Showing papers on "Ribosomal DNA published in 1995"

Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments

Abstract: Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genus Thiomicrospira. To identify these 'phylotypes' in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three described Thiomicrospira species, viz., Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate, Thiomicrospira sp. strain MA2-6. All Thiomicrospira species except Tms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria. Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated with Thiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivision Thiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence of Tms. crunogena. Identifying a dominant molecular isolate as Tms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another 'phylotype' represented a new Thiomicrospira species, phylogenetically in an intermediate position between Tms. crunogena and Tms. pelophila. The third 'phylotype' was identified as a Desulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.

Bidirectional interlocus concerted evolution following allopolyploid speciation in cotton (Gossypium)

Abstract: Polyploidy is a prominent process in plant evolution; yet few data address the question of whether homeologous sequences evolve independently subsequent to polyploidization. We report on ribosomal DNA (rDNA) evolution in five allopolyploid (AD genome) species of cotton (Gossypium) and species representing their diploid progenitors (A genome, D genome). Sequence data from the internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S gene indicate that rDNA arrays are homogeneous, or nearly so, in all diploids and allopolyploids examined. Because these arrays occur at four chromosomal loci in allopolyploid cotton, two in each subgenome, repeats from different arrays must have become homogenized by interlocus concerted evolution. Southern hybridization analysis combined with copy-number estimation demonstrate that this process has gone to completion in the diploids and to completion or near-completion in all allopolyploid species and that it most likely involves the entire rDNA repeat. Phylogenetic analysis demonstrates that interlocus concerted evolution has been bidirectional in allopolyploid species--i.e., rDNA from four polyploid lineages has been homogenized to a D genome repeat type, whereas sequences from Gossypium mustelinum have concerted to an A genome repeat type. Although little is known regarding the functional significance of interlocus concerted evolution of homeologous sequences, this study demonstrates that the process occurs for tandemly repeated sequences in diploid and polyploid plants. That interlocus concerted evolution can occur bidirectionally subsequent to hybidization and polyploidization has significant implications for phylogeny reconstruction, especially when based on rDNA sequences.

Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species

Abstract: In order to assess the effect of genome size and number of 16S rRNA genes (rDNAs) on the quantities of PCR-generated partial 16S rDNA fragments, equimolar amounts of DNA from pairs of different species for which these parameters are known were subjected to gene amplification. The experimentally determined ratio of PCR products obtained, as determined by image analysis of SYBR-Green I-stained amplification products, corresponded well with the predicted ratio calculated from the number of rrn genes per equimolar amounts of DNA in mixtures of Escherichia coli and "Thermus thermophilus" and of Pseudomonas aeruginosa and "T. thermophilus." The values for the pair of Bacillus subtilis and "T. thermophilus" showed greater deviations from the predicted value. The dependence of the amount of 16S rDNA amplification product on these two parameters makes it impossible to quantify the number of species represented in 16S rDNA clone libraries of environmental samples as long as these two parameters are unknown for the species present.

A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences

Abstract: Partial sequences of the 16S rRNA genes of the type strains of nine species of the genus Nocardia were determined following the isolation and cloning of the amplified genes. These sequences were aligned with the sequences of representatives of the genera Corynebacterium, Gordona, Mycobacterium, Rhodococcus, and Tsukamurella, and phylogenetic trees were inferred by using the Fitch-Margoliash and neighbor-joining methods. The genus Nocardia formed a distinct clade that was most closely associated with the genus Rhodococcus. The average level of sequence similarity found among the type strains of the Nocardia species was 97.2 ± 0.7%. Two sublines were recognized within the Nocardia clade; one encompassed Nocardia asteroides and related species, and the other encompassed Nocardia otitidiscaviarum and allied taxa. Separation of the two sublines is based on differences in helix 37–1. The results of isoprenoid quinone analyses provided evidence that nocardiae can be distinguished from all other actinomycete taxa on the basis of their characteristic menaquinone profiles. Nocardiae typically contain hexahydrogenated menaquinones with eight isoprene units in which the two end units are cyclized.

Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution

Abstract: The internal transcribed spacers (ITS) of nuclear ribosomal DNA of 33 species of genus Paeonia (Paeoniaceae) were sequenced. In section Paeonia, different patterns of nucleotide additivity were detected in 14 diploid and tetraploid species at sites that are variable in the other 12 species of the section, suggesting that reticulate evolution has occurred. Phylogenetic relationships of species that do not show additivity, and thus ostensibly were not derived through hybridization, were reconstructed by parsimony analysis. The taxa presumably derived through reticulate evolution were then added to the phylogenetic tree according to additivity from putative parents. The study provides an example of successfully using ITS sequences to reconstruct reticulate evolution in plants and further demonstrates that the sequence data could be highly informative and accurate for detecting hybridization. Maintenance of parental sequences in the species of hybrid origin is likely due to slowing of concerted evolution caused by the long generation time of peonies. The partial and uneven homogenization of parental sequences displayed in nine species of putative hybrid origin may have resulted from gradients of gene conversion. The documented hybridizations may have occurred since the Pleistocene glaciations. The species of hybrid origin and their putative parents are now distantly allopatric. Reconstruction of reticulate evolution with sequence data, therefore, provides gene records for distributional histories of some of the parental species.

Cycloclasticus pugetii gen. nov., sp. nov., an Aromatic Hydrocarbon-Degrading Bacterium from Marine Sediments

Abstract: Three heterotrophic bacterial strains were isolated from different locations in Puget Sound, Washington, by using biphenyl as the principal carbon source. These strains grow by using a limited number of organic compounds, including the aromatic hydrocarbons naphthalene, phenanthrene, anthracene, and toluene, as sole carbon sources. These aerobic, gram-negative rods are motile by means of single polar flagella. Their 16S rRNA sequences indicate that they are all members of the γ subdivision of the Proteobacteria. Their closest known relatives are the genera Methylobacter and Methylomonas (genera of methane-oxidizing bacteria), uncultured sulfur-oxidizing symbionts found in marine invertebrates, and clone FL5 containing 16S ribosomal DNA amplified from an environmental source. However, the Puget Sound bacteria do not use methane or methanol as a carbon source and do not oxidize reduced sulfur compounds. Furthermore, a 16S rRNA base similarity comparison revealed that these bacteria are sufficiently different from other bacteria to justify establishment of a new genus. On the basis of the information summarized above, we describe a new genus and species, Cycloclasticus pugetii, for these bacteria; strain PS-1 is the type strain of C. pugetii.

Genetic comparisons reveal the same unknown bacterial lineages in Atlantic and Pacific bacterioplankton communities

Abstract: The phylogenetic diversity of oligotrophic bacterioplankton communities was compared with 16s ribosomal RNA genes cloned from natural populations. The data reported here extend a previous analysis of a bacterioplankton 16s rRNA clone library with 15 additional nucleic acid clone sequences, to provide information on 60 16s rDNA clones from hydrostation S in the Sargasso Sea. The data were compared to partial sequences of 37 Bacterial 16s rDNA clones reported from a surface picoplankton population collected at the Aloha station in the North Pacific gyre, and partial sequences of 29 Bacterial 16s rRNA clones obtained from sites near Bermuda and the western California Current. The results support reports of diverse groups of previously unknown a-proteobacteria, y-proteobacteria, and cyanobacteria in oceanic surface samples. Three novel lineages (SAR 12 1,125, 145) of proteobacteria were found. Several genes cloned from the Sargasso Sea were nearly identical to genes cloned from the Pacific samples, suggesting that these previously unrecognized bacteria groups (SAR 11, SAR 122, SAR86) are distributed widely in the surface waters of subtropical oceans. Two gene clones closely matched nucleotide sequences from the cultivated bacterial species Photobgxterium phosphoreum and Alteromonas haloplanktis. Oceanic biogeochemical processes are mediated by communities of microorganisms, which have often been viewed as “black boxes” in which most of the organisms present are not defined in taxonomic or phylogenetic terms. Uncertainties about the diversity and genetic structure of these natural bacterioplankton populations stem from the limitations of common microbial cultivation techniques, which typically support the growth of only a small subset of cells from the original sample (Ferguson et al. 1984; Jannasch and Jones 1959; Kogure et al. 1979). The use of ribosomal RNAs as markers for species diversity is providing a fresh approach for investigating the structure of microbial communities (Ward et al. 1992). Biases in the filtration and cloning procedures used in these studies are not yet well understood, but they clearly appear to be less limiting than the biases associated with microbial cultivation. Phylogenetic analyses provide information on bacterioplankton population diversity, as well as nucleic acid probes, which can be used to establish the significance of cultured microorganisms and to study the temporal and spatial distributions of microbial taxa in the oceans (DeLong 1992; Giovannoni et al. 1990a). The general utility of nucleic acid probes for the study of

Multiple origins of lichen symbioses in fungi suggested by SSU rDNA phylogeny.

Abstract: Phylogenetic hypotheses provide a context for examining the evolution of heterotrophic lifestyles. The lichen lifestyle, which is the symbiotic association of fungi with algae, is found in various representatives of Dicaryomycotina, both Ascomycetes and Basidiomycetes. A highly resolved parsimony analysis of small subunit ribosomal DNA (SSU rDNA) sequences suggests at least five independent origins of the lichen habit in disparate groups of Ascomycetes and Basidiomycetes. Because lichen associations arose from parasitic, mycorrhizal, or free-living saprobic fungi, neither mutualism nor parasitism should be construed as endpoints in symbiont evolution.

Transition in Specification of Embryonic Metazoan DNA Replication Origins

Abstract: In early Xenopus embryos, in which ribosomal RNA genes (rDNA) are not transcribed, rDNA replication initiates and terminates at 9- to 12-kilobase pair intervals, with no detectable dependence on specific DNA sequences. Resumption of ribosomal RNA (rRNA) synthesis at late blastula and early gastrula is accompanied by a specific repression of replication initiation within transcription units; the frequency of initiation within intergenic spacers remains as high as in early blastula. These results demonstrate that for rRNA genes, circumscribed zones of replication initiation emerge in intergenic DNA during the time in metazoan development when the chromatin is remodeled to allow gene transcription.

Identification of ribosomal DNA polymorphisms among and within spores of the Glomales: application to studies on the genetic diversity of arbuscular mycorrhizal fungal communities

Abstract: summary Little information currently exists on species diversity in communities of arbuscular mycorrhizal fungi (AMF), mainly owing to difficulties in identification of field extracted spores on the basis of morphology. The possibility was explored to identify individual AMF spores from the field on the basis of a molecular marker, namely the nuclear ribosomal DNA encoding the highly conserved 5.8S rRNA with the two flanking internal transcribed spacers (ITS region), known to vary between species. A technique involving polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR–RFLP) was developed to amplify and characterize the ITS region from single AMF spores. PCR reactions with extracts from single spores of three AMF species, raised under glasshouse conditions, yielded reproducibly a single amplification product of the ITS region in sufficient amounts to allow cleavage with several restriction enzymes. The size of the ITS region, c. 600 base pairs, varied only slightly between species. Digestion of the PCR products with the restriction enzymes Hinfl and Taq I resulted in banding patterns that were reproducible for different individual spores of a given species, but showed clear differences between the three species tested. The sum of the fragment sizes was sometimes greater than the size of the original PCR product, e.g. in Glomus mosseae. Clones of the amplification product from a single spore of this fungus were obtained and sequenced. This yielded two closely related but different sequences, indicating that two different ITS regions co-existed in the spore. The RLFP pattern of the amplification product of the spore was a result of an amalgamation of these two sequences. The technique was applied to AMF spores collected from a species-rich grassland. Spores were sorted into morphological groups on the basis of their colour, size, and shape, and then subjected to PCR–RFLP analysis. In some morphological groups, a large percentage of spores failed to yield an amplification product, probably because they had lost their contents. A group of Glomus spores yielding amplification products in the majority of cases was further investigated: PCR RFLP analysis on 10 individual spores from the field produced 10 different patterns. Similar results were obtained with other groups of spores. The results suggest that the diversity in natural AMF communities and the genetic diversity within individual spores might he much greater than previously thought.

Nuclear rDNA ITS sequence variation in the trematode genus Echinostoma : an aid to establishing relationships within the 37-collar-spine group

Abstract: The taxonomic history of members of the 37-collar-spine group within the genus Echinostoma has been very confused. We obtained DNA sequence data from the nuclear rDNA ITS1, 5.8S and ITS2 of 7 nominal species belonging to this group, Echinostoma trivolvis (Cert, 1914), E. revolutum (Frolich, 1802), E. caproni Richard, 1964, E. acaproni arasasingam et nl. 1952, E. paraelzsei Lie & Basch, 1967, two African isolates, E. sp.I and E. sp.II, and of one 28-collar-spined echinostome, E. holtense (,4sada, 1926). Five of the eight species n;ere clearly distinguishable using ITS data. Sequences from the remaining three tasa, E. cnpl olzi, E. sp.ll and E. liel mere identical to one another and the group containing these laxa was distant from other 3'7-collar-spine species on a phylogenetic tree. E. tvizrolzris and E. pavaensei form a second, but less distinct group within the 35-collar-spine group. The resolution obtained using DNA sequencing will assist in the current reclassification of the group. It also provides a model for future work on sibling species.

Bacterial community fingerprinting of amplified 16S and 16–23S ribosomal DNA gene sequences and restriction endonuclease analysis(ARDRA)

Abstract: The 16S and 23S rRNA genes have been utilized for phylogenetic analysis of both prokaryotic and eukaryotic organisms (see Section 3). In addition to direct comparison of the nucleic acid sequences [9], numerous groups have used the rapid method of polymerase chain reaction (PCR) amplification of this gene [6] as well as the complete rRNA locus [3, 4, 8] for a simple method for identification of bacterial genera and species. In these latter procedures, the amplified ribosomal gene (rONA) is subjected to restriction endonuclease digestion; this has been termed ARDRA (Amplified Ribosomal DNA Restriction Analysis [8]). The resulting restriction fragment pattern is then used as a fingerprint for the identification of bacterial genomes. This method is based on the principle that the restriction sites on the RNA operon are conserved according to phylogenetic patterns.

Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences

Abstract: Over 250 species have been described in Ganoderma. Species identification and species circumscription are often unclear and taxonomic segregation of the genus remains controversial. In this study w...

Molecular systematics of the Hypocreales: a teleomorph gene phylogeny and the status of their anamorphs

Abstract: Phylogenetic relationships among 40 species in the Hypocreales and Clavicipitales were inferred from sequence data obtained from the nuclear large-subunit ribosomal DNA. Cladistic analysis of these...

Identification and characterization of three Encephalitozoon cuniculi strains

Abstract: Microsporidia are increasingly recognized as causing opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi is probably the most studied mammalian microsporidian that infects insects and mammals, including man. In this study, 8 E. cuniculi isolates were compared and were found to fall into 3 strains. Strain type I includes the rabbit type isolate, as well as isolates from an additional rabbit, a dwarf rabbit, and a mouse. Strain type II includes 2 murine isolates and strain type III includes 2 isolates obtained from domestic dogs. By SDS-PAGE, the 3 strains differ primarily in the molecular weight range of 54-59 kDa where strain type I displays an apparent broad singlet at 57 kDa, strain type II displays an apparent doublet at 54 and 58 kDa, and strain type III displays an apparent broad band at 59 kDa. Antigenic differences were detected in the molecular weight regions of 54-58 kDa as well as 28-40 kDa by Western blot immunodetection using murine antisera raised against E. cuniculi, Encephalitozoon hellem, and the Encephalitozoon-like Septata intestinalis. Polymerase chain reaction (PCR) products containing only small subunit rDNA sequences from the different E. cuniculi isolates formed homoduplexes whereas PCR products containing intergenic rRNA gene sequences formed heteroduplexes in mobility shift analyses. Fok I digestion of the PCR products containing the intergenic rRNA gene region resulted in unique restriction fragment length polymorphism patterns, and DNA sequencing demonstrated that in the intergenic spacer region, the sequence 5'-GTTT-3' was repeated 3 times in strain type I, twice in strain type II, and 4 times in strain type III. This study indicates that there exist at least 3 E. cuniculi strains which may become important in the epidemiology of human E. cuniculi infections. Furthermore, as additional E. cuniculi isolates are characterized, these strains will be named or reclassified once the criteria for taxonomy and phylogenetic tree construction for microsporidia become better defined.

Phylogenetic relationships of the monogenomic species of the wheat tribe, Triticeae (Poaceae), inferred from nuclear rDNA (internal transcribed spacer) sequences.

Abstract: Phylogenetic relationships of 30 diploid species of Triticeae (Poaceae) representing 19 genomes were estimated from the sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The ITS sequence phylogeny indicated that: (i) each genome group of species is monophyletic, concordant with cytogenetic evidence; (ii) Hordeum (I) and Critesion (H) are basal; (iii) Australopyrum (W) is closely related to Agropyron (P); (iv) Peridictyon (G), Heteranthelium (Q), and Dasypyrum (V) are closely related to Pseudoroegneria (S); (v) most of the annuals, Triticum s.l. (A, B, D), Crithopsis (K), Taeniatherum (T), Eremopyrum (F), Henrardia (O), Secale (R), and two perennials, Thinopyrum (J) and Lophopyrum (E), all of Mediterranean origin, are a monophyletic group. However, phylogenetic trees based on morphology group these Mediteranean species with various perennial lineages of the Arctic-temperate region. The molecular data and biogeography of the tribe suggest that the Mediterranean lineage is derived from the Arctic-temperate lineage and that the two lineages have evolved in parallel. Extensive morphological parallelism apparently obscures the true genealogical history of the tribe when only morphology is considered.

Small Subunit Ribosomal DNA Phylogeny of Various Microsporidia with Emphasis on AIDS Related Forms

Abstract: Phylogenetic analysis of the small subunit ribosomal DNA of a broad range of representative microsporidia including five species from humans (Enterocytozoon bieneusi, Nosema corneum, Septata intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi), reveals that human microsporidia are polyphyletic in origin. Septata intestinalis and E. hellem are very similar to the mammalian parasite E. cuniculi. Based on the results of our phylogenetic analysis, we suggest that S. intestinalis be designated Encephalitozoon intestinalis. Furthermore, analysis of our data indicates that N. corneum is much more closely related to the insect parasite Endoreticulatus schubergi than it is to other Nosema species. This finding is supported by recent studies which have shown a similarity between E. schubergi and N. corneum based on the origin and development of the parasitophorous vacuole. Thus these opportunistic microsporidian parasites can originate from hosts closely or distantly related to humans. Finally, the phylogeny based on small subunit ribosomal DNA sequences is highly inconsistent with traditional classifications based on morphological characters. Many ofthe important morphological characters (diplokaryon, sporophorous vesicle, and meiosis) appear to have multiple origins.

Phologeny of Alternaria fungi known to produce host-specific toxins on the basis of variation in internal transcribed spacers of ribosomal DNA

Abstract: The internal transcribed spacer regions (ITS1 and ITS2) of ribosomal DNA from Alternaria species, including seven fungi known to produce host-specific toxins, were analyzed by polymerase chain reaction-amplification and direct sequencing. Phylogenetic analysis of the sequence data by the Neighbor-joining method showed that the seven toxin-producing fungi belong to a monophyletic group together with A. alternata. In contract, A. dianthi, A. panax, A. dauci, A. bataticola, A. porri, A. sesami and A. solani, species that can be morphologically distinguished from A. alternata, could be clearly separated from A. alternata by phylogenetic analysis of the ITS variation. These results suggest that Alternaria pathogens which produce host-specific toxins are pathogenic variants within a single variable species, A. alternata.

Positions of multiple insertions in SSU rDNA of lichen-forming fungi.

Abstract: Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.

Is Penicillium monophyletic? An evaluation of phylogeny in the family Trichocomaceae from 18S, 5.8S and ITS ribosomal DNA sequence data.

Abstract: Using ribosomal DNA sequences from 17 fungi, we evaluated the monophylly of Penicillium and the phylogeny in the ascomycete family Trichocomaceae (= Eurotiaceae). We determined the 5.8S and interna...

The diversity of Malassezia yeasts confirmed by rRNA sequence and nuclear DNA comparisons

Abstract: One hundred and fourMalassezia strains (52 isolated from humans and 52 from animals) were compared using large subunit (LSU) ribosomal RNA sequence similarity and nuclear DNA complementarity. Eight groups of strains were recognized as genetically distinct species. Each taxon was confirmed by a homogeneous mole % GC and percentages of DNA/DNA reassociations higher than 85%. The non-lipid-dependentMalassezia yeasts were maintained as the unique taxonM. pachydermatis. In contrast, lipid-dependent strains were shown to be distributed among seven species:M. furfur, M. sympodialis andM. species 1–5. These taxa matched remarkably well with morphological and serological differences documented by previous investigators. The LSU rRNA sequences allowed a further intraspecific resolution with most of genomic taxa represented by several closely related sequences:M. pachydermatis counted up to seven sequences,M. furfur four sequences,M. species 1 comprised three sequences andM. species 2 andM. species 5 two sequences. Three species,M. sympodialis, M. species 3 andM. species 4, displayed a unique type of sequence. Thus, the present report demonstrates the usefulness of sequencing for both taxonomic and epidemiological purposes.

Detection of Putative Periodontal Pathogens in Subgingival Specimens by 16S Ribosomal DNA Amplification with the Polymerase Chain Reaction

Abstract: The utility of the 16S ribosomal RNA-based polymerase chain reaction (PCR) for detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola was examined and compared with that of anaerobic culture. Primer pairs consisting of 20-27 nucleotides amplified 404- to 688-bp regions of 16S ribosomal RNA genes of these organisms. This method had a lower detection limit of 50 target cells in a background of 10(7) cells. Its specificity for B. forsythus, P. gingivalis, and T. denticola seemed high. The primers for A. actinomycetemcomitans, C. rectus, and P. intermedia cross-reacted with some closely related species but did not reveal amplification products in tests with more distantly related organisms. The primers for E. corrodens did not seem to cross-react with oral organisms. This PCR technique was sensitive, reproducible, and easy to perform. PCR-based amplification may prove valuable for the detection of some periodontal pathogens in crude subgingival specimens.

Genomic heterogeneity of strains nodulating chickpeas (Cicer arietinum L.) and description of Rhizobium mediterraneum sp. nov.

Abstract: The genetic diversity of chickpea strains was studied by using 30 isolates obtained from nodules on chickpeas growing in uninoculated fields over a wide geographic range. The following taxonomic approaches were used: DNA-DNA relatedness analysis, restriction fragment length polymorphism analysis of the amplified 16S ribosomal DNA (rDNA) intergenic spacer (IGS), and total 16S rRNA sequence analysis. The division of chickpea-infective strains into two major phylogenetic groups (groups A and B) that has been described previously was confirmed by the polymorphism of the 16S IGS rDNA. We identified a total of five genomic species, including the previously described species Rhizobium ciceri. All of the group B strains except one were homogeneous and belonged to a single genomic species corresponding to R. ciceri. Group A was heterogeneous, containing three genomic species and five strains that remained unclassified, and its members had very different PCR restriction fragment length polymorphism profiles. The complete 16S rRNA sequences of strains representing the two major groups. R. ciceri UPM-Ca7T (T = type strain) and genomic species 2 strain UPM-Ca36T, exhibited 19 mismatches. Both of these strains belonged to the Rhizobium loti-Rhizobium huakuii branch; R. ciceri UPM-Ca7T was closely related to R. loti, and strain UPM-Ca36T was clearly separated from R. ciceri and closely related to R. huakuii. Thus, genomic species 2 could be distinguished from R. ciceri by its 16S rRNA sequence, by DNA relatedness data, by the polymorphism of the 16S IGS rDNAs, and by previously described multilocus enzyme electrophoresis results and phenotypic characteristics. Therefore, we propose that strains belonging to genomic species 2 should be classified in a new species, Rhizobium mediterraneum, and that strain UPM-Ca36 should be the type strain.

Discriminatory power of three DNA-based typing techniques for Pseudomonas aeruginosa.

Abstract: We assessed the capacity of three DNA typing techniques to discriminate between 81 geographically, temporally, and epidemiologically unrelated strains of Pseudomonas aeruginosa. The methods, representing powerful tools for hospital molecular epidemiology, included hybridization of restricted chromosomal DNA with toxA and genes coding for rRNA (rDNA) used as probes and macrorestriction analysis of SpeI-digested DNA by pulsed-field gel electrophoresis. The probe typing techniques were able to classify all strains into a limited number of types, and the discriminatory powers were 97.7 and 95.6% for toxA and rDNA typing, respectively. Strains that were indistinguishable on the basis of both toxA and rDNA types defined 12 probe type homology groups. Of these, one contained five strains, three contained three strains each, and eight groups were represented by two strains each. Strains in 10 of the homology groups had the same O serotype. SpeI macrorestriction patterns discriminated between all strains with at least four band differences, which corresponded to a similarity level of 85%. Fifteen pairs of strains were similar at a level of > 75% and differed by only four to seven bands. Of these pairs, 11 belonged to the same probe type homology group, indicating their clonal relatedness. We conclude that macrorestriction analysis of P. aeruginosa with SpeI provides the best means of discrimination between epidemiologically unrelated strains. However, DNA probe typing with either toxA or rDNA reveals information on the strain population structure and evolutionary relationships.

Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA

Abstract: The PCR was used to amplify a targeted region of the ribosomal DNA from 84 Candida isolates. Unique product sizes were obtained for Candida guilliermondii, Candida (Torulopsis) glabrata, and Candida pseudotropicalis. Isolates of Candida albicans, Candida tropicalis, Candida stellatoidea, Candida parapsilosis, and Candida krusei could be identified following restriction digestion of the PCR products.