Analysis of the peptidoglycan from a morphological variant strain of W7 revealed that the altered peptidoglycan composition in slowly growing W7 cells was not correlated with the observation that these cells, due to their decreased cell length, were relatively enriched in polar material. In fact, our results suggested that peptidoglycan forming cell poles is chemically identical to that forming lateral wall.     

Cross-linking of the proteins in the outer membrane of Escherichia coli.

1. The organization of the proteins in the outer membrane of Escherichia coli was examined by the use of cross-linking agents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of protein A-peptidoglycan complexes with dithiobis(succinimidyl propionate) or glutaraldehyde produced the dimer, trimer, and higher oligomers of protein A. Both forms of this protein, proteins A1 and A2, produced similar cross-linking products. No cross-linking of protein A to the peptidoglycan was detected. 2. The proteins of the isolated outer membrane varied in their ease of cross-linking. The heat-modifiable protein, protein B, was readily cross-linked to give high molecular weight oligomers, while protein A formed mainly the dimer and trimer under the same conditions. The pronase resistant fragment, protein Bp, derived from protein B was not readily cross-linked. No linkage of protein A to protein B was detected. 3. Cross-linking of cell wall preparations, consisting of the outer membrane and peptidoglycan, showed that protein B and the free form of the lipoprotein, protein F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. 4. These results suggest that specific protein-protein interactions occur in the outer membrane.