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A comparative analysis of 16S ribosomal RNA sequences of all Peptostreptococcus species revealed that most members of the genus Peptostreptococcus should be divided into many different genera. The relationship between clostridia and peptostreptococci was analysed to find the phylogenetic position of peptostreptococci.
DNA coding for the 16S rRNA of an intracellular bacterium was directly amplified from lysed cells of a host amoebae using the polymerase chain reaction and primers specific for eubacteria. The amoebae had been used to recover an uncultured bacterium observed in the sputum of a patient with pneumonia. The amplified DNA was sequenced directly and compared with published 16S rRNA sequences. The analysis revealed that the intracellular bacterium is a member of the genus Legionella and that it is different from species, including L. pneumophila, for which 16S ribosomal RNA sequence data are available.
Other workers have found that clinical isolates of Helicobacter pylori exhibit very extensive DNA sequence polymorphisms when they are examined by ribotyping or some other genomic sequence characterization technique. In fact, it is rare to find similar clones, much less identical ones, among isolates. We found that the levels of divergence between the 16S ribosomal DNA sequences of individual organisms and the consensus sequence of the five isolates which we examined ranged from 0.2 to 0.5%. In contrast, other workers have shown that levels of divergence between the 16S ribosomal DNA sequence of H. pylori and the 16S ribosomal DNA sequences of four other Helicobacter species range from 2.7 to 8.0%. Our results show that the H. pylori 16S ribosomal DNA is not very polymorphic and support the conclusion that H. pylori is a unique species.
The phylogenetic positions of six Vibrio costicola strains were determined by direct sequencing and analysis of their PCR-amplified 16S ribosomal DNAs. A comparative analysis of the sequence data revealed that the moderate halophile V. costicola forms a monophyletic branch that is distinct from other Vibrio species and from moderately halophilic species of other genera. These results complement phenotypic and genotypic data determined previously. The molecular evidence, together with several phenotypic differences, distinguishes V. costicola from species of the genus Vibrio and other species belonging to the gamma subclass of the Proteobacteria and indicates that V. costicola should be placed in a new and separate genus. The name Salinivibrio costicola gen. nov., comb. nov. is proposed for this bacterium. The guanine-plus-cytosine content of the DNA is 49.4 to 50.5 mol%. The type strain of S. costicola is strain NCIMB 701 (= ATCC 33508).
The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A. pleuropneumoniae. Amplification and sequence analysis of the 16S-23S rDNA ribosomal intergenic sequence (RIS) from the three species showed the existence of two RIS's, differing by about 100 bp. Both sequences contained a region resembling the ribonuclease III cleavage site found in Escherichia coli. The smaller RIS contained a Glu-tRNA gene, and the larger one contained genes encoding Ile-tRNA and Ala-tRNA. These tRNA's showed a high sequence homology to the respective tRNA genes found in E. coli. Sequence analysis of the RIS's showed a high degree of genetic similarity of 24 strains of A. pleuropneumoniae. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ hybridization experiments to detect A. pleuropneumoniae in biopsies of diseased porcine lungs.
In order to develop a PCR method to detect Fusobacterium prausnitzii in human feces and to clarify the phylogenetic position of this species, its 16S rRNA gene sequence was determined. The sequence described in this paper is different from the 16S rRNA gene sequence is specific for F. prausnitzii, and the results of this assay confirmed that F. prausnitzii is the most common species in human feces. However, a PCR assay based on the original GenBank sequence was negative when it was performed with two strains of F. prausnitzii obtained from the American Type Culture Collection. A phylogenetic tree based on the new 16S rRNA gene sequence was constructed. On this tree F. prausnitzii was not a member of the Fusobacterium group but was closer to some Eubacterium spp. and located between Clostridium "clusters III and IV" (M.D. Collins, P.A. Lawson, A. Willems, J.J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J.A.E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994).
Since nonfermenting, Gram negative bacilli recovered from patients with cystic fibrosis could be misidentified with phenotypic procedures, we used partial 16S ribosomal RNA gene (16S gene) sequencing to identify these "Pseudomonas-like" isolates. 473 isolates were recovered from 66 patients in 2003. Sequencing was used to identify 29 (from 24 patients) of the 473 isolates, showing unclear results with routine tests. PCR with specific primers was carried out to amplify a 995 bp fragment, which was then sequenced. The sequences were analyzed with GenBank database for species assignment. Phenotypic and genotypic results were concordant for 20/29 isolates (10 Pseudomonas aeruginosa, 5 Burkholderia cepacia, 3 Stenotrophomonas maltophilia, 2 Achromobacter xylosoxidans). However, 3 of the 5 B. cepacia isolates were then identified as Burkholderia multivorans with a PCR-RFLP procedure. Phenotypic misidentification was observed for 9/29 isolates: 4 A. xylosoxidans, 1 P. aeruginosa, 1 Bordetella petrii, 1 Bordetella bronchiseptica, 1 Ralstonia respiraculi and 1 Ralstonia mannitolilytica. Partial 16S gene sequencing improved the identification of "Pseudomonas-like" isolates from cystic fibrosis patients, but the accuracy to distinguish between genomovars of the B. cepacia complex was inadequate.
Previously, virtually complete 16S ribosomal ribonucleic acid sequences were determined for 54 strains of species in the family Pasteurellaceae. The sequences for 16 additional strains have been determined, bringing the total number of strains sequenced to 70. The additional strains include: Actinobacillus hominis, A. muris, A. salpingitis, Pasteurella bettyae, P. mairii, P. testudinis, and Bisgaard taxa 2, 3, 5, 6, 7, 8, 9, 13, and 14 (2 strains). A phylogenetic tree was constructed based upon sequence similarity using the Neighbor-Joining method. The additional sequence information and phylogenetic analysis generally supported our previously described phylogenetic structure for the family Pasteurellaceae. Cluster 1, containing Haemophilus sensu stricto, was unchanged. P. mairii was closely related to P. aerogenes and Bisgaard taxon 6 was related to H. somnus in Cluster 2. A. salpingitidis and Bisgaard taxa 2, 3, 7, and 13 fell in Cluster 3 which contains Pasteurella sensu stricto. A. hominis was closely related to Actinobacillus sensu stricto species in Cluster 4A. Bisgaard taxa 5, 8, 9 and P. bettyae fell in Cluster 4B. A. muris was related to P. pneumotropica in Cluster 5. Haemophilus parainfluenzae strains branched deeply as a 6th cluster. Bisgaard taxon 14 and P. testudinis formed a 7th cluster which branched deeper than any previously described clusters in the family Pasteurellaceae. The branching was extremely complex and taxonomic division of the family into phylogenetically and phenotypically coherent genera will be difficult.
The phylogenetic relationships of xenodontine snakes are inferred from sequence analyses of portions of two mitochondrial genes (12S and 16S ribosomal RNA) in 85 species. Although support values for most of the basal nodes are low, the general pattern of cladogenesis observed is congruent with many independent molecular, morphological, and geographical data. The monophyly of xenodontines and the basal position of North American xenodontines in comparison with Neotropical xenodontines are favored, suggesting an Asian-North American origin of xenodontines. West Indian xenodontines (including endemic genera and members of the genus Alsophis) appear to form a monophyletic group belonging to the South American clade. Their mid-Cenozoic origin by dispersal using ocean currents is supported. Within South American mainland xenodontines, the tribes Hydropsini, Pseudoboini, and Xenodontini are monophyletic. Finally, our results suggest that some morphological and ecological traits concerning maxillary dentition, macrohabitat use, and foraging strategy have appeared multiple times during the evolution of xenodontine snakes.
North American freshwater unionacean bivalves are a diverse group of nearly 300 species. Unionaceans exhibit an array of conchological, anatomical, life history, and reproductive characteristics that have figured prominently in proposed classification schemes. Recently, two very different classifications of North American unionaceans have been proposed. Depending on the classification system utilized, a very different evolutionary trajectory of anatomical and reproductive features is obtained. The lack of a robust, well corroborated phylogeny of North American unionacean bivalves hinders the progress of evolutionary and ecological studies involving these species. Here we present a mitochondrial DNA (mtDNA) based phylogeny for North American unionacean mussels and compare it to previously proposed classifications. In addition, we present a 'total evidence' phylogeny which incorporates both the mtDNA sequence data and available morphological data. The molecular and total evidence phylogenies agree largely with the conclusions of a previous study based largely on immunoelectrophoretic data. North American unionaceans can be divided into two families: the Unionidae, which is comprised of most of the species and the Margaritiferidae. Within the Uniondae are two subfamilies, the Anodontinae and Ambleminae. The resultant phylogeny was used to examine the evolution of several key anatomical features including the number of gills (demibranchs) used by females to brood developing embryos, incubation length (bradytictic vs tachytictic), larval (glochidial) tooth structures, and shell texture. Both molecular and total evidence phylogenies indicate several of the aforementioned characters evolved independently or were subsequently lost or gained in several lineages.
Disseminated neoplasia is a leukemia-like disease that occurs in many species of bivalve molluscs worldwide, including the bay mussel (Mytilus trossulus). The etiology of the disease is undetermined, but an early report proposed that the anomalous bivalve cells were actually an invasive parasite rather than cancerous cells of host origin. Comparison of partial sequences of small subunit rRNA from normal and putative cancer cells was performed to resolve this issue. These studies showed a close phylogenetic relationship of the different forms of cancer cells to each other (similarity coefficient, 0.982), to the normal hemocytes (similarity coefficient, 0.990, 0.992), and to the oyster, Crassostrea virginica (similarity coefficient, 0.895-0.927). A large phylogenetic distance separates all 3 mussel hemocyte types from several representative protists (similarity coefficient, 0.702-0.761). These results indicate that the disseminated neoplastic cells in mussels are indeed proliferative host cells and not unicellular parasites.
Eight Campylobacter isolates that were able to grow at 25 degrees C and 42 degrees C and had the same biochemical profile were isolated from the blood of eight immunocompromised patients. Conventional biochemical tests were unable to determine whether they were isolates of thermo-tolerant C. fetus, H2S-negative C hyointestinalis, or a new Campylobacter species. Sequencing of the 16S ribosomal RNA genes showed that all eight isolates had the same nucleotide sequence and this was identical to that of C. fetus (GenBank accession no. AF219233). All eight patients had underlying disease and two died despite antibiotic treatment. Because of the ability of C fetus to grow over a wide range of temperatures and a higher incidence of bacteraemia by this organism than C. jejuni in the past 5 years in Hong Kong, thermo-tolerant C fetus may be an emerging pathogen in immunocompromised patients in the years to come.
The many-celled magnetotactic prokaryote (MMP) is an uncultivated, highly motile aggregate of 10-30 cells containing numerous chains of greigite (Fe(3)S(4)) magnetosomes. It is unique to marine environments and is abundant in slightly sulfidic sediments of the Little Sippewissett salt marsh (Falmouth, MA). We sequenced 16s rDNA genes from a natural population of MMP and found five lineages separated by at least 5% sequence divergence. Fluorescent in situ hybridization probes for three of these lineages showed significant variation in their relative abundances across a seasonal cycle in marsh productivity. The MMP should therefore be considered a separate genus in the delta-proteobacteria rather than a single species as previously thought. All cells in each aggregate express identical SSU rRNAs, suggesting that the aggregates are composed of a single MMP phylotype. This observation supports a model of the MMP as comprised of clonal cells which reproduce by binary fission of the aggregate.
A significant proportion of oral bacteria are unable to undergo cultivation by existing techniques. In this regard, the microbiota from root canals still requires complementary characterization. The present study aimed at the identification of bacteria by sequence analysis of 16S rDNA clone libraries from seven endodontically infected teeth. Samples were collected from the root canals, subjected to the PCR with universal 16S rDNA primers, cloned and partially sequenced. Clones were clustered into groups of closely related sequences (phylotypes) and identification to the species level was performed by comparative analysis with the GenBank, EMBL and DDBJ databases, according to a 98% minimum identity. All samples were positive for bacteria and the number of phylotypes detected per subject varied from two to 14. The majority of taxa (65.2%) belonged to the phylum Firmicutes of the Gram-positive bacteria, followed by Proteobacteria (10.9%), Spirochaetes (4.3%), Bacteroidetes (6.5%), Actinobacteria (2.2%) and Deferribacteres (2.2%). A total of 46 distinct taxonomic units was identified. Four clones with low similarity to sequences previously deposited in the databases were sequenced to nearly full extent and were classified taxonomically as novel representatives of the order Clostridiales, including a putative novel species of Mogibacterium. The identification of novel phylotypes associated with endodontic infections suggests that the endodontium may still harbour a relevant proportion of uncharacterized taxa.
We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042-5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 x 10(7) to 2.5 x 10(8) cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.
Bacterial diversity in unimproved and improved grassland soils was assessed by PCR amplification of bacterial 16S ribosomal DNA (rDNA) from directly extracted soil DNA, followed by sequencing of ~45 16S rDNA clones from each of three unimproved and three improved grassland samples (A. E. McCaig, L. A. Glover, and J. I. Prosser, Appl. Environ. Microbiol. 65:1721-1730, 1999) or by denaturing gradient gel electrophoresis (DGGE) of total amplification products. Semi-improved grassland soils were analyzed only by DGGE. No differences between communities were detected by calculation of diversity indices and similarity coefficients for clone data (possibly due to poor coverage). Differences were not observed between the diversities of individual unimproved and improved grassland DGGE profiles, although considerable spatial variation was observed among triplicate samples. Semi-improved grassland samples, however, were less diverse than the other grassland samples and had much lower within-group variation. DGGE banding profiles obtained from triplicate samples pooled prior to analysis indicated that there was less evenness in improved soils, suggesting that selection for specific bacterial groups occurred. Analysis of DGGE profiles by canonical variate analysis but not by principal-coordinate analysis, using unweighted data (considering only the presence and absence of bands) and weighted data (considering the relative intensity of each band), demonstrated that there were clear differences between grasslands, and the results were not affected by weighting of data. This study demonstrated that quantitative analysis of data obtained by community profiling methods, such as DGGE, can reveal differences between complex microbial communities.
Traditional methods for microbial identification require the recognition of differences in morphology, growth, enzymatic activity, and metabolism to define genera and species. Full and partial 16S rRNA gene sequencing methods have emerged as useful tools for identifying phenotypically aberrant microorganisms. We report on three bacterial blood isolates from three different College of American Pathologists-certified laboratories that were referred to ARUP Laboratories for definitive identification. Because phenotypic identification suggested unusual organisms not typically associated with the submitted clinical diagnosis, consultation with the Medical Director was sought and further testing was performed including partial 16S rRNA gene sequencing. All three patients had endocarditis, and conventional methods identified isolates from patients A, B, and C as a Facklamia sp., Eubacterium tenue, and a Bifidobacterium sp. 16S rRNA gene sequencing identified the isolates as Enterococcus faecalis, Cardiobacterium valvarum, and Streptococcus mutans, respectively. We conclude that the initial identifications of these three isolates were erroneous, may have misled clinicians, and potentially impacted patient care. 16S rRNA gene sequencing is a more objective identification tool, unaffected by phenotypic variation or technologist bias, and has the potential to reduce laboratory errors.
Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli is described. The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95% of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented. In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described.