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PubMed1997

Phylogeny of the genus Chlorobium based on 16S rDNA sequence.

A comparative analysis of 16S ribosomal DNA sequences from 18 strains belonging to different species of the genus Chlorobium has been made in order to investigate their phylogenetic relationships. All studied strains with brown pigmentation, belonging to species C. phaeobacteroides and C. phaeovibrioides, were clustered together and separated from green species. Most of the C. limicola strains formed a consistent group and were clustered next to C. tepidum. But, despite their identical morphology, C. vibrioforme strain 6030 (= DSM 260T) and C. vibrioforme strain 8327 (= DSM 263) were clearly differentiated on the basis of their 16S rDNA sequence. On the other hand, morphologically distinctive strains of C. chlorovibrioides, C. vibrioforme and C. limicola presented sequences with a high degree of similarity. Since morphological characters do not seem to be sufficient to adequately classify this group, these findings help to set up the basis for a revision of the taxonomy of the genus Chlorobium.

Base Sequence↗
PubMed1993

Assignment of the agent of Tyzzer's disease to Clostridium piliforme comb. nov. on the basis of 16S rRNA sequence analysis.

The small-subunit rRNA (16S rRNA) sequence of Tyzzer's bacillus (also known as "Bacillus piliformis") was elucidated by using the polymerase chain reaction followed by reverse transcriptase sequencing. By using maximum-likelihood analysis, a phylogenetic tree was constructed from this and other 16S rRNA sequences available from the first release of the Ribosomal Database Project (G. J. Olsen, R. Overbeek, N. Larsen, T. L. Marsh, M. J. McCaughey, M. A. Maciukenas, W.-M. Kuan, T. J. Macke, Y. Xing, and C. R. Woese, Nucleic Acids Res. 20:2199-2200, 1992). Tyzzer's bacillus grouped with a specific set of anaerobic bacteria, most of which are Clostridium spp. The closest identified relatives are Clostridium coccoides, Clostridium oroticum, Clostridium clostridiiforme, Clostridium symbiosum, and Streptococcus hansenii. Clostridium amino-valericum and "Acetitomaculum ruminis" are also solidly allied with this ensemble. We propose that Tyzzer's bacillus be reclassified as Clostridium piliforme on the basis of its 16S rRNA sequence.

3T3 Cells↗
PubMed2001

Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences.

The partial sequences (1465 bp) of the 16S rDNA of Clostridium novyi types A, B and C and Clostridium haemolyticum were determined. C. novyi types A, B and C and C. haemolyticum clustered with Clostridium botulinum types C and D. Moreover, the 16S rDNA sequences of C. novyi type B strains and C. haemolyticum strains were completely identical; they differed by 1 bp (level of similarity > 99.9%) from that of C. novyi type C, they were 98.7% homologous to that of C. novyi type A (relative positions 28-1520 of the Escherichia coli 16S rDNA sequence) and they exhibited a higher similarity to the 16S rDNA sequence of C. botulinum types D and C than to that of C. novyi type A. These results suggest that C. novyi types B and C and C. haemolyticum may be one independent species generated from the same phylogenetic origin.

Clostridium↗
PubMed1993

Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences.

A fluorescently labeled version of a population-specific oligonucleotide hybridization probe was used to monitor the enrichment and isolation of a sulfate-reducing bacterium from a multispecies anaerobic bioreactor. The organism was originally identified as a molecular isolate that was phylogenetically related to Desulfovibrio vulgaris by amplification and sequencing of part of its 16S rRNA sequence. The sequence, in turn, was used to design a population-specific probe. The anaerobic medium used for the organism's enrichment and isolation was based on the physiological properties of the its closest relatives as identified by sequence comparisons. Of 30 isolates examined, only 3 hybridized with the probe. Nearly complete 16S rRNA sequences determined for each of these three isolates (i) had no mismatches with the probe target site, (ii) were identical to the amplified partial sequence of about 500 nucleotides and to one another in all other positions, and (iii) were 93.9% similar to that of D. vulgaris. In addition, one isolate chosen for further study (strain PT-2) had a substrate specificity comparable to that of D. vulgaris. These results confirmed that polymerase chain reaction amplification of 16S rRNA sequences from environmental samples can be accurate and can also provide phylogenetic information from which aspects of a population's physiology can be inferred.

Base Sequence↗
PubMed2000

Analysis of ammonia-oxidizing bacteria from hypersaline Mono Lake, California, on the basis of 16S rRNA sequences.

Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the beta subdivision of the division Proteobacteria (beta-subdivision Proteobacteria) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different beta-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured Nitrosomonas strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the Nitrosomonas europaea group that are acclimated to alkaline, high-salinity environments. Primers specific for Nitrosococcus oceanus, a marine ammonia-oxidizing bacterium in the gamma subdivision of the Proteobacteria, did not amplify target from any samples.

Ammonia↗
PubMed1997

Phylogenetic diversity of the deinococci as determined by 16S ribosomal DNA sequence comparison.

16S ribosomal DNA (rDNA) sequences were determined for the five species of the genus Deinococcus (Deinococcus erythromyxa, Deinococcus proteolyticus, Deinococcus radiodurans, Deinococcus radiophilus, and Deinococcus radiopugnans) and the single species of the genus. Deinobacter (Deinobacter grandis). With the exception of Deinococcus erythromyxa, the deinococci form a coherent phylogenetic cluster which is related to the Thermus-Meiothermus lineage. An analysis of the 16S rDNA sequence of Deinococcus erythromyxa revealed that this organism is an actinomycete and a member of the genus Kocuria. Deinobacter grandis falls within the radiation of the genus Deinococcus and phylogenetically can be considered a member of this genus. The results of the phylogenetic analyses are consistent with chemotaxonomic data. On the basis of our data, Deinobacter grandis is transferred to the genus Deinococcus as Deinococcus grandis comb. nov., the description of the genus Deinococcus is emended accordingly, and Deinococcus erythromyxa is transferred to the genus Kocuria as Kocuria erythromyxa comb. nov. The description of the family Deinococcaceae is emended to include organisms with rod-shaped cells, and a set of 16S rDNA signature nucleotides is designated for this group. On the basis of the distinct phylogenetic position of the Deinococcus lineage and a set of 16S rDNA signature nucleotides, the order Deinococcales ord. nov. is described.

DNA, Bacterial↗
PubMed2006

Phylogenetic relationship of Pasteurella pneumotropica isolates from laboratory rodents based on 16S rDNA sequence.

A 1344 bp fragment of the 16S ribosomal DNA (rDNA) sequence was used to determine the genetic relationship of Pasteurella pneumotropica isolates from laboratory rodents. A total of 30 nucleotide sequences of P. pneumotropica, including 24 wild strains, 3 reference strains, and 3 nucleotide sequences deposited in GenBank, were examined for heterogeneity of their 16S rDNA sequences. Phylogenetic analysis based on 16S rDNA sequence discriminated 5 types of branching lineages. Of these 5 types, 3 types had significant associations with mice or rats, and 2 had significant associations with the beta-hemolytic phenotype. These results suggest that 16S rDNA sequencing of P. pneumotropica isolates demonstrates genetic heterogeneity and phylogenetic discrimination in terms of their hemolytic phenotype and host associations.

Animals↗
PubMed2001

Identification of symbiotic bacteria (Photorhabdus and Xenorhabdus) from the entomopathogenic nematodes Heterorhabditis marelatus and Steinernema oregonense based on 16S rDNA sequence.

Two species of entomopathogenic nematodes, Heterorhabditis marelatus and Steinernema oregonense, were described recently from the west coast of North America. It is not known whether the bacterial symbionts of these nematodes are also unique. Here we compared partial 16S rRNA sequences from the symbiotic bacteria of these two nematodes with sequence from previously described Photorhabdus and Xenorhabdus species. The 16S sequence from the new Xenorhabdus isolate appears very similar to, although not identical to, that of X. bovienii, the common symbiont of S. feltiae. The new Photorhabdus isolate appears to be very distinct from other known Photorhabdus species, although its closest affinities are with the P. temperata group. We also verified a monoxenic association between each isolate and its nematode by amplifying and sequencing bacterial 16S sequence from crushed adult and juvenile nematodes and from bacterial cultures isolated from infected hosts.

Animals↗
PubMed2003

Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences.

High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected approximately 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative alpha-proteobacteria); two sequences each clustered with Simonsiella (beta-proteobacteria) and Serratia (gamma-proteobacteria); and three sequences each clustered with Bartonella (alpha-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6-kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees.

Animals↗
PubMed1996

Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences.

Twenty-two extremely halophilic aerobic archaeal strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago. The isolates were phenotypically clustered into five different groups, and a representative from each group was chosen for further study. Almost the entire sequences of the 16S rRNA genes of these representatives, and of Haloarcula hispanica ATCC 33960, were determined to establish their phylogenetic positions. The sequences of these strains were compared to previously published sequences of 27 reference halophilic archaea (members of the family Halobacteriaceae) and two other archaea, Methanobacterium formicicum DSM 1312 and Methanospirillum hungatei DSM 864. Phylogenetic analysis using approximately 1,400 base comparisons of 16S rRNA-encoding gene sequences demonstrated that the five isolates clustered closely to species belonging to three different genera--Haloferax, Halobacterium, and Haloarcula. Strains E1 and E8 were closely related and identified as members of the species Haloferax volcanii, and strain E12 was closely related and identified as a member of the species Halobacterium salinarum. However, strains E2 and E11 clustered in the Haloarcula branch with Haloarcula hispanica as the closest relative at 98.9 and 98.8% similarity, respectively. Strains E2 and E11 could represent two new species of the genus Haloarcula. However, because strains of these two new species were isolated from a single source, they will not be named until additional strains are isolated from other sources and fully characterized.

Archaea↗
PubMed1991

An automated RNA extraction procedure and application for 16S rRNA sequencing of Leuconostoc amelobiosum.

The determination of 16S ribosomal ribonucleic acid (16S rRNA) primary structures by in vitro reverse transcription requires the extraction of rRNA in pure form. Although a number of high reliable techniques have been developed for the purpose most are fairly complex, involving numerous steps and the wasting of large volumes. It describes here a RNA extraction and purification method, suitable for automatic extractors, which consistently yields reasonable amounts of pure total RNA from prokaryotes, free of DNA and RNases. The rRNA from the type strain Leuconostoc amelobiosum was isolated using this procedure. Its 16S rRNA sequence was determined and a comparative analysis with those from all currently described leuconostocs, including several atypical lactobacilli, revealed very high sequence homology with Leuconostoc citreum confirming Leuconostoc amelobiosum is phylogenetically a member of the genus Leuconostoc sensu stricto.

Base Sequence↗
PubMed1992

Phylogenetic relationship of Cowdria ruminantium, agent of heartwater, to Anaplasma marginale and other members of the order Rickettsiales determined on the basis of 16S rRNA sequence.

The phylogenetic relationship between Cowdria ruminantium and representative members of the orders Rickettsiales and Chlamydiales has been examined on the basis of the sequence of the 16S rRNA. Phylogeny reconstruction by using both parsimony and distance methods supports the conclusion that C. ruminantium is closely related to the Rickettsiales and in particular to the family Anaplasmataceae. A signature of nine base substitutions delineated the linkage of Anaplasma marginale with C. ruminantium and differentiated these two species from the 45 other members of the alpha group of Proteobacteria examined, and five of these base substitutions were unique among all members of the class Proteobacteria examined to date.

Alphaproteobacteria↗
PubMed2001

On the status of the Serranid fish genus Epinephelus: evidence for paraphyly based upon 16S rDNA sequence.

Historically, attempts to elucidate evolutionary relationships among members of the genus Epinephelus (Teleostei: Serranidae), commonly known as groupers, have been hindered by the overwhelming number of species (98, sensu stricto), a pan global distribution, and the lack of morphological specializations traditionally used in ichthyological classification. To date, no comprehensive phylogenetic study, morphological or molecular, to evaluate the monophyly of this genus has been presented. In this study, previous hypotheses regarding the relationships among the American grouper species and the allied genera were evaluated by examination of mitochondrial DNA sequences of the 16S ribosomal DNA region. A 590-bp region of the 16S rDNA gene was amplified using a universal primer pair for 42 serranid species, including members of the genera Epinephelus, Mycteroperca, and Paranthias from the New World and selected Indo-Pacific congeners. Maximum-parsimony criteria and neighbor-joining analysis dispute the monophyly of the American Epinephelus species as previously hypothesized. The data support the monophyly of Cephalopholis only with the inclusion of the morphologically distinct Paranthias and the monophyly of Mycteroperca with the inclusion of the Indo-Pacific Anyperodon leucogrammicus.

Animals↗
PubMed2005

Evaluation of 16S rRNA sequencing and reevaluation of a short biochemical scheme for identification of clinically significant Bacteroides species.

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. In this study, we evaluated the utility of 16S rRNA gene sequencing as a means of identifying clinically important Bacteroides species. We sequenced 231 clinical isolates that had been identified by a short biochemical scheme. Based on the sequence analysis, 192 clinical isolates were assigned to an established species, with the other 39 clinical strains revealing five unique sequences that may represent five novel species. This is in contrast to identification obtained from a short biochemical scheme, by which only 73.5% (172 of 231) of isolates were correctly identified to species level. Based on the solid identification obtained from 16S rRNA gene sequencing, the short biochemical scheme was modified and improved to provide clinical laboratories with an inexpensive and simple alternative for the identification of isolates of clinically significant Bacteroides species.

Bacterial Typing Techniques↗
PubMed2001

Genetic diversity and evolution of Mycoplasma capricolum subsp. capripneumoniae strains from eastern Africa assessed by 16S rDNA sequence analysis.

Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae), the causal agent of contagious caprine pleuropneumonia (CCPP), is a member of the so-called Mycoplasma mycoides cluster. These mycoplasmas have two rRNA operons in which intraspecific variations have been demonstrated. The sequences of the 16S rRNA genes of both operons from 13 field strains of M. capripneumoniae from three neighbouring African countries (Kenya, Ethiopia, and Tanzania) were determined. Four new and unique polymorphism patterns reflecting the intraspecific variations were found. Two of these patterns included length differences between the rrnA and rrnB operons. The length difference in one of the patterns was caused by a two-nucleotide insert (TG) in the rrnB operon and the length difference in the other pattern was due to a three-nucleotide deletion, also in the rrnB operon. Another pattern was characterised by a polymorphic position caused by a mutation that is known to cause streptomycin resistance in other bacterial species. The strain with this pattern was also found to be resistant to streptomycin. Streptomycin resistant clones were selected from four M. capripneumoniae strains to further investigate the correlation of this mutation to streptomycin resistance. Mutations in the 16S rRNA genes had occurred in two of these strains. The fourth pattern included a new polymorphism in position 1059. The results show that polymorphisms in M. capripneumoniae strains can be used as epidemiological markers for CCPP in smaller geographical areas and to study the molecular evolution of this species.

Animals↗
PubMed1993

Phylogenetic affiliation of BEV, a bacterial parasite of the leafhopper Euscelidius variegatus, on the basis of 16S rDNA sequences.

The phylogenetic relationship of a nonflagellated, Gram-negative, rod-shaped intracellular bacterial parasite (BEV) of the leafhopper Euscelidius variegatus to other bacteria within the class Proteobacteria was determined by sequence analysis of 16S rDNAs. The presence of specific signature nucleotides showed this bacterium to be a member of the gamma-3 subdivision of the Proteobacteria. Phylogenetic analysis based on maximum parsimony placed BEV within a clade in the Enterobacteriaceae, which includes a number of bacteria that are facultative symbiotes of insects and have a common ancestor with Proteus vulgaris. Within this clade, BEV is most closely related to a bacterium identified as the secondary endosymbiote of another homopteran, the pea aphid, Acyrthosiphon pisum.

Animals↗
PubMed1995

Phylogenetic analysis of the genus Corynebacterium based on 16S rRNA gene sequences.

The 16S rRNA gene sequences of 30 strains representing 23 validated Corynebacterium species and 7 currently non-valid Corynebacterium species were determined. These sequences were aligned with the sequences of other Corynebacterium species and related actinomycete taxa. A comparative sequence analysis revealed that there is considerable phylogenetic depth and internal structure in the genus Corynebacterium. Turicella otitidis and the amycolate species Corynebacterium amycolatum were located at the periphery of the genus Corynebacterium. It was evident that the species of the genus Corynebacterium form a monophyletic association and, together with other chemotype IV and mycolic acid-containing taxa (including the genera Dietzia, Gordona, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella), form a natural suprageneric group.

Base Sequence↗
PubMed2002

Sequencing of 16S rRNA gene: a rapid tool for identification of Bacillus anthracis.

In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax.

Anthrax↗