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PubMed2003

16S ribosomal DNA sequence-based analysis of clinically significant gram-positive anaerobic cocci.

Sequence analysis of the 16S rRNA gene represents a highly accurate and versatile method for bacterial classification and identification, even when the species in question is notoriously difficult to identify by phenotypic means. However, its use for identification based on public sequence databases is not without limitation due to the presence of ambiguous data in the databases. In this study, we evaluated the utility of 16S ribosomal DNA sequencing as a means of identifying clinically important gram-positive anaerobic cocci (GPAC) by sequencing 13 type strains of established GPAC species and 156 clinical isolates that had been studied only by phenotypic tests. Among the 13 type strains of GPAC species we tested, only 4 gave a "perfect" match with their corresponding sequences in GenBank, whereas the other 9 had lower sequence similarities (<98%). This indicates that data in the public database may be inaccurate at times. Based on the sequences of the 13 type strains obtained in this study, 84% (131 of 156) of the clinical isolates were accurately identified to species level, with the remaining 25 clinical strains revealing nine unique sequences that may represent eight novel species. This finding is in contrast to the phenotypic identification results, by which only 56% of isolates were correctly identified to species level.

Anaerobiosis↗
PubMed1998

Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval.

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10(7) per g of dry soil for the H2-CO2-utilizing methanogens and of up to 10(6) for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H2-CO2, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 microM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant.

Acetates↗
PubMed2003

16S ribosomal RNA sequence-based identification of veterinary clinical bacteria.

This study evaluated 16S rRNA gene sequence analysis methods as tools for identification of 22 phenotypically difficult to identify veterinary clinical bacterial isolates in a veterinary diagnostic laboratory. The study compared 16S rRNA gene sequencing and conventional phenotypic identification methods. Using 16S rRNA full-gene sequencing, 95% (21/22) of the isolates were identified to the genus level and 86% (19/22) to the species level. The conventional or commercially available manual identification phenotypic characterization methods presumptively identified 91% (20/22) of the isolates to the genus level and 1 isolate to the species level. However, only 55% (12/22) or 4.5% (1/22) of the phenotypic identifications were correct at the genus or species level when they were compared with the 16S rRNA full-gene sequencing. This study also compared 16S rRNA full-gene and partial-gene sequencing. The results demonstrated that the best 16S rRNA gene-sequencing approach is full-gene sequencing because it gives the most precise species identification. Sequencing of the variable regions 1, 2, and 3 of the 16S rRNA gene could be used for tentative identification because the ability of this sequencing to identify bacteria to the genus level is similar to that of the 16S rRNA full-gene sequencing. This method identified only 14% (3/22) isolates differently to the species level compared with the 16S rRNA full gene sequence. Sequencing of the variable regions 7, 8, and 9 is not recommended because it gives more ambiguous identifications. The cost of a 16S RNA full-gene-sequencing analysis was Can 160 dollars and Can 60 dollars for a partial 16S rRNA gene sequence, i.e., sequencing of variable regions 1, 2, and 3 or variable regions 7, 8 and 9.

Bacteria↗
PubMed2001

rRNA sequences reflect the ecophysiology and define the toxic cyanobacteria of the genus Nodularia.

Nodularia, a member of the order Nostocales, is a bloom-forming filamentous cyanobacterium that possesses the ability to form toxic blooms. The toxin produced by Nodularia, nodularin, is a hepatotoxin, similar in structure to the heptapeptide toxin microcystin. Twenty-one strains of Nodularia, representing the species Nodularia spumigena, Nodularia harveyana and Nodularia sphaerocarpa, were analysed for toxin production by protein phosphatase inhibition assay and sequenced over the 16S rDNA region. Phylogenetic analysis of Nodularia 16S rDNA sequences found that Nodularia clustered into two main groups. An N. spumigena cluster was distinct from the benthic species N. harveyana and N. sphaerocarpa. There was no distinction between strains isolated from globally diverse locations. Nodularin-producing species were restricted to the single, evolutionally distinct cluster of N. spumigena. This observation has enabled the design of a specific 16S rRNA PCR for the rapid detection of nodularin-producing strains. Alignment of 16S rDNA sequences from toxic and non-toxic Nodularia with other members of the cyanobacteria allowed the design of both Nodularia generic and toxic N. spumigena-specific primers.

Bacterial Toxins↗
PubMed1991

Phylogeny of the phototrophic rhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment.

A 260-bp segment of the DNA that encodes 16S rRNA, corresponding to positions 44 to 337 in the Escherichia coli sequence, was amplified by the polymerase chain reaction and sequenced from each of 13 bacteria (rhizobia and purple phototrophs) in the alpha subdivision of the class Proteobacteria. The phylogenetic tree calculated from differences in the sequenced segment conforms well to our expectations based on other previously published data. The sequence from BTAi1 (a recently described phototrophic symbiont of the legume Aeschynomene) and that from the free-living phototroph Rhodopseudomonas palustris both fall within the range of variation found among strains of the soybean symbiont Bradyrhizobium japonicum. This suggests that it would be appropriate to include all of these organisms in a single genus.

Bacteria↗
PubMed1997

Molecular phylogenetic study of Tephritidae (Insecta: Diptera) using partial sequences of the mitochondrial 16S ribosomal DNA.

We analyzed sequence data from 925 base pairs within the mitochondrial 16S ribosomal DNA from 34 tephritid species and two outgroup taxa. DNA sequences were amplified by the polymerase chain reaction and sequenced by the dideoxy chain-termination method. Patterns of nucleotide composition and substitution were assessed based on sequence comparison. Phylogenetic relationships among the included taxa were inferred using neighbor-joining and maximum parsimony methods. The trees were congruent with the morphological classification and suggested the following phylogenetic relationships: (1) monophyly of the tribe Trypetini, s. str., excluding Parastenopa; (2) a possible sister group relationship between Platyparea and the subfamily Tephritinae; (3) a sister group relationship between Hexachaeta and the tribe Toxotrypanini; (4) monophyly of the Dacinae and possible relationship of Oedicarena to this subfamily; (5) monophyly of the tribe Adramini; and (6) the nonmonophyletic nature of the genus Rhagoletis. We propose that the 16S rDNA shows great promise for helping to create a phylogenetically based higher classification of the Tephritidae.

Animals↗
PubMed1995

Phylogeny of the Mycobacterium chelonae-like organism based on partial sequencing of the 16S rRNA gene and proposal of Mycobacterium mucogenicum sp. nov.

The Mycobacterium chelonae-like organism (MCLO) is a recently described member of the Mycobacterium fortuitum complex which causes posttraumatic skin infections and catheter sepsis. This taxon is a distinct group biochemically and has a unique mycolic acid profile as determined by high-performance liquid chromatography. Its phylogenetic relationships to other mycobacteria, however, have not been studied previously. We sequenced 1,062 bp of the 16S rRNA genes from three MCLO strains obtained from the American Type Culture Collection and compared our results with the sequences of previously described taxa of rapidly growing and slowly growing mycobacteria. Two biochemically typical strains (ATCC 49650T [T = type strain] and ATCC 49651) had identical sequences, while the sequence of a biochemically atypical strain (ATCC 49649) differed by 4 bp from the sequence of the two typical strains. The Hamming distances between these MCLO strains and related rapidly growing mycobacteria are comparable to the Hamming distances among taxa of rapidly growing mycobacteria established as species by DNA-DNA hybridization. We propose the name Mycobacterium mucogenicum sp. nov. for this new taxon because of the highly mucoid nature of most isolates on solid media.

Base Sequence↗
PubMed2000

Application of sequence-specific labeled 16S rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization.

DNA sequence information for the small-subunit rRNA gene (16S rDNA) obtained from cyanobacterial cultures was used to investigate the presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study. The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of oligonucleotide probes targeted to 16S rDNA and subsequent hybridization of the labeled probes to their respective complements spotted onto a solid support (DNA array). Ten probes were established to determine the relative abundances of the discernible cyanobacteria encountered in the selected lakes. The probes were generally specific for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and hydrochemical analyses. Qualitatively, there were relatively good correlations among the groups of organisms (Nostoc, Microcystis, and Planktothrix species) found in the different lakes. The levels of correlation were lower for the quantitative data. This may, however, be due to differences in sample processing technique. The conclusions from these comparisons are that the genetic abundance profiles may provide a foundation for separating and quantifying genetically distinct groups of cyanobacteria in their natural habitats.

Cyanobacteria↗
PubMed1996

PCR detection of colonization by Helicobacter pylori in conventional, euthymic mice based on the 16S ribosomal gene sequence.

Many animal models of Helicobacter infection have been described, including infection in rhesus monkeys, ferrets, gnotobiotic piglets, and mice. These animal models utilize a combination of detection methods, including culture, urease testing, and histopathology, all of which may be unreliable, insensitive, or labor-intensive. Development of new animal models of Helicobacter pylori requires new methods of detection with increased sensitivity and specificity. We have developed sensitive and specific PCR primers based on the 16S ribosomal gene sequence of H. pylori. The primers detected single-copy 16S DNA representing 0.2 cell of pure H. pylori (2 cells in the presence of mouse stomach mucosal DNA) and did not cross-react with closely related bacteria. We were able to detect colonization by H. pylori in conventional, euthymic, outbred mice up to 4 weeks postinoculation with a high percentage of isolates tested. One isolate of H. pylori was detected by PCR in 100% of the mice at 6 months and 60% of the mice 1 year after inoculation. Approximately 10(3) to 10(4) H. pylori cells per stomach were detected by utilizing this PCR methodology semiquantitatively. These primers and PCR methodology have facilitated detection of H. pylori colonization in conventional, euthymic mice, colonization which may not have been detectable by other methods.

Animals↗
PubMed1998

Archaeal cold-adapted proteins: structural and evolutionary analysis of the elongation factor 2 proteins from psychrophilic, mesophilic and thermophilic methanogens.

To identify structural features important for low temperature activity in archaeal proteins, elongation factor 2 (EF-2) genes (aef2) were sequenced from psychrophilic, mesophilic and thermophilic methanogens. Scatter plots were used to compare evolutionary distances for EF-2 amino acid sequences vs. 16S-rRNA sequences from methanogens growing at diverse temperatures. The absence of a temperature bias for the rate of protein vs. nucleic acid evolution demonstrated the importance of comparing closely related proteins in order to identify changes indicative of thermal adaptation. Three-dimensional modelling of the new EF-2 sequences enabled the identification of amino acid residues that may be important for conferring low temperature activity and included greater structural flexibility produced by fewer salt bridges, less packed hydrophobic cores and the reduction of proline residues in loop structures.

Adaptation, Physiological↗
PubMed1997

Endocarditis caused by Haemophilus parainfluenzae identified by 16S ribosomal RNA sequencing.

Haemophilus parainfluenzae, a human commensal, is an infrequent cause of serious disease. A case of endocarditis caused by this organism in a five year old boy with complex congenital heart disease is reported. The course of this disease was very aggressive, leading to heart failure, disseminated intravascular coagulation and multiorgan failure in spite of appropriate antibiotics and surgical intervention. The difficulties in the detection and identification of H parainfluenzae using conventional culture based technology, and the potential role of molecular techniques, are highlighted.

Anti-Bacterial Agents↗
PubMed1984

Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma capricolum.

The nucleotide sequences of the rrnB 16S ribosomal RNA gene and its 5'-and 3'-flanking regions from Mycoplasma capricolum have been determined. The coding sequence is 1521 base pairs long, being 21 base pairs shorter than that of the Escherichia coli 16S rRNA gene. The 16S rRNA sequence of M. capricolum reveals 74% and 76% identify with that of E. coli and Anacystis nidulans, respectively. The secondary structure model constructed from the M. capricolum 16S rRNA gene sequence resembles that proposed for E. coli 16S rRNA. A large stem structure can be constructed between the 5'- and 3'-flanking sequences of the 16S rRNA gene. The flanking regions are extremely rich in AT.

Base Sequence↗
PubMed1978

Application of a rapid gel method to the sequencing of fragments of 16S ribosomal RNA from Escherichia coli.

A gel sequencing method has been applied to two 5' end-labelled fragments of the 16S ribosomal RNA from E. coli. The procedure involves partial enzymatic hydrolysis by ribonucleases T1, U2 or A, in order to generate series of end-labelled subfragments terminating in guanine, adenine, or pyrimidine residues, respectively. The two fragments concerned were approximately 75 and 90 nucleotides in length, and both arose from the 3' region of the 16S RNA. The sequences deduced are compared with the published sequence of 16S RNA, and contribute information to the final ordering of the ribonuclease T1 oligonucleotides in the latter, as well as revealing some probable errors.

Base Sequence↗
PubMed2000

Pseudoalteromonas peptidolytica sp. nov., a novel marine mussel-thread-degrading bacterium isolated from the Sea of Japan.

A new bacterial species belonging to the genus Pseudoalteromonas is described on the basis of phenotypic characterization, and sequence analysis of its 16S rRNA-coding and gyrase B (gyrB) genes. Ten strains, isolated from sea water of Yamato Island, Sea of Japan, were Gram-negative, yellow, motile, polarly flagellated, aerobic, rod-shaped eubacteria and had a G + C content of 42 mol%. Analysis of the 16S rDNA sequence revealed a clear affiliation between these strains and members of the gamma-Proteobacteria. High similarity values were found with members of the genus Pseudoalteromonas and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain F12-50-A1T and Pseudoalteromonas piscicida was very high (99.1%). However, molecular characterizations employing small subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving gyrB gene were performed. Our assertion that this strain represents a distinct bacterial species within the genus Pseudoalteromonas was supported by both of these molecular analyses. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of F12-50-A1T-like strains, thereby confirming the species. As these strains cleave complex protein compounds of the Mytilus edulis foot by secreting proteases, the name Pseudoalteromonas peptidolytica sp. nov. is proposed, with strain F12-50-A1T (= MBICC F1250A1T) as the type strain.

Animals↗
PubMed1996

Phylogenetic diversity of intra-amoebal legionellae as revealed by 16S rRNA gene sequence comparison.

Following a recent phylogenetic analysis of all 39 species of the genus Legionella and related organisms, the name Legionella lytica has been proposed for bacteria previously considered either as Sarcobium lyticum or as 'Legionella-like amoebal pathogens' (LLAPs). To investigate the phylogenetic integrity of this newly proposed species, we determined the 16S rRNA gene sequences of 10 LLAPs isolated from various environmental sources. All 10 isolates clustered within a monophyletic group containing all other members of the genus Legionella. Eight of the 10 isolates formed a monophyletic subgroup within the genus which also included the two previously characterized L. lytica strains. Four of these 10 isolates shared a specific and very close relationship with the L. lytica type strain (> 99% sequence similarity). However, although clearly legionellae, the remaining two LLAP strains bore no specific evolutionary relationship to either L. lytica or any other Legionella species (< 96% sequence similarity). Both isolates lay on their own relatively deep-rooted branches within the radiation of the Legionella cluster. LLAPs do not, therefore, represent a unique species or even a single line of descent within the genus, and investigation of more isolates may reveal them to be as evolutionarily diverse as the other presently recognized Legionella species.

Acanthamoeba↗
PubMed2002

Widespread distribution in polar oceans of a 16S rRNA gene sequence with affinity to Nitrosospira-like ammonia-oxidizing bacteria.

We analyzed the phylogenetic compositions of ammonia-oxidizing bacteria of the beta subclass of Proteobacteria from 42 Southern Ocean samples. We found a Nitrosospira-like 16S rRNA gene sequence in all 20 samples that yielded PCR products (8 of 30 samples from the Ross Sea and 12 of 12 samples from the Palmer Peninsula). We also found this sequence in Arctic Ocean samples, indicating a transpolar, if not global, distribution; however, slight differences between Arctic and Antarctic sequences may be evidence of polar endemism.

Ammonia↗
PubMed1999

Correlation of 16S ribosomal DNA signature sequences with temperature-dependent growth rates of mesophilic and psychrotolerant strains of the Bacillus cereus group.

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance.

Bacillus↗
PubMed2004

Novel relationships among hyloid frogs inferred from 12S and 16S mitochondrial DNA sequences.

Advanced frogs (Neobatrachia) are usually divided into two taxa, Ranoidea (the firmisternal frogs) and Hyloidea (all other neobatrachians). We investigated phylogenetic relationships among several groups of Hyloidea using 12S and 16S rRNA mitochondrial gene sequences and tested explicit relationships of certain problematic hyloid taxa using a sample of 93 neobatrachians. Parsimony, maximum likelihood, and Bayesian inference methods suggest that both the Ranoidea and Hyloidea are well-supported monophyletic groups. We reject three hypotheses using parametric bootstrap simulation: (1) Dendrobatidae lies within the Ranoidea; (2) The group containing Hylidae, Pseudidae, and Centrolenidae is monophyletic; and (3) Brachycephalus is part of Bufonidae.

Animals↗