Fractionation of the Providencia stuartii cell envelope

Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.     

Lipopolysaccharides from thermosensitive mutants of Escherichia coli K12 (author's transl)]

Thermosensitive mutants of Escherichia coli K12 were grown at 30 degrees C and 40 degrees C. The serologic properties and the composition of their lipopolysaccharides were investigated. The inhibition of hemagglutination by the lipopolysaccharides of various mutant strains was tested against the anti-E. coli K12 CR34 system. An inhibition was observed with all the mutants but one, CR34 T83 which had no inhibitory effect. Chemical analysis of lipopolysaccharides and mass spectrometric analysis of their methylated derivatives indicated the presence of the same components in the various lipopolysaccharides: glucose, galactopyranose, galactofuranose, heptopyranose and heptofuranose. However the 2,3,4 tri-O-methyl glucose is missing in the lipopolysaccharide of the mutant T83. This result agrees with the absence of a substituent on the 6-position of the non-reducing core-terminal glucose. The lipopolysaccharide of the T83 mutant has the complete core-type of E. coli K12. The relations of mutations with modifications of the composition of inner and outer envelopes in various mutants are discussed.     

The effect of zwitterionic detergents on the extraction and functional properties of cartilage proteoglycans

A range of structurally related zwitterionic detergents, Zwittergents 3-06, 3-08, 3-10, and 3-12, and a derivative of cholic acid (Chaps) were examined for their ability to enhance the extraction of newly synthesized, intracellular proteoglycans and for their effect on the functional properties of cartilage proteoglycan. Although none of the detergents could extract greater than 4% of the intracellular proteoglycans when used alone, Zwittergents 3-10, 3-12, and Chaps proved equally as effective when used in combination with 4 M guanidine HCl extracting greater than 90% of newly synthesized proteoglycans. Rate zonal centrifugation of aggregates containing either 3H-link protein or 3H-monomer, which had been incubated with 2% (w/v) detergent indicated that none of the test detergents caused a disassembly of intact aggregates. However, both Zwittergents 3-10 and 3-12 prevented the reaggregation of components dissociated with 4 M guanidine HCl. Similar to the finding with aggregate, none of the detergents caused a disassembly of monomer-link protein complexes prepared from purified 3H-link protein and proteoglycan monomer, while Zwittergents 3-10 and 3-12 prevented their assembly from free link protein and monomer. However, monomer-link protein complexes once formed were able to associate with hyaluronic acid to form link-stable ternary complexes in the presence of all detergents tested including Zwittergents 3-10 and 3-12.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

Characterization of biological activities of Brucella melitensis lipopolysaccharide

Biological activities of lipopolysaccharide (LPS) from Brucella melitensis 16M were characterized in comparison with LPS from Escherichia coli O55. LPS extracted from B. melitensis was smooth type by electrophoretic analysis with silver staining. The endotoxin-specific Limulus activity of B. melitensis LPS was lower than that of E. coli LPS. There was no significant production of tumor necrosis factor-alpha and nitric oxide in RAW 264.7 macrophage cells stimulated with B. melitensis LPS, although E. coli LPS definitely induced their production. On the other hand, B. melitensis LPS exhibited a higher anti-complement activity than E. coli LPS. B. melitensis LPS as well as E. coli LPS exhibited a strong adjuvant action on antibody response to bovine serum. The characteristic biological activities of B. melitensis are discussed.     

Extraction of membrane antigens from Brucella ovis and an assessment of their serological activity by immunoblotting

The efficacy and selectivity of chaotropic and phase-partitioning procedures for the extraction of membrane proteins from Brucella ovis were compared with a standard Sarkosyl method. Major group 1, 2 and 3 outer-membrane proteins (OMPs) of B. ovis stained by Coomassie blue in SDS-PAGE gels had, respectively, apparent molecular masses of 81/82 kDa, 39-41 kDa and 30-32 kDa. The presence of these bands in the Sarkosyl extract of total membrane vesicles (TMVs) indicate that the procedure failed to selectively solubilize only inner-membrane proteins (IMPs). SDS-PAGE analyses also revealed the presence of OMPs and other additional bands following extraction of B. ovis TMVs by butanol phase-partitioning or with extraction solutions based on the chaotropic reagents potassium thiocyanate (KSCN), sodium salicylate (SSC) and lithium acetate (LAE). OMPs are therefore not selectively extracted by any one of these procedures. Based on the number and staining intensity of extracted membrane-associated polypeptides, the efficacy of different extraction procedures could be graded in decreasing order as follows: KSCN, SSC, butanol and LAE. Both butanol and SSC were particularly effective in extracting group 3 OMPs. Sera from chronic excretor rams were used to identify zones of seroreactivity in immunoblots. Essentially, two reactivity patterns were seen: strong antibody binding against polypeptides in zones A (46-85 kDa), C (28-32 kDa) and D (18-22 kDa) in one, and additional reactivity against zones B (34-44 kDa) and E (13-18 kDa) polypeptides in the other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological detection of heterogeneous O-antigen containing lipopolysaccharides in Escherichia coli

The components of the cell envelopes of Escherichia coli O1:K1, O7:K1, O18:K1 and O83:K1 strains were separated on SDS-polyacrylamide gels. Longitudinal slices (50 microns thick) of the gel were incubated with typing sera for E. coli O1, O7, O18 and O83, followed by detection of the bound antibodies with 125I-labelled protein A and autoradiography. The antisera reacted with many cell envelope components of strains both with the homologous O-serotype and heterologous O-serotypes. With O-typing sera cross-reactions with heterologous cells and cells boiled for 2 h were found. Up to 40 serotype-specific bands at regular positions with molecular weights between 12000 and 100000 were demonstrated. Since these bands were also observed when purified lipopolysaccharide and unabsorbed homologous O-typing sera were used, it was concluded that these bands represented lipopolysaccharide molecules with increasing molecular weight, all of which contained O-antigen specific immunodeterminants. The band patterns were not influenced by the growth conditions of the cells or the various isolation procedures for the cell envelopes. Comparison of various strains serotyped as O18 revealed strain differences with respect to their lipopolysaccharide band patterns. In the case of O21- and O83-serotyped strains lipopolysaccharide cross-reactions, which were detected by agglutination, were analysed in detail using the gel immunoradioassay method. These cross-reactions appeared to be caused by the presence of common determinants on their lipopolysaccharides and polysaccharide-like material. The cross-reacting antibodies could be removed by cross-absorption. It is concluded that the immunological detection of lipopolysaccharides and other components of E. coli in gels is an important tool in (1) the control of the specificity of typing antisera, (2) the study of the nature of cross-reacting antigens and (3) the study of the nature and uniformity of the various O- and K-serotypes.     

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol-water extraction procedure was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure: (formula; see text) The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4, 6-dideoxy-alpha-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.     

Comparison of methods used to separate the inner and outer membranes of cell envelopes of Campylobacter spp

The outer membrane of Campylobacter coli, C. jejuni and C. fetus cell envelopes appeared as three fractions after sucrose gradient centrifugation. Each outer membrane fraction was contaminated with succinate dehydrogenase activity from the cytoplasmic membrane fraction. Similarly the inner membrane fraction was contaminated with 2-ketodeoxyoctonate and outer membrane proteins including the porin(s). The separation of these two membranes was not facilitated by variations in lysozyme treatment, cell age, presence or absence of flagella, or longer lipopolysaccharide chain length. Sodium lauroyl sarcosinate extraction resulted in an outer membrane fraction which contained some inner membrane contamination and produced multiple bands upon sucrose gradient centrifugation. Triton X-100 extraction removed the inner membrane from the outer membrane and Triton X-100/EDTA treatment extracted lipopolysaccharide-rich regions of the outer membrane which contained almost exclusively the Campylobacter porin(s). These data indicated that the inner and outer membranes of the Campylobacter cell envelope were very difficult to separate, possibly because of extensive fusions between these two membranes.     

Isolation and characterization of Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor.

Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor (ConI-) were isolated and characterized. These mutants are specific in that they are conjugation proficient with other types of donor strains. They have an altered susceptibility to phages and detergents. Chemical analysis of the cell envelopes of mutant strains has shown that the lipopolysaccharide (LPS) is altered and that one major outer-membrane protein is absent. Conjugation experiments in which LPS from wild-type cells was added to a mating mixture, made up with wild-type donor and recipient cells, showed inhibition in transconjugant formation when an I-type donor, but not an F-type donor, was used. This strongly suggests that LPS of the recipient cell is directly involved in the ability to mate with an I-type donor but not with an F-type donor. The mutations are located in the 78- to 82-min region of the E. coli map, with one exception where the mutation maps near or in the galactose operon.     

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.     

Immunological activity of lipopolysaccharide of Helicobacter pylori on human peripheral mononuclear blood cells in comparison to lipopolysaccharides of other intestinal bacteria

Abstract Lipopolysaccharide of Helicobacter pylori was tested for its mitogenicity and for its ability to stimulate cytokine release in human peripheral blood mononuclear cells (PBMC) of healthy and H. pylori -infected blood donors. Mitogenicity in PBMC induced by H. pylori LPS was similar to that induced by Campylobacter jejuni lipopolysaccharide, but lower than that induced by Escherichia coli lipopolysaccharide in the H. pylori negative blood donor group. Furthermore, H. pylori LPS was able to induce tumour necrosis factor (TNF) interleukin 1 (IL-1) and interleukin 6 (IL-6) secretion of PBMC. Compared with the ability of C. jejuni and E. coli lipopolysaccharides to stimulate cytokine release, H. pylori lipopolysaccharide induced a significantly lower TNF and IL-1 secretion of PBMC than the other tested bacterial lipopolysaccharides. Similar amounts of IL-6 release were obtained by stimulation of PBMC with H. pylori and C. jejuni lipopolysaccharides, whereas a higher IL-6 release was measured by stimulation with E. coli lipopolysaccharide. The results of this study suggest that H. pylori lipopolysaccharide has a lower immunological activity than lipopolysaccharides of other intestinal bacteria. This is probably due to its unusual acylation and phosphorylation pattern of lipid A.     

Sensitivity of Escherichia coli cell membranes to various classes of detergents

The mechanisms of interaction between non-ionic or cationic surfactants with Escherichia coli K-12 cell membranes were studied using an approach based on the registration of changes in the membrane permeability to ethidium bromide, a fluorescent dye for nucleic acids. Triton X-100, a non-ionic detergent, was shown to exert no effect on the permeability of intact cell membranes. Triton X-100 interacted with the bacteria only after treatment with EDTA, a complexing agent for bivalent cations. Cetyltrimethyl ammonium bromide increased the permeability to ethidium bromide and the action of this cationic detergent did not require the pretreatment with the complexing agent. SDS, an anionic detergent, damaged E. coli K-12 and this could be registered by the lowering of intensity of light scattering by the bacterial suspension. The surface charge of E. coli K-12 cells was shown to influence the interaction of ionic detergents with bacterial cell membranes. Its variation by changing the pH of the incubation medium did not make E. coli K-12 sensitive to Triton X-100.     

Isolation and biological activities of endotoxin from Leptospira interrogans.

Endotoxins extracted with ethylenediaminetetraacetate (EDTA) from Leptospira interrogans serovars icterohaemorrhagiae and canicola and Leptospira biflexa serovar patoc were tested for various biological activities characteristic of endotoxins. The presence of lipopolysaccharide biological activity was demonstrated by the Limulus amoebocyte lysate test, pyrogenicity in rabbits, complement interaction inhibiting the erythrocyte lysis, and chicken-embryo lethality. The lipopolysaccharides did not induce the local Shwartzman reaction. The lipopolysaccharides of serovars icterohaemorrhagiae and canicola were immunogenic in rabbits and were cytotoxic to chicken-embryo fibroblasts.     

Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5.

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkyline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.     

Biological properties of lipopolysaccharides from Bordetella species

Biological activities of lipopolysaccharides (LPS) extracted from Bordetella pertussis, B. parapertussis and B. bronchiseptica were compared with those of Escherichia coli LPS. The LPS preparations from B. pertussis showed biological activities comparable to those of E. coli LPS in terms of lethal toxicity in galactosamine-sensitized mice, pyrogenicity in rabbits, mitogenicity in C3H/He spleen cell cultures, macrophage activation, and induction of tumour necrosis factor. All the activities of LPS preparations from B. parapertussis, except mitogenicity, were lower than those of E. coli LPS. LPS from B. parapertussis gave the greatest mitogenic action of all those tested. Biological activities stronger than or comparable to those of E. coli LPS were observed for LPS from B. bronchiseptica.     

Proteins Released from Cell Envelopes of Pseudomonas aeruginosa on Exposure to Ethylenediaminetetraacetate: Comparison with Dimethylformamide-Extractable Proteins

Isolated cell envelopes of Pseudomonas aeruginosa were treated with N,N'-dimethylformamide (DMF) or with ethylenediaminetetraacetate (EDTA). DMF solubilized 73% of the dry weight of the cell envelope, 76% of the protein, 78% of the carbohydrate, and 76% of the phosphorus. Electron microscopy showed that DMF caused extensive alterations in the appearance of the cell envelope with blebs and bleblike vesicles predominating. After incubation with EDTA, the cell envelopes appeared to have lost material, but still retained the cell-like morphology. Analysis of DMF-solubilized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 16 protein bands. There were three major proteins that predominated, however, with molecular masses of 43,000 (protein A), 16,500 (protein B), and 72,000 daltons (protein C). Evidence is presented that protein A and protein B are glycoproteins. Gel electrophoresis of EDTA-solubilized material revealed that a number of proteins were released from the cell envelope. However, electrophoresis of an isolated protein-lipopolysaccharide complex released by EDTA showed that protein A and protein B were the major protein components of this complex. These data suggest that protein A and protein B are components of the outer cell wall membrane of P. aeruginosa. There is suggestive evidence that these proteins may play a role in maintaining the structural integrity of the cell envelope. Whether these proteins also have enzymatic activity could not be discerned from the present study, although it is possible that they may be associated with the terminal stages of lipopolysaccharide synthesis.     

Detergent induced inhibition of eukaryotic RNA polymerase B activity and amanitin binding

We studied the effect of detergents on the binding of amanitin to RNA polymerase and on enzymatic activity. SDoS, Sarkosyl and deoxycholate were most inhibitory. Cholate and non-ionic detergents were less inhibitory. Evidence is presented that Sarkosyl inhibits chain elongation. The inhibition of amanitin binding was most influenced by the hydrophilicity of the detergent.     

Avian air sac and plasma proteins that bind surface polysaccharides of Escherichia coli O2

Some serovars of Escherichia coli, mainly O2 and O78, are responsible for air sac and systemic infections in farm-raised turkeys (Meleagris gallopavo) and chickens (Gallus gallus). We looked in air sac surface fluid from young turkeys to identify proteins that bind surface polysaccharides of pathogenic respiratory E. coli O2. Turkey air sac surface fluid was subjected to affinity chromatography on Toyopearl AF-Epoxy-650M, coupled with either lipopolysaccharide (LPS) or lipid-free polysaccharide (LFP) purified from an avian pathogenic E. coli O2 isolate. A multimeric protein termed lipid-free polysaccharide binding protein-40 (LFPBP-40) composed of six covalently associated subunits of approximately 40 kDa was isolated by elution from LFP by EDTA or L-rhamnose. An analogous protein in air sac fluid proteins bound to intact E. coli O2 and eluted with L-rhamnose or N-acetylglucosamine (GlcNAc). The N-terminal amino acid sequence of LFPBP-40 DINGGGATLPQHLYLTPDV was related to the N-terminus of fragment 3 of a partially characterized human protein possessing T cell stimulation activity in synovial membrane of rheumatoid arthritis patients. However, endogenous amino acid sequences were unrelated to other known proteins. LFPBP-40 was immunoreactively distinct from pulmonary collectins and ficolins. These studies demonstrate a novel avian respiratory soluble lectin that can bind surface polysaccharides of pathogenic E. coli responsible for respiratory disease.     

Accelerated Adsorption of Bacteriophage T5 to Escherichia coli F, Resulting from Reversible Tail Fiber-Lipopolysaccharide Binding

A dual specificity for phage T5 adsorption to Escherichia coli cells is shown. The tail fiber-containing phages T5(+) and mutant hd-3 adsorbed rapidly to E. coli F (1.2 x 10(-9) ml min(-1)), whereas the adsorption rate of the tail fiber-less mutants hd-1, hd-2, and hd-4 was low (7 x 10(-11) ml min(-1)). The differences in adsorption rates were due to the particular lipopolysaccharide structure of E. coli F. Phage T4-resistant mutants of E. coli F with an altered lipopolysaccharide structure exhibited similar low adsorption for all phage strains with and without tail fibers. The same held true for E. coli K-12 and B which also differ from E. coli F in their lipopolysaccharide structures. Only the tail fiber-containing phages reversibly bound to isolated lipopolysaccharides of E. coli F. Infection by all phage strains strictly depended on the tonA-coded protein in the outer membrane of E. coli. We assume that the reversible preadsorption by the tail fibers to lipopolysaccharide accelerates infection which occurs via the highly specific irreversible binding of the phage tail to the tonA-coded protein receptor. The difference between rapid and slow adsorption was also revealed by the competition between ferrichrome and T5 for binding to their common tonA-coded receptor in tonB strains of E. coli. Whereas binding of T5(+) to E. coli K-12 and of the tail-fiber-less mutant hd-2 to E. coli F and K-12 was inhibited 50% by about 0.01 muM ferrichrome, adsorption of T5 to E. coli F was inhibited only 40% by even 1,000-fold higher ferrichrome concentrations.