Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope

Cell envelopes of Salmonella typhimurium and Escherichia coli were disrupted in a French pressure cell and fractionated by successive cycles of sedimentation and floatation density gradient centrifugation. This permitted the identification and isolation of several membrane fractions in addition to the major inner membrane and murein-outer membrane fractions. One of these fractions (fraction OML) accounted for about 10% of the total cell envelope protein, and is likely to include the murein-membrane adhesion zones that are seen in electron micrographs of plasmolyzed cells. Fraction OML contained inner membrane, murein, and outer membrane in an apparently normal configuration, was capable of synthesizing murein from UDP-[3H]N-acetylglucosamine and UDP-N-acetylmuramylpentapeptide and covalently linking it to the endogenous murein of the preparation, and showed a labeling pattern in [3H]galactose pulse-chase experiments that was consistent with its acting as an intermediate in the movement of newly synthesized lipopolysaccharide from inner membrane to outer membrane. The fractionation procedure also identified two new minor membrane fractions, with characteristic protein patterns, that are usually included in the region of the major inner membrane peak in other fractionation procedures but can be separated from the major inner membrane fraction and from contaminating flagellar fragments by the subsequent floatation centrifugation steps.     

Specific location of penicillin-binding proteins within the cell envelope of Escherichia coli.

This communication deals with the location of penicillin-binding proteins in the cell envelope of Escherichia coli. For this purpose, bacterial cells have been broken by various procedures and their envelopes have been fractioned. To do so, inner (cytoplasmic) and outer membranes were separated by isopycnic centrifugation in sucrose gradients. Some separation methods (Osborn et al., J. Biol. Chem. 247:3962-3972, 1972; J. Smit, Y. Kamio, and H. Nikaido, J. Bacteriol. 124:942-958, 1975) revealed that penicillin-binding proteins are not exclusively located in the inner membrane. They are also found in the outer membrane (A. Rodríguez-Tébar, J. A. Barbas, and D. Vásquez, J. Bacteriol. 161:243-248, 1985). Under the milder conditions for cell rupture used in this work, an intermembrane fraction, sedimenting between the inner and outer membrane, can be recovered from the gradients. This fraction has a high content of both penicillin-binding proteins and phospholipase B activity and may correspond to the intermembrane adhesion sites (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-769, 1982). We postulate that this intermembrane fraction is a labile structure that contains a high amount of all penicillin-binding proteins which are usually found in both the inner and outer membranes when the adhesion sites are destroyed by the cell breakage and fractionation procedures.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. I. Electrophoretic and immunochemical analyses

We have developed a fast and reliable method for the separation of two membrane fractions respectively enriched in outer and inner envelope membranes from isolated, intact, purified spinach chloroplasts kept in a hypertonic medium (0.6 M mannitol). This separation was achieved by osmotically shrinking the inner envelope membrane, thus widening the intermembrane space, and then subsequently removing the "loosened" outer envelope membrane by applying low pressure to the shrunken chloroplasts and slowly extruding them through the small aperture of a Yeda press under controlled conditions. By centrifugation of the mixture obtained through a discontinuous sucrose gradient, we were able to separate two membrane fractions having different densities (fraction 2 or light fraction, d = 1.08 g/cm3, and fraction 3 or heavy fraction, d = 1.13 g/cm3). The recent characterization of polypeptides localized on the outer envelope membrane from spinach chloroplasts, E10 and E24 (Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, N.-H., and Douce, R. J. Biol. Chem., 258, 10000-10006) enabled us to characterize our two membrane fractions. Analyses of the polypeptides by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and immunoblotting have shown that fraction 2 (light fraction) was completely devoid of polypeptide E30, which is involved in the transport of phosphate across the inner envelope membrane, but was enriched in polypeptides E10 and E24. The reverse was true for fraction 3 (heavy fraction). Under these conditions, it is clear that fraction 2 is strongly enriched in outer envelope membrane whereas fraction 3 consisted mostly of inner envelope membrane. Indeed, by immunoelectrophoresis, we were able to demonstrate that, on a protein basis, fraction 2 contained about 90% of outer membrane, whereas fraction 3 contained about 80% of inner membrane. Further characterization of the outer envelope membrane was achieved by using thermolysin, a nonpenetrant protease.     

Chemical cross-linking of thioredoxin to hybrid membrane fraction in Escherichia coli

Thioredoxin was cross-linked to a membrane fraction in vivo using the heterobifunctional photoreactive cross-linking reagent p-azidophenacyl bromide, chosen to couple thioredoxin via its highly reactive thiol. Under mild reaction conditions, a significant amount of thioredoxin (30%) was rapidly cross-linked to the crude membrane fraction. The cross-linking reaction was selective, with thioredoxin purified 15-fold in the cross-linked membrane fraction. Membrane fractionation studies showed that thioredoxin associated with the inner membrane and with a hybrid membrane fraction. This hybrid membrane fraction banded at a density between the inner and outer membranes. This result is consistent with the localization of thioredoxin in association with the bacterial membrane adhesion sites first described by Bayer (Bayer, M. (1968) J. Gen. Microbiol. 53, 395-404). Association of thioredoxin with the membrane adhesion sites defines a structure corresponding to the osmotically sensitive cytoplasmic compartment (Lunn, C. A., and Pigiet, V. (1982) J. Biol. Chem. 257, 11424-11430).     

Interactions of the origin of replication (oriV) and initiation proteins (TrfA) of plasmid RK2 with submembrane domains of Escherichia coli.

It has been possible to locate a submembrane domain representing less than 10% of the total membrane that appears to be responsible for sequestering some essential components required for plasmid RK2 DNA replication. This subfraction, whose cellular location in the membrane prior to extraction is still unknown, is derived from the inner membrane fraction, since it possesses enzyme marker activity (NADH oxidase) exclusively associated with the inner membrane. The subfraction was detected by a modification of the methods of Ishidate et al. (K. Ishidate, E. S. Kreeger, J. Zrike, S. Deb, B. Glauner, T. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) in which low pressure in a French pressure cell and lysozyme were used to preserve the supercoil plasmid DNA template during cell disruption. This was followed by successive cycles of sucrose gradient sedimentation and flotation density gradient centrifugation to reveal a number of subfractions, including the one of interest. The characteristics of plasmid interaction with the subfraction include the presence of supercoil DNA after extraction, the binding of the origin of plasmid replication (oriV) in vitro, and the association of the two plasmid-encoded initiation (TrfA) proteins (encoded by overlapping genes). However, another peak, the outer membrane fraction, also binds oriV in vitro, contains plasmid DNA in vivo, and associates with the TrfA initiation proteins. Nevertheless, it contains much less of the initiation proteins, and the specific activity of binding oriV is also much reduced compared with the other subfraction. There is a strong correlation between the association of the TrfA initiation proteins with a particular membrane fraction and the binding of oriV in vitro or plasmid DNA in vivo. Since the proteins are known to bind to repeated sequences in oriV (S. Perri, D. R. Helinski, and A. Toukdarian, J. Biol. Chem. 266:12536-1254, 1991; M. Pinkney, R. Diaz, E. Lanka, and C. M. Thomas, J. Mol. Biol. 203: 927-938, 1988), it appears that the initiation proteins themselves could be responsible, at least in part, for the association of plasmid DNA to the membrane.     

TrfA-dependent inner membrane-associated plasmid RK2 DNA synthesis and association of TrfA with membranes of different gram-negative hosts

TrfA, the replication initiator protein of broad-host-range plasmid RK2, was tested for its ability to bind to the membrane of four different gram-negative hosts in addition to Escherichia coli: Pseudomonas aeruginosa, Pseudomonas putida, Salmonella enterica serovar Typhimurium, and Rhodobacter sphaeroides. Cells harboring TrfA-encoding plasmids were fractionated into soluble, inner membrane, and outer membrane fractions. The fractions were subjected to Western blotting, and the blots were probed with antibody to the TrfA proteins. TrfA was found to fractionate with the cell membranes of all species tested. When the two membrane fractions of these species were tested for their ability to synthesize plasmid DNA endogenously (i.e., without added template or enzymes), only the inner membrane fraction was capable of extensive synthesis that was inhibited by anti-TrfA antibody in a manner similar to that of the original host species, E. coli. In addition, although DNA synthesis did occur in the outer membrane fraction, it was much less extensive than that exhibited by the inner membrane fraction and only slightly affected by anti-TrfA antibody. Plasmid DNA synthesized by the inner membrane fraction of one representative species, P. aeruginosa, was characteristic of supercoil and intermediate forms of the plasmid. Extensive DNA synthesis was observed in the soluble fraction of another representative species, R. sphaeroides, but it was completely unaffected by anti-TrfA antibody, suggesting that such synthesis was due to repair and/or nonspecific chain extension of plasmid DNA fragments.     

Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili

Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with [35S]methionine and fractionated by a variety of techniques. A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein. Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space. The 29,000-dalton polypeptide was shown to be processed in E. coli minicells. The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions. E. coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants. The possible functions of the adh cistron products are discussed.     

Isolation of an inner membrane-derived subfraction that supports in vitro replication of a mini-RK2 plasmid in Escherichia coli

Previous results have demonstrated that the inner, but not the outer, membrane fraction of Escherichia coli is the site of membrane-associated DNA replication of plasmid RK2, a broad-host-range plasmid capable of replication in a wide variety of gram-negative hosts (K. Michaels, J. Mei, and W. Firshein, Plasmid 32:19-31, 1994). To resolve the inner membrane replication site further, the procedure of Ishidate et al. (K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, G. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) was used to separate the inner membrane into a number of subfractions, of which only one, a small subfraction containing only 10% of the entire membrane, was found to synthesize DNA inhibited by antibody prepared against the plasmid-encoded initiation protein TrfA. This is the same subfraction that was also found to bind oriV and TrfA to the greatest extent in filter binding assays (J. Mei, S. Benashski, and W. Firshein, J. Bacteriol. 177:6766-6772, 1995).     

Morphogenesis of the bacterial division septum: identification of potential sites of division in lkyD mutants of Salmonella typhimurium.

It previously has been shown that lkyD mutants of Salmonella typhimurium form large blebs of outer membrane over the septal and polar regions of dividing cells. To determine whether the outer membrane blebs are formed over potential sites of division even in the absence of septal ingrowth, lkyD strains were studied under conditions in which ingrowth of inner membrane and murein was prevented by inactivation of the envA gene product. In aseptate filaments of the LkyD EnvA strain, outer membrane blebs occurred with the usual frequency and were preferentially located over regions where new septa were formed when cell division was subsequently permitted to resume. The results indicate that the outer membrane blebs of the LkyD strain are markers for potential sites of cell division, implying that an alteration in association of outer membrane and murein exists in these sites before the initiation of septal ingrowth. This localized change in cell envelope organization is independent of the septation-inducing effects of the envA gene product.     

Mutations in genes cpxA and cpxB alter the protein composition of Escherichia coli inner and outer membranes.

Mutations in chromosomal genes cpxA and cpxB altered the protein composition of the inner and outer bacterial membranes. Electrophoretic analyses of membrane proteins from isogenic strains differing only at their cpx loci and of spontaneous cpxA+ revertants of a cpxA cpxB double mutant showed that the alterations define a pattern that is uniquely attributable to the cpx mutations. Two major outer membrane proteins, the OmpF matrix porin and the murein lipoprotein, were deficient or absent from the outer membrane of mutant cells, whereas the quantities of two other major outer membrane proteins, the OmpC matrix porin and the OmpA protein, were not significantly altered. The cpx mutations did not generally alter the functional or chemical properties of the cell envelope. In the electron microscope, mutant cells appeared ovoid, but individual cells showed no surface irregularities to suggest gross defects in the cell envelope. These observations suggest that the primary effect of the mutations is to alter selectively the synthesis or translocation of certain envelope proteins.     

Insertion of a MalE β-Galactosidase Fusion Protein into the Envelope of Escherichia coli Disrupts Biogenesis of Outer Membrane Proteins and Processing of Inner Membrane Proteins

The synthesis of a membrane-bound MalE β-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.     

Isolation and partial characterization of membrane vesicles carrying markers of the membrane adhesion sites.

At areas of adhesion between outer membrane (OM) and inner membrane (IM) in gram-negative bacteria, newly synthesized membrane constituents are inserted, and bacteriophage infection occurs. We describe here the isolation of these sites from cell membrane fractions of Salmonella anatum. Sucrose density gradients yielded membrane vesicles of the OM and IM; their mutual cross-contamination was low, as measured by 2-keto-3-deoxyoctonate and beta-NADH-oxidase activities. To mark the areas of lipopolysaccharide synthesis in the envelope (the adhesion sites), we infected S. anatum with phage epsilon 15, which causes a rapid change (conversion) in the cell's O-antigenic composition from serogroup E1 to E2; lipopolysaccharide of type E2 also serves as receptor for phage epsilon 34. We found that the fractions of intermediate density (Int. M) from briefly converted cells bound both phage epsilon 34 and E2-specific antibody. In the electron microscope, epsilon 34 was seen to have absorbed with a high degree of significance to the Int. M fraction of briefly converted cells, but not to the Int. M fraction of unconverted cells. Furthermore, the Int. M fractions of briefly converted cells coagglutinated anti-E2-coated Staphylococcus aureus, whereas the OM and IM fractions showed comparatively little agglutination. In addition, Int. M material exhibited elevated phospholipase A1 and A2 activities comparable to those of the OM fraction; the IM was essentially phospholipase free. Our data indicate that this membrane fractionation allows one to isolate from Int. M regions a variety of activities associated with adhesion sites.     

Caulobacter crescentus cell envelope: effect of growth conditions on murein and outer membrane protein composition

The murein and membrane protein compositions of Caulobacter crescentus strains CB13B1a and CB15 have been characterized, and the influence on cell envelope constituents of culture conditions which affect morphogenesis have been studied. Amino acid and sugar analysis of murein sacculi revealed a simple A1gamma murein configuration typical of gram-negative bacteria. The membranes of C. crescentus had low levels of 2-keto-3-deoxyoctonate relative to enteric bacteria, in addition to the absence of lipid A components (Shapiro et al., Science 173:884-892, 1971; Chow and Schmidt, J. Gen. Microbiol, 83:369-373, 1974). Nevertheless, C. crescentus membranes could be fractionated into inner and outer membrane components by sucrose density gradient centrifugation procedures developed for Escherichia coli. The proteins of the outer membrane were distributed between three major (I, II, and III) and two minor (IV and V) protein classes. Class I proteins were greater than or equal to 74,000 daltons and constituted the primary proteins of the outer membrane. Class I proteins were separated into approximately 50 polypeptides by two dimensional gel electrophoresis; the protein composition of thi s class was affected by culture conditions in both CB13B1a and CB15. Class II (47,000 to 39,000 daltons) and III (20,000 to 11,500 daltons) proteins differed in each strain in composition and response to culture conditions.     

Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells

In previous studies we demonstrated that peripheral-type benzodiazepine receptors (PBR) were coupled to steroidogenesis in several adrenocortical and Leydig cell systems (Mukhin, A.G., Papadopoulos, V., Costa, E., and Krueger, K.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9813-9816; Papadopoulos, V., Mukhin, A.G., Costa, E., and Krueger, K.E. (1990) J. Biol. Chem. 265, 3772-3779). The current study elucidates the specific step in the steroid biosynthetic pathway by which PBR mediate the stimulation in steroid hormone production. The adrenocorticotropin (ACTH)-responsive Y-1 mouse adrenocortical cell line was used to compare the mechanisms by which ACTH and PK 11195 (a PBR ligand) stimulate steroidogenesis. The effects of these agents were studied at three stages along the steroid biosynthetic pathway: 1) secretion of 20 alpha OH-progesterone by Y-1 cell cultures; 2) pregnenolone production by isolated mitochondrial fractions; 3) quantities of cholesterol resident in outer and inner mitochondrial membrane fractions. Steroid synthesis stimulated by ACTH was blocked by cycloheximide, an effect documented by other laboratories characterized by an accumulation of mitochondrial cholesterol specifically in the outer membrane. In contrast, PK 11195-stimulated steroidogenesis was not inhibited by cycloheximide, and the magnitude of the stimulation was markedly enhanced when the cells were pretreated with cycloheximide and ACTH. When isolated mitochondria were used, stimulation of pregnenolone production by PK 11195 was largely independent of exogenously supplied cholesterol, indicating that PBR act on cholesterol already situated within the mitochondrial membranes. This phenomenon was found to be the result of a translocation of cholesterol from outer to inner mitochondrial membranes induced by the PBR ligand. These studies therefore suggest that mitochondrial intermembrane cholesterol transport in steroidogenic cells is mediated by a mechanism coupled to PBR.     

Characterization of F-pilin as an inner membrane component of Escherichia coli K12.

Antipeptide antibodies were used to detect, purify, and characterize nonfilament F-pilin in the cell envelope of an F'tra+ strain of Escherichia coli. Affinity-purified goat antibodies raised against a peptide corresponding to the amino-terminal 14 amino acids of F-pilin detected F-pilin in immuno-overlay ("Western") blots of electrophoretically separated inner and outer membrane proteins. As expected, the molecule was absent from inner membrane preparations of F- or F'traA[Am] strains. Immunoreactive material was purified from inner membrane fractions and shown to be F-pilin by amino acid analysis. The anti-peptide antibodies also detected membrane forms of F-pilin produced by cells containing plasmid pTG801 (Grossman, T. & Silverman, P. (1989) J. Bacteriol. 171, 650-656). Most cell envelope pilin was in the inner membrane fraction, but a significant quantity fractionated with the outer membrane as well. The hydropathy profile of F-pilin suggested that the molecule is an integral membrane protein with two membrane-spanning domains. In confirmation, F-pilin and pTG801 pilins in inner membrane preparations were solubilized by a single extraction with the nonionic detergents Nonidet P-40 (2%) or Triton X-100 (2%), but not by 2 M KCl or 0.1 M NaOH. Moreover, analysis of traA'-'phoA constructs indicated that both the amino and carboxyl termini of F-pilin face the periplasm. The periplasmic location of the amino terminus was confirmed by immunoelectron microscopy of spheroplasts from F' and pTG801 strains, using a monoclonal antibody that recognizes an amino-terminal epitope. These data suggest a specific structure for membrane F-pilin. We discuss that structure in relation to the probable structure of filament F-pilin.     

Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.     

Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane

A DNA/membrane complex extracted from a miniplasmid derivative of the broad host range plasmid RK2 cultured in Escherichia coli capable of synthesizing new plasmid supercoiled DNA in vitro was treated with antibodies that were made against or reacted with the dnaA and dnaK host-encoded proteins, respectively. Anti-dnaA protein antibody inhibited total plasmid DNA synthesis significantly and the synthesis of supercoil plasmid DNA almost completely. In contrast, anti-dnaK protein antibody and nonimmune serum had little or no effect on total plasmid DNA synthesis. Both proteins were found to be present in the inner but not outer membrane fraction of E. coli. A variety of miniplasmid-encoded proteins which had previously been found in the DNA/membrane complex have also been localized to the inner but not outer membrane fraction. These include an essential initiation protein of 32 kDa (and an overlapping protein of 43 kDa coded for by the same gene), as well as a 30-kDa protein that may be linked to incompatibility functions. Various extraction methods were used to distinguish between the associated and the integral nature of the plasmid-encoded proteins. The results demonstrated that the essential replication proteins (32 and 43 kDa) as well as the 30-kDa protein was tightly bound to the inner membrane. Computer analysis of the amino acid sequence of the 32 (and 43)-kDa protein revealed a hydrophobic region that is only half that normally required to span the membrane. Other interactions are discussed with respect to attaching this protein to the membrane.     

Changes in the fine structure of microbial cells induced by chaotropic salts

The electron microscopic examination of the thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37 degrees C for 3-5 h or at 100 degrees C for 5-6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

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.