The use of RAPD genomic fingerprinting to study relatedness in strains of Acidithiobacillus ferrooxidans

Twelve strains of Acidithiobacillus ferrooxidans were recovered from acid mine drainage (AMD) sites from three different geographical locations: Copper Cliff, Ontario, Canada; Mineral City, OH, USA; and Cornwall, England. The spread-plate technique and various culture media were used to isolate and purify all strains. DNA was extracted from each purified culture and amplified using PCR and twenty, 10-mer primers. Amplification products were separated by gel electrophoresis and photographed under UV light. The RAPD (Randomly Amplified Polymorphic DNA) profiles were compared on the basis of the presence or absence of each DNA band and a data matrix was constructed. Strain diversity was calculated using the Jaccard's coefficient and UPGMA (Unweighted Pair-Group Arithmetic Average Clustering) cluster analysis. The variations in the banding patterns indicated genomic variability among the twelve A. ferrooxidans strains tested. The primers used in this study grouped the twelve strains into five major groups. Similarities between the strains ranged from 5.49% to 85.14%. These results show that the strains have a high degree of genomic diversity and that the RAPD procedure is a powerful technique to assess strain variability in this bacterium.     

Characterization of Colletotrichum gloeosporioides isolates from avocado and almond fruits with molecular and pathogenicity tests.

One hundred twenty isolates of Colletotrichum gloeosporioides from avocado (6 U.S. and 57 Israeli isolates) and almond (57 Israeli isolates) fruits were compared by various molecular methods and a pathogenicity assay in order to determine the genetic diversity and host specificity between and among the different populations. DNA from eight additional U.S. almond anthracnose isolates were also compared. PCR amplification of genomic DNA with four primers produced uniform banding patterns for all the Israeli almond isolates from different geographic locations in Israel. DNAs from the U.S. almond isolates were distinct from DNAs of the Israeli isolates. In contrast, the avocado isolates from Israel and the United States were more diverse, with numerous arbitrarily primed-PCR phenotypes being observed. HaeIII digestion patterns of A+T-rich DNA distinguished between the almond and avocado isolates. Southern hybridization of the repetitive nuclear-DNA element GcpR1 to PstI-digested genomic DNA of almond and avocado isolates revealed no polymorphic fragments among the almond isolates, whereas polymorphic fragments were observed among the avocado isolates. Amplification and subsequent restriction enzyme digestion of the internal transcribed spacer 4 and 5 regions between the small and large nuclear subunits of DNA encoding rRNA failed to distinguish between C. gloeosporioides isolates from a diverse host range. In artificial inoculations, avocado isolates produced various lesions on avocado and almond fruits, whereas the almond isolates infected both fruits at a lower rate.     

Characterization of Pseudoalteromonas citrea and P. nigrifaciens isolated from different ecological habitats based on REP-PCR genomic fingerprints

DNA primers corresponding to conserved repetitive interspersed genomic motifs and PCR were used to show that REP, ERIC and BOX-like DNA sequences are present in marine, oxidative, gram-negative Pseudoalteromonas strains. REP, ERIC and BOX-PCR were used for rapid molecular characterization of both the type species of the genus and environmental strains isolated from samples collected in different geographical areas. PCR-generated genomic fingerprint patterns were found to be both complex and strain specific. Analysis of the genotypic structure of phenotypically diverse P. citrea revealed a geographic clustering of Far Eastern brown-pigmented, agar-digesting strains of this species. Marine isolates of P. nigrifaciens with 67-70% DNA relatedness generated genomic patterns different from those of the type strain and formed a separate cluster. It is concluded that REP, ERIC and BOX-PCR are effective in generating strain specific patterns that can be used to elucidate geographic distribution, with these genomic patterns providing a valuable biogeographic criterion.     

用识别四个GC对的限制酶研究高等担子菌线粒体DNA

本文报道了用识别四个GC对的限制性内切酶HaeⅢ、CofI、MsPI酶切高等担子菌伞菌目（Agaricales）、非褶菌目（Aphyllophorales）12个菌株的总DNA,在琼脂糖凝胶上显现线粒体DNA带谱的结果。通过对带型、累加分子量、酶切片段数和同源性的分析表明,不同种类高等担子菌的线粒体DNA变化较大．研究还显示,以多种识别四个GC对的限制酶分析高等担子菌的线粒体DNA及其它们的RFLP,比只用HaeⅢ更灵敏有效。     

Different strategies for molecular differentiation of Mycobacterium bovis strains isolated in Sardinia, Italy

Different genetic markers were used to analyze 22 Mycobacterium bovis strains isolated from cattle in Sardinia and one human isolate. IS6110 DNA fingerprinting differentiated the strains into six patterns, whereas with enterobacterial repetitive consensus sequence primers produced seven clusters. PCR ribotyping followed by digestion with HaeIII and PvuII produced five and seven patterns, respectively. PCR with the (GTG)5 oligonucleotide primer showed the best discriminatory power, generating eight clusters among the strains analyzed.     

Linkage in human heterochromatin between highly divergent Sau3A repeats and a new family of repeated DNA sequences (HaeIII family)

The hybridization of human DNA with three non-cross-hybridizing monomers (68 bp in length) of the heterochromatic Sau3A family of DNA repeats, indicates the coexistence within a Sau3A-positive genomic block of divergent Sau3A units as well as of unrelated sequences. To gain some insight into the structure of these human heterochromatic DNA regions, three previously cloned Sau3A-positive genomic fragments (with a total length of approximately 1900 base-pairs (bp] were sequenced. The analysis of the sequences showed the presence of clustered Sau3A units with different degrees of divergence and of two DNA regions of approximately 100 bp and 291 bp in length, unrelated to the family of repeats. A consensus sequence derived from the 24 identified Sau3A monomers presents, among highly variable regions, two less variant regions of 8 bp and 10 bp in length, respectively. The Sau3A-unrelated DNA fragment 291 bp in length, used as a probe on genomic DNA digested with a series of restriction enzymes, defines a "new" family of DNA repeats possessing periodicities for HaeIII (HaeIII family). Sau3A and HaeIII repeats display a high degree of linkage in a collection of Sau3A-positive genomic recombinant phages.     

Biotype and macromolecular profiles of cytotoxin-producing strains of Helicobacter pylori from antral gastric mucosa

Biotype, genome, protein and plasmid profile diversity amongst 40 epidemiologically unrelated strains of Helicobacter pylori was studied. Strains were API Zym biotypes II, III and IV but most (87%) were biotype II. Four subsets of strains were defined on a combination of motility (56% positive) and cytotoxin production (44% positive). A close association (P = 0.45) between these two features was observed for 69% of strains. Each strain of H. pylori had a unique DNA type defined by either HaeIII or HindIII total digest patterns and by ribopatterns, except for DNA of the rare strains not cut by these endonucleases. Strain diversity was confirmed by one-dimensional SDS-PAGE electrophoretic protein patterns. No consistent associations between cytotoxin activity and overall ribopattern or band subsets within a ribopattern were detected. Some strains (39%) contained a plasmid but the presence of plasmids was not consistently associated with either cytotoxin activity, biotype, motility or ribopattern. We conclude that the cytotoxin-producing strains of H. pylori were genomically as diverse as the non-cytotoxin producing strains.     

Differentiation of ruminal and human Streptococcus bovis strains by DNA homology and 16s rRNA probes

Streptococcus bovis is commonly present in the rumen, but strains of S. bovis have also occasionally been isolated from human blood or fecal samples. Studies were undertaken with 16s rRNA gene sequences and DNA hybridizations to define the genetic relationships between these two groups of strains. Ruminal strains were found to yield genomic DNA restriction endonuclease digest patterns different from human strains when either the 16s rRNA gene amplified from ruminal S. bovis strain JB1 or a conserved universal 23s rRNA fragment was used as probes. A DNA probe based on the V1 region of the 16s rRNA of S. bovis JB1 was found to hybridize to DNAs of other ruminal S. bovis strains K27FF4, 21-09-6C, five new ruminal isolates, and weak hybridization was found with DNAs from S. bovis 33317 (type strain), S. equinus 9812, and six other ruminal isolates. No hybridization occurred with strains representing different major human biotypes/homology groups (43143, 43144, 27960, V1387). All ruminal S. bovis strains had a guanosine plus cytosine DNA content of 37.4–38.8 mol% and, based on DNA-DNA genomic hybridizations, could be separated into two homology groups, one of which included S. equinus 9812 and S. bovis 33317. Both ruminal groups had less than 38% DNA homology to the human strains, indicating ruminal strains are clearly two separate species distinct from the human strains.     

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.     

Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp

We present a simple, efficient extension of denaturing gradient gel electrophoresis that allows the detection of nearly any sequence change in a defined fragment of DNA. The fragment can be obtained either by means of the polymerase chain reaction or by restriction digestion of genomic DNA. With restriction fragments of genomic DNA, sequence information is not required, and covalent modifications in genomic DNA that are lost in a PCR, such as methylation, are detectable. We describe how a GC clamp (an arbitrary, G+C-rich sequence of 30 to 60 bp) can be attached to a selected restriction fragment present in a digest of genomic DNA. The GC clamp alters the melting properties of the fragment; this change greatly increases the fraction of possible mutations that is detectable. In a 272-bp HaeIII fragment from the human beta-globin gene, we were able to detect 13 of 13 mutations tested in human genomic DNA. Four additional mutations in cloned plasmids were analyzed. The data agree with a simple theoretical model for DGGE, which predicts how two fragments, differing at a single (specified) base pair, are resolved in a gradient gel as a function of running time for the gel. The calculation assists in the design of probes and gel conditions that aid in the detection of sequence changes.     

Genetic and phenotypic characterization of a Pseudomonas aeruginosa population with high frequency of genomic islands

Various genomic islands, PAPI-1, PAPI-2, PAGI-1, PAGI-2, PAGI-3, and PAGI-4, and the element pKLC102 have been characterized in different P. aeruginosa strains from diverse habitats and geographical locations. Chromosomal DNA macroarray of 100 P. aeruginosa strains isolated from 85 unrelated patients hospitalized in an intensive care unit was created to assess the occurrence of these genomic islands (GEIs). The macroarray was then hybridized with labeled probes derived from each genomic island. In addition, PFGE patterns with SpeI, frequency of virulence genes, and antimicrobial resistance patterns of the strains were studied. Our results showed that almost all P. aeruginosa strains presented up to eight virulence genes. By SpeI macrorestriction fragment analysis we were able to identify 49 restriction patterns; 35 patterns correspond to single strains and the remaining 14 to strains subgroup (a-n). Most of the strains showed variation in number or composition of GEIs and a specific antimicrobial pattern indicating that each strain was an unrelated isolate. In terms of the number of genomic islands per strain, 7 GEIs were found in 34% of the strains, 6 in 18%, 5 in 12%, 4 in 14%, 3 in 10%, 2 in 7%, and 1 in 4%; only one isolate did not present any GEI. The genomic islands PAPI-1 and PAPI-2 and the element pKLC102 were the most frequently detected. The analysis of the location of each GEI in the chromosome of two strains show that the islands PAGI-3, PAPI-1, PAPI-2 and pKLC102 are present in the insertion site previously reported, but that PAGI-2 and PAGI-4 are inserted in another chromosome place in a site not characterized yet. In conclusion our data show that P. aeruginosa strains exhibited an epidemic population structure with horizontal transfer of DNA resulting in a high frequency of GEIs.     

Ribosomal RNA gene restriction fragment diversity amongst Lior biotypes and Penner serotypes of Campylobacter jejuni and Campylobacter coli

Diversity based on ribosomal RNA gene-restriction endonuclease digest patterns was detected amongst 42 strains of Campylobacter jejuni and 18 strains of C. coli including representatives of 53 different Penner serotypes. HaeIII ribopatterns were coded for numerical analysis which showed that all except two were different including those of several strains of the same serotype (P2 and P20). At the 30% similarity level, four groupings were formed in the analysis of which three corresponded to C. jejuni, C. coli and C. lari phenotypes respectively. Eight strains (13%) were atypical as their phenotypic and ribopattern associations did not correspond. Ribopattern fragments of 3.0, 5.0 and 9.3 kb were characteristic of the majority of C. jejuni, whereas 1.5, 2.2-, 2.3- and 4.7-kb fragments were commonly present in C. coli. These fragments provided novel species-specific markers. We conclude that HaeIII ribotyping was as discriminatory as Penner serotyping of C. jejuni and C. coli and may even provide a basis for distinguishing between strains of the same serotype and for identifying new groups within the thermophilic campylobacters.     

DNA-DNA hybridization and analysis of restriction endonuclease and rRNA gene patterns of atypical (catalase-weak/negative) Campylobacter jejuni from paediatric blood and faecal cultures.

Sixteen strains of atypical (catalase-weak or negative), hippurate-hydrolysing campylobacters from paediatric blood and faecal cultures were identified by DNA-DNA slot hybridization. All were closely related (greater than or equal to 67%) to Campylobacter jejuni and representative strains had G + C contents of 30 +/- 1 mol%. Numerical analysis of chromosomal DNA HaeIII digest patterns revealed two clusters of strains at the 55%S level corresponding to C. jejuni subsp. jejuni and C. jejuni subsp. doylei; most strains belonged to the latter subspecies. No two strains had identical patterns but within each subspecies two subgroups were identifiable, corresponding to Lior biotypes I and II. Southern blot hybridization analysis with a 16 + 23S rRNA cistron probe from Escherichia coli also showed differences between the various strains, and in a numerical analysis three groupings were formed at 70%S corresponding to C. jejuni subsp. jejuni Lior biotypes I and II, and C. jejuni subsp. doylei. Four of the subspecies doylei strains contained a 3.4-MDa plasmid. These analyses showed that catalase-negative C. jejuni subsp. jejuni were genomically distinguishable from C. jejuni subsp. doylei as were Lior biotypes within subsp. jejuni. Ability to produce catalase is not a feature common to all C. jejuni strains, and our results confirm that some strains of subspecies jejuni may be negative in that character although typical in other respects. DNA pattern heterogeneity was consistent with serological differences between strains.     

Identification of 'Campylobacter upsaliensis' and other catalase-negative campylobacters from paediatric blood cultures by numerical analysis of electrophoretic protein patterns

Twenty-one strains of catalase-negative campylobacters from paediatric blood cultures were characterized by one-dimensional SDS-PAGE of cellular proteins. A further 11 Campylobacter strains were included for reference purposes. The partial protein patterns were used as the basis of a numerical analysis, which showed that 17 hippurate-negative strains had a high similarity to 'C. upsaliensis' (r greater than or equal to 0.82) irrespective of their geographical location, and that three hippurate-positive isolates had a high similarity (r greater than or equal to 0.87) to C. jejuni subsp. jejuni. One hippurate-positive CNW strain was not identified. The analysis of SDS-PAGE protein patterns proved an excellent method of characterizing these thermophilic campylobacter as they were difficult to identify by traditional methods.     

BliI, a restriction endonuclease from Bacillus licheniformis

From Bacillus licheniformis a site-specific restriction endonuclease, named BliI, has been purified and characterized. BliI was able to digest lambda DNA at pH 9.1 over a wide temperature range (25-65 degrees C). Digestion of lambda and psi X174 DNAs with BliI produced banding patterns identical to those seen with HaeIII. Therefore, BliI and HaeIII endonculeases are isoschizomers.     

Overlapping deletion in two spontaneous phase variants of Coxiella burnetii

Chromosomal DNA from the Nine Mile phase I strain of Coxiella burnetii (CB9MIC7) was cloned into the cosmid vector pHC79. The resulting gene library was probed with a radiolabelled HaeIII fragment present in the parental strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). The insert, which includes the missing HaeIII fragment, was 38.5 kb in length. When DNA from this cosmid clone was hybridized to genomic DNA of the parental CB9MIC7 and its derivative CB9MIIC4, a number of fragments were missing or altered in the latter strain. Restriction mapping localized the fragments to a contiguous portion of the chromosomal DNA fragment. The data were consistent with an 18 kb deletion in the chromosome of CB9MIIC4. Another intrastrain spontaneous derivative, CB9MI514, also lacked the sentinel HaeIII fragment and carried a deletion of approximately 29 kb within the same cloned insert. Both deletions appeared to share a common terminus, within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed intact. The strains examined were representative of various stages of phase variation. The relationship between the observed deletions and the mechanism of phase transition in Nine Mile strains is discussed.     

Stability of related human and chicken Campylobacter jejuni genotypes after passage through chick intestine studied by pulsed-field gel electrophoresis

The genomic stability of 12 Campylobacter jejuni strains consisting of two groups of human and chicken isolates was studied by analysis of their PFGE (pulsed-field gel electrophoresis) patterns after passage through newly hatched chicks' intestines. The patterns of SmaI, SalI, and SacII digests remained stable after intestinal passage, except for those of two strains. One originally human strain, FB 6371, changed its genotype from II/A (SmaI/SacII) to I/B. Another strain, BTI, originally isolated from a chicken, changed its genotype from I/B to a new genotype. The genomic instability of the strains was further confirmed by SalI digestion and ribotyping of the HaeIII digests. In addition, heat-stable serotype 57 of strain FB 6371 changed to serotype 27 in all isolates with new genotypes but remained unchanged in an isolate with the original genotype. Serotype 27 of strain BTI remained stable. Our study suggests that during intestinal colonization, genomic rearrangement, as demonstrated by changed PFGE and ribopatterns, may occur.     

Genetic and biological diversity among populations of Leishmania major from Central Asia, the Middle East and Africa

Evidence is provided for genetic and biological variation among Leishmania major strains that correlates with their geographical origin. The host-parasite relationship also appears to be specific. Great gerbils, Rhombomys opimus, and fat sand rats, Psammomys obesus, are the main reservoir hosts in Central Asia and the Middle East, respectively. However, the Central Asian parasite failed to infect the Middle Eastern rodent host in the laboratory, and vice versa. A permissively primed intergenic polymorphic (PPIP)-PCR and a single-stranded conformation polymorphism (SSCP)-PCR exposed genetic polymorphism among 30 strains of L. major from different geographical regions. This was verified by subsequent sequencing of DNA from the same strains using four genomic targets: (a) the NADH-dehydrogenase (NADH-DH) gene, (b) the 6-phosphogluconate dehydrogenase (6PGD) gene, (c) the ribosomal internal transcribed spacers, and (d) an anonymous DNA sequence originally amplified with random primers. All the genetic markers indicated that the nine Central Asian strains were a separate homogenous genetic group. The Middle Eastern strains formed another geographical group that displayed heterogeneity corresponding with their different Middle Eastern locations. Molecular markers and host-parasite relationships confirmed that Central Asian and Middle Eastern strains are genetically and biologically distinct sub-populations of L. major. Three African strains of L. major were genetically closer to the Middle Eastern strains, and a representative one did infect fat sand rats, but they had distinct permissively primed inter-genic polymorphic PCR patterns and internal transcribed spacer 2 types.     

Mobile dispersed repeated DNA elements in the Drosophila genome

The molecular and cytogenetic organizations of 19 nonhomologous dispersed repeated sequence families were studied in 15 different laboratory strains of Drosophila melanogaster. Elements from each of the families appear to undergo transposition within the Drosophila genome, because there were striking differences in both the number and chromosomal locations of these elements between strains. A significant fraction (greater than 1%) of Drosophila DNA therefore has an unstable genomic organization. Each middle repetitive family exhibited similar variations in the chromosomal distribution of elements between the strains. Although the movements of these elements are not limited to a small number of genomic sites, there are chromosomal regions where elements from the different dispersed repeated DNA families appear to be clustered. The locations of such preferred integration sites are different in each of the D. melanogaster strains examined.     

Analysis of the clonal relationships between strains of Neisseria meningitidis by pulsed field gel electrophoresis.

Fingerprint patterns were generated from strains of Neisseria meningitidis by digestion of chromosomal DNA samples with 'rare-site' restriction endonucleases and resolution of the resultant fragments by pulsed field gel electrophoresis (PFGE). The potential of this technique for the rapid establishment of the clonal relationships between different isolates of the meningococcus was investigated. The fingerprint patterns from various serogroup A strains, previously assigned to clonal subgroups on the basis of their electrophoretic types (ETs), were compared. Fingerprints generated with the endonucleases SfiI, SpeI and NheI each gave distinctive patterns for the clonal subgroups I-IV of serogroup A. Further, the endonucleases SpeI and, particularly, NheI were capable of resolving differences between various subgroup III strains isolated at different times and geographical locations. Strains isolated during the 'new wave' pandemic, which was associated with the Haj, from Europe, America, and Africa, had a characteristic fingerprint pattern and appeared to be distinct from 'old wave' pandemic strains. The PFGE technique is a relatively rapid and sensitive method for establishing clonal relationships among epidemic strains of N. meningitidis.