Restriction endonuclease analysis of four Borrelia burgdorferi strains

Abstract A restriction endonuclease analysis was performed on four strains of Borrelia burgdorferi : one isolated from man (SF), one from Ixodes dammini (B31) and two form I. ricinus (BITS in Italy and B45 in Germany). Digestion by Taq I and Hae III gave the best resolution of the DNA fragments. Three different restriction patterns were obtained: BITS and B45 showed only one band difference. These results correlate with the reactivity of the four strains with monoclonal antibodies.     

Restriction endonuclease analysis of four Borrelia burgdorferi strains

A restriction endonuclease analysis was performed on four strains of Borrelia burgdorferi: one isolated from man (SF), one from Ixodes dammini (B31) and two from I. ricinus (BITS in Italy and B45 in Germany). Digestion by Taq I and Hae III gave the best resolution of the DNA fragments. Three different restriction patterns were obtained: BITS and B45 showed only one band difference. These results correlate with the reactivity of the four strains with monoclonal antibodies.     

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.     

DNA homology of surface protein antigen A gene in mutans streptococci

1. A recombinant plasmid, pYA724, containing an 8.45 kb DNA fragment encoding surface protein antigen A (spaA) from Streptococcus sobrinus 6715 was used to examine the DNA homology of the spaA gene with chromosomal DNA of various mutans streptococci strains. 2. Restriction endonuclease BamHI-digested pYA724 DNA was radio-labeled by nick-translation, and a DNA-DNA hybridization experiment was carried out. pYA724 DNA hybridized with chromosomal DNA of serotypes a, c, d, e, f and g strains, but not with b by dot DNA-hybridization and Southern blot DNA hybridization. 3. Chromosomal DNAs were isolated from several serotype c Streptococcus mutans strains, digested with BamHI, and analyzed by Southern blot DNA hybridization. pYA724 DNA hybridized with different sizes and numbers of BamHI-digested DNA fragments of the chromosomal DNAs. 4. These data indicated that all mutans streptococci strains except serotype b have DNA homologous with the spaA gene, although within the same serotype strain the spaA gene has a diversity of arrangement within the chromosome.     

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.     

Mineralization of 2-chloro- and 2,5-dichlorobiphenyl by Pseudomonas sp. strain UCR2

Pseudomonas sp. strain UCR2 was isolated from a multi-chemostat mating experiment between a chlorobenzoate-degrader, Pseudomonas aeruginosa strain JB2, and a chlorobiphenyl-degrader, Arthrobacter sp. strain B1Barc. Strain UCR2 differed from either of the parental organisms in that it grew on both 2-chloro- and 2,5-dichlorobiphenyl with concomitant release of chloride. Phenotypic typing by the Biolog system indicated that strain UCR2 shared greater similarity with strain JB2 (88%) than strain B1Barc (3%). In DNA:DNA hybridization experiments, genomic DNA from strain UCR2 hybridized with both strain JB2 and strain B1Barc, with the former pairing yielding a much stronger signal than the latter. In contrast, no hybridization whatsoever was observed when the parental organisms strains JB2 and B1Barc were probed against each other.     

Genomic analysis of a virulent and a less virulent strain of the entomopathogenic fungus Beauveria bassiana, using restriction fragment length polymorphisms.

The genomic DNA of two strains of the entomopathogenic fungus Beauveria bassiana, strain GK2016, a "wild type" (virulent), and strain GK2051, a less virulent mutant to grasshoppers, was digested with 12 restriction endonucleases. Gel electrophoresis conditions were established to show restriction fragment length patterns visually in the digested DNA stained with ethidium bromide. The less virulent mutant was generated by ultraviolet illumination of conidiospores at a 95% lethal dose. Both strains of the fungi were identical in morphology as well as in 16 of 22 API-ZYM kit enzyme assays. Differences in levels of total enzyme activity were observed for esterase, esterase-lipase, beta-galactosidase, chitinase, and protease. A Neurospora crassa beta-tubulin gene (heterologous gene) and two homologous DNA probes (pJK16 and pJK18) hybridized to several specific DNA bands in B. bassiana strain GK2016 but not in strain GK2051. Strain GK2051 gave different restriction fragment length pattern when compared with its parent strain. Taken together, the data show restriction fragment length differences between the genomic DNA of the two strains, including the loss of some DNA sequences from the mutant strain, which may be involved in pathogenicity. Finally, B. bassiana GK2016 contains a beta-tubulin gene with at least partial homology to that of N. crassa.     

Molecular analysis of the rat MHC. I. Delineation of the major regions in the MHC and in the grc

Southern blot analysis with liver DNA from a unique series of recombinant (R10, R11, R16, R18, R21, and R22), congenic (Y0.1U.grc+, Y0.1U.grc+/Y0.1L.grc, and Y0.1L.grc) and inbred rats has been performed to examine the restriction fragment length polymorphisms of class I genes. After digestion with Xba I or Eco RI, the genomic DNA was resolved on agarose gels, was transferred to nitrocellulose membranes, and was hybridized with murine H-2 cDNA probes. Eighteen to 25 bands of varying intensities could be clearly resolved in any given strain. Analysis of these hybridization patterns detected restriction fragment length polymorphisms that permitted the assignment of 17 specific fragments to regions within the major histocompatibility complex: RT1.A, RT1.B/D, and the RT1.E-grc-T1 alpha region. Fragments have been identified that are specific for grc, grc+, and RT1.E, and mark the junction sites between these loci. In addition, several markers identify the region around the sites of recombination in some strains. The hybridization pattern of the R18 recombinant had a unique band that specified a point of recombination within the grc. The recombinant R11 presented a unique restriction pattern unrelated to either of the parental strains or other related strains. This result suggests that R11 arose from a recombination event(s) undetected by conventional serologic methods.     

Genome Stability of Bacillus thuringiensis subsp. israelensis Isolates

Swedish soil isolates biochemically classified as Bacillus thuringiensis subsp. israelensis were further examined for genetic diversity by multilocus enzyme electrophoresis (MLEE), random amplified polymorphic DNA analysis (RAPD), pulse field gel electrophoresis (PFGE), and Southern blotting, and were compared with reference strains. All the tested strains belonging to the Bt. israelensis serotype H14 were found to be identical, as judged from the RAPD analysis. MLEE analysis gave a similar result; only one H14 strain was found to differ from the remaining H14 strains by one null allele. PFGE analysis confirmed a very close relationship between the H14 strains but revealed an SfiI restriction fragment of variable size. Southern blot analyses were carried out with probes for the chromosomally encoded flagellin gene(s) and the plasmid-encoded mosquitocidal toxins. All probes gave similar hybridization patterns in the H14 strains. The mosquito toxin probes hybridized only to the H14 strains, except for one probe hybridizing to strain 6:3, which was originally isolated from the same soil sample as strains 6:11 and 6:12. Because the RAPD, MLEE, and PFGE analyses showed that strain 6:3 appears to be unrelated to strains 6:11 and 6:12, the presence of a mosquito toxin sequence in strain 6:3 may suggest that gene transfer has occurred. Received: 8 July 1999 / Accepted: 9 August 1999     

Strain-dependent variation in ribosomal DNA arrangement in Absidia glauca

Restriction analysis of total DNA from the zygomycete Absidia glauca reveals a pattern of prominent bands on a homogeneous background. By Southern blot analysis with 32P-end-labelled ribosomal RNA most of these bands could unequivocally be identified as repetitive copies of ribosomal DNA. There are marked differences in restriction patterns of rDNA between all seven strains tested, even of strains belonging to mating type pairs, presumably isolated from the same location. By using purified rRNAs as probes in hybridization experiments, evidence is presented that 5S rRNA is part of the ribosomal repetitive unit. A more detailed analysis of one strain pair [A. glauca CBS 100.48 (+)/101.48 (-)] provided evidence that the (+) strain, in addition to one rDNA repeat unit common to both strains, contains a second one, derived from the common form by a small deletion.     

Polymorphism in Brucella spp. due to highly repeated DNA.

The species of Brucella are very closely related, but Brucella ovis does not express detectable amounts of a protein, designated BCSP31, that is common to the other species. We studied the lack of expression of BCSP31 by Southern analysis. DNAs from the B. ovis culture collection strains and field isolates were probed with a 1.3-kb HindIII fragment encoding BCSP31 of Brucella abortus. The probe hybridized to a 1.6-kb HindIII fragment of all B. ovis strains tested, showing that the gene is present in B. ovis but occurs on a larger restriction fragment. DNA linkage studies and restriction mapping of the cloned polymorphic region of B. ovis showed that the polymorphism was due to a DNA insertion of approximately 0.9 kb at a site downstream of the BCSP31-coding region. When the 1.6-kb polymorphic B. ovis fragment was used to probe a HindIII Southern blot of cellular DNA of strains of B. ovis and of B. abortus, at least 24 fragments of B. ovis and 6 fragments of B. abortus hybridized to the inserted DNA. Specimens of B. ovis collected over a 30-year period on two continents had similar hybridization patterns. The large difference between B. ovis and B. abortus in the number of copies of the repeated DNA is interesting in the context of the closeness of the Brucella species.     

Characterization of Dutch porcine Serpulina (Treponema) isolates by restriction endonuclease analysis and DNA hybridization.

Genomes of 55 Dutch porcine Serpulina (Treponema) hyodysenteriae and non-pathogenic Serpulina isolates were characterized by restriction endonuclease analysis (REA) and DNA hybridization. The Dutch porcine isolates were compared with American Type Culture Collection (ATCC) strains of S. hyodysenteriae and S. innocens and isolates of S. hyodysenteriae with known serotypes (reference strains). REA of the Dutch S. hyodysenteriae isolates resulted in two main patterns, while the non-pathogenic isolates had many distinct REA patterns, all different from the S. hyodysenteriae strains. The S. hyodysenteriae reference strains all had distinct REA patterns, different from the Dutch strains. Upon Southern hybridization with a S. hyodysenteriae DNA fragment encoding a flagellar protein, all S. hyodysenteriae strains could be divided in two groups. The non-pathogenic Serpulina strains had many distinct hybridization patterns and hybridized less intensely. Upon hybridization with a S. hyodysenteriae DNA fragment encoding a haemolysin, DNA of all S. hyodysenteriae strains reacted in the same band. DNA of non-pathogenic Dutch Serpulina strains and S. innocens did not hybridize. It was concluded that there are two main genotypes of S. hyodysenteriae in the Netherlands. This could be of importance for recombinant DNA vaccine development.     

Common physical map of four Brucella bacteriophage genomes

Abstract DNAs isolated from four strains of Brucella bacteriophages were studied by restriction endonuclease mapping and Southern blot analysis. In all strains the genome was composed of a 38 kb (25.1 × 10⁶ dalton) double-stranded circular DNA. The physical map was the same for the four genomes and Southern blot hybridization of restriction endonuclease fragments with the Tbilissi strain DNA as a probe showed complete homology between the four DNAs. Thus, the four phage strains appear to be identical, the specific host range of each originating from minor changes in phage or Brucella receptors or both.     

Characterization of Lactobacillus sake isolates from dry-cured sausages by restriction fragment length polymorphism analysis of the 16S rRNA gene

Lactobacillus sake strains originally isolated from dry-fermented sausages were characterized by phenotypic and genotypic methods, including DNA-DNA hybridization, restriction fragment length polymorphism (RFLP), and 16S rDNA sequencing analysis, in order to establish their taxonomic position and relation to well defined reference species. Initially, isolates of Lact. sake showing a characteristic phenotype (melibiose-positive, maltose- and arabinose-negative) were identified by DNA-DNA hybridization. Subsequently, RFLP studies using Eco RI and Hin dIII as restriction enzymes, and cDNA from Escherichia coli or 16S rDNA from Lact. sake strains as probes, showed distinct polymorphism levels. Thus, Eco RI-digested DNA probed with cDNA from E. coli disclosed the presence of a unique cluster for the meat isolates tested, allowing their differentiation from the reference type strain. When Hin dIII-digested DNA was hybridized with the cDNA probe, strain-specific patterns were obtained, showing a higher discrimination power. Considerable strain differentiation was also observed when Eco RI and Hin dIII digests were hybridized with 16S rDNA probes. Finally, sequence analysis of the 16S rDNA from one isolate also revealed a certain degree of genetic variability with respect to the reference strain of Lact. sake .     

Conservation of the 2,4-diacetylphloroglucinol biosynthesis locus among fluorescent Pseudomonas strains from diverse geographic locations.

The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (PHL) is a major determinant in the biological control of a range of plant pathogens by many fluorescent Pseudomonas spp. A 4.8-kb chromosomal DNA region from Pseudomonas fluorescens Q2-87, carrying PHL biosynthetic genes, was used as a probe to determine if the PHL biosynthetic locus is conserved within PHL-producing Pseudomonas strains of worldwide origin. The phl gene probe hybridized with the genomic DNA of all 45 PHL-producing Pseudomonas strains tested, including well-characterized biocontrol strains from the United States and Europe and strains isolated from disease-suppressive soils from Switzerland, Washington, Italy, and Ghana. The PHL producers displayed considerable phenotypic and genotypic diversity. Two phenotypically distinct groups were detected. The first produced PHL, pyoluteorin, and hydrogen cyanide and consisted of 13 strains from almost all locations sampled in the United States, Europe, and Africa. The second produced only PHL and HCN and consisted of 32 strains from the U.S. and European soils. Analysis of restriction patterns of genomic DNA obtained after hybridization with the phl gene probe and cluster analysis of restriction patterns of amplified DNA coding for 16S rRNA (ARDRA) and randomly amplified polymorphic DNA (RAPD) markers indicated that the strains that produced both PHL and pyoluteorin were genetically highly similar. In contrast, there was more diversity at the genotypic level in the strains that produced PHL but not pyoluteorin. ARDRA analysis of these strains indicated two clusters which, on the basis of RAPD analysis, split into several subgroups with additional polymorphisms. In general, the occurrence of phenotypically and genotypically similar groups of PHL producers did not correlate with the geographic origin of the isolates, and highly similar strains could be isolated from diverse locations worldwide.     

Homology of variable major protein genes between Borrelia hermsii and Borrelia miyamotoi

Abstract Antigenic variation has been studied in detail for the etiological agent of relapsing fever, Borrelia hermsii . The variable major proteins (vmps) are found at its cell surface, enabling it to avoid the host's immune response. We have cloned and sequenced the vmp -gene ( vmp )-like sequences from the Borrelia miyamotoi strains HT31 and FR64b and the deduced amino acid sequences were compared with the published vmp proteins vmp3, vmp24, and vmp33 of B. hermsii . The sequences were aligned and revealed pairwise sequence identities ranging from 45 to 51%, and differences were scattered throughout the sequences. Southern hybridization using the cloned vmp -like sequence of strain HT31 as a probe suggested that the vmp homologues reside on the linear plasmids of B. miyamotoi . The probe hybridized weakly with B. hermsii linear plasmids and restriction digests. These results suggest that B. miyamotoi has sequences resembling the vmp genes in B. hermsii .     

Estimation of ribosomal RNA operon (rrn) copy number in Acinetobacter isolates and potential of patterns of rrn operon-containing fragments for typing strains of members of this genus

The copy number of the rrn operon in 70 strains of Acinetobacter including the type strains of almost all the genomic species with validated names was estimated after digestion of their genomic DNA by the restriction enzymes BglII and PstI, and Southern blotting. Copy number estimates varied between and among species, with between 3 and 7 rrn operon copies detected. Copy number estimates obtained from the same strain with the two enzymes sometimes varied. BglII generated RFLP patterns of the rrn containing fragments obtained from Southern blots after agarose gel electrophoresis were examined for their value in identifying Acinetobacter isolates. This method was very reproducible with the same fragment pattern always generated from the same isolate on repeated analysis. Often multiple strains of the same genomic species gave identical or very similar patterns (e.g. Acinetobacter baylyi), clustering closest together on the dendrogram generated after numerical analysis of these patterns. However, with some, like BG5 and BG8, the patterns derived from the different strains, some of which had been placed in this genomic species from DNA:DNA hybridization data, varied considerably to each other and to the type strain. Little similarity was seen when relationships between these strains based on these patterns were compared to those using DNA:DNA hybridization data. Often these patterns could be used to question earlier identification of strains using phenotypic characters. Thus, strain AB82 thought to belong to genomic species 5 gave an identical pattern to A. bouvetii(T) (DSM 14964). In some cases this pattern analysis suggested that novel species of Acinetobacter might exist among the strains examined.     

Comparison of the spirochete Borrelia burgdorferi S. L. isolated from the tick Ixodes scapularis in southeastern and northeastern United States

Thirty-five strains of the Lyme disease spirochete Borrelia burgdorferi sensu lato (B. burgdorferi s. l.) were isolated from the blacklegged tick vector Ixodes scapularis in South Carolina, Georgia, Florida, and Rhode Island. They were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. PCR-RFLP analysis indicated that the strains represented at least 3 genospecies (including a possible novel genospecies) and 4 different restriction patterns. Thirty strains belonged to the genospecies B. burgdorferi sensu stricto (B. burgdorferi s. s.), 4 southern strains were identified as B. bissettii, and strain SCCH-5 from South Carolina exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Complete sequences of rrf-rrl intergenic spacers from 14 southeastern and northeastern strains were determined and the phylogenetic relationships of these strains were compared. The 14 strains clustered into 3 separate lineages on the basis of sequence analysis. These results were confirmed by phylogenetic analysis based on 16S rDNA sequence analysis.