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PubMed1991

Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA.

The primary structures of the 16S rRNAs of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were determined by using the reverse transcription-dideoxy sequencing method. All of the strains exhibited very high levels of sequence similarity (greater than 99%) that were consistent with the close relationships shown by previous DNA hybridization studies. The sequences of B. anthracis Sterne and B. cereus emetic strain NCTC 11143 were found to be identical for a continuous stretch of 1,446 bases and differed from the sequence of B. cereus NCDO 1771T (T = type strain) by only a single nucleotide. The 16S rRNA sequences of B. mycoides and B. thuringiensis differed from each other and from the sequences of B. anthracis and B. cereus by four to nine nucleotides.

Bacillus anthracis↗
PubMed1998

16S rDNA sequence diversity of a culture-accessible part of an anaerobic digestor bacterial community.

A bacterial culture-based inventory with 16S rDNA identification of the isolates was carried out on an anaerobic digestor microbial ecosystem to compare to the 16S rDNA sequences directly retrieved from the ecosystem by a molecular inventory previously made in our laboratory. Twenty OTUs (Operational Taxonomic Units) belonging to five of the major bacterial groups were identified from 338 isolated colonies. The sequences of 13 of the 20 OTUs were not closely related to any hitherto published sequences (less than 96% sequence identity). Six OTUs out of 20 were found to have sequences similar to sequences of the molecular inventory. Despite the biases expected to be associated with the molecular and culture-based methods, the distribution of the isolated OTUs into the different bacterial phyla was similar to that of the molecular OTUs.

Journal Article↗
PubMed1993

Assignment of fatty acid-beta-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16S rRNA sequence analyses.

After enrichment from Chinese rural anaerobic digestor sludge, anaerobic, sporing and nonsporing, saturated fatty acid-beta-oxidizing syntrophic bacteria were isolated as cocultures with H2- and formate-utilizing Methanospirillum hungatei or Desulfovibrio sp. strain G-11. The syntrophs degraded C4 to C8 saturated fatty acids, including isobutyrate and 2-methylbutyrate. They were adapted to grow on crotonate and were isolated as pure cultures. The crotonate-grown pure cultures alone did not grow on butyrate in either the presence or the absence of some common electron acceptors. However, when they were reconstituted with M. hungatei, growth on butyrate again occurred. In contrast, crotonate-grown Clostridium kluyveri and Clostridium sticklandii, as well as Clostridium sporogenes, failed to grow on butyrate when these organisms were cocultured with M. hungatei. The crotonate-grown pure subcultures of the syntrophs described above were subjected to 16S rRNA sequence analysis. Several previously documented fatty acid-beta-oxidizing syntrophs grown in pure cultures with crotonate were also subjected to comparative sequence analyses. The sequence analyses revealed that the new sporing and nonsporing isolates and other syntrophs that we sequenced, which had either gram-negative or gram-positive cell wall ultrastructure, all belonged to the phylogenetically gram-positive phylum. They were not closely related to any of the previously known subdivisions in the gram-positive phylum with which they were compared, but were closely related to each other, forming a new subdivision in the phylum. We recommend that this group be designated Syntrophomonadaceae fam. nov.; a description is given.

Bacteria, Anaerobic↗
PubMed1999

Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA.

The downstream box (DB) is a sequence element that enhances translation of several bacterial and phage mRNAs. It has been proposed that the DB enhances translation by base pairing transiently to bases 1469-1483 of 16S rRNA, the so-called anti-DB, during the initiation phase of translation. We have tested this model of enhancer action by constructing mutations in the anti-DB that alter its mRNA base-pairing potential and examining expression of a variety of DB-containing mRNAs in strains expressing the mutant anti-DB 16S rRNA. We found that the rRNA mutant was viable and that expression of all tested DB-containing mRNAs was completely unaffected by radical alterations in the proposed anti-DB. These findings lead us to conclude that enhancement of translation by the DB does not involve mRNA-rRNA base pairing.

Bacterial Proteins↗
PubMed1998

Tularemia in a common marmoset (Callithrix jacchus) diagnosed by 16S rRNA sequencing.

We report a case of tularemia in a common marmoset (Callithrix jacchus) diagnosed by determination of the isolate's 16S ribosomal RNA (rRNA) gene sequence. Pathological examination of the animal revealed a multifocal acute necrotizing hepatitis, interstitial nephritis, splenitis, and lymphangitis of the mandibular, retropharyngeal, and cervical and mesenteric lymph nodes. Moreover, multiple foci of acute necrosis were found in the epithelium of the jejunum and the interstitium of the lung. Bacteriological investigations revealed a septicemia. The isolated infectious agent was uncommon, not routinely diagnosed in our laboratory and therefore difficult to identify by conventional tools in a reasonable time and effort. thus, we decided to perform a genetic analysis based on the 16S rRNA gene sequence. Thereby, an infection with Francisella tularensis, the causative agent of tularemia, was unambiguously diagnosed. This shows the great advantage 16S rRNA gene sequencing has as a general identification approach for unusual or rare isolates.

Animals↗
PubMed2001

Taxonomic characterization of two rubber degrading bacteria belonging to the species Gordonia polyisoprenivorans and analysis of hyper variable regions of 16S rDNA sequences.

Two cis-1,4-polyisoprene (isoprene rubber) degrading bacteria, strains VH2 and Y2K, were identified as strains of the species Gordonia polyisoprenivorans belonging to the Corynebacterineae, a suborder of the order Actinomycetales. Both showed characteristic growth and degradation of isoprene rubber as described previously for the type strain of G. polyisoprenivorans Kd2 (DSM 44302(T)). For strain VH2 the chemotaxonomic properties were investigated, and DNA-DNA hybridization experiments with the type strain revealed the affiliation to the species G. polyisoprenivorans. The comparison of the 16S rDNA sequences, and especially hyper variable regions of these, led to the classification of strain Y2K to the same species. At present, the species G. polyisoprenivorans comprises three different isolates which share the ability to degrade isoprene rubber potently but which were obtained from different geographic regions.

Actinomycetales↗
PubMed2002

Improved molecular identification of Thermoactinomyces spp. associated with mushroom worker's lung by 16S rDNA sequence typing.

Mushroom worker's lung (MWL) is a hypersensitivity pneumonitis or allergic alveolitis caused by a type III IgG-mediated immunopathogenic inflammatory reaction in the host due to the inhalation of several thermophilic organisms, including Thermoactinomyces spp. It is difficult to distinguish phenotypically the eight species of this genus; therefore, this study sought to develop an improved molecular means of identifying Thermoactinomyces spp. associated with MWL by partial 16S rDNA PCR amplification and direct sequencing. Hypervariable regions within the 16S rRNA gene, which could be employed as signature sequences of the eight individual species, were identified and employed with highly conserved flanking primers to allow initial PCR amplification, before direct DNA sequencing of the 16S rDNA amplicons. A novel 24-mer 16S rDNA oligonucleotide upstream primer was designed from in silico alignments of all Thermoactinomyces spp. and was employed in combination with downstream (reverse) 16S rDNA primers. This permitted the successful identification of all four isolates associated with mushroom workers' lung. The method may be useful in the identification of Thermoactinomyces spp. associated with allergic alveolitis or pneumonitis associated with occupational exposure in agricultural and horticultural environments.

Agricultural Workers' Diseases↗
PubMed2003

Molecular diagnosis of a vascular prosthesis infection, due to Propionibacterium acnes, by amplification and sequencing of 16S rDNA.

We describe a case of indolent vascular prosthesis infection due to Propionibacterium acnes. The microorganism was identified only by amplification and sequencing of 16S rDNA, while standard cultures remained negative. This observation underscores the usefulness of molecular techniques for the diagnosis of infection caused by fastidious microorganisms.

Anti-Bacterial Agents↗
PubMed2006

Validation of 16S rDNA sequencing in microdissected bowel biopsies from Crohn's disease patients to assess bacterial flora diversity.

The bowel flora is implicated in Crohn's disease (CD) pathogenesis but its precise role is still unclear. Several non-mutually exclusive hypotheses have been proposed: an unidentified persistent pathogen; excessive bacterial translocation; an immune system abnormality in response to normal bacteria; or a breakdown in the balance between protective and harmful bacteria. These hypotheses can be tested by identifying bacteria in specific microscopic bowel structures or lesions. The present paper describes a novel technique to assess bacterial flora diversity in bowel biopsies, by combining laser capture microdissection with broad-range 16S rDNA sequencing. Fifty-four samples comprising histologically normal and pathological mucosa, MALT, ulcers, submucosal lymphangiectasias, epithelioid granulomas, and lymph nodes were microdissected out of 30 bowel biopsies from five CD patients. Bacterial 16S rDNA was successfully amplified by PCR in all samples, and PCR products from 15 samples were selected for cloning and sequence analysis. A total of 729 bacterial DNA sequences were analysed, which could be attributed to six different phyla (Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria, and Planctomycetes). DNA from typical bowel bacteria (Enterobacteriaceae, Clostridiales, Bacteroidetes, Fusobacteria) was detected in all microdissected areas. It was thus convincingly demonstrated that 16S rDNA sequencing can be combined with microdissection to study the bowel flora. However, no specific persistent pathogen causal for CD was identified. The results suggest that Enterobacteriaceae may initiate or colonize ulcers in CD. Translocation of bacteria through established mucosal lesions or as a result of increased permeability may be involved in the evolution towards chronic inflammation and in the establishment of persistent lesions. Further study is needed to confirm these preliminary findings.

Adolescent↗
PubMedDate not supplied

[Extensive complementarity of the Shine-Dalgarno region and 3'-terminal sequence of 16S ribosomal RNA is inefficient for translation in vivo].

Translation initiation in Escherichia coli involves as a rule complementary interactions between a Shine-Dalgarno (SD) sequence upstream of the initiation codon and a highly conserved 3'-end sequence of 16S rRNA (anti-SD). The translation efficiency is believed to be directly affected by the affinity of the ribosome to the mRNA initiation region. Earlier, high-affinity RNA ligands to E. coli ribosomes were selected by the SELEX approach, with the ligands containing an extended SD-sequence well represented. In this work, we examined the ability of artificial ribosome binding sites (RBSs) containing such an extended (10-nt) SD-sequence (superSD) to drive translation in vivo, as well as its ability to form the translation initiation complex in vitro. Toe print experiments showed the formation of a ternary initiation complex on mRNA comprising superSD. Moreover, they proved the formation of an extended SD-duplex in the binary 30S-mRNA complex. Nevertheless, the superSD appeared to be inefficient in translation in vivo. We believe that the initiation complex involving a superSD-element is too stable to be functional; it may impede the transition from initiation to elongation, thus disrupting the transcription-translation coupling and inhibiting the formation of polysomes.

Codon, Initiator↗
PubMed1997

Phylogeny and classification of bacteria in the genera Clavibacter and Rathayibacter on the basis of 16s rRNA gene sequence analyses.

A phylogenetic analysis by parsimony of 16S rRNA gene sequences (16S rDNA) revealed that species and subspecies of Clavibacter and Rathayibacter form a discrete monophyletic clade, paraphyletic to Corynebacterium species. Within the Clavibacter-Rathayibacter clade, four major phylogenetic groups (subclades) with a total of 10 distinct taxa were recognized: (I) species C. michiganensis; (II) species C. xyli; (III) species R. iranicus and R. tritici; and (IV) species R. rathayi. The first three groups form a monophyletic cluster, paraphyletic to R. rathayi. On the basis of the phylogeny inferred, reclassification of members of Clavibacter-Rathayibacter group is proposed. A system for classification of taxa in Clavibacter and Rathayibacter was developed based on restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA sequences. The groups delineated on the basis of RFLP patterns of 16S rDNA coincided well with the subclades delineated on the basis of phylogeny. In contrast to previous classification systems, which are based primarily on phenotypic properties and are laborious, the RFLP analyses allow for rapid differentiation among species and subspecies in the two genera.

Actinomycetales↗
PubMed1997

Bacterial diversity in Adirondack mountain lakes as revealed by 16S rRNA gene sequences.

Bacterial communities of seven lakes in the Adirondack Mountains of New York State were characterized by amplification and sequencing of 16S ribosomal DNA. Analysis of over 100 partial sequences revealed a diverse collection of lineages, largely of the class Proteobacteria (19% alpha subdivision, 31% beta subdivision, and 9% gamma subdivision), the phylum Cytophaga-Flavobacteria-Bacteroides (15%), and the order Actinomycetales (18%). Additionally, a number of the sequences were similar to those of the order Verrucomicrobiales. However, few of the sequence types are closely related to those of characterized species. The relative contributions of the groups of sequences differed among the lakes, suggesting that bacterial population structure varies and that it may be possible to relate aquatic bacterial community structure to water chemistry.

Bacteria↗
PubMed2005

Clostridium sardiniense Prévot 1938 and Clostridium absonum Nakamura et al. 1973 are heterotypic synonyms: evidence from phylogenetic analyses of phospholipase C and 16S rRNA sequences, and DNA relatedness.

Clostridium sardiniense Prévot 1938 and Clostridium absonum Nakamura et al. 1973 have long been considered similar in terms of their biological and biochemical properties, but their taxonomic positions have not been clarified by DNA-DNA hybridization studies or rigorous analysis of 16S rRNA genes. In the present study, DNA-DNA hybridization analysis revealed that C. absonum strains DSM 599(T), DSM 600 and KZ 1544 shared 83.0-86.3 % DNA relatedness with C. sardiniense DSM 2632(T). 16S rRNA gene sequence analysis showed that the C. absonum strains also shared high identity with C. sardiniense DSM 2632(T) (99.7, 99.3 and 99.8 % for DSM 599(T), DSM 600 and KZ 1544, respectively), implying that C. absonum and C. sardiniense are synonyms. In addition, alignment of the inferred amino acid sequences for phospholipase C (PLC) indicated 96.5 % identity between PLCs from C. sardiniense and C. absonum, but relatively low identity with other clostridial species. These results strongly suggest that the species C. sardiniense and C. absonum should be united, with the name C. sardiniense having priority.

Bacterial Proteins↗
PubMed1993

Phylogenetic relationships of marine bacteria, mainly members of the family Vibrionaceae, determined on the basis of 16S rRNA sequences.

The phylogenetic relationships of 50 reference strains, mostly marine bacteria which require Na+ for growth, were determined on the basis of 600 16S rRNA nucleotides by using reverse transcriptase sequencing. Strains belonging to 10 genera were included (four genera of the family Vibrionaceae, the genus Aeromonas of the family Aeromonadaceae, and the genera Alteromonas, Marinomonas, Shewanella, Pseudomonas, and Deleya). The sequences were aligned, the similarity values and evolutionary distance values were determined, and a phylogenetic tree was constructed by using the neighbor-joining method. On the basis of our results, the family Vibrionaceae was separated into at least seven groups (genera and families). Vibrio marinus clearly was on a line of descent that was remote from other vibrios. As determined by the similarity and evolutionary distance values, V. marinus is more distantly related to the family Vibrionaceae than the members of the Aeromonadaceae are. Also, Vibrio cholerae strains formed a separate group with Vibrio mimicus at the genus level. Of 30 species of the Vibrionaceae, 17 formed a large phylogenetic cluster. The genus Listonella was found to be a heterogeneous group, and the species were distributed in various subgroups of the Vibrionaceae. The separation of the family Aeromonadaceae from the family Vibrionaceae and the separation of the genera Marinomonas and Shewanella from the genus Alteromonas were confirmed in this phylogenetic study. However, a marine Pseudomonas species, Pseudomonas nautica, was clearly separated from two terrestrial Pseudomonas species. Each group that was separated by the phylogenetic analysis had characteristic 16S rRNA sequence patterns that were common only to species in that group. Therefore, the characteristic sequences described in this paper may be useful for identification purposes.

Base Sequence↗
PubMed1994

Oligonucleotide probes for Bordetella bronchiseptica based on 16S ribosomal RNA sequences.

Bordetella bronchiseptica 16S ribosomal RNA (rRNA) gene was cloned and identified. On the basis of information from computer-assisted sequence comparison of the B. bronchiseptica 16S RRNA sequences with that of other bacterial species, we constructed B. bronchiseptica-specific oligonucleotide probes complementary to variable regions in the 16S rRNA molecule. Specificity of these 32P-labeled oligo-nucleotide probes was tested in a RNA/DNA hybridization with B. bronchiseptica strains and other bacterial strains. Probe BB4 was more specific than three other oligonucleotide probes. This probe BB4 was sensitive enough to be able to detect 10(4) bacterial cells.

Base Sequence↗