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The outer membrane protein (OMP) profiles of 135 isolates of Yersinia ruckeri, obtained from nine European countries (100 isolates), North America (23 isolates), Australia (six isolates) and South Africa (two isolates), and including four reference strains, were examined by SDS-PAGE. Outer membranes were isolated by selective solubilisation of the cytoplasmic membrane with 0.5% (w/v) sodium N-lauroyl sarcosinate (Sarkosyl). Outer membrane proteins were stable after in vitro passage and there was no variation in OMP profiles due to colony selection. With the exception of a 39.5 kDa peptidoglycan-associated protein there was also no variation at different stages of the growth cycle. The 39.5 kDa protein was not produced during logarithmic growth phase but increased in abundance as the stationary phase progressed. Interstrain variation occurred in the possession of a 36.5 or 38 kDa heat-modifiable protein and in the possession of peptidoglycan-associated proteins in the molecular weight range 36.5 to 40.5 kDa. Based on variation of these proteins five OMP-types, designated OMP-types 1-5, were identified among the 135 isolates examined. Outer membrane protein analysis was demonstrated to be useful in epidemiological studies of Y. ruckeri.

Cell extracts of Yersinia ruckeri (serotype I) were examined by SDS-PAGE and Western blotting. An unusual band, termed heat-sensitive factor (HSF) was observed in extracts of virulent strains only. It is thought to be lipid in nature; no differences could be detected in the region of the band in protein profiles of virulent and avirulent strains. When trout were infected either by intraperitoneal injection or bath immersion, mortalities occurred only with HSF+ strains. The HSF appears to be an important virulence determinant of Y. ruckeri.

Double diffusion in agar showed that yersinia enterocolitica O-serotypes 3, 7, 8 and 9 each contained a type-specific precipitinogen. Serotype 6 contained several specific precipitinogens and was heterogeneous. The type-specific precipitinogens were shown to be lipopolysaccharides (LPS). Acid hydrolysis of the isolated LPS followed by gel filtration separated the material into two peaks, the first of which contained the serologically active fractions. The active fraction of serotype 3 contained relatively large amounts of a terminally linked deoxyhexose, probably 6-deoxy-L-altrose. Fucose was found to be present in serotype 7.

The stretches of Yersinia enterocolitica strain Ye 62 DNA which show base sequence homology with Escherichia coli strain B DNA were removed from a preparation of tritiated Y. enterocolitica DNA by DNA reassociation techniques. This DNA preparation was then used in reassociation reactions with DNA from various selected strains of Yersinia and coliform bacteria. The results suggest that the bulk of the genes, which are in common between Y. enterocolitica and E. coli, are different from those shared by Y. enterocolitica and other species of Yersinia.

The composition of outer membrane (OM) proteins of Y. enterocolitica, Y. intermedia, Y. frederiksenii and Y. kristensenii was investigated. POMP proteins were described from Y. enterocolitica and Y. intermedia. In all the studied bacteria the presence of two to four major proteins, that is YOMP-C, YOMP-F, YOMP-A and protein with molecular weight 47 kDa was demonstrated. The position of 47 kDa protein on polyacrylamide gel (SDS-PAGE) corresponds to maltoporins in Escherichia coli. This protein may be induced by the addition of maltose to the medium, and in the case of Y. intermedia also maltodextrins. The amount of 47 kDa protein in the OM of all the examined strains does not change after addition of Ca++ ions, it increases, however, under conditions of increased osmolarity. Y. enterocolitica is an exception since its synthesis of the above mentioned protein is independent on the medium osmolarity.

With a single exception among 11 strains, each strain of Y. enterocolitica representing one of the six O-groups possesses, as judged by the sugar composition of its lipopolysaccharides, a particular chemotype. All 14 of the strains examined possessed glucosamine, KDO, two heptoses and glucose. In the most complicated chemotype 10 sugars were demonstrable. Of three Y. enterocolitica strains not included in the 6 O-groups, two had the same chemotype as the representatives of the O-groups IV and VI. Details can be seen in the two tables.

Pyrolysis gas-liquid chromatography (PGLC) was used to differentiate between HeLa cell-invasive and noninvasive strains of Yersinia enterocolitica and between Sereny-positive and -negative strains. A temperature-programmed gas-liquid chromatograph, equipped with a high-resolution Carbowax 20M coated capillary column, separated the volatiles from pyrolyzed whole cells preparations and cell wall fractions. The resulting pyrolysis elution patterns (pyrograms) were divided into 313 30-s time interval areas. The time interval areas were normalized in relation to the entire pyrogram area and were evaluated by stepwise linear discriminant analysis. The results of the PGLC-statistical analyses showed good correlation in prediction of the HeLa cell invasivity test. The technique of PGLC coupled with statistical analyses is objective, in contrast to traditional methods of determining pathogenicity of Y. enterocolitica.

A peptidoglycan layer of Y. pseudotuberculosis cell wall was isolated from cells after treatment with sodium dodecylsulphate and digestion with pronase. The isolated peptidoglycan contained glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in the approximate molar ratio of 1:0.8:2:1:1.

Heat-stable enterotoxin (ST) of Yersinia enterocolitica was produced under defined conditions. It was first detected in the culture supernatant of the late-logarithmic phase of growth and increased lineally during the the stationary phase of growth. The ST level became maximum at the decline phase of growth, and the ST was not detected in the lysate of bacteria obtained from the decline phase of growth. The ST was extensively purified from the culture supernatant, and about a 1,905-fold purification was achieved with a yield of 8.9%. The minimal effective dose of the purified ST was approximately 25 ng in the suckling mouse assay. The purified ST gave a single 280-nm absorbing peak on polyacrylamide disc gel electrophoresis and had a maximum absorption at 272 nm, and its molecular weight was 9,700 by Sephadex G-75 superfine gel filtration. The biological activity of the purified ST was lost by treatment with 2-mercaptoethanol, suggesting that the ST contained disulfide bridges in the molecule which were required for the development of toxic activity. The purified ST was heat stable at 100 degrees C for 10 min between pH 2.2 and 8.0, but not at pH values greater than 9.0 or in 2 N HCl. The treatment of the ST with trypsin resulted in a retarded elution of the ST activity by Sephadex F-75 superfine gel filtration and a passage through a UM-20 membrane filter.

By using a suckling mouse assay, heat-stable enterotoxin (ST) was purified from the culture filtrate of Yersinia enterocolitica isolated from a diarrheal patient. The purification procedures involve ultrafiltration with an Amicon HIP-10 hollow fiber, ethanol fractionation, protamine sulfate treatment, diethylaminoethyl-Sephacel and hydroxylapatite column chromatographies, and Sephacryl S-200 superfine gel filtration. About 408-fold purification was achieved, with a yield of 12.0%. The minimal effective dose of purified ST was about 110 ng in the suckling mouse assay. The molecular weight of purified ST was 9,000 by Sephadex G-100 superfine gel filtration. The purified ST was stable to heating (100 degrees C for 20 min, 121 degrees C for 20 min) and did not lose its toxicity after treatment with protease, trypsin, lipase, phospholipase C, ribonuclease, deoxyribonuclease, beta-glucosidase, and neuraminidase. The purified ST was separated by isoelectric focusing into two active fractions, with pI's of 3.29 (ST-1) and 3.00 (ST-2), respectively. Antiserum from guinea pigs immunized with the purified ST neutralized the activity of both Y. enterocolitica ST and Escherichia coli ST.

D-3-Dodecanoyltetradecanoic acid has been separated from the lipid A of Yersinia pseudotuberculosis and its structure has been established by chromato-mass-spectrometry and 13C NMR spectroscopy, by comparison with authentic samples.

The polysaccharide of the O-specific side chain of the lipopolysaccharide from the Yersinia pseudotuberculosis VA serovar has been isolated and studied. The structural pattern of the chemical repeating unit of the specific polysaccharide has been proposed as follows: (formula; see text).

The fatty acid composition of lipopolysaccharides of the strains of Y. enterocolitica, Y. intermedia, Y. frederiksenii and Y. ruckeri studied during cultivation on meat-peptone agar is characterized by the predominance of 3-hydroxytetradecanoic and dodecanoic acids. Closely related to the mentioned bacteria is the strain of Y. kristensenii which is distinguished only by its higher level of hexadecanoic acid. The strains of Y. pseudotuberculosis and the vaccine strain of Y. pestis have a uniform fatty acid composition of lipopolysaccharides with predominance of 3-hydroxytetradecanoic acid. Their relatively low level of dodecanoic acid conditions the characteristic fatty acid spectrum of lipopolysaccharides which differs from that of the above mentioned group of Yersinia. The peculiarities of the fatty acid composition of lipopolysaccharides of both groups of Yersinia are preserved during growth on meat-peptone broth, but the increase in the level of hexadecanoic acid balances the differences between Y. kristensenii, the other Y. enterocolitica-like bacteria and Y. ruckeri. The obtained results confirm close relationship of Y. pseudotuberculosis and Y. pestis, and also of Y. enterocolitica and Y. enterocolitica-like bacteria, showing propinquity of Y. ruckeri to the latter.

Lipid A was isolated from lipopolysaccharide of Yersinia pseudotuberculosis S form (strain 341, subtype IB) using mild hydrolysis with acetic acid. The purified material (yield about 25%, molecular weight about 2900) contained D-glucosamine (11%), fatty acids (54%), protein concomitant (9.7%) and phosphorus (approximately 2%). Dodecanoic and 3-hydroxy-tetradecanoic acids in a molar ratio of 1 : 3.6 were detected as major fatty acid constituents. The hydroxyl groups of D-glucosamine were acylated with the residues of both fatty acids, while the amino groups were substituted with the residue of 3-hydroxy-tetradecanoic acid. Such a simple fatty acid composition is reminiscent of that found in lipid A in Y. pestis.

Yersinia enterocolitica serotype O3 was found to produce a haemolytic substance which could be released from the bacterial cells by sonic disintegration. The substance was non-dialysable, thermolabile, antigenic, and sensitive to trypsin. Chromatographic studies indicated a high molecular weight. Erythrocytes from different mammalian species differed in sensitivity to the haemolytic substance. Y. enterocolitica serotypes O8 and O9 produced no haemolytic substance.

The chemical properties and the general biological activities of lipopolysaccharide (LPS) and Boivin-type endotoxin obtained respectively by phenol-water and trichloroacetic acid extraction from Yersinia enterocolitica serotypes O3 and O9 were studied. The yield of LPS from the O9 strain was about 10% of the O3 strain possibly because of the lower solubility of O9-LPS in aqueous phase. However, the chemical composition of O9-LPS was similar to that of O3-LPS in the proportions of reducing sugar, glucosamine, heptose, KDO, and lipid A. In pyrogenicity and local Shwartzman reactivity in rabbits and lethality for mice, there was also no difference between O3 and O9-LPS. The anthrone-positive carbohydrate and lipid A contents of Boivin-type endotoxin from O3 were higher than those of the endotoxin from O9. The biological activities of Boivin-type endotoxin from O3 were also remarkably higher than those of the endotoxin from O9. It seems that endotoxin of Y. enterocolitica serotype O3 may play an important role in infection by this organism.

A procedure is described for fractionating Yersinia grown in small cultures into inner and outer membranes and soluble cytoplasmic proteins. The procedure was applied to the three recognized species of the genus grown under conditions simulating mammalian intracellular fluid with respect to Ca2+ and Mg2+. These conditions are known to elicit the production of the plague virulence antigen V. Isolates capable of making this antigen were compared with virulence-antigen-negative derivatives by two-dimensional electrophoresis. The V antigen was localized to the soluble protein fraction as a peptide that comigrates with the major component of a specific immunoprecipitate. This peptide had an apparent molecular weight of 38,000 and was not found in either apparent molecular weight of 38,000 and was not found in either membrane fraction. The comparison of virulence antigen-producers and nonproducers of Y. pseudotuberculosis and Y. enterocolitica revealed large qualitative and quantitative differences in outer membrane protein patterns, whereas the same comparison for Y. pestis showed only minor differences. The complexity of changes in the various protein fractions corroborate data in the literature indicating that extensive physiological changes occur in virulent organisms cultivated under simulated intracellular conditions.

The outer membrane proteins (OMP) of fifteen strains of Yersinia were separated from the inner membrane (IMP) and intracytoplasmic (ICP) proteins. The molecular composition of these fractions was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The OMP of all fifteen strains exhibited a uniform pattern markedly distinct from the IMP and ICP. There was, however, heterogeneity in the OMP among the various bacteria, based on the apparent molecular weights of several of the major proteins. The two most prominent major proteins are easily distinguished because their apparent molecular weights varied substantially depending on the temperature at which the membranes were solubilized. Using the apparent molecular weights of these two proteins as a basis for comparison, the Yersinia organisms of serotype 3 presented a unique electrophoretic pattern, different from those of non-serotype 3 Yersinia organisms. Four of the nine serotype 3 organisms analyzed here were isolated from patients who developed arthritis subsequent to infection. However, no obviously unique features could be distinguished. A major focus of research effort is currently being devoted to the examination of the immune response generated against the Yersinia organisms by patients with Yersinia-induced arthritis. Most of the studies, so far, have utilized whole bacteria as the antigens. The data presented in this paper provide the basis of future analysis of such immune responses to individual molecules of Yersinia.

Disc electrophoresis of phenol:acetic acid:water extracts of Yersinia pestis, Y produced pseudotuberculosis, Y enterocolitica, Y philomiragia and Y ruckeri protein separation patterns characteristic of each species. The patterns produced by Y pestis, Y pseudotuberculosis, Y enterocolitica and Y ruckeri showed features in common. Those given by Y philomiragia strains were quite distinctive and showed little similarity to the other Yersinia species. All Y enterocolitica strains produced similar patterns irrespective of biochemical properties. Strains classified as Y intermedia or Y enterocolitica-like produced patterns indistinguishable from those given by Y enterocolitica.

The expression of the temperature-inducible plasmid-coded outer membrane proteins (YOPs) of Yersinia pseudotuberculosis was studied. These proteins were not recovered in the outer membrane fraction when the strain was grown in minimal medium at 37 degrees C, but they were expressed under these conditions. A strict correlation was found between Ca2+ dependency in the virulent strain, YPIII(pIB1), and ability to express YOPs. Ca2+-independent plasmid mutants or RNA-polymerase mutants harboring the virulence plasmid were unable to express YOPs, in contrast to the wild-type strain. These strains were also found to be avirulent. Sera recovered from patients or animals undergoing infection with either Y. pseudotuberculosis, Y. pestis, or Y. enterocolitica possessed antibodies directed against YOPs, indicating that they were expressed in all three pathogenic Yersinia species during infection. The YOPs of the three different species showed high immunological relatedness.

The fine structure of isolated lipopolysaccharides (LPS) from the rough and smooth form of an Yersinia enterocolitica strain (Ye 75 R/Ye 75 S) and from smooth forms of Salmonella typhimurium (S 1010) and Escherichia coli (Essen) were examined electron-microscopically by negative staining. Partial denaturation of LPS in Tris-buffer with acid and/or polymyxin B treatment revealed a common structure of strandlike LPS. Electron-microscopically, LPS-strands were found to consist of two identical sub-strands which form a double helix. High resolution electron microscopy permitted the identification of a total of four longitudinal fibrils (diameter approximately 20 A); therefore, each sub-strand consists of two longitudinal fibrils. These results were correlated with those obtained with positive staining procedure and chemical fixation technique. The development of the "double-track"-profile of the outer membrane was interpreted to result from the projection of the helical longitudinal fibrils into the image plane.

A strain of Yersinia pseudotuberculosis which harbors a 63-kilobase plasmid was found to cause a lethal infection in Swiss albino mice. The rate of infection paralleled the ability of the pathogenic organism to attach to a monolayer of HeLa cells. One novel outer membrane protein (protein 1) with a molecular weight of 140,000 was found to be associated with the possession of the 63-kilobase plasmid not at 26 degrees C, and expression was moderately affected by the concentration of calcium in the growth medium. Moreover, it was found that synthesis of protein 1 associated outer membrane protein showing similar properties was also found to be expressed in plasmid-containing strains of Yersinia enterocolitica. The properties of protein 1 indicate that it could be identical to the previously described virulence W antigen.

Deoxyribonucleic acid (DNA) relatedness among members of the genus Yersinia and the biochemical reactions of these organisms were compared. These data were used to reassess speciation within the genus Yersinia, and three new species were proposed. These are Yersinia ruckeri sp. n., formerly called the redmouth bacterium; Yersinia intermedia, sp. n., formerly a rhamnose-positive, raffinose-positive, melibiose-positive biogroup within Yersinia enterocolitica; and Yersinia frederiksenii sp. n., formerly a rhamnose-positive, raffinose-negative, melibiose-negative biogroup within Y. enterocolitica.

We have previously shown that under iron limitation, different Yersinia species synthesize new polypeptides. Two of them, the high-molecular-weight proteins (HMWPs), are expressed only by the highly pathogenic strains. In the present study, the HMWPs from Y. enterocolitica serovar O:8 were purified by gel filtration, and specific antibodies were obtained. Using these antibodies, we show that the two polypeptides were synthesized de novo during iron starvation and that they were found essentially in the bacterial outer membrane fractions, although the majority of the molecules were not exposed on the cell surface. We also demonstrate that the two proteins had common epitopes and that the HMWPs of the high-virulence-phenotype species Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serovar O:8 (a strain different from the one used to purify the proteins) are antigenically related. The less pathogenic and nonpathogenic strains did not exhibit cross-reacting material, suggesting that these strains do not synthesize even an altered form of the HMWPs.