Ribosomal DNA

About: Ribosomal DNA is a research topic. Over the lifetime, 7266 publications have been published within this topic receiving 407281 citations.

16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates

Abstract: Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of > or =97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of > or =99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

Revised Classification Scheme of Phytoplasmas based on RFLP Analyses of 16S rRNA and Ribosomal Protein Gene Sequences

Abstract: RFLP analyses of 16S rDNA nested PCR products from 34 phytoplasma strains with 17 restriction enzymes delineated distinct pattern types. Based on similarity coefficients derived from RFLP analyses, the 34 representative phytoplasma strains were differentiated into 14 major groups (termed 16Sr groups) and 32 sub-groups. The similarity coefficients of RFLP patterns between distinct groups were 90% or below. By including additional groups and sub-groups from which RFLP analyses were not performed but for which 16S rDNA sequence data were available to predict restriction sites, a total of 14 groups and 41 sub-groups were proposed. By combined RFLP analyses of 16S rRNA and ribosomal protein gene sequences, thus far, a total of 46 subgroups have been recognized. The phytoplasma 16Sr groups were consistent with the phylogenetic groups (subclades) defined by phylogenetic analysis of near-full-length 16S rRNA gene sequences, indicating that the RFLP-based groups are phylogenetically valid. The approach using RFLP analyses of PCR-amplified 16S rDNA (and ribosomal protein gene sequences) provides a simple, reliable and rapid means for differentiation and classification of unknown phytoplasmas.

New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region

Abstract: Medical microbiology is extremely reliant on the culture of bacteria from clinical specimens and their subsequent identification by biochemical and phenotypic characteristics for the diagnosis of disease. Following determination of the structure of DNA by Watson & Crick (1953), studies in bacteriology have seen a major shift from functional to molecular techniques for identifying bacteria (Towner & Cockayne, 1993). This review will deal with the 16s-23s spacer region of the rRNA operon (Fig. 1) and its use in the identification of micro-organisms at the species and strain (typing) levels.

Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

Abstract: Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519–539, E969–983, E1063–1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.