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PubMed1992

Amplification of 16S rRNA sequences to detect Mycobacterium paratuberculosis.

A probe based on 16S ribosomal RNA (rRNA) sequences was developed to detect Mycobacterium paratuberculosis, the causative agent of Johne's disease in cattle. Three universal primers were used to sequence the amplified fragments of the 16S rRNA gene of various species of mycobacteria. When the nucleotide sequences were analysed, a deletion was detected in the sequence of the fast-growing species. An oligonucleotide probe (P) directed to this region was synthesised and hybridised directly with total RNA of various mycobacterial strains in a dot-spot assay. The probe detected M. paratuberculosis, some other slow-growing mycobacteria of the M. avium-intracellulare (MAI) complex, and one atypical strain, M. gordonae. To increase the sensitivity of the probe, a 413-bp fragment of the 16S rRNA gene of M. paratuberculosis between P and a second oligonucleotide primer was amplified and hybridised with a M. paratuberculosis/M. avium-specific probe. When faecal samples of cattle were tested, all culture-positive samples were positive in the PCR assay.

Animals↗
PubMed1994

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt.

We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema. It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.

Animals↗
PubMed2002

Intraspecific diversity of Brevibacterium linens, Corynebacterium glutamicum and Rhodococcus erythropolis based on partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy.

The intraspecific diversity of 31 strains of Brevibacterium linens, 27 strains of Corynebacterium glutamicum and 29 strains of Rhodococcus erythropolis was determined by partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy. As a prerequisite for the analyses, 27 strains derived from culture collections which had carried invalid or wrong species designations were reclassified in accordance with polyphasic taxonomical data. FT-IR spectroscopy proved to be a rapid and reliable method for screening for similar isolates and for identifying these actinomycetes at the species level. Two main conclusions emerged from the analyses. (1) Comparison of intraspecific 16S rDNA similarities suggested that R. erythropolis strains have a very low diversity, B. linens displays high diversity and C. glutamicum occupies an intermediate position. (2) No correlation of FT-IR spectral similarity and 16S rDNA sequence similarity below the species level (i.e. between strains of one species) was observed. Therefore, diversification of 16S rDNA sequences and microevolutionary change of the cellular components detected by FT-IR spectroscopy appear to be de-coupled.

Bacterial Typing Techniques↗
PubMed1992

Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification.

The 16S rRNA gene from various bacterial cultures was amplified by the polymerase chain reaction without DNA purification, and sequenced directly by using a laser fluorescent DNA sequencer and Tth polymerase with a cycle sequencing protocol. The described procedures provide almost complete 16S rDNA sequence data within a couple of days and facilitate systematic studies.

Autoanalysis↗
PubMed2001

Relationship of 16S rRNA sequence similarity to DNA hybridization in prokaryotes.

The relationship between 16S rRNA sequence similarity (S) and the extent of DNA hybridization (D) was well described by the equation In(-InD) = 0.53 [In(-InS)]+2.201 when D was determined by either the S1 nuclease or membrane filter methods. When the presence of nonultrametric rRNA sequences and differences between genera or families were controlled, this relationship accounted for 78% of the variability of D given S, and it was possible to estimate the distribution of D from S with a known precision. Thus, D<0.70 was expected to occur 50, 95 and 99% of the time when S was 0.998, 0.992 and 0.986, respectively. The relationship between D and S varied between prokaryotic taxa even within the same subphylum, and more precise estimates of D could be made when the relationship for a particular taxon was known. The relationship between D and S was not significantly different between the prokaryotic domains, and S appeared to be a quasi-molecular clock of approximately constant rate when averaging effects and stochastic factors were taken into account. The relationship between logD and logS was nonlinear, and D provided a very poor measure of relatedness for distantly related organisms. For instance, within the range 1.0 >S>> 0.95, D decreased from 1.0 to 0.15; and within the range 0.95 >S> 0.90, D decreased from 0.15 to 0.06. Lastly, at least some of the rRNA sequences from about one-third of the taxa examined had nonultrametric properties where S was much lower than expected from the value of D. For these taxa, S was a poor indicator of relatedness for closely related strains. Thus, the ultrametric properties of rRNA sequences should be tested before making taxonomic or phylogenetic conclusions based upon S.

Archaea↗
PubMed1997

Rapid identification of Medicago nodulating strains by using two oligonucleotide probes complementary to 16S rDNA sequences.

Symbiotic bacteria associated with the Medicago genus are separated into two closely related species named Sinorhizobium meliloti and Sinorhizobium medicae. To discriminate rapidly between these two bacterial species, two 15-base DNA probes, 16Smfs and 16Smed, were designed from the alignment of 16S rDNA sequences to differentiate S. meliloti from S. medicae. Their specificities were evaluated by dot-blot hybridization experiments on 25 reference strains representing 13 species of Rhizobium and Sinorhizobium, and by comparison with all 16S rDNA sequences available in the GenBank data base. No cross-reaction was found with 16Smed, which was thus considered species specific for S. medicae. By contrast, as expected according to the 16S rDNA sequence alignment, the labeled 16Smfs probe cross-hybridized with the DNAs of S. meliloti, Sinorhizobium fredii, and Sinorhizobium saheli but not with the DNA of S. medicae. Since S. saheli and S. fredii do not nodulate Medicago, 16Smed and 16Smfs can be routinely used to characterize the two Sinorhizobium species nodulating Medicago from pure cultures or from Medicago root nodules. Fifty strains isolated from eight annual Medicago species were then characterized by using colony hybridizations. Sinorhizobium meliloti was more frequently obtained (> 80% isolates) than was S. medicae. Both Sinorhizobium species seemed to be trapped by annual Medicago and no plant-host specificity was detected.

Base Sequence↗
PubMed2002

Phylogeny of green sulfur bacteria on the basis of gene sequences of 16S rRNA and of the Fenna-Matthews-Olson protein.

The phylogeny of green sulfur bacteria was studied on the basis of gene sequences of the 16S rRNA and of the Fenna-Matthews-Olson (FMO) protein. Representative and type strains (31 strains total) of most of the known species were analyzed. On the basis of fmoA gene sequences from Chlorobium tepidum ATCC 49652(T) and Chlorobium limicola DSM 249(T) available from the EMBL database, primers were constructed that allowed sequence analysis of a major part of the fmoAgene. The largely congruent phylogenetic relationship of sequences of the fmoA gene and of 16S rDNA gives considerable support to the phylogeny of green sulfur bacteria previously suggested on the basis of 16S rDNA sequences. Distinct groups of strains were recognized on the basis of 16S rDNA and FMO sequences and supported by characteristic signature amino acids of FMO. Marine strains formed clusters separate from freshwater strains. The resulting phylogenetic grouping and relationship of the green sulfur bacteria do not correlate with their current taxonomic classification.

Amino Acid Sequence↗
PubMed1994

Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy.

Primers specific to conserved and variable regions in the 16S rRNA sequence were selected from the partially sequenced 16S rRNA genes of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, and Staphylococcus epidermidis. The PCR assay was divided into two DNA amplifications. The first resulted in a general bacterial amplicon, and the second resulted in a species-specific amplicon. The high specificity of the PCR assay was documented after testing bacteria of 28 different species (133 strains). A total of 304 clinical cerebrospinal fluid samples, including 125 samples from patients with bacterial meningitis, were assayed to investigate the diagnostic sensitivity and specificity for bacterial meningitis. The assay showed high sensitivity (0.94) and specificity (0.96) with the clinical samples, although some false results were obtained, the reasons for which are discussed. With agarose gel electrophoresis for detection of the PCR products, the detection limit for meningococci in cerebrospinal fluid was 3 x 10(2) CFU/ml.

Base Sequence↗
PubMed2005

At least 1 in 20 16S rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies.

A new method for detecting chimeras and other anomalies within 16S rRNA sequence records is presented. Using this method, we screened 1,399 sequences from 19 phyla, as defined by the Ribosomal Database Project, release 9, update 22, and found 5.0% to harbor substantial errors. Of these, 64.3% were obvious chimeras, 14.3% were unidentified sequencing errors, and 21.4% were highly degenerate. In all, 11 phyla contained obvious chimeras, accounting for 0.8 to 11% of the records for these phyla. Many chimeras (43.1%) were formed from parental sequences belonging to different phyla. While most comprised two fragments, 13.7% were composed of at least three fragments, often from three different sources. A separate analysis of the Bacteroidetes phylum (2,739 sequences) also revealed 5.8% records to be anomalous, of which 65.4% were apparently chimeric. Overall, we conclude that, as a conservative estimate, 1 in every 20 public database records is likely to be corrupt. Our results support concerns recently expressed over the quality of the public repositories. With 16S rRNA sequence data increasingly playing a dominant role in bacterial systematics and environmental biodiversity studies, it is vital that steps be taken to improve screening of sequences prior to submission. To this end, we have implemented our method as a program with a simple-to-use graphic user interface that is capable of running on a range of computer platforms. The program is called Pintail, is released under the terms of the GNU General Public License open source license, and is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

Bacteroides↗
PubMed1998

Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pylori-associated gastritis?

The aim of the present study was to correlate molecular evidence of the presence of Helicobacter pylori in gastric biopsy samples, based on analysis of 16S rDNA, vacuolating toxin (vacA), urease A (ureA) and cagA genes, with the clinical, histological and serological findings in patients with H. pylori-associated gastritis. Fresh biopsy samples were collected from the gastric antrum and corpus of 22 asymptomatic volunteers with or without H. pylori-associated gastritis. Total DNA was extracted from the biopsy material and subjected to 16S rDNA PCR amplification, Southern blotting and 16S rDNA sequence analysis of the PCR products. The vacA, ureA and cagA genes were characterised by PCR amplification and Southern blot analysis. Based on partial 16S rDNA sequence analysis, DNA belonging to the genus Helicobacter was detected in gastric biopsy samples from 20 of 22 subjects, including seven of nine histologically and serologically normal controls. Six of 20 partial 16S rDNA sequences revealed variations within variable regions V3 and V4 that deviated from those of the H. pylori type strain ATCC 4350T and, therefore, possibly represented other species of Helicobacter. VacA genes identical with those of the type strain were found predominantly in the subjects with H. pylori gastritis, and all the patients except one were found to be cagA-positive. There was no evidence of false positive PCR reactions. In conclusion, the PCR-based molecular typing methods used here were apparently too sensitive when applied to the detection of H. pylori in human gastric tissues. The lack of quantitative analysis makes them inappropriate as clinical tools for the diagnosis of H. pylori-associated gastritis, despite the fact that they provide a qualitative and sensitive tool for the detection and characterisation of H. pylori in the gastrointestinal tract.

Adult↗
PubMed2006

Study of bacterial communities in Antarctic coastal waters by a combination of 16S rRNA and 16S rDNA sequencing.

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to gamma-Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with beta-Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities.

Antarctic Regions↗
PubMed2000

Molecular systematics of the order anaspidea based on mitochondrial DNA sequence (12S, 16S, and COI).

Fragments from three mitochondrial genes (12S, 16S, and COI) were sequenced to reconstruct a molecular phylogeny of the opisthobranch order Anaspidea. The molecular phylogeny supports the placement of the genus Akera, a taxon previously regarded by some authors as a cephalaspidean, within the Anaspidea. Incongruence between the molecular data and the classifications based on morphology suggests that some of the taxonomic characters (i.e., shell, parapodia fusion) traditionally used for the classification of sea hares must be reevaluated, since they may be homoplastic. The ancestral nature of Notarchus based on the molecular evidence suggests that homoplasy may be an explanation for the morphological resemblance of this species to the more derived sea hares with highly fused parapodia and concentrated nerve ganglia. Finally, examples are given of how comparative studies of the evolution of learning mechanisms in the anaspidean clade will benefit from the phylogenetic hypothesis presented in this paper.

Animals↗
PubMed1993

The use of 16S rDNA sequence analysis to investigate the phylogeny of Leptospiraceae and related spirochaetes.

The 16S rDNA sequences from 15 Leptospiraceae were determined by automated PCR-directed cycle sequencing. Nucleotide comparisons, including those from published sequences for Leptospira canicola Moulton and Serpulina spp., were used to construct phylogenetic trees. Serpulina hyodysenteriae and S. innocens were related to each other but were distinct from the Leptospiraceae comprising Leptospira parva incertae sedis (Turneria parva H), Leptonema illini and Leptospira spp. The pathogenic and the saprophytic leptospires were distinct and separated from each other. Leptospira inadai occupied an intermediate position between the two forms. The pathogens formed three groups. Group I was represented by L. interrogans sensu stricto and L. kirschneri, Group II by L. weilii, L. borgpetersenii and L. santarosai, and Group III comprised L. noguchii and L. meyeri. The saprophytic species, L. wolbachii and L. biflexa sensu stricto shared about 99% sequence similarity. The freshwater isolates were distinct from the marine isolate L. biflexa sensu lato ancona Ancona Porto.

Base Sequence↗
PubMed2004

Differentiation of Micromonospora isolates from a coastal sediment in Wales on the basis of Fourier transform infrared spectroscopy, 16S rRNA sequence analysis, and the amplified fragment length polymorphism technique.

A number of actinomycetes isolates were recovered from coastal sediments in Aberystwyth (Wales, United Kingdom) with standard isolation techniques. Most of them were putatively assigned to the genera Streptomyces and Micromonospora on the basis of their morphological characteristics, and there appeared to be no difference whether the isolation media contained distilled water or seawater. A group of 20 Micromonospora isolates was selected to undergo further polyphasic taxonomic investigation. Three approaches were used to analyze the diversity of these isolates, 16S rDNA sequencing, fluorescent amplified fragment length polymorphism (AFLP), and Fourier transform infrared spectroscopy (FT-IR). The 16S rDNA sequence analysis confirmed that all of these isolates should be classified to the genus Micromonospora, and they were analyzed with a group of other Micromonospora 16S rDNA sequences available from the Ribosomal Database Project. The relationships of the 20 isolates were observed after hierarchical clustering, and almost identical clusters were obtained with these three techniques. This has obvious implications for high-throughput screening for novel actinomycetes because FT-IR spectroscopy, which is a rapid and reliable whole-organism fingerprinting method, can be applied as a very useful dereplication tool to indicate which environmental isolates have been cultured previously.

Bacterial Typing Techniques↗
PubMedDate not supplied

[Diversity of bacteria at various depths in the southern part of lake Baikal as detected by 16S rRNA sequencing].

Phylogenetic analysis of the bacterial community inhabiting the water of Lake Baikal was performed on the basis of 16S rRNA sequencing. The composition of the bacterial community was shown to vary significantly with depth. Cyanobacteria were dominant species at the surface of the lake. At a moderate depth (400 m), actinomycete relatives were most abundant. At a great depth and near the bottom, the community was composed mainly of proteobacteria and cyanobacteria (the latter are probably brought from the surface layers by vertical near-shore water fluxes). Most of the bacterial 16S rRNA sequences detected exhibited low similarity to those known and formed separate clusters in the phylogenetic tree, which may indicate the endemic nature of the corresponding bacteria.

Bacteria↗
PubMed2005

Retrieval of nearly complete 16S rRNA gene sequences from environmental DNA following 16S rRNA-based community fingerprinting.

16S rRNA-based fingerprinting techniques allow rapid analyses of overall bacterial community structure but suffer from a lack of phylogenetic information hitherto retrievable from the short 16S rRNA gene sequences obtained from excised bands. An approach is presented that allows nearly complete 16S rRNA gene sequences to be retrieved for abundant components of the bacterial community as obtained by the community fingerprint, i.e. those reflected by major fingerprint bands. This was achieved by designing a pair of highly specific primers derived from the sequence of an excised band. Combined with universal 16S rRNA primers, these specific primers were applied directly to environmental DNA serving as template. This procedure allowed the generation of a nearly complete 16S rRNA gene sequence of the target taxon by specific polymerase chain reaction (PCR) followed by cycle sequencing down to a relative abundance of at least 1.5% of the environmental DNA. The procedure was exemplified for an epsilonproteobacterium related to Thiomicrospira denitrificans occurring in the central Baltic Sea. This approach is based only on PCR without any cloning step involved. It allows focussing on specific target taxa and is thus rather efficient. This approach should be applicable in general to 16S rRNA or 16S rRNA gene-based fingerprinting techniques and their respective environmental DNA.

DNA Fingerprinting↗
PubMed1994

Helicobacter sp. strain Mainz isolated from an AIDS patient with septic arthritis: case report and nonradioactive analysis of 16S rRNA sequence.

A campylobacter-like organism was isolated from an effusion of the left knee joint of an AIDS patient 2 weeks after bacteremia with a morphologically identical organism. Amplified genomic 16S rRNA sequences were analyzed by a nonradioactive blotting technique. The closest match was found with Helicobacter fenelliae (97.7% homology). Sequence data and phenotype suggest that the isolate may represent a so far unrecognized species of the genus Helicobacter.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Identification of a third genomic group of Borrelia burgdorferi through signature nucleotide analysis and 16S rRNA sequence determination.

As part of a continuing effort to assess genetic variation among isolates of Borrelia burgdorferi we have determined the 16S rRNA signature nucleotide makeup of two tick isolates from the USSR. Signature nucleotides were identified via reverse transcriptase primer extension sequencing of select regions of the 16S rRNA molecule. In addition, the near complete 16S rRNA sequence of one of the isolates, R-IP3, was determined and utilized in a phylogenetic assessment. The sequence was aligned with the 16S rRNA sequences of other B. burgdorferi isolates as well as with other Borrelia species. Distance matrix analyses were performed and a phylogenetic tree was constructed. These analyses demonstrate that these isolates belong to a third previously unidentified genomic group of B. burgdorferi.

Base Sequence↗