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YscU is an essential component of the export apparatus of the Yersinia injectisome. It consists of an N-terminal transmembrane domain and a long cytoplasmic C-terminal domain, which undergoes auto-cleavage at a NPTH site. Substitutions N263A and P264A prevented cleavage of YscU and abolished export of LcrV, YopB and YopD but not of Yop effectors. As a consequence, yscU(N263A) mutant bacteria made needles without the LcrV tip complex and they could not form translocation pores. The graft of the export signal of the effector YopE, at the N-terminus of LcrV, restored LcrV export and assembly of the tip complex. Thus, YscU cleavage is required to acquire the conformation allowing recognition of translocators, which represent an individual category of substrates in the hierarchy of export. In addition, yscU(N263A) mutant bacteria exported reduced amounts of the YscP ruler and made longer needles. Increasing YscP export resulted in needles with normal size, depending on the length of the ruler. Hence, the effect of the yscU(N263A) mutation on needle length was the consequence of a reduced YscP export.

Type III secretion is used by many Gram-negative pathogenic bacteria to inject effector proteins into eukaryotic host cells. Effector delivery requires a secretion apparatus, called an injectisome or needle complex, and the assembly of a translocation pore in a target-cell membrane. Recent work provides evidence that enlightens the view of how pore assembly might occur and of how the injectisome and the pore might be linked.

The interaction of Staphylococcus aureus, Yersinia enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. frederikseni, Y. kristenseni and erythrocytes was studied with the use scanning electron microscopy. Highly adhesive and moderately adhesive Staphylococcus and Yersinia strains displayed both individual coated bacterial cells and groups of cells interconnected by common intercellular matrix on the surface of erythrocytes. In nonadhesive Staphylococcus and Yersinia strains no coating was detected on the surface of bacterial cells. Some of Staphylococcus and Yersinia cells interacting with erythrocytes were at the stage of heteromorphism with different manifestations of L-transformation (cells with cell wall defects, spheroplasts and protoplasts). Heteromorphic cells did not adhere to the surface of erythrocytes.

The morphological picture of different bacteria (Salmonella typhimurium, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa, Yersinia enterocolitica O3, Y.pseudotuberculosis 1, Y.frederiksenii, Y.intermedia, Y.kristensenii) on environmental objects was studied with the use of scanning electron microscopy (SEM). Bacteria adhered to the surface of pieces of fodder, egg shell, cabbage leaves and form microcolonies, whose morphology was similar to colonies, grown on nutrient media. The cells produced extracellular substances, seen in SEM as integuments. These integuments were gourd to protect the population from the action of unfavorable factors.

One the cell surface of smooth of Yersinia enterocolitica 75 (Ye 75 S), and electron-lucent layer was identified in thin sections after incubation with Ye 75 S-antiserum. It is adjacent to the "double track" of the outer membrane. This layer does not appear on the analogously treated rough mutant (Ye 75 R), whose lipopolysaccharide (LPS) does not contain O-specific polysaccharides. It could be shown that this layer is formed by the O-specific side chains of LPS, which are unstained by routine methods. As the side chains of LPS obviosly make it difficult to visualize the LPS-strands in the outer membrane of rough strands was investigated. On Ye 75 R, the LPS-strands could be demonstrated only after treatment of cells with polymyxin B. After short time of exposure, the LPS appeared as contigous strand-like structures (diameter roughly 60 A) on the cell surface of cells which were negatively stained or dried by the critical-point-method. After prolonging polymyxin B treatment, "blebs" or "protrusions" appeared on the surface of Ye 75 cells, which were in negatively stained preparations and in thin sections identified as partly loosened LPS. The demonstration of LPS-strands in untreated cells was possible only on a "deep rough" mutant (Ye 161-45 a R), whose LPS contains only glucosamine and KDO besides lipid A. It is suggested that the LPS form a layer of contiguous strands (mid-to-distance 62 +/- 3 A) on the surface of negatively stained cells. According to this view, on the Ye 75 S, the O-specific side chains extend from this layer into the medium.

The use of the erythrocyte agglutination test for characterizing the properties of Y. pseudotuberculosis led to the detection of a highly adhesive strain belonging to serotype III and capable of forming pili at 37 degrees C. The adhesion of the cells provided with pili was completely inhibited by the mixture of gangliosides, specific antibodies, and the preliminary treatment of erythrocytes with neuraminidases sharply enhanced the effectiveness of adhesion. The adhesion pili consisted of protein subunits with a molecular weight of 16 800 daltons and had the isoionic point at pH 4.1.

Thirty-nine strains of Yersinia enterocolitica and 10 strains of Yersinia pseudotuberculosis were studied for the presence of fimbriae, plasmids and two plasmid associated phenotypic expressions, autoagglutination and Ca2+ dependent growth at 37 degrees C. All of the strains studied became fimbriated, which was confirmed by electron microscopy and haemagglutination tests. Fimbriation was not correlated with the presence or absence of plasmids.

Yersinia enterocolitica RIMD 2501003 grown at 25 C avidly adhered to various kinds of cultured epithelial cell lines (HeLa, FL, Y-1 adrenal, human intestine, human conjunctiva) but the bacteria grown at 37 C did not adhere. This phenomenon paralleled the temperature-dependent motility of the bacteria. To clarify the adherence mechanism, we obtained two kinds of mutants, an immobile mutant and a nonadherent mutant, by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The immobile mutant did not move on soft agar but retained the capacity to adhere to cultured epithelial cells when grown at 25 C. The nonadherent mutant did not adhere to cultured epithelial cells but retained the ability to move on soft agar when grown at 25 C. When the bacteria were killed by heat, ultraviolet light irradiation or formaldehyde they lost their capacity to adhere to the cultured epithelial cells. Antiserum against Y. enterocolitica RIMD 2501003 grown at 25 C was absorbed with the bacteria grown at 37 C, with the bacteria grown at 25 C, with the nonadherent mutant grown at 25 C and with the bacteria killed by various means. Only the antiserum absorbed with bacteria grown at 37 C inhibited the adherence of bacteria. These data indicate that motility does not correlate with adherence of Y. enterocolitica. It appears that the adherence factor involves both a temperature-dependent surface factor and a factor synthesized de novo during the interaction of susceptible cells with the bacteria.

Strains of Yersinia enterocolitica from different O serogroups were tested for their ability to adhere to cultured epithelial cells (H.Ep 2) after growth of bacteria in broth at 22 C or 37 C. Strains that adhered to H.Ep 2 cells generally did so better after growth at 22 C than at 37 C. The data indicate that the ability to produce either of the mannose-resistant (MR) hemagglutinins, MR/Y or K1, each associated with fimbriae, does not correlate with ability to adhere to epithelial cells in this particular in vitro model.

When cultivated at 37 degrees C in static broth, human clinical isolates of Yersinia enterocolitica (serogroups O:3, O:8, and O:9) and Yersinia pseudotuberculosis (serogroup O:III) produced numerous nonflagellar surface appendages, which appeared as a lawn of fine fibrillae, each having a diameter of 1.5 to 2.0 nm and a length of 50 to 70 nm. Cultivation at 22 degrees C resulted in complete disappearance of the fibrillae. The phenotypic expression of these appendages was correlated with the presence of the 40- to 48-megadalton virulence plasmid and was strongly affected by the growth medium. Evidence is presented which suggests that these plasmid-mediated, temperature-inducible surface fibrillae are responsible for autoagglutination and are related to production of one prominent, Sarkosyl-insoluble polypeptide of ca. 180 kilodaltons in the bacterial outer membrane.

A total of 1242 strains of Y. enterocolitica, 104 strains of Y. frederiksenii, 95 strains of Y. kristensenii and 85 strains of Y. intermedia were serotyped with antisera against 56 O antigens and 19 H antigens according to the extended antigenic scheme of Wauters, and with additional antisera against 4 somatic and 19 flagellar antigens not previously described. H antigens of Y. frederiksenii, Y. kristensenii, and Y. intermedia turned out to be rather homogeneous without distinct subfactors. In these species the scope of identified serovars was narrow. Flagellar antigens of Y. enterocolitica were mostly composed of several subfactors, leading to a total of 117 serovars identified in the species. A number of cross-reactions between Yersinia H antigens were observed which could be avoided by absorption without significant lowering of the titre. Flagellar antigens of Yersinia were monophasic, and species specific. The antigens remained stable after storage in agar stabs and repeated subcultures. The epidemiological value of serotyping is demonstrated by strains from three different sources. It is suggested serotyping of Yersinia strains should be performed in three steps: O typing of the prevailing enteropathogenic Y. enterocolitica serogroups in the medical routine laboratory; O and H typing of Y. enterocolitica by National Reference Centres applying a typing scheme reduced to this species; and O and H typing of Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia by specialized International Centres using an extended typing scheme. The need for international standards comparable to those established for Salmonella is emphasized.

The influence of six widely used media on the motility of 100 isolates of Yersinia enterocolitica grown at 25 and 37 degrees C was investigated. Seven isolates were motile at 25 and 37 degrees C in all media, and the presence of flagellated cells was demonstrated by a flagellum stain. Six isolates were nonmotile at both temperatures in all media, and no flagella were observed. Identification schemes for Y. enterocolitica should reflect these possibilities.

Chemical, physical and immunological properties of the fimbrial antigen of two different genera of Enterobacteriaceae, namely Salmonella (S. enteritidis) and Yersinia (Y. enterocolitica) were analysed. Only a few strains of these two bacterial genera possess a fimbrial antigen which proved to be identical in all tests. Purified fimbriae are unstable in buffer solutions. They tend to form aggregates, patterns of which are between 240000 and 270000. Submitted to solubility testing by various methods these aggregates easily dissociate into particles of different molecular size. Treatment with 1% sodium dodecyl sulphate (SDS) in 1% mercaptoaethanol at 37 degrees C for 60 minutes gave constant reproducible results. The fimbrial protein prepared by this method proved to be an antigenically active unit with a molecular weight of 23450 for S. enteritidis and 23350 for Y. enterocolitica. The electron microscope revealed that purified fimbriae of these two genera consist of extremely thin filaments (1.5 to 2.7 nm), covering the bacterial cell as an envelope. They are supposed to be composed of no more than 2 to 3 peptid chains. The presence of the amino acids tyrosine, phenylalanine and tryptophane may be responsible of the particular behaviour of the fimbriae which according to different external conditions react with different states of dissociation. Probably the filaments are not stable in a solution as single filaments but tend to form oligomer, secondary and tertiary structures. Experiments of isoelectric focusing revealed that both antigens consist of a pure protein component (composed of 17 amino acids) which appears at pH 3.9.

An SEM study upon the developmental cycle of Yersinia enterocolitica added further confirmation to its being an L-cycle. It was not found easy to demonstrate flagella by the method, but the conclusion was reached that it has potential as a method for the rapid identification of bacterial L-forms.

40 cases of bacteriologically proved Yersinia enterocolitica infections in children under 15 years were reviewed. Most children presented with abdominal symptoms, and diarrhoea was present in 80% of them. In half of those with diarrhoea the stools were mucoid and gross blood was often present. Faecal leucocytes were found in 4 out of the 5 children studied. The clinical findings are consistent with the enteroinvasive pathogenic mechanism proposed for Y. enterocolitica. 29 of 30 faecal isolates of Y. enterocolitica were found invasive for human epithelial cells in vitro. Nine strains produced an enterotoxin demonstrable in newborn mouse assay. Toxin production may be an additional pathogenic mechanism in human yersiniosis.

The ultrastructural changes in 3 strains Yersinia pseudotuberculosis with different virulence after treatment with sodium lauryl sulfate (SLS) and petroleum ether were studied. The ultrafine sections after treatment with SLS show heavy destructive changes, concerning the cell wall, the cytoplasmic membrane and the inner structure of the cell. It was established that the same cells Y. pseudotuberculosis after cultivation on a medium with glycerol show a tendency to recover their ultrastructure. The cells treated with petroleum ether did not exhibit any notable ultrastructural changes.

In Y. enterocolitica strain, serovar 0:10, the capacity for the formation of pili inducing the mannose-resistant hemagglutination (MRHA) of formolated sheep red blood cells was due to the presence of plasmid pYE10. MRHA-inducing pili differed serologically from Y. pestis and Y. tuberculosis adhesion pili. Plasmid pYE10 was immobilized for transfer to cells of Escherichia coli strain HB101 (rec A) by means of pRP 4. The expression of MRHA-inducing pili in the new host the rec A-independent character of the synthesis. Y. enterocolitica cells containing pYE10 agglutinated in tissue-culture media with 10% of serum added at 37 degrees C.

The comparative electron-microscopic study of early stages of the interaction of Y. pseudotuberculosis virulent strain (No. 282) with "professional" (macrophages) and "nonprofessional" (HeLa cells) phagocytes has been carried out. The character of the intimate mechanism of this interaction has been found to be essentially different. The common feature for both systems is the adsorption of bacteria and their penetration into cells due to phagocytosis. But the subsequent fate of Y. pseudotuberculosis is different. In HeLa cells they are isolated from the cytoplasm by multilayer membrane structures, thus remaining morphologically intact. In macrophages the destruction of the microbe in phagolysosomes occurs.

Rabbits were immunized with the enterobacterial common antigen (ECA)-immunogenic strain Escherichia coli F470. ECA-specific antiserum was obtained by absorbing the resulting antisera with the genetically closely related ECA-negative strain E. coli F1283. These two strains also served as positive and negative controls in the localization study of ECA in Yersinia enterocolitica strain 75, smooth and rough forms (Ye75S and Ye75R), by the indirect immunoferritin technique. Cells of Ye75S grown at 22 degrees C showed no labeling with ferritin after treatment with the ECA-specific antiserum and subsequent ferritin-conjugated goat anti-rabbit antibodies. If the cells were grown at 40 degrees C, however, most of the cells showed weak ferritin labeling. At this higher growth temperature, the lipopolysaccharide of this strain contains less O-specific chains (6-deoxy-L-altrose), as was shown in a previous study. The rough mutant Ye75R, which lacks O-specific chains completely, showed denser labeling with ferritin. These results indicate that ECA on the cell surface of Ye75S is covered by O-specific chains of the lipopolysaccharide if grown at 22 degrees C and is therefore not accessible to ECA antibodies. It becomes accessible, however, when O-chains are lacking (R mutants) or when they are reduced in size or amount (growth at 40 degrees C).

The production of bacteriocin-like substances by strains of Yersinia frederiksenii, Y. kristensenii and Y. intermedia in broth culture was established. These substances showed a selective activity against Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia strains. Electron micrographs revealed the presence of phage tails in culture media. The production of these substances was detected in cultures grown at 25 degrees C but not in those grown at 37 degrees C, while these bacteriocin-like substances were active at 25 and 37 degrees C. Y. enterocolitica serogroups 0:3 and 0:9 were more susceptible to these bacterin-like substances than strains of Yersinia isolated from environmental sources.

Virulent Yersinia pestis was grown on heart infusion blood agar and examined by scanning electron microscopy, exposing the fraction 1 envelope antigen on cell surfaces as a lumpy coating that spilled into the surrounding milieu. The amount of antigen depended on both the duration and temperature of incubation. Comparison of the cultures grown at 37 degrees C for 24, 48, and 72 h with those grown at 22 degrees C for identical periods demonstrated that the antigen increased in amount with the length of incubation, and that the overall production of antigen was much greater of 37 degrees C than at 22 degrees C. These experiments visually confirmed the findings of our previous immunological studies. Moreover, we established that the closely related, virulent organism Y. pseudotuberculosis bears no such antigenic coating at any temperature or incubation period. In addition, the emergence of multiple flagella was noted when Y. pseudotuberculosis was grown at 22 degrees C in a liquid medium, whereas Y. pestis remained without these organelles. These observations preceptibly corroborated the absence of fraction 1 envelope antigen and the presence of flagella, respectively, for distinguishing Y. pseudotuberculosis from Y. pestis.

Rapid killing of Yersinia enterocolitica strain 75 in smooth form (Ye 75 S) was observed in the presence of serum or of lysozyme-free serum whereas the killing activity of EGTA-serum was slow, and absent in heated (30 min 56 degree C) serum. Similarly, complement (C) activation by Ye 75 S was rapid in serum and lysozyme-free serum but slow via the alternative pathway (EGTA-serum). These data suggest that C is sufficient for killing of the cells and most active via an intact classical pathway. Electronmicroscopic studies were performed on bacterial killed by serum (C + lysozyme) or by lysozyme-free serum (C). In these experiments cell fragmentation and spheroplast formation were seen after exposure of Ye 75 S to serum; in bacteria incubated with lysozyme-free serum "blebs" formation was observed as the most prominent alteration. These blebs most likely originate from the outer membrane as a result of C activation on the cell surface. The deposition of activated C (C3b) on Ye 75 S was analyzed kinetically in the presence of serum or EGTA-serum. With serum (30 vol%) massive C3b deposition was observed within 20--30 min whereas with EGTA-serum (30 vol%) the deposition of C3b was slower and less complete. Experiments with EGTA-serum also revealed that the deposition of C3b started at single sites mainly located in the region of the cell poles; from these sites spreading of C3b occurred until large areas of the cell surface were covered. These data suggest that C activation via the alternative pathway is restricted to certain regions of the bacterial surface.

Phenol-water-extracted lipopolysaccharide (LPS) from Yersinia enterocolitica (strain 75 in smooth form; O-group I), after growth at 10 degrees C, contains approximately 40% (w/w) 6-deoxy-L-altrose. By increasing the cultivation temperature a significant decrease in the O-specific sugar is observed. LPS from cells grown at 40 degrees C contains about 12% 6-deoxy-L-altrose. Thin-section micrographs of cells grown at lower temperatures reveal a distinct layer corresponding to the O-specific side chains of LPS in contrast to cells grown at higher temperatures where a decrease in thickness is observed. It is assumed that this observation is due to the decreasing amount of the O-specific sugar at a higher cultivation temperature. The results obtained by the indirect immunoferritin test indicate that the O-specific polysaccharide layer covers the cell surface completely up to 37 degrees C. However, this layer no longer covers the entire surface of all cells as soon as an incubation temperature of 40 degrees C is applied.

The envelope antigen K1 found in the majority of Yersinia enterocolica (Y. ent.) strains belonging to the O group 10 as described by WAUTERS, was re-examined under the electron microscope, and identified as being fimbriae. These fimbriae envelop the bacterial cell with a dense fringe. Single filaments show a width of 2.5 to 1.7 nm whilst their length is variable, often many times the size of the bacterial cell. Fimbriated Y. ent. cultures do not form pellicle, and the haemagglutination test is negative. They are O and fimbriae agglutinable. Test results for the acknowledged 3 antigenic differences, i.e. agglutinability, agglutinin-binding capacity and agglutinogenic capacity showed that: 1. there is a certain thermo-lability as to the agglutinability. For its total suppression, half-an-hour's boiling at 120 degrees C of the culture fim+ is necessary. Chemical treatment of such a culture hardly impairs the agglutinability of fimbriae. 2. the agglutinin-binding (= absorbing) capacity of the fimbrial antigen is little affected by continuous heating, even up to 3 hrs. However, at least half-an-hour's boiling at 120 degrees C or alternatively chemical treatment with 50% alcohol or nHC1 practically destroys this ability. 3. The heating process has a significant and progressive effect on the agglutinogenic capacity of the Y. ent. fimbriae which is destroyed irrevocably after 30 mins. boiling at 120 degrees C. Chemical treatment hardly affects this character. It is recommended to use a formalised culture Y. ent. fim+ for the preparation of a specific Y. ent. fimbrial serum. For the elimination of O antibodies, this antiserum should be absorbed with a strain fim- of the same O group. Provided the strain was incubated at 37 degrees C, no H absorption is required, because Y. ent. strains do not form flagella at that incubation temperature.

A comparative study of various procedures of a lipopolysaccharide-protein complex (LPPC) from Yersinia pseudotuberculosis was carried out. The materials obtained were fractionated by molecular-sieve chromatography on Sepharose 2B resulting in highly aggregated complexes with antigen activity. LPPC aggregates dissociated in the presence of sodium dodecylsulphate (SDS) and urea. The chemical composition and serologic properties of fractions obtained are under consideration. The protein component of the complex consists of two major polypeptides (molecular weights--45,000 and 20,000) and some minor ones. The LPS component appeared to give 2--3 narrow bands in gel under conditions of SDS-polyacrylamide gel electrophoresis. It is suggested that such fractionation is caused by LPS association-dissociation in the course of electrophoresis.

A fresh human isolate of Yersinia enterocolitica serotype 03, and its derivative that had lost the virulence-associated 46-Md plasmid, were grown under defined conditions and compared for their outer membrane protein and cell surface structure. Under these conditions, the virulent strain grown at 37 degrees C expressed one major outer membrane protein (47 kd) not present in the plasmidless strain or in either strain grown at room temperature. A 200-kd protein also seen in the same preparations was shown to be an oligomer composed of the 47-kd protein subunits. Four different electron microscopic techniques showed tack-like projections covering the surface of those bacteria that expressed the 47-kd protein. These were specifically labeled with antibody to the 47-kd protein. This surface structure appeared to mediate aggregation (auto-agglutination) of the bacteria bringing their surfaces into unusually close apposition.