Immunochemical characteristics and serologic properties of lipopolysaccharides isolated from various Brucella species using various extraction methods

The results of the comparative analysis of LPS isolated by different methods of extraction from the cultures of several Brucella species differing in their virulence are presented. Purified LPS preparations have been obtained from Brucella virulent and vaccine strains by using such methods as water-phenol extraction, Boivin's method, mild alkaline hydrolysis of the antigen according to Boivin's procedure. The presence of certain relationship between the method used for the extraction of Brucella LPS to be compared and their chemical composition, immunological characteristics and serological activity has been established. As shown in this investigation, in the process of the preparation of a highly sensitive diagnosticum for the passive hemagglutination test the use of LPS obtained from Brucella virulent strains, but not from the vaccine strain, by the method of mild alkaline hydrolysis according to Boivin's procedure is expedient. The data presented in this work indicate that the soluble complex of lipid A obtained from Brucella LPS has been found to possess serological activity. The results of the study of the serological properties of lipid A indicate that the lipid component may also play a certain role in the manifestation of the serological activity of Brucella LPS.     

Preparation of tritiated lipopolysaccharides from Escherichia coli K12

Tritiated lipopolysaccharide (LPS) from E. coli K12 was prepared by coupling [3H]ethanolamine to the LPS core residue ketodeoxyoctonate (KDO) via activation of its carboxylic function with N-hydroxysuccinimide or N-hydroxy-sulfosuccinimide. Specific activities of 1.5 microCi/mg and 9 microCi/mg were obtained, respectively. Experiments comparing the activity of native and derivatized LPS suggested that the preparation of the radiolabelled LPS did not alter the structural properties of E. coli K12 LPS. This probe will be useful for studying the interactions between LPS and proteins.     

Isolation and various properties of nucleoside monophosphate kinases from Escherichia coli

A technique is proposed for isolation of nucleosidemonophosphate kinases--AMP-kinase (EC 2.7.4.11), GMP-kinase (EC 2.7.4.8), CMP-kinase (EC 2.7.4.14), UMP-kinase (EC 2.7.4.14) and TMP-kinase (EC 2.7.4.9)--from E. coli MRE-600. It involves cell destroying, precipitation of nucleic acids with polyethyleneimine, fractionation with ammonium sulphate followed by chromatography on different carriers (DEAE-Toyopearl-650 M, Matrex gel Blue A, Matrex gel Red A). The technique enables all the five enzymes to be obtained separately and without contaminations with nucleotide dephosphorylating enzymes. For all the enzymes the pH optimum was found to range from 6.5 to 8.0, and Mg2+ ions were found to be the best activator for all the enzymes studied. The substrate specificity was investigated with respect to acceptors and donors of the phosphate groups. The enzymes showed strict specificity to the heterocyclic base of the acceptor phosphate group. AMP-, GMP- and CMP-kinases phosphorylated the corresponding deoxynucleoside monophosphates less effectively than ribonucleoside monophosphates. ATP was found to be the most effective phosphate donor for all the enzymes under study.     

Characterization of lipopolysaccharides from Escherichia coli K-12 mutants.

Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.     

Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli.

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates (preferential growth along the a axis) or solid columns (preferential growth along the c axis), when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4 degrees C for 10 days. Analyses of crystals suggested that they consist of hexagonal lattices with the a axis (a side of the lozenge as a unit cell on the basal plane) of 0.462 nm for all these three kinds of LPSs and the c axes (perpendicular to the basal plane) of 5.85, 8.47, and 8.75 nm for S. minnesota Re and Ra LPSs and E. coli K-12 LPS, respectively, and that hydrocarbon chains of the lipid A portion play the leading part in crystallization, whereas the hydrophilic part of the lipid A (the disaccharide backbone) and R core exhibit a disordered structure or are in a random orientation. The phenomenon of doubling of the a axis to 0.924 nm was observed with crystals of S. minnesota Re LPS when they were incubated in 70% ethanol for an additional 180 days, but not with crystals of S. minnesota Ra LPS or E. coli K-12 LPS. S. minnesota S-form LPS possessing the O-antigen-specific polysaccharide and S. minnesota free lipid A obtained by acid hydrolysis of Re LPS did not crystallize under the same experimental conditions.     

The DNA binding properties of the MutL protein isolated from Escherichia coli.

The mutL gene of Escherichia coli, which is involved in the repair of mispaired and unpaired nucleotides in DNA, has been independently cloned and the gene product purified. In addition to restoring methyl-directed DNA repair in extracts prepared from mutL strains, the purified MutL protein binds to both double and single stranded DNA. The affinity constant of MutL for unmethylated single stranded DNA was twice that of its affinity constant for methylated single stranded DNA and methylated or unmethylated double stranded DNA. The binding of MutL to double stranded DNA was not affected by the pattern of DNA methylation or the presence of a MutHLS-repairable lesion.     

Analysis of electrophoretically heterogeneous lipopolysaccharides of Escherichia coli by immunoblotting

Abstract The purified lipopolysaccharide (LPS) from each of four Escherichia coli serogroups which was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by periodic acid-silver staining, exhibited a different distribution of bands. The same serogroup-specific banding patterns were evident on immunoblots developed with homologous but not heterologous O-antisera. Thus, the electrophoretic heterogeneity of LPS was confirmed and the multiple bands seen on SDS-PAGE further shown to contain O-serogroup-specific antigenic determinants.     

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.     

Characteristics of chromosomal DNA isolated from Escherichia coli spheroplasts

The chromosomal DNA of Escherichia coli spheroplasts induced by penicillin G was studied biochemically and electron microscopically. Although the spheroplasts were unable to divide, they continued to synthesize chromosomal DNA for several hours even in the presence of penicillin G. Some differences were observed between the chromosomal DNA of the parent cells and that of the spheroplasts in sucrose gradient centrifugation and electron microscopy; two types of chromosomal DNA, a slower sedimenting form and a faster sedimenting form, were released from the gently lysed parent cells. The former was membrane-free folded chromosome and the latter was membrane-associated chromosome. In contrast, the chromosome from the spheroplast showed a single intermediate value of sedimentation coefficient between those of the chromosomal DNA from the parent cell. Cytochrome spreading for electron microscopy showed that the spheroplast chromosomal DNA formed an aggregated mass consisting of several chromosome-molecules of the parent cell.     

Immunobiological properties of lipopolysaccharides isolated from Fusobacterium nucleatum and F. necrophorum

Lipopolysaccharides (LPSs) were isolated from Fusobacterium nucleatum ATCC 10953 and F. necrophorum ATCC 25286 by the hot phenol/water procedure. F. nucleatum LPS was composed of 16% (w/w) carbohydrate, 10% (w/w) hexosamine and 40% (w/w) fatty acid, while F. necrophorum LPS was composed of 26% (w/w) carbohydrate, 12% (w/w) hexosamine and 28% (w/w) fatty acid. These LPS preparations induced mitogenic responses in spleen cells of BALB/c, BALB/c (nu/nu) and C3H/HeN mice, and these responses were suppressed by the addition of polymyxin B. The preparations also induced the polyclonal responses of C3H/HeN spleen cells. In addition, enhanced glucose utilization and interleukin-1 production by murine peritoneal macrophages were demonstrated. Neither spleen cells nor macrophages from the 'LPS-nonresponsive' C3H/HeJ mouse were activated by LPSs from the Fusobacterium species.     

Acquisition of apparently intact and unmodified lipopolysaccharides from Escherichia coli by Bdellovibrio bacteriovorus.

The ability of Bdellovibrio bacteriovorus to relocalize the OmpF major outer membrane porins from its Escherichia coli prey to its own outer membranes is diminished in prey expressing smooth lipopolysaccharide (S-LPS). Since porins exist in the membrane complexed with LPS, we examined the LPS associated with relocalized porin to determine whether it had been acquired intact, mixed or replaced with Bdellovibrio LPS, or derivatized by the bdellovibrios. The relocalized trimers were found associated with the same LPS originally bound to them in the E. coli. The bulk-phase LPS from bdellovibrios grown on various chemotypes of rough prey was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine whether more than the trimer-bound LPS was acquired by the bdellovibrios. This analysis revealed bands of Bdellovibrio LPS matching the LPS chemotype of the prey. One or two other bands were identical in migration to the LPS of prey-independent mutants of B. bacteriovorus and represented bdellovibrio-synthesized LPS. The LPS of bdellovibrios grown on prey with radiolabeled lipid A showed radioactivity only in gel band positions identical with those of the prey's LPS. The amount of this prey-derived LPS was shown by enzyme-linked immunosorbent assay to reach a constant value during the purification of the bdellovibrios, and it represented approximately 25% of the total Bdellovibrio LPS. Immunoelectron microscopy confirmed the presence of prey-derived LPS on the cell surface of bdellovibrios, and no evidence could be found for bdellovibrio-induced modifications of the relocalized prey LPS.     

Phage typing of Escherichia coli isolated from chickens.

Ten different bacteriophages were isolated from untreated city sewage water. These phages were stable at 57 degrees C for 40 min. A modified agar layer technique was used to obtain high titre phages. Ninety-four of a stock of 101 cultures of Escherichia coli, which were isolated from inflamed portions of intestines of chickens, were lysed by one or more of these phages. The E. coli of a known serological grouping were phage typed.