Changes in the electrical characteristics of the internal surface of cytoplasmic membrane of bacteria Escherichia coli after the binding of microdoses of copper ions by its external surface

The effect of microdoses of copper ions bound with the external surface of the cytoplasmic membrane of Escherichia coli spheroplasts on the charge density of its superficial structures was investigated by the ESR method. Positively charged spin probes of KAT(n)-type and the newly synthesized KAT15 were used. The experimental data were analyzed using the formalism of Gouy-Chapman theory. The binding of microdoses of copper ions by the external surface of the cytoplasmic membrane changed the value of charge density on the membrane internal surface.     

Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state

Changes in the outer membrane subproteome of Escherichia coli along the transition to the viable but nonculturable state (VBNC) were studied. The VBNC state was triggered by exposure of E. coli cells to adverse conditions such as aquatic systems, starvation, suboptimal temperature, visible light irradiation and seawater. The subproteome, obtained according to Molloy et al ., was analysed at the beginning of exposure (inoculum, phase 1), after a variable exposure time (95% of population culturable, phase 2) and when populations were mainly in the VBNC state (95% of cells VBNC, phase 3). Proteome changes were dependent on adverse conditions inducing the transition and were detected mainly in phase 2. The permanence of E. coli cells in seawater under illumination conditions entailed a dramatic rearrangement of the outer membrane subproteome involving 106 new spots, some of which could be identified by peptide fingerprinting. However, proteins exclusive to the VBNC state were not detected.     

EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli

Abstract A gene product with an apparent molecular mass of approximately 39000 Da can be identified in the cytoplasmic membrane of Escherichia coli upon expression of cloned envC . In this communication we report that the product was labelled with [³H]glycerol and [³H]palmitic acid, and a precursor molecule of increased molecular mass was accumulated when cells were treated with globomycin, a specific inhibitor for the prolipoprotein signal peptidase. The same precursor molecule was encoded by an envC mutant gene, in which the cysteine residue in a pentapeptide sequence, Leu-Ile-Ala-Gly-Cys²⁴ within the amino terminal region of EnvC, was replaced by tryptophane (Trp²⁴). This protein was not labelled with [³H]glycerol. The results demonstrate that the envC gene product represents a new lipoprotein of the cytoplasmic membrane of E. coli .     

Identification of EaeA protein in the outer membrane of attaching and effacing Escherichia coli O45 from pigs

Abstract We have previously reported that the production of attaching and effacing lesions by Escherichia coli O45 isolates from pigs is associated with the eaeA ( E. coli attaching and effacing) gene. In the present study, expression of the EaeA protein, the eaeA gene product, among swine O45 E. coli isolates was examined. The majority (20/22) of attaching and effacing positive, eaeA⁺ E. coli O45 isolates, but none of ten attaching and effacing negative, eaeA⁻ or eaeA⁺ isolates, expressed a 97-kDa outer membrane protein as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Amino-terminal amino acid sequencing demonstrated a high homology between this 97-kDa protein of swine E. coli O45 and the EaeA protein (intimin) of human enteropathogenic E. coli and enterohemorrhagic E. coli . In addition, a serological relationship between the EaeA proteins of swine O45, rabbit (RDEC-1) and human (E2348/69) attaching and effacing E. coli strains was observed. Our results indicate an association between expression of the EaeA protein and attaching and efficacing activity among O45 E. coli isolates. The data also suggest an antigenic relatedness of the EaeA proteins of swine, rabbit, and human attaching and effacing E. coli .     

Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.     

Immunological cross-reactivity between outer membrane pore proteins of Campylobacter jejuni and Escherichia coli

Immunocrossreactivity between the major outer membrane protein (MOMP) of Campylobacter jejuni 85H and the OmpC porin of Escherichia coli K-12 was observed. These results indicate that a common antigenic domain is conserved in both MOMP and OmpC. This antigenic region is detected only after a 96 degrees C treatment suggesting that it is buried in the native conformation of the respective porins. In addition, differences were observed between the major outer membrane proteins from various C. jejuni strains. About 60% of the C. jejuni pathogenic strains tested contained a protein exhibiting a similar electrophoretic profile to the 85H porin.     

Three phosphatidylglycerol-phosphate phosphatases in the inner membrane of Escherichia coli

The phospholipids of Escherichia coli consist mainly of phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin. PG makes up ～25% of the cellular phospholipid and is essential for growth in wild-type cells. PG is synthesized on the inner surface of the inner membrane from cytidine diphosphate-diacylglycerol and glycerol 3-phosphate, generating the precursor phosphatidylglycerol-phosphate (PGP). This compound is present at low levels (～0.1% of the total lipid). Dephosphorylation of PGP to PG is catalyzed by several PGP-phosphatases. The pgpA and pgpB genes, which encode structurally distinct PGP-phosphatases, were identified previously. Double deletion mutants lacking pgpA and pgpB are viable and still make PG, suggesting the presence of additional phosphatase(s). We have identified a third PGP-phosphatase gene (previously annotated as yfhB but renamed pgpC) using an expression cloning strategy. A mutant with deletions in all three phosphatase genes is not viable unless covered by a plasmid expressing either pgpA, pgpB, or pgpC. When the triple mutant is covered with the temperature-sensitive plasmid pMAK705 expressing any one of the three pgp genes, the cells grow at 30 but not 42 °C. As growth slows at 42 °C, PGP accumulates to high levels, and the PG content declines. PgpC orthologs are present in many other bacteria.     

The binding of colonization factor antigens of enterotoxigenic Escherichia coli to intestinal cell membrane proteins

We examined the binding of colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli to electrophoretically separated membrane components of rabbit intestinal brush borders or human intestinal (and non-intestinal) cell lines using an immunoblotting technique. Both CFA/I and CFA/II bound to distinct membrane components which seemed to be identical in rabbit brush borders and in a human intestinal cell line; these binding structures were mainly missing in membranes from epithelial cell lines of non-intestinal origin. Both shared and specific binding components were identified for CFA/I and the different subcomponents of CFA/II (CS1, CS2 and CS3), respectively. Chloroform-methanol extraction of lipids from the cell membranes did not change the binding pattern for either CFA/I or CFA/II suggesting that the binding occurred to (glyco)proteins rather than to (glyco)lipids.     

Biosynthetic requirements for the repair of sublethal membrane damage in Escherichia coli cells after pulsed electric fields

AIMS: The aim was to evaluate the biosynthetic requirements for the repair of sublethal membrane damages in Escherichia coli cells after exposure to pulsed electric fields (PEF). METHODS AND RESULTS: The partial loss of the barrier and homeostatic functions of the cytoplasmic membrane was examined by adding sodium chloride to the recovery media. More than 4 log10 cycles of survivors were sublethally injured after PEF. Repair of such sublethal membrane damages occurred when survivors to PEF were incubated in peptone water for 2 h. Two different types of sublethally injured cells were detected. Whereas a small proportion (<5%) repaired after PEF in less than 2 min, the repair of the remaining 95% injured cells lasted 2 h and was dependent on biosynthetic requirements. The addition of inhibitors such as chloramphenicol, cerulenin, penicillin G, rifampicin and sodium azide to the liquid repair medium showed that the repair required energy and lipid synthesis, and was not dependent on protein, peptidoglican or RNA synthesis. CONCLUSIONS: Cell survival after PEF is dependent on the repair of the cytoplasmic membrane. Requirement of lipid synthesis for the repair of sublethally injured cells confirms that the cytoplasmic membrane is a target directly involved in the mechanism of inactivation by PEF. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about the damages inflicted by PEF might help in the design of more efficient treatments.     

The EnvZ protein of Salmonella typhimurium LT-2 and Escherichia coli K-12 is located in the cytoplasmic membrane

Abstract The osmoregulated expression of the porin proteins OmpC and OmpF in S. typhimurium and E. coli is dependent on the regulatory proteins OmpR and EnvZ. The function of the EnvZ protein is not clear. In order to establish the cellular location of EnvZ two different methods of buoyant sucrose density centrifugation was employed. The presence of EnvZ in the different fractions was visualised by immunoblotting. It was conclusively shown that the EnvZ protein is located in the cytoplasmic membrane fraction. The result is in agreement with the available sequence data which shows that the EnvZ polypeptide contains two long hydrophobic stretches.     

Conformation of Escherichia coli outer membrane protein OmpA produced in Bacillus subtilis: Influence of lipopolysaccharide

Abstract The conformation of the outer membrane protein OmpA of Escherichia coli produced in Bacillus subtilis and solubilized in Sarkosyl was studied by measuring its ability to bind OmpA-specific phage K3 and to inhibit F-mediated conjugation. The partially purified protein was inactive in both these assays. Refolding of the protein in the presence of lipopolysaccharide resulted in preparations with full phage-binding and conjugation-inhibiting capacity, indicating the formation of surface-exposed loops of OmpA of native conformation. The finding is of importance for the potential use of outer membrane proteins of Gram-negative bacteria as vaccines.     

Role of the carboxy-terminal region of the outer membrane protein AatA in the export of dispersin from enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. AatA, an outer-membrane protein that is a homolog of E. coli TolC, facilitates the export of the dispersin protein Aap across the outer membrane in EAEC. To identify which amino acids are important for this export activity, site-directed mutagenesis of the carboxy terminus was performed. An insertional mutant of aatA was complemented with each of several deletion mutants, and was examined for Aap secretion. The results showed that three nonpolar amino acids at positions 381-383 (Phe-Leu-Leu) were required for the activity, and these residues were located at the base of carboxy-terminal elongation in the equatorial domain of AatA.     

The outer membrane protein of enteropathogenic Escherichia coli, described as the ''localised adherence factor'', is OmpF and probably not involved in adhesion to HEp-2 cells

Strains of enteropathogenic Escherichia coli (EPEC) were examined for a factor, described as an outer membrane protein (OMP) of 32 kilodaltons (kDa) and reported to be involved in the adhesion of EPEC to HeLa cells. A comparable OMP of 35 kDa was detected in strains of EPEC, although expression of this protein was not related to the ability of strains to adhere to HEp-2 cells. The 35 kDa OMP was found to be heat-modifiable and peptidoglycan associated, and considered to be the porin protein OmpF.     

Combined fluorescent antibody assay and viability staining for the assessment of the physiological states of Escherichia coli in seawaters

AIMS: A comparison of methods that combine the use of immune sera with specific fluorescent probes for testing viability at single cell level was performed in order to estimate different living attributes of Escherichia coli in natural seawater samples. METHODS AND RESULTS: Cell culturability was assayed by plate method, respiratory activity and membrane integrity were determined by an indirect fluorescent antibody assay, combined with 5-cyano-2, 3 ditolyl tetrazolium chloride and propidium iodide, respectively. Results showed the coexistence of different physiological states within the E. coli population, of which a large fraction (46%) of cells was actively respiring. CONCLUSIONS: The methodological approach used offer interesting perspectives in water pollution monitoring, particularly when the differentiation between dead and living E. coli cells is required for a more precise assessment of the bacteriological quality of seawaters. SIGNIFICANCE AND IMPACT OF THE STUDY: The study suggests the importance of knowledge of the viability status of faecal bacteria in aquatic environments as a fundamental issue for the preservation of public health; the availability of rapid analytical procedures for this purpose may find significant applications in the evaluation of the sanitary risk consequent to water use.     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

Epitope mapping by cysteine mutagenesis: Identification of residues involved in recognition by three monoclonal antibodies directed against LamB glycoporin in the outer membrane of Escherichia coli

Abstract Site-directed mutagenesis of the lamB gene was used to introduce individual cysteine substitutions at 20 sites in two regions (surface loops L7 and L8) of LamB protein significant in antibody recognition. Characterisation of cysteine mutants involved immunoblotting with three surface-specific monoclonal antibodies (mAb72, mAb302, mAb347) before and after incubation with thiol-specific reagents. In contrast to an earlier study that showed no amino acid changes affecting recognition by all three antibodies, changes at six amino acids were found to influence a common core epitope. These core sites included one residue (T336) in the predicted loop L7 containing amino acids 329–342 and four (Y379, N387, N389, K392, F398) in the large surface loop involving residues 370–412. Individual antibodies made additional but distinct contacts within the two studied regions, with mAb347 binding the most different and affected by seven substitutions in the 328–338 regions. The lamB mutants were also tested for phage λ receptor activity and starch binding before and after thiol modification and were useful in extending previous maps of these ligand binding sites.     

Synergistic effect of enterocin AS-48 in combination with outer membrane permeabilizing treatments against Escherichia coli O157:H7

AIMS: To determine the effects of outer membrane (OM) permeabilizing agents on the antimicrobial activity of enterocin AS-48 against Escherichia coli O157:H7 CECT 4783 strain in buffer and apple juice. METHODS AND RESULTS: We determined the influence of pH, EDTA, sodium tripolyphosphate (STPP) and heat on E. coli O157:H7 CECT 4783 sensitivity to enterocin AS-48 in buffer and in apple juice. Enterocin AS-48 was not active against intact cells of E. coli O157:H7 CECT 4783 at neutral pH. However, cells sublethally injured by OM permeabilizing agents (EDTA, STPP, pH 5, pH 8.6 and heat) became sensitive to AS-48, decreasing the amount of bacteriocin required for inhibition of E. coli O157:H7 CECT 4783. CONCLUSIONS: The results presented indicate that enterocin AS-48 could potentially be applied with a considerably wider range of protective agents, such as OM permeabilizing agents, with increased efficacy in inhibiting E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study support the potential use of enterocin AS-48 to control E. coli O157:H7 in combination with other hurdles.     

Incompatibility of outer membrane proteins OmpA and OmpF of Escherichia coli with secretion in Bacillus subtilis: Fusions with secretable peptides

The secretion of the outer membrane proteins OmpA and OmpF of Escherichia coli has previously been found to be blocked at an early intracellular step, when these proteins were fused to a bacillar signal sequence and expressed in Bacillus subtilis. We have now fused these proteins to long secretable polypeptides, the amino-terminal portions of alpha-amylase or beta-lactamase. In spite of this, no secretion of the fusion proteins was detected in B. subtilis. With the exception of a small fraction of the beta-lactamase fusion, the proteins were cell-bound with uncleaved signal sequences. Protease accessibility indicated that the fusion proteins were not even partially exposed on the outer surface of the cytoplasmic membrane. Thus there was no change of the location compared to the OmpA or OmpF fused to the signal sequence only. We conclude that, like OmpA and OmpF, the fusion proteins fold into an export-incompatible conformation in B. subtilis before the start of translocation, which we postulate to be a late post-translational event.