Release of outer membrane fragments from wild-type Escherichia coli and from several E. coli lipopolysaccharide mutants by EDTA and heat shock treatments.

EDTA-induced outer membrane losses from whole cells of wild-type Escherichia coli (O111:B4) and several lipopolysaccharide (LPS) mutants derived from E. coli K-12 D21 were analyzed. EDTA treatment induced losses of LPS (up to 40%), outer membrane proteins OmpA, OmpF/C, and lipoprotein, periplasmic proteins, and phosphatidylethanolamine. The extent of these releases was strain specific. Successively more EDTA was necessary to induce these losses from strains containing LPS with increasing polysaccharide chain length. An additional heat shock immediately following the EDTA treatment had no effect on LPS release, but it decreased the release of outer membrane proteins and reduced the leakage of periplasmic proteins, suggesting that the temporary increase in outer membrane "permeability" caused by Ca2+-EDTA treatment was rapidly reversed by the redistribution of outer membrane components, a process which is favored by a mild heat shock. The fact that the material released from E. coli C600 showed a constant ratio of lipoprotein, OmpA, and phosphatidylethanolamine at all EDTA concentrations tested suggests that the material is lost as specific outer membrane patches. The envelope alterations caused by EDTA did not result in cell lysis.     

Purification of protein A, an outer membrane component missing in Escherichia coli K-12 ompA mutants.

Outer membrane materials prepared from an Escherichia coli ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA+ strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA+ strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II or B purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II or B that is difficult to solubilize is identical with the more readily solubilized fraction.     

Two-component nature of bacteriophage T4 receptor activity in Escherichia coli K-12.

Escherichia coli bacteriophage T4 uses the lipopolysaccharide of the outer cell envelope membrane as a receptor. Lipopolysaccharide from E. coli K-12 required a major outer membrane protein, polypeptide Ib, for phage inactivation.     

Bacteriophage T4-Mediated Release of Envelope Components from Escherichia coli

When Escherichia coli B, labeled by prior growth in ¹⁴C-glucose, are infected with T4 phage there is a rapid release of ¹⁴C-nondialyzable material into the medium. About half of this material is derived from the cell envelope as evidenced by its content of phospholipid and lipopolysaccharide and its buoyant density upon isopycnic ultracentrifugation of 1.19 g/cm³. It is similar in its gross chemical and physical properties to envelope material released at a lower rate from growing uninfected cells or from cells whose protein synthesis is inhibited by chloramphenicol (22). The rate of release of this envelope material at a multiplicity of infection (MOI) of 10 is greatest in the first minute after infection, and release is completed by 4 min. The rate of its release, as a function of MOI at 2 min after infection, is greatest at low MOI (e.g., MOI 2 and 4); in addition, the release does not continue above MOI 30. The main conclusion derived from the data is that phage, as part of the process of adsorption and injection of DNA, cause an increased release of envelope substance from the cells. With the assumption that all of the envelope material released is derived from the outer envelope, it is estimated that uninfected cells release 20 to 30% of their outer envelope per hour, whereas infected cells release 30% in 2 min at MOI 30. Further, because release does not continue at high MOI, this phenomenon is not considered to be a direct cause of lysis from without. Data are also presented on the amounts of other non-dialyzable ¹⁴C-components released and on the differences in the kinetics of release from chloramphenicol-treated cells compared to phage-infected cells. To avoid the possibility that the release is due to phage lysozyme which is an adventitious “contaminant” of wild-type phage, a phage mutant (T4BeG59s) devoid of this enzyme was used in these experiments.     

Involvement of phage T5 tail proteins and contact sites between the outer and inner membrane of Escherichia coli in phage T5 DNA injection.

The penetration of phage T5 DNA into the Escherichia coli envelope takes place through ion channels (Boulanger, P., and Letellier, L. (1992) J. Biol. Chem. 267, 3168-3172). To identify putative phage protein(s) involved in the formation of these channels, E. coli cells were infected at 37 degrees C with radioactively labeled phage and their envelopes were fractionated. After a flotation gradient, proteins belonging to the phage tail were recovered both in fractions containing the contact sites between the inner and outer membranes and in the outer membrane. The electrophoretic banding pattern of phage proteins indicates that the contact sites were enriched in the protein pb2. Moreover, infected cells were significantly enriched in contact sites as compared to intact cells. There was no enrichment of contact sites and very little radioactivity was found in this fraction and in the outer membrane when the cells were infected at 4 degrees C (i.e. under conditions where the phage does not inject its DNA). These results suggest that both contact sites and pb2 may play a central role in the translocation of phage T5 DNA.     

Transport of vitamin B12 in Escherichia coli: common receptor system for vitamin B12 and bacteriophage BF23 on the outer membrane of the cell envelope.

We showed previously that the outer membrane of the Escherichia coli cell envelope normally contains about 200 to 250 B12 receptors, and that these receptors function both in B12 transport and as receptors for the E colicins. This paper shows that this receptor system is also shared with bacteriophage BF23. A strong positive correlation was observed between the number of B12 receptors per cell and the rate of adsorption of BF23. Cells from mutant strains that lacked B12 receptors did not adsorb BF23 particles. The rate of adsorption of BF23 to cells of a merodiploid strain (RK4151), with about 550 B12 receptors per cell, was approximately double that to cells of a normal, haploid strain. The adsorption of BF23 to hole cells, cell envelopes, outer membrane particles, and solubilized outer membranes was inhibited by vitamin B12, with 50% inhibition at B12 concentrations in the range of 0.5 to 2.0 nM. These values are close to the observed KS for B12 binding to the B12 receptors. Vitamin B12 concentrations as high as 100 nM did not inhibit adsorption of bacteriophages T5, T6, and lambdacI to cells of sensitive strains of E. coli. Bacteriophage BF23 inhibited B12 transport by whole cells and was shown to be a competitive inhibitor of B12 binding to isolated cell envelope particles. The B12/BF23 receptors from E. coli strains KBT069 (btuB69) and RK4104 (btuB69) were fully active, but the number per cell was reduced to an average value of about 0.5.     

Systematic identification of the subproteome of Escherichia coli cell envelope reveals the interaction network of membrane proteins and membrane-associated peripheral proteins

Membrane proteins of Gram-negative bacteria are key molecules that interface the cells with the environment. Despite recent proteomic identification of numerous oligomer proteins in the Escherichia coli cell envelope, the protein complex of E. coli membrane proteins and their peripherally associated proteins remain ill-defined. In the current study, we systematically analyze the subproteome of E. coli cell envelope enriched in sarcosine-insoluble fraction (SIF) and sarcosine-soluble fraction (SSF) by using proteomic methodologies. One hundred and four proteins out of 184 spots on 2D electrophoresis gels are identified, which includes 31 outer membrane proteins (OMPs). Importantly, our further proteomic studies reveal a number of previously unrecognized membrane-interacting protein complexes, such as the complex consisting of OmpW and fumarate reductase. This established complete proteomic profile of E. coli envelope also sheds new insight into the function(s) of E. coli outer envelope.     

Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli.

Thermal damage to the outer membrane of Escherichia coli W3110 was studied. When E. coli cells were heated at 55 degrees C in 50 mM Tris-hydrochloride buffer at pH 8.0, surface blebs were formed on the cell envelope, mainly at the septa of dividing cells. Membrane lipids were released from the cells during the heating period, and part of the released lipids formed vesicle-like structures from the membrane. This vesicle fraction had a lipopolysaccharide to phospholipid ratio similar to that of the outer membrane of intact cells, whereas it had a lower content of protein than the isolated outer membrane. After heating bacterial cells at 55 degrees C for 30 min, the resulting leakage from the cells of a periplasmic enzyme, alkaline phosphatase, amounted to 52% of the total activity, whereas no release of a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, was detected. The results obtained suggest that surface blebs formed by heat treatment almost completely consist of the outer membrane and that the blebs may be gradually released from the cell surface into the heating menstruum to partially form vesicles.     

Molecular sieve mechanism of selective release of cytoplasmic proteins by osmotically shocked Escherichia coli

Escherichia coli cells, the outer membrane of which is permeabilized with EDTA, release a specific subset of cytoplasmic proteins upon a sudden drop in osmolarity in the surrounding medium. This subset includes EF-Tu, thioredoxin, and DnaK among other proteins, and comprises approximately 10% of the total bacterial protein content. As we demonstrate here, the same proteins are released from electroporated E. coli cells pretreated with EDTA. Although known for several decades, the phenomenon of selective release of proteins has received no satisfactory explanation. Here we show that the subset of released proteins is almost identical to the subset of proteins that are able to pass through a 100-kDa-cutoff cellulose membrane upon molecular filtration of an E. coli homogenate. This finding indicates that in osmotically shocked or electroporated bacteria, proteins are strained through a molecular sieve formed by the transiently damaged bacterial envelope. As a result, proteins of small native sizes are selectively released, whereas large proteins and large protein complexes are retained by bacterial cells.     

Preferential release of new outer membrane fragments by exponentially growing Escherichia coli

We have examined whether the outer membrane fragments released by normally growing Escherichia coli contain relatively old or new outer membrane.Double-label experiments show that after incorporation of radioactive leucine into E. coli protein, there is a preferential release of outer membrane material which contains a high percentage of newly labeled protein. This implies that outer membrane fragments are preferentially released from those regions where newly synthesized proteins are inserted into the outer membrane. We estimate that these insertion regions cover no more than 13% of the total outer membrane, and that newly inserted proteins diffuse in the plane of the outer membrane with a diffusion constant ? 5 · 10^?13 cm²/s.     

Attachment of Flagellar Basal Bodies to the Cell Envelope: Specific Attachment to the Outer, Lipopolysaccharide Membrane and the Cytoplasmic Membrane

A procedure is described for the purification of the Escherichia coli outer membrane (lipopolysaccharide or L membrane) with flagella still attached. The resulting lipopolysaccharide membrane was in the form of vesicles that had a trilaminar structure in thin section and contained about 55% lipopolysaccharide and 45% protein. T2 or T4 phage preadsorbed to E. coli were found attached to the purified lipopolysaccharide membrane. Flagella were bound to the purified lipopolysaccharide membrane specifically at the basal body ring closest to the hook (the L ring). The cytoplasmic membrane in preparations from osmotically lysed E. coli spheroplasts or Bacillus subtilis protoplasts was specifically attached to flagella at the basal body ring farthest from the hook (the M ring). In the E. coli preparation, lipopolysaccharide membrane was also present and was attached to the L ring. From these data and a knowledge of the structure and dimensions of the E. coli flagellar basal body and cell envelope, a model for flagellar attachment is deduced.     

The antibacterial effect of attacins from the silk moth Hyalophora cecropia is directed against the outer membrane of Escherichia coli.

The attacins are antibacterial proteins which accumulate in the hemolymph of the giant silk moth, Hyalophora cecropia, in response to a bacterial infection. Here we show that the permeability barrier function of the outer membrane is affected shortly after addition of attacin to growing cultures of Escherichia coli. Specifically, the penetration through the outer membrane of beta-lactam antibiotics, chicken egg white lysozyme and the detergent Triton X-100 was found to be facilitated. The sensitivity of E. coli to cecropin B, another antibacterial protein present in the hemolymph of H. cecropia, was also found to be increased after treatment with attacin. The results suggest that the target of the attacins in E. coli is the outer membrane. Other effects of the attacins which have been observed are likely to be indirect consequences of the alteration in the properties of the outer membrane. These effects include changes in the cell shape, irregular patterns of cell division and lysis. The minimal concentration at which the attacins affected the growth of E. coli was 1 and 0.5 microM for the neutral (pI 7) and basic (pI 9) attacins, respectively, which corresponds to less than 2% of the concentration of the attacins in the hemolymph of infected pupae.     

Zinc uptake and incorporation into proteins in T4D bacteriophage-infected Escherichia coli.

The metabolism of Zn2+ in Escherichia coli infected with T4D bacteriophage and various T4D mutants has been examined. E. coli B infected with T4D, and all T4D mutants except T4D 12-, took up zinc ions at a rate identical to that of uninfected cells. E. coli B infected with T4D 12- had a markedly decreased rate of zinc uptake. The incorporation of zinc into proteins of infected cells has also been studied. T4D phage infection was found to shut off the synthesis of all bacterial host zinc metalloproteins while allowing the formation of viral-induced zinc proteins. The amount of zinc incorporated into viral proteins was affected by the absence of various T4D gene products. Cells infected with T4D 12-, and to a much less extent those infected with T4D 29-, incorporated the least amount of zinc into proteins, while cells infected with T4D 11- and T4D 51- incorporated increased amounts of zinc into the zinc metalloproteins. In cells infected with T4D 11- and 51- most of the zinc protein was found to be the product of gene 12. The marked effect of infection of E. coli with T4D 12- on both zinc uptake and zinc incorporation into protein supports the conclusion that T4D gene 12 protein is a zinc metalloprotein. Additionally, these observations have indicated that this metalloprotein interacts with host cell membrane.     

Outer membrane vesicle production by Escherichia coli is independent of membrane instability

It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the sigma(E) cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.     

Localization of the dicarboxylate binding protein in the cell envelope of Escherichia coli K12

Examination of the localization of the dicarboxylate binding protein (DBP) in the cell envelope of Escherichia coli K12 reveals that this protein is present on the cell surface, and also in the inner and outer regions of the periplasmic space. The cell surface DBP is release by treating the cells with EDTA. This protein can be surface labeled by lactoperoxidase radioiodination, and by diazo[125I]iodosulfanilic acid in whole cells. It also binds tightly, but not covalently, to lipopolysaccharide. The DBP located in the outer region of the periplasmic space is released when the outer membrane is dissociated by EDTA-osmotic shock treatment. The DBP located in the inner region of the periplasmic space is released only when the EDTA-osmotic shocked cells are subjected to lysozyme treatment. At the moment, it is not certain whether this protein is bound to or trapped by the peptidoglycan network. This protein cannot be surface labeled in whole cells or in EDTA-osmotic shock treated cells; and it is not associated with lipopolysaccharide. Analysis of transport mutants indicates that these DBP are coded by the same gene.     

Phospholipase-A-independent damage caused by the colicin A lysis protein during its assembly into the inner and outer membranes of Escherichia coli

The requirement for the activation of phospholipase A by the colicin A lysis protein (Cal) in the efficient release of colicin A by Escherichia coli cells containing colicin A plasmids was studied. In particular, we wished to determine if this activation is the primary effect of Cal or whether it reflects more generalized damage to the envelope caused by the presence of large quantities of this small acylated protein. E. coli tolQ cells, which were shown to be leaky for periplasmic proteins, were transduced to pldA and then transformed with the recombinant colicin A plasmid pKA. Both the pldA and pldA+ strains released large quantities of colicin A following induction, indicating that in these cells phospholipase A activation is not required for colicin release. This release was, however, still dependent on a functioning Cal protein. The assembly and processing of Cal in situ in the cell envelope was studied by combining pulse-chase labelling with isopycnic sucrose density gradient centrifugation of the cell membranes. Precursor Cal and lipid-modified precursor Cal were found in the inner membrane at early times of chase, and gave rise to mature Cal which accumulated in both the inner and outer membrane after further chase. The signal peptide was also visible on these gradients, and its distribution too was restricted to the inner membrane. Gradient centrifugation of envelopes of cells which were overproducing Cal resulted in very poor separation of the membranes. The results of these studies provide evidence that the colicin A lysis protein causes phospholipase A-independent alterations in the integrity of the E. coli envelope.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipase activity in bacteriophage-infected Escherichia. II. Activation of phospholipase by T4 ghost infection.

The release of free fatty acids from the phospholipids of Escherichia coli is initiated immediately after the attachment of T4 ghosts. A similar accumulation of free fatty acids is observed if the cells are infected with T4 phage in the presence of chloramphenicol or puromycin. An early accumulation of free fatty acids, however, is not observed in T4 infections in which chloramphenicol or puromycin are not present, nor does it occur if the E. coli are infected with T4 phage before ghost infection, suggesting that phage products can prevent the phospholipid deacylation. If E. coli is infected with T4 ghosts before T4 phage infection, the accumulation of free fatty acids is not suppressed. When phospholipase-deficient E, coli are infected with T4 ghosts the appearance of free fatty acids is not observed, suggesting that T4 ghost attachment can activate the phospholipase of wild-type E. coli. Although the formation of free fatty acid apparently is a consequence of activation of the detergent-resistant phospholipase of the outer membrane, it is not observed in mutants deficient in the detergent-sensitive phospholipase.     

Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Accumulation of a murein-membrane attachment site fraction when cell division is blocked in lkyD and cha mutants of Salmonella typhimurium and Escherichia coli.

Membrane fractionation studies were performed on Salmonella typhimurium lkyD(Ts) and E. coli cha(Ts) mutants that appeared to be blocked at a late stage of the cell division cycle. In both cases growth of the mutant strains at nonpermissive temperatures was associated with accumulation of a characteristic cell envelope fraction (fraction OML) that contained inner membrane, murein, and outer membrane components. The isolated fraction corresponded in composition and bouyant density to a fraction from wild-type strains that had previously been suggested (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-767, 1982; K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, B. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) to contain adhesion sites between inner membrane, murein, and outer membrane. The accumulation of OML in LkyD- and Cha- cells was prevented by treatments that blocked DNA synthesis. The effects of interference with DNA synthesis did not appear to involve the SOS response.