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The dephosphorylation of p-nitrophenyl phosphate by Yersinia protein-tyrosine phosphatase (PTPase) and by the rat PTP1 has been examined by measurement of heavy-atom isotope effects at the nonbridge oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom [18(V/K)bridge], and the nitrogen atom in the leaving group 15(V/K). The effects were measured using an isotope ratio mass spectrometer by the competitive method and thus are effects on V/K. The results for the Yersinia PTPase and rat PTP1, respectively, are 1.0142 +/- 0.0004 and 1.0152 +/- 0.0006 for 18(V/K)bridge; 0.9981 +/- 0.0015 and 0.9998 +/- 0.0013 for 18(V/K)nonbridge; and 1.0001 +/- 0.0002 and 0.9999 +/- 0.0003 for 15(V/K). The magnitudes of the isotope effects are similar to the intrinsic values measured in solution, indicating that the chemical step is rate-limiting for V/K. The transition state for phosphorylation of the enzyme is dissociative in character, as is the case in solution. Binding of the substrate is rapid and reversible, as is the binding-induced conformational change which brings the catalytic general acid into the active site. Cleavage of the P-O bond and proton transfer from the general acid Asp to the leaving group are both far advanced in the transition state, and there is no development of negative charge on the departing leaving group. Experiments with several general acid mutants give values for 18(V/K)bridge of around 1.0280, 15(V/K) of about 1.002, and 18(V/K)nonbridge effects of from 1.0007 to 1.0022. These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion but suggest more nucleophilic participation than in the solution reaction.

Protein tyrosine phosphatases (PTPases) play critical roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. The structures of several different PTPases have revealed a conserved active site architecture in which a phosphate-binding loop, together with an invariant arginine, cradle the phosphate of a phosphotyrosine substrate and poise it for nucleophilic attack by an invariant cysteine nucleophile. We previously reported that binding of tungstate to the Yop51 PTPase from Yersinia induced a loop conformational change that moved aspartic acid 356 into the active site, where it can function as a general acid. This is consistent with the aspartic acid donating a proton to the tyrosyl leaving group during the initial hydrolysis step. In this report, using a similar structure of the inactive Cys 403-->Ser mutant of the Yersinia PTPase complexed with sulfate, we detail the structural and functional details of this conformational change. In response to oxyanion binding, small perturbations occur in active site residues, especially Arg 409, and trigger the loop to close. Interestingly, the peptide bond following Asp 356 has flipped to ligate a buried, active site water molecule that also hydrogen bonds to the bound sulfate anion and two invariant glutamines. Loop closure also significantly decreases the solvent accessibility of the bound oxyanion and could effectively shield catalytic intermediates from phosphate acceptors other than water. We speculate that the intrinsic loop flexibility of different PTPases may be related to their catalytic rate and may play a role in the wide range of activities observed within this enzyme family.

Protein-tyrosine phosphatases (PTPases) are believed to exhibit restricted specificity toward phosphotyrosine. I demonstrate here that both the Yersinia PTPase and rat PTP1 can dephosphorylate alkyl phosphates such as flavin mononucleotide, pyridoxal 5'-phosphate, D-glucose 6-phosphate, DL-alpha-glycerophosphate, O-phospho-L-serine, and O-phospho-L-threonine. The kcat values for alkyl phosphates are orders of magnitude slower than those for aryl phosphates such as p-nitrophenyl phosphate and O-phospho-L-tyrosine, reflecting the intrinsic lower chemical reactivity of the alkyl phosphates. In addition, the kcat values for the PTPase-catalyzed hydrolysis of alkyl phosphates are similar to the kcat values for the PTPase-catalyzed 18O exchange reaction between inorganic phosphate and water. I conclude that the rate-limiting step for the hydrolysis of alkyl phosphates has changed to the phosphorylation of the PTPases, i.e. the formation of the phosphoenzyme intermediate. The implications of the results described in this report in terms of studying the PTPase catalytic mechanism and their potential application in developing selective PTPase inactivators are discussed.

The Yersinia protein tyrosine phosphatase (PTPase) was identified in the genus of bacteria responsible for the plague or the Black Death and was shown to be essential for pathogenesis. The three-dimensional structure of the catalytic domain of the Yersinia PTPase has been solved, and this information along with a detailed kinetic analysis has led to a better understanding of the catalytic mechanism of the PTPase. Mutational and chemical modification experiments have established that an invariant Cys residue (Cys403) is directly involved in formation of a covalent phosphoenzyme intermediate. We have shown that Arg409 plays a critical role in PTPase action and that the Cys(X)5Arg active site motif forms a phosphate-binding loop which appears to represent the essential features necessary for catalysis by the PTPases, the dual specific phosphatases, and the low molecular weight acid phosphatases.

Predictions of protein structure are best tested without prior knowledge of the protein three-dimensional structure. Three-dimensional atomic models will soon be determined by X-ray crystallography for the alpha-subunit of human blood clotting factor XIII and members of the family of protein tyrosine specific phosphatases. Accordingly, we here present secondary structure predictions for each of these proteins. The secondary structure predictions were generated from aligned sets of protein sequences. This technique has previously provided reliable predictions for the Annexins and the SH2 domains. The factor XIII alpha prediction contains 39 regions predicted in strand conformation (34% of the protein) with only 3 helices (4%). The protein tyrosine phosphatases have 12 predicted strands and 5 helices (30 and 17%, respectively). We expect greater reliability from regions of alignments that show clear patterns of residue conservation (61% of factor XIII alpha and 57% of the protein tyrosine phosphatases). The aligned protein tyrosine phosphatases show two regions (L39-L80 and I138-E253) with clear patterns of residue conservation separated by a region of variable amino acid composition. We suggest this indicates that the tyrosine phosphatase fold comprises two domains separated by an exposed linker. Potential phosphate binding sites are identified in the protein tyrosine phosphatases.

Product inhibition and 18O exchange experiments suggest that the Yersinia protein-tyrosine phosphatase-catalyzed phosphate monoester hydrolysis proceeds through at least two different chemical steps, i.e. the formation and breakdown of a covalent phosphoenzyme intermediate. The pH dependence of kcat values is bell-shaped, with the apparent pKa derived from the acidic limb of the profile at 4.6 for both p-nitrophenyl phosphate and beta-naphthyl phosphate, whereas the apparent pKa derived from the basic limb of the profile is substrate-dependent, with apparent pKa values of 5.2 and 5.8 for p-nitrophenyl phosphate and beta-naphthyl phosphate, respectively. Twelve aryl phosphates with leaving groups having pKa values from approximately 7 to 10 are also examined as substrates at two pH values. At pH 4.0, the beta lg values is effectively zero, whereas at pH 7.5, a beta lg value of 0.16 is observed. Collectively, our results suggest that the rate-determining step under acidic conditions corresponds to the breakdown of the phosphoenzyme intermediate, whereas under more alkaline conditions, substrate effects also contribute to the rate-limiting step. A model is proposed for the mechanism of the Yersinia protein-tyrosine phosphatase-catalyzed reaction.

The structural requirements of substrates for two recombinant protein tyrosine phosphatases (PTPases) are probed using various-sized synthetic phosphotyrosine (pY)-containing peptides corresponding to the autophosphorylation site in EGF receptor (EGFR) at Y992. The peptide EGFR988-998 (DADEpYLIPQQG) is chosen as a template due to its favorable kinetic constants. The contribution of individual amino acids on both sides of pY to binding and catalysis was assessed by kinetic analysis using a continuous, spectrophotometric assay. For both Yersinia PTPase and a soluble recombinant mammalian PTPase of 323 amino acid residues (rat PTP1), efficient binding and catalysis required six amino acids including the pY residue, i.e., four residues N-terminal to pY and one residue C-terminal to pY. Thus, PTPase substrate specificity is primarily dictated by residues to the N-terminal side of pY. The pY moiety and the rest of the peptide interact with PTPases in a cooperative manner. The presence of pY in the peptide substrate is necessary but not sufficient for high-affinity binding, since phosphotyrosine and other simple aryl phosphates exhibit weak binding, and dephosphorylated peptides do not bind to PTPases. Two variations on the pY moiety are also examined in order to assess their utility in PTPase inhibitor design. It is demonstrated that the thiophosphoryl analog in which one of the phosphate oxygens is replaced by sulfur can be hydrolyzed by PTPases, whereas the phosphonomethylphenylalanine analog in which the tyrosyl oxygen is replaced by a CH2 group is a competitive and nonhydrolyzable inhibitor, with Ki values of 18.6 and 10.2 microM, respectively, for the Yersinia PTPase and the rat PTP1.

Unrestricted protein tyrosine phosphatase (PTPase) activity may play a role in pathogenesis. For instance, the virulence determinant gene, yopH, of Yersinia pseudotuberculosis encodes a PTPase. The phosphatase activity of the YopH protein is essential for the pathogenesis of Y. pseudotuberculosis. Yersinia pestis, the bacterium which causes the bubonic plague, also contains a gene closely related to yopH. The action of YopH on host proteins appears to break down signal transduction mechanisms in many cell types including those of the immune system. This may contribute to the ability of the bacterium to escape effective surveillance by the immune system. The vaccinia virus VH1 gene, like yopH in the Yersinia bacteria, encodes a protein phosphatase. The VH1 PTPase defines a new class of phosphatases capable of dephosphorylating both phosphoserine/threonine and tyrosine containing substrates. Proteins sharing sequence identity to this dual-specificity phosphatase have been identified from other viruses, yeast and man. Although a complete understanding of the function of these dual-specificity phosphatases is not presently available, they clearly play important roles in cell cycle regulation, growth control and mitogenic signaling mechanisms. The unique catalytic properties of the dual specificity phosphatases suggest that these catalysts constitute a distinct subfamily of phosphatases.

The adherence and invasive capacities as well as the pathobiological activities exhibited by Yersinia ruckeri were examined. Although adhesive ability was dependent on the cell-line employed, all the strains showed moderate adhesion and invasiveness in the salmon cell-line CHSE-214. With regard to the extracellular products (ECP) all of them were strongly toxic for fish with LD50 ranging from 2 to 9.12 micrograms protein per g fish. In addition, all the ECP samples showed caseinase, gelatinase, amylase, lipase and phospholipase activities, hydrolysed esculin and displayed hemolytic activities for trout, salmon, sheep and human erythrocytes. Heat treatment (100 degrees C for 10 min) caused the loss of all these biological activities except the hydrolysis of gelatin. On the other hand, SDS-PAGE analysis of the LPS and protein components of the ECP revealed variations among strains depending on the serotype. The lack of lethal effects of the LPS present in the ECP was also demonstrated.

In this report, we demonstrated that the Yersinia protein tyrosine phosphatase (PTPase) could be inactivated by the alkylating agent iodoacetate. The enzyme modification was selective, and the covalent attachment was stoichiometric. The residue that was labeled by iodoacetate was shown to be Cys403, which was the same catalytically essential residue identified by site-directed mutagenesis [Guan, K. L., & Dixon, J.E. (1990) Science 249, 553-556]. The rate of iodoacetate modification decreased as the ionic strength of the media increased. There was no significant D2O solvent isotope effect associated with the inactivation of the enzyme, suggesting that thiol anion of Cys403 reacted as a nucleophile. The Yersinia PTPase also displayed differential reactivity (940-fold) toward iodoacetate over iodoacetamide. This indicates that residues within the active site of the enzyme are positively charged. The pKa of the active site thiol group was determined to be 4.67. The low pKa value suggests that ionic interactions are important in stabilizing the thiolate anion. One candidate residue for this stabilization is the invariant histidine (His402) found in all PTPases. Substitutions of His402 with Asn or Ala altered the active site thiol pKa to 5.99 and 7.35, respectively. Interestingly, the active site thiol in the mutants also showed enhanced reactivity toward iodoacetate. The second-order rate constants for the inactivation of the wild-type enzyme, H402N, and H402A were 59.7, 3305, and 1763 M-1 min-1, respectively.

The substrate specificity of a recombinant protein tyrosine phosphatase (PTPase) was probed using synthetic phosphotyrosine-containing peptides corresponding to several of the autophosphorylation sites in epidermal growth factor receptor (EGFR). The peptide corresponding to the autophosphorylation site, EGFR988-998, was chosen for further study due to its favorable kinetic constants. The contribution of individual amino acid side chains to the binding and catalysis was ascertained utilizing a strategy in which each amino acid within the undecapeptide EGFR988-998 (DADEpYLIPQQG) was sequentially substituted by an Ala residue (Ala-scan). The resulting effects due to singular Ala substitution were assessed by kinetic analysis with two widely divergent homogeneous PTPases. A "consensus sequence" for PTPase recognition may be suggested from the Ala-scan data as DADEpYAAPA, and the presence of acidic residues proximate to the NH2-terminal side of phosphorylation is critical for high-affinity binding and catalysis. The Km value for EGFR988-998 decreased as the pH increased, suggesting that phosphate dianion is favored for substrate binding. The results demonstrate that chemical features in the primary structure surrounding the dephosphorylation site contribute to PTPase substrate specificity.

Multilocus enzyme electrophoresis was used to analyze 244 strains of nine Yersinia species isolated from the environment, animals, and humans at 18 genes encoding metabolic enzymes. All 18 enzymes were polymorphic. Among the 137 electrophoretic types (ETs) distinguished, the mean allelic diversity per locus was 0.531. Yersinia frederiksenii ETs were divided into three major clusters that were separated by a large genetic distance, and one ET was more closely related to Yersinia enterocolitica. Thus, strains classically identified as Y. frederiksenii may represent more than one species. Furthermore, two strains identified as Yersinia kristensenii proved to be more closely related to Yersinia mollaretii. Environmental strains formed independent groups. A very interesting ET consisting of as many as 61 isolates of Yersinia enterocolitica was detected, and the epidemiologic relevance of this ET is discussed. Human strains of Y. enterocolitica biotype 4 and Yersinia pseudotuberculosis were recognized as being closely related to animal strains of the same species. Therefore, animal strains of these two species may be considered potential human pathogens.

Thirty-seven strains of Yersinia isolated from human and environmental sources were investigated for virulence and molecular characteristics. Clinical isolates of Y. enterocolitica serotypes 0:3 and 0:9, harbouring the 45 MDa virulence plasmid were Ca++ dependent and responded positively to the CV-binding assay. The environmental Y. kristensenii strain, although harbouring a 45 MDa plasmid proved to be negative for both assays. All the environmental strains showed pyrazinamidase activity, while isolates from human sources proved negative. Two different plasmid profiles were disclosed: one Y. enterocolitica strain (serotype 0:7, 8) showed one plasmid of 67 MDa and one plasmid of 1.5 MDa. Eleven human isolates of Y. enterocolitica and one environmental strain of Y. krinstensenii showed only one plasmid of 45 MDa. Restriction endonuclease analysis of the 45 MDa plasmids supported the hypothesis that the environmental Y. krinstensenii plasmid is unrelated to the family of homologous virulence plasmids of Y. enterocolitica.

The Yersinia protein tyrosine phosphatase (PTPase) Yop51, a C235R point mutation (Yop51*), and a protein lacking the first 162 amino acids at the NH2 terminus (Yop51*delta 162) have been overexpressed in Escherichia coli and purified to homogeneity through the use of CM Sephadex C25 cation exchange chromatography followed by Sephadex G-100 gel filtration. Greater than 50 mg of homogeneous Yop51* and Yop51*delta 162 can be obtained from a single liter of bacterial culture, whereas the same procedure yields only 5 mg of pure Yop51. Large, diffraction-quality crystals have been obtained for Yop51*delta 162. Size exclusion chromatography, sedimentation equilibrium, and enzyme concentration dependence experiments have established that the Yersinia PTPases exist and function as monomers in solution. Yop51 and Yop51* display identical UV, CD, and fluorescence spectra and have identical kinetic and structural stability properties. These full-length Yersinia PTPases have 31% alpha-helix, an emission maximum of 342 nm, a turn-over number of 1200 s-1 at pH 5.0, 30 degrees C, and an unfolding delta G value of 6 kcal/mol at 25 degrees C. Yop51*delta 162 has very similar kinetic and fluorescence characteristics to the full-length molecules, whereas its CD and UV spectra show noticeable differences due to the elimination of 162 NH2-terminal residues. The Yersinia PTPases are by far the most active PTPases known, and their kinetic parameters are extremely sensitive to the ionic strength of reaction medium.

The importance of parasite-directed phosphatases in such diseases as smallpox and the bubonic plague emphasizes the need to understand the molecular events associated with the normal function of protein tyrosine phosphatase in eukaryotic cells.

The intra- and inter-species differentiation of 90 strains of Yersinia belonging to six species were studied independently by computer-assisted statistical analysis of data from enzyme electrophoretic polymorphism and ribosomal DNA (rDNA) restriction fragment length polymorphism. Two correspondence analyses (CA) demonstrated the concordance between the bacterial classification obtained from enzymatic and genomic data. This concordance was reinforced by an algorithm of the correspondence established between the two dendrograms drawn from previous computations. Comparison of CA with similarity analysis (also called "numerical taxonomy") indicated that the intra- and inter-species differentiation obtained by the two methods are similar. The advantage of CA is that it gives a synthetic geometrical representation of the results (factorial planes), displaying both the main features of the clusters of strains (location and dispersion) and their essential character (i.e.: enzyme electrophoretic variant, rDNA fragment size).

A method for qualitative assessment of glucose-6-phosphate dehydrogenase in Yersinia is suggested. The reaction mixture was prepared by mixing the ingredients in the ratios as follows: 10 microliters of 0.01 M glucose-6-phosphate, 10 microliters of 0.0075 M NADP, 30 microliters of 0.75 M tris HCl pH 7.8, 10 microliters of 0.2 M MgCl2, 20 microliters of distilled water. The reaction was triggered by adding 20 microliters of cell-free bacterial extract. After 110-130 min incubation 10-20 microliters aliquots w re collected on filter paper (Whatman No 1) strips. The stains were dried at ambient temperature and examined in UV light at 365 nm with the use of a routine UV lamp. This method shows intensive fluorescence of the stain in M. tuberculosis and very poor fluorescence in Y. pestis. This technique is convenient when the scope of identification tests and differentiation between these two agents is high.

Yersinia is the genus of bacteria that is the causative agent in plague or the black death, and on several occasions this organism has killed a significant portion of the world's population. An essential virulence determinant of Yersinia was shown to be a protein tyrosine phosphatase. The recombinant 50-kilodalton Yersinia phosphatase had a specificity for removal of phosphate from Tyr-containing as opposed to Ser/Thr-containing phosphopeptides and proteins. Site-directed mutagenesis was used to show that the Yersinia phosphatase possesses an essential Cys residue required for catalysis. Amino acids surrounding an essential Cys residue are highly conserved, as are other amino acids in the Yersinia and mammalian protein tyrosine phosphatases, suggesting that they use a common catalytic mechanism.

Ribosomal DNA (rDNA) polymorphism was compared with electrophoretic enzyme polymorphism for the intra- and interspecies differentiation of Yersinia enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. aldovae, Y. frederiksenii and Y. kristensenii. DNA from 90 strains previously classified into six zymotypes (Y. enterocolitica and Y. frederiksenii) and into distinct enzyme electrophoretic patterns (the four other species) was digested with EcoRI or HindIII and analysed by Southern blotting. The six species were clearly differentiated from each other. In Y. enterocolitica, the subclassification of biotype 1 into zymotypes 1A and 1B was also reflected in the rDNA and the four other bio-zymotypes gave four different classes of restriction pattern. In Y. frederiksenii, both EcoRI and HindIII gave five distinct riboclasses which correlated with the zymotypes. In the four other species, the phenotype polymorphism appeared to be better correlated with the restriction fragment length polymorphism data in some enzymes than others. The data demonstrate that the inter- and intraspecies classification by rDNA polymorphism using two restriction enzymes is similar to that based on electrophoretic enzyme polymorphism. The analysis could be refined for taxonomic and epidemiological purposes by using other restriction enzymes.

A modified nutrient medium, made of reagents and standard media manufactured in this country, tried in practice, is suggested for Yersinia pyrazinamidase test. Tisamid (Finland), a pyrazinamide antituberculosis agent was used as substrate. A positive test is characteristic of nonpathogenic and a negative one for pathogenic Yersinia. The test and the medium may be effectively used in laboratory diagnostic studies for identification of Yersinia.

The presence of hexokinase, aldolase, glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase has been detected in Y. enterocolitica, which indicates the glycolytic and pentosophosphate pathways of glucose catabolism. Y. enterocolitica can be classified as an opportunistic anaerobic microorganism on the basis of the whole complex of its characteristics (growth in both aerobic and anaerobic conditions, the reduction of nitrates into nitrites, the presence of higly active glycolytic enzymes).

Sensitivity of 33 foreign and 10 native strains of Y. enterocolitica to tetracycline, aminoglycosides, penicillins, levomycetin and polymyxin was studied. All the strains proved to be resistant to penicillin, oxacillin and ampicillin: they produced penicillinase. The level of resistance to penicillin did not always correlate with penicillin activity. The ability of the native strains to acquire R-factor in vitro from Coli bacteria was shown.

The presence of high levels of isocitrate lyase activity in Yersinia pestis grown on blood agar base medium, as compared with low levels of this enzyme in Yersinia pseudotuberculosis and Yersinia enterocolitica, suggested that the differences in the levels of this enzyme could be used for the presumptive identification of Y. pestis. A modified, semiquantitative assay for isocitrate lyase activity is described which requires no expensive instrumentation, utilizes readily available chemicals and substrates, and requires only 20 min for completion. This test yielded positive results with all 108 isolates of Y. pestis tested and negative results with all strains of Y. pseudotuberculosis (68 isolates) and Y. enterocolitica (202 isolates) tested. Less than 2% of the approximately 1,300 non-Yersinia isolates from the family Enterobacteriaceae and none of the 93 isolates from the family Pseudomonadaceae yielded positive results. We conclude that this test provides for rapid identification of Y. pestis and should be useful in the initial screening of isolates from rodent and flea populations and in the presumptive identification of this organism from suspected cases of human plague.

Esterases of 32 strains of Y. enterocolitica and of 16 strains of Y. Pseudotuberculosis were compared by electrophoresis in polyacrylamide-agarose gel and by electrophoresis in a 4 to 30% polyacrylamide gradient gel. Differences observed in electrophoretic distributions and in molecular weights of esterases defined distinct esterase patterns for these two species.

The activity of enzymes, inactivating levomycetin and penicillin in the cells of plague and pseudotuberculosis microbes bearing extrachromosomal determinants resistant to a number of antibiotics was studied as dependent on some cultivation parameters: population age, aeration rate and temperature. It was shown that the highest capacity for levomycetin acetylation was characteristic of the cells in the late logarithmic and early stationary growth phages. Accumulation of levomycetin O-acetothers in the incubation medium markedly increased, when the cells were grown under the conditions of intensive aeration. An increase in the cultivation temperature up to 37 degrees C was accompanied by a reliable decrease in the activity of levomycetin acetylase in the transconjugant plague and pseudotuberculosis microbes though no correlation with the resistance levels in the same strains to the above antibiotics was observed. Optimal conditions for penicillinase production were determined. The maximum levels of penicillinase were found in the cells of Y. pestis 556/106 Rn with the episotic resistance type in the early exponential developmental phase under the aeration conditions and the temperature of 28 degrees C.

The beta-lactamase isoelectric focusing patterns of 37 strains of Yersinia enterocolitica from various serological and biochemical groups were examined. Strains of different serological groups generally had different patterns, but those of serological groups 1, 2, 3 and 9 were identical.

1) The phage typing of Y. enterocolitica has been made on more than 4,000 strains of various origins by means of 2 sets of phages. The 1st one, composed of 12 phages extracted from lysogenic strains of the same species has permitted to distinguish 10 phages types numbered from I to VIII and from IXa to IXb among the 3,323 lysotypable strains on the 4,366 examinated. 2) The search of the beta-galactosidase has differentiated the phage type IXa in its 2 varieties, the IXa 1, beta-galactosidase negative and the IXa 2, beta-galactosidase positive. 3) The 1,043 untypable strains by the 1st set were forming 2 very different groups. One of them, the X was insensitive to the 12 phages of the 1st set; the other XI, showed such a variety of lytic reactions to the phages of the 1st set that it was impossible to consider the rare samples of each of them as phage types. 4) The group X, submitted to a "complementary phage typing" by means of a second set of phages isolated from sewage water, has been subdivised into about 20 undergroups, from which only, 7 have been studies up to now, numbered from X 1 to X 7...

Two beta-lactamases, A and B, have been shown to be present in a strain of Yersinia enterocolitica (w222). Beta-Lactamase A hydrolyses a variety of penicillins and cephalosporins. This enzyme is sensitive to thiol reagents, is only partially inhibited by 0-1 mM-cloxacillin and has a molecular weight of approximatley 20,000.beta-Lactamase B shows strong cephalosporinase activity but does not hydrolyse some of the penicillins. It is more resistant than beta-lactamase A to thiol reagents, is completely inhibited by 0-1 mM-cloxacillin and has a molecular weight of about 34,000. With cephaloridine as a substrate, which is readily hydrolysed by both enzymes, about 85% of the total activity of a cell extract is due to beta-lactamase A and 15% to B. Addition of 6-aminopenicillanic acid to the culture during growth results in a 2-to4-fold selective increase in the amount of beta-lactamase B. Two beta-lactamases similar to enzymes A and B have been found in five other strains of Y. enterocolitica. In contrast, only one beta-lactamase, similar to enzyme B, has been detected in a different strain of Y. enterocolitica (H66), which is abnormal in that it is sensitive to ampicillin. Addition of 6-aminopenicillanic acid to cultures of this strain results in an 8-to 10-fold increase in beta-lactamase production.

The influence of incubation temperature (37, 30 and 22 degrees C) on antibiotic susceptibility, beta-lactamase activity and growth characteristics was studied on 43 unselected strains of Y. enterocolitica (Serovar O:3 and O:9) freshly isolated from cliical specimens. Antibiotic susceptibility was measured by the disc diffusion technique and by a broth dilution test (MIC). Beta-lactamase activity was detected with chromogenic cephacetrile using standard curves prepared for 37, 30 and 22 degrees C. Continuous increase of beta-lactamase activity was found when incubation temperatures were lowered. All strains were found to be resistant by ampicillin and cephalothin at the three temperatues tested. Some strains showed an intermediate susceptibility to carbenicillin in the disc diffusion test. A temperature reduction of 37 to 30 degrees C significantly decreased the inhibitory zone diameters for the beta-lactam antibiotics ampicillin, carbenicillin and cephalothin, but also for other substances like tetracycline, chloramphenicole and cotrimoxazole. This suggests, that the observed decrease is caused by a better growth of Y. enterocolitica at 30 degrees C rather than increased beta-lactamase production. From 30 to 22 degrees C a further decrease in inhibitory zone diameters was only seen with ampicillin and carbenicillin. This seems to be mainly due to the increased B-lactamase activity observed at 22 degrees C. In contrast the resistance to cephalothin was apparently not influenced by this additional beta-lactamase activity. Resistance to cephalothin therefore depends probably more on other, not beta-lactamase-related, factors such as permeability variations of the outer membrane or modifications of binding proteins involved in the peptidolycan biosynthesis. The correlation between beta-lactamase activity at various incubation temperatures and resistance to beta-lactam antibiotics was less pronounced when the broth dilution test (MIC) was applied. Only carbenicillin showed significantly increasing MIC values from 30 to 22 degrees C. All the Y.e. strains investigated could be divided into two groups with respect to their beta-lactamase production characteristics. The first group showed continuously increasing beta-lactamase values at lower incubation temperatures. In the second group generally lower amounts of beta-lactamase values were found and temperature dependence was not observed. On the other hand variations in cell wall permeability, resulting in a diminished accessability of the cell wall bound enzymes must also be considered.

The paper describes a number of tests for the rapid detection of glycosidases including alpha-glucosidase, beta-glucosidase, beta-glucuronidase, beta-xylosidase and alpha-fucosidase. The methods use heavy suspensions of viable but non-multiplying bacteria in a buffered solution of a chromogenic substrate. The results of the tests are readable within 4 h. The application of these tests to a collection of 633 strains of Enterobacteriaceae and Vibrionaceae demonstrates that some of the tests may be valuable additions to the present tests available for the identification of bacteria belonging to these families. beta-glucuronidase activity was observed only in strains of the Escherichia-Shigella group. 97 per cent of the Escherichia strains possessed beta-glucuronidase activity. beta-xylosidase activity was almost completely restricted to strains of the Klebsiella-Enterobacter group in addition to Yersinia strains. None of the strains possessed alpha-fucosidase activity.

Raw milk in southern Ontario frequently contains Yersinia enterocolitica. The potential for transmission of this organism by cheese manufactured from unpasteurized milk was evaluated by examination of milk and cheese curd samples from cheese manufacturing plants and finished cheddar and Italian cheeses. The incidence of Y. enterocolitica was lower in cheese curd samples (9.2%) than in raw milk (18.2%). Most of the curd samples showed a positive phosphatase test, indicating production from raw milk. One curd sample yielded Y. enterocolitica after 4 weeks of storage at 4 degrees C but was negative after 8 weeks. All samples of cheddar and Italian cheeses, most of which showed a positive phosphatase test, were negative for Y. enterocolitica. One out of 265 samples (0.4%) of pasteurized fluid dairy products contained Y. enterocolitica.

The specific activity of urease, nitrogenase, hialuronidase and neuraminidase in Y. pseudotuberculosis grown in different culture media and at different temperature has been studied. These enzymes have been found capable of functioning at both relatively low (2-8 degrees C) and high (37 degrees C) temperatures. The thermoadaptive properties of Y. pseudotuberculosis within a wide range of temperatures are ensured by the constant presence of isoenzymes, functioning only at low temperatures or only at high temperatures, in the microbial cells. Low temperature in combination with a definite culture medium triggers the activity of certain enzymatic systems, which explains, to some extent, the biochemical mechanisms of the psychrophilic properties of Y. pseudotuberculosis.

To study the ecology of Yersinia, culture media, the pool method and the algorithm for their isolation and identification are proposed. The correct selection of typical Yersinia colonies grown in special media is the major step. This permits the study of culture pools (their amount reaching 12 and more from each sample) and makes it expedient to use the economical dish method for obtaining pure cultures and identifying Yersinia by the minimal set of key signs. The use of these methods makes it possible to study a great number of cultures and to obtain valuable information on local Yersinia populations with the least expenditures.

Allelic variation in the chromosomal genome of 81 isolates of Yersinia enterocolitica and single isolates of Yersinia intermedia, Yersinia frederiksenii, Yersinia mollaretii, and Yersinia kristensenii was assessed by analysis of electrophoretically demonstrable polymorphism in 21 genes encoding metabolic enzymes. Eighteen distinctive multilocus genotypes (electrophoretic types [ETs]) were identified. Clustering of the ETs from a matrix of pairwise genetic distances, based on the 21 enzyme loci, confirmed the genetic distinctness of serogroup 3 isolates of Y. intermedia, Y. frederiksenii, Y. mollaretii, and Y. kristensenii and identified another serogroup 3 isolate that was also not a member of Y. enterocolitica. The 13 ETs of Y. enterocolitica clustered into two groups: cluster A, which included eight ETs represented by isolates of serogroups 1; 2; 3; 5,27; and 9, and cluster B, which included four ETs represented by isolates of serogroups 8, 13, and 21. Clones of cluster A were found to be distributed worldwide, but those of cluster B were largely restricted to North America. Isolates of genotypes belonging to cluster B were lethal to mice, whereas those of cluster A were not, suggesting an influence of the chromosomal background on the virulence of Y. enterocolitica.

Esterases of 53 strains of Yersinia enterocolitica sensu stricto, including five previously defined biotypes, and 30 strains of Yersinia pseudotuberculosis were analysed by horizontal polyacrylamide-agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. Esterase bands were defined by their range of activity towards several synthetic substrates, their resistance to heat and to di-isopropyl fluorophosphate. The two species were characterized by distinct electrophoretic patterns of their esterases. The apparent molecular weights of the heat-resistant esterase of Y. enterocolitica and of the major heat-resistant esterase of Y. pseudotuberculosis, as determined by polyacrylamide gradient gel electrophoresis, were estimated to be 52 000 and 250 000, respectively. On the basis of electrophoretic mobilities and isoelectric points of esterases produced by strains of Y. enterocolitica, five principal zymotypes were observed: two for strains of biotype 1, two for strains of biotypes 2 and 3, respectively, and only one for strains of both biotypes 4 and 5. The zymotypes of strains of biotypes 2, 3, 4 and 5 appeared to be more closely related to one another than to zymotypes of strains of biotype 1. Variations in number or mobility of bands observed within each biotype of Y. enterocolitica and within some serotypes of Y. pseudotuberculosis could represent an additional marker for epidemiological analysis.

The expression of gamma-glutamyltransferase activity was studied among 87 strains representing all the Yersinia species. Y. pestis lacked constantly this enzyme, while y. pseudotuberculosis expressed it as a rule. This test, when positive, is useful for the exclusion of a suspected Y. pestis diagnosis. All the other Yersinia species, like most of the Enterobacteriaceae, showed a gamma-glutamyltransferase activity.

Isocitrate lyase and malate synthase, the two unique enzymes of the glyoxylate cycle, were detected in crude extracts of Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica. Y. pestis, unlike Escherichia coli and the other yersiniae tested, yielded two forms of isocitrate lyase during growth on acetate. These forms differed in electrophoretic mobility and temperature optima. One form (A) was present during growth on acetate, but was absent during growth on alternate carbon sources such as glucose. The second form (B) was not constitutive, but was found during growth on acetate, glucose, xylose, or other complex carbon sources. Itaconate, a succinate analog which inhibited both forms of isocitrate lyase in crude extracts, did not affect the growth of Y. pestis under conditions where little isocitrate lyase activity was detected. This inhibitor, however, retarded the growth of Y. pestis under conditions where acetate was provided as the primary carbon and energy source as well as under all conditions in which either form of isocitrate lyase was evident. This suggests that the B form may play an important role in the growth of this bacterium under conditions where a requirement for the classical anaplerotic sequence involving this enzyme is not apparent.

Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.

The authors demonstrated the presence of neuraminidase in Past. pestis. Optimal conditions for its formation and detection were chosen. Extracellular form of neuraminidase was revealed. An increase of cultivation temperature led to the elevation of the neuraminidase activity in Past. pestis cells. Neuraminidase was revealed in bacteria affiliated to Past. pestis (causative agents of pseudotuberculosis and Y. enterocolitica).

Yersinia enterocolitica W222 (serological group 3) synthesized two different intracellular beta-lactamases, called A and B. Enzyme B was more sensitive than A to inhibition by cloxacillin. The minimum inhibitory concentrations of various beta-lactam antibiotics for strains of Y. enterocolitica of groups 3 and 9 and the effect of cloxacillin on these concentrations suggested differential roles for beta-lactamases of types A anb B in penicillin and cephalosporin resistance. Type B enzymes protected Y. enterocolitica against cephalothin and cephalosporin C, wheras type A enzymes protected very efficiently against carbenicillin. Protection against other beta-lactam antibiotics was exerted by both enzymes. However, while both enzymes readily hydrolysed cephaloridine and showed no crypticity with this substrate, they only conferred a very weak protection against it. This may be because cephaloridine reached its target quickly, before it was degraded. The resistance of strains of Y. enterocolitica from groups I, 2 and I6 was also explicable in terms of a two-enzyme system, whereas strains belonging to group 5b produced only a type B lactamase and were sensitive to carbenicillin.