Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases

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.     

Restriction map of virulence plasmid in Yersinia enterocolitica O:3

Restriction map of the 72-kb virulence plasmid isolated from Yersinia enterocolitica O:3 was generated using EcoRI, BamHI, HindIII, and XbaI restriction enzymes. The mapping was done after cloning all of the 13 BamHI fragments of the plasmid in Escherichia coli. In addition, the restriction enzyme analysis revealed two types of virulence plasmids (types I and II) in Y. enterocolitica O:3. No functional differences between the strains bearing type I or type II plasmid were observed.     

Ribosomal DNA (rDNA) spacer polymorphism in mole rats

Genetic differentiation of ribosomal DNA (rDNA) nontranscribed-spacer (NTS) polymorphism was analyzed in 50 individuals from 13 populations among the four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of subterranean mole rats of the Spalax ehrenbergi complex in Israel. Southern blot analysis with a mouse rDNA probe and two restriction enzymes, EcoRI and BamHI, revealed various sizes of major restriction fragments. The assumption that this variation is due to length polymorphism of NTS DNA was supported by the construction of restriction-site maps. On the basis of the EcoRI and BamHi fragment lengths, we could characterize the major types of NTS rDNA repeating units in each individual. Each member of the central population in the four chromosomal groups of mole rats has a characteristic combination of the NTS types, suggesting that the karyotype groups were genetically diverged. Some near-hybrid-zone populations reflect similarities with the rDNA spectra of a neighbor chromosomal group. This might have resulted from gene flow across the hybrid zones.      

Assessment of the degree and the type of restriction fragment length polymorphism in barley (Hordeum vulgare)

Summary In order to determine the extent of polymorphism in barley (Hordeum vulgare), DNA from 48 varieties was analyzed with 23 genomic, single-copy probes, distributed across all seven chromosomes. Upon hybridization to wheat-barley addition lines, the probes showed different degrees of homology compared to the wheat genome. Polymorphisms were detected in the barley genome at a frequency of 43% after digestion with EcoRI, BamHI, and HindIII. Subgroups of spring and winter barley and of two- and six-rowed types showed less diversity which, in most cases, was due to shifts in allelic frequencies. One probe (MWG1H504) hybridized to an EcoRI restriction fragment exclusively observed in winter barley. A comparison of six different restriction enzymes revealed clear differences with regard to their efficiency in detecting polymorphisms. The respective frequencies were between 13% (HindIII) and 37% (EcoRV). A significant correlation between the efficiency of a restriction enzyme and the mean fragment size detected by the different probes identified insertion/deletion events as the major factor causing polymorphism in barley.     

Structure and variability of nuclear ribosomal genes in the genus Helianthus

Summary The restriction map of the rDNA unit of Helianthus annuus was constructed using EcoRI, BamHI, HindIII, KpnI and SacI restriction enzymes. Variations in this map among 61 ecotypes representing 39 species of the genus Helianthus were analyzed. The sizes of the rDNA unit ranged from 9.8 to 11.0 kbp, due to a length-repeat heterogeneity of the external non-transcribed spacer by increments of 200 base pair segments. Lengthrepeat heterogeneity and restriction polymorphism were found to be characteristic of populations or species of Helianthus. Restriction patterns and thermal melting with probes of a cloned H. annuus ENTS segment allowed us to differentiate species from each other. However, most lines of the cultivated sunflower were found to be identical on the basis of the physical properties of their ribosomal DNA.     

Characterization of Staphylococcus species by ribosomal RNA gene restriction patterns

The rRNA gene restriction pattern sof 110 strains belonging to 12 staphylococcal species have been determined. The strains, isolated from various sources, were epidemiologically unrelated. Total DNA was cleaved with restriction enzymes HindIII and EcoRI, electrophoretically separated and probed with radiolabelled 16S rDNA from Bacillus subtilis inserted in a plasmid vector, pBR322. Fourty-four distinct HindIII patterns and 44 distinct EcoRI patterns were observed. Strains belonging to different species had different patterns. Although distinct patterns were also observed with some species, a core of common bands could be discerned within each species or subspecies. Analysis of the patterns revealed two taxa in Staphylococcus xylosus which were not evident using phenotypic characteristics. Of 18 strains which were difficult to identify using phenotypic schemes, 15 showed patterns typical of known species. The three remaining atypical strains showed unusual patterns and may belong either to a known species, not included in the study, or to a new species. Since various patterns were observed within some species (e.g.S.aureus and S. epidermidis), rRNA gene restriction patterns may have epidemiological, as well as taxonomic interest.     

Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus

A 1.35-kb EcoRI fragment of Lytechinus variegatus DNA containing a single 5S rRNA gene has been cloned into the plasmid vector pACYC184. Four clones from different transformation experiments contain 5S rDNA inserts of about the same size and have the same restriction enzyme digestion patterns for the enzymes HaeIII, HinfI, HhaI, and AluI. One EcoRI site near the HindIII site of the plasmid vector pACYC184 is missing in all the four clones. By DNA sequencing, the missing EcoRI ws found to be EcoRI site, d(AAATTN)d(TTTAAN) in pLu103, one of the four 5S rDNA clones. The structure of pLu103 was determined by restriction mapping and blot hybridization. Three restriction fragments, 1.0-kb HaeIII/HaeIII, 0.375-kb AluI/AluI and 0.249-kb MboII/MboII, which contain the 5S rRNA coding region, have been subcloned into the EcoRI site of the plasmid pACYC184. The organization of 5S rRNA genes in the sea urchin genome was also investigated. It was found that restriction endonuclease HaeIII has a single recognition site within each 5S rDNA repeat, and yields two fragment lengths, 1.2 and 1.3 kb. The behavior of these 5S rRNA genes when total L. variegatus DNA is partially digested with HaeIII is consistent with an arrangement of 5S rRNA genes in at least two tandemly repeated, non-interspersed families. Both the coding region and spacer region of the 5S rRNA gene in pLu103 hybridize to 1.2 and 1.3-kb rDNA families. This indicates that the cloned EcoRI fragment of 5S rDNA in pLu103 represents one single repeat of 5S rDNA in the genome.     

Distinctive electrophoretic and isoelectric focusing patterns of esterases from Yersinia enterocolitica and Yersinia pseudotuberculosis

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.     

Comparative restriction endonuclease analysis and molecular cloning of plastid DNAs from wild species and cultivated varieties of the genus Beta (L.)

Summary A phyletic tree of the genus Beta has been constructed based on EcoRI and PstI plastid DNA restriction patterns of eight species from three sections of the genus. In contrast to the remarkable morphological variability of the varieties of B. vulgaris the restriction patterns of the plastid DNA of this species were found to be almost identical. The comparison of plastic DNAs of B. vulgaris crassa fertile and sterile lines with 13 different restriction enzymes revealed only a single fragment polymorphism in the HindIII patterns. Hybridization analyses in the plastidal rDNA region revealed an interesting loss of an EcoRI restriction site in all cultivated B. vulgaris varieties in contrast to wild species. The results of the construction of clone banks for SalI and BamHI fragments of plastid DNA from fertile B. vulgaris crassa are reported and difficulties in the cloning of specific fragments are discussed.     

Identification of Yersinia enterocolitica within the genus Yersinia

In this report we describe a PCR strategy for the unambigous identification of biochemically presumptive typed Yersinia (Y.) enterocolitica. A total of 269 isolates belonging to ten species of the genus Yersinia were investigated. In a first PCR only isolates classified as Y. enterocolitica (n = 113) gave rise to a specific amplification resulting in a sensitivity and a specificity of 100%. By sequencing the 269 amplicons of a second pan-Yersinia PCR spanning a distinct 16S rRNA gene region, 20 different sequence clusters could be identified within the genus. By this, Y. enterocolitica isolates of American and European origin could be distinguished safely and already described sequence clusters of the species Y. frederiksenii were confirmed. New 16S rRNA gene sequence clusters were detected for the species Y. frederiksenii, Y. intermedia, Y. mollaretii, Y. aldovae, Y. kristensenii, and Y. rohdei.     

Comparative electrophoretic profiles of esterases, and of glutamate, lactate and malate dehydrogenases, from Aeromonas hydrophila, A. caviae and A. sobria

Esterases, and glutamate, lactate and malate dehydrogenases of 64 Aeromonas hydrophila, A. caviae and A. sobria strains, were analysed by polyacrylamide agarose gel electrophoresis and by thin layer isoelectrofocusing. On the basis of the isoelectric points of malate dehydrogenase from the three species and the mobility of lactate dehydrogenase from A. sobria, 8 species specific zymotypes were defined: three for A. hydrophila strains, three for A. caviae strains and two for A. sobria strains. These zymotypes correlated with previously established DNA hybridization groups. The other electrophoretic data were found to be less useful for distinction between A. hydrophila and A. sobria strains, but supported differentiation into zymotypes for A. caviae strains. The two-dimensional electrophoretic profile established by plotting isoelectric point against electrophoretic mobility of the major esterase illustrated the degree of enzyme polymorphism among the strains of the three species. Variation in electrophoretic patterns within A. hydrophila and A. caviae might provide useful epidemiological markers.     

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

A cloned chromosomal DNA fragment which differentiates Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica

Chromosomal DNA from reference Yersinia strains was digested individually with 9 restriction endonucleases. DNA fragments were separated and analyzed by electrophoresis through agarose gels. The clearest fragment patterns were obtained when EcoRI was employed. The Y. pestis fragment pattern obtained after the use of this enzyme showed the presence of a unique DNA fragment with molecular mass 1400 bp. This DNA fragment was cloned, purified, labeled with 32P and then used to probe EcoRI digests of all three Yersinia species. A strong hybridization signal was obtained with Y. pestis strain. No such signal was found with Y. pseudotuberculosis or Y. enterocolitica. These results indicate that the DNA fragment is species specific and could be used as a diagnostic DNA probe for Y. pestis.     

Intraspecies polymorphism for the structural organization of ribosomal gene cluster in Blattella germanica

Intraspecific HindIII restriction fragment length polymorphism (RFLP) for the structural organization of the ribosomal gene cluster was described in Blattella germanica. In this species, homologous chromosomes were shown to contain ribosomal DNA (rDNA) repeats that differ in structure. The pattern of inheritance was determined for various structural variants of rDNA.     

DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism.

We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical maps of bovine papillomavirus type 1 and type 2 genomes.

Physical maps of bovine papillomavirus type 1 and type 2 (BPV-1 and BPV-2) DNA were constructed from analysis of the electrophoretic mobilities of restriction endonuclease cleavage fragments from dual digests. BPV-1 DNA was sensitive to Hind III, HindIII, EcoRI, HpaI, AND BamHI, with all but HindII yielding single scissions. BPV-2 DNA was resistant to EcoRI, and HindIII had one cleavage site whereas HpaI, BamHI, and HindII yielded multiple fragments. Of four BPV-1 isolates examined, DNA from one isolate was resistant to HindIII, and another DNA isolate was resistant to BamHI. The three BPV-2 isolates examined were uniformly sensitive to the restriction endonucleases employed.     

DNA sequence heterogeneity in the gene encoding a 60-kilodalton extracellular protein of Listeria monocytogenes and other Listeria species.

Chromosomal DNA sequences from the 60 kilodalton protein gene of Listeria monocytogenes, amplified by the polymerase chain reaction, were used for restriction fragment length polymorphism differentiation of L. monocytogenes serotypes and other Listeria species. All 24 strains of L. monocytogenes examined produced an extracellular protein of molecular weight 60,000 (p60) as determined by Western blot analysis. Four of six other Listeria species had a protein that cross-reacted to antibodies to p60, but all differed in molecular weight, ranging from approximately 50,000 to 65,000. The gene encoding p60 was amplified from chromosomal DNA in all strains using polymerase chain reaction with a single primer pair. Restriction enzyme digestion with HindIII of the amplified product revealed a restriction pattern that was distinct between serotypes 1/2a and either 4b or 1/2b of L. monocytogenes. Of the other Listeria species, four strains that produced a cross-reacting protein likewise produced a polymerase chain reaction amplification product with the primer pair. Listeria innocua alone had a restriction pattern similar to that of Listeria monocytogenes serotype 4b and 1/2b. Genotypic heterogeneity, as revealed by DNA amplification and restriction endonuclease digestion of the p60 open reading frame, correlates with "electrophoretic type" grouping and may be related to differences in virulence mechanisms of Listeria monocytogenes and other Listeria species.     

Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites

Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonuclease analysis were examined in four Japanese Black cows, three Japanese Shorthorn cows, and six Holstein cows. Seventeen restriction enzymes which recognize six base pairs and two restriction enzymes which recognize four base pairs were used in this study. Polymorphism was observed with three restriction enzymes, HindIII, TaqI, and MspI, and was detected within the breeds. Nucleotide substitution was determined in the HindIII polymorphic site by DNA cloning and sequencing; this is C----T at position 10126 of the URF-3 region. Furthermore, the MspI and TaqI polymorphic sites were located on the physical map.     

Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene

Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific. Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.     

Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys.

Total cellular DNA from 28 strains of Listeria monocytogenes isolated from food implicated in food-borne illness and from patients with listeriosis was digested with the restriction endonucleases HindIII, HaeIII, and EcoRI. Following agarose gel electrophoresis, the fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for classifying L. monocytogenes strains, and the resulting subtypes were compared with serotyping and multilocus enzyme electrophoresis classification schemes. A total of 15 distinct and identical groups were obtained when genomic DNA was digested with either HindIII or HaeIII. The most discriminating enzyme for ribotyping of strains was EcoRI, which divided the 28 strains of L. monocytogenes into 6 ribotype groups. DNA fingerprinting and ribotyping differentiated L. monocytogenes from other Listeria spp., including L. ivanovii, L. welshimeri, and L. innocua as well as the lactic acid bacteria Lactococcus lactis subsp. lactis and subsp. cremoris. L. monocytogenes strains isolated from four independent food-borne illness incidents were analyzed by all typing methods. Patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis. DNA fingerprinting was the only method capable of differentiating these strains, or conversely, of proving relatedness of patient-product pairs of isolates. This method was a relatively simple, sensitive, reproducible, and highly discriminating method for epidemiological tracking of L. monocytogenes implicated in food-borne illness.