Speciation in bacteria: comparison of the 16S rRNA gene for closely related Enterococcus species

Sequencing of the 16S rRNA genes from enterococcal strains used as starters suggested the existence of specialized taxa of lactic acid enterococci within the species Enterococcus durans and E. faecium and a new species, E. lactis. Comparisons showed that the 16S rRNA genes of closely related species have the same sets of variable positions with different combinations of nucleotides. The presence of identical combinations of nucleotide substitutions in different species was assumed to result from a transfer of genetic information via gene conversion between different rRNA operons. Such events were presumably associated with speciation in bacteria.     

Variation of 3'-terminal fragment of 16S rRNA gene in closely related species of Drosophila virilis group

Primary sequence of 3-terminal fragment of the mitochondrial 16S rRNA gene has been determined in 12 Drosophila species of the virilis group. The functionally important elements in secondary structure of the RNA product were defined. The region corresponding to the peptidyltransferase center has been localized. Variation of the 3'-terminal region of 16S rRNA gene has been described in 12 species of the virilis group. Phylogeny of the Drosophila virilis species group is discussed.     

Comparison of partial 16S rRNA gene sequences obtained from activated sludge bacteria

The cultivated and uncultivated bacterial communities of an activated sludge plant were studied. Two samples were taken and a total of 516 bacterial isolates were classified into groups using their whole-cell protein patterns. The distribution of bacteria into protein-pattern groups differed significantly between the two samples, suggesting variation in culturable bacterial flora. Partial 16S rRNA gene sequences were determined for representatives of the commonest protein-pattern groups. Most of the sequences obtained were previously unknown, but relatively closely related to known sequences of organisms belonging to the α, β or γ subclasses of the proteobacteria, the first two subclasses being predominant. This classification of bacteria isolated on a diluted nutrient-rich medium differed from recent culture-dependent studies using nutrient-rich media. The uncultivated bacterial community was studied by analyzing ten partial 16S rRNA gene sequences cloned directly from activated sludge. None of the cloned sequences was identical to those determined for culturable organisms; or to those in the GenBank database. They were, however, related to the α or β subclasses of the proteobacteria, or to the gram-positive bacteria with a high G+C DNA content. Received: 4 November 1996 / Received revision: 24 February 1997 / Accepted: 28 February 1997     

Identification of Comamonas species using 16S rRNA gene sequence

A bacterial strain Bz02 was isolated from a water sample collected from river Gomti at the Indian city of Lucknow. We characterized the strain using 16S rRNA sequence. Phylogenetic analysis showed that the strain formed a monophyletic clade with members of the genus Comamonas. The closest phylogenetic relative was Comamonas testosteroni with 95% 16S rRNA gene sequence similarity. It is proposed that the identified strain Bz02 be assigned as the type strain of a species of the genus Comamonas (Comamonas sp Bz02) based on 16S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases together with the phylogenetic tree analysis. The sequence is deposted in GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ211417","term_id":"209164434"}}FJ211417.     

Bacteria closely related to Phyllobacterium trifolii according to their 16S rRNA gene are discovered in the nodules of Hungarian sainfoin

The population genetic diversity and phylogeny of the bacteria entering the symbiosis with sainfoin that grows on the Chesnokovskaya Mountain, Ufa region, Republic of Bashkortostan, have been studied. RAPD analysis of DNA polymorphism of the microbial strains grown from the nodules of 20 plants using several random primers detected a high degree of genetic homogeneity in their population as compared with the populations of rhizobia of other leguminous plants growing at the same site. Sequencing of 16S rRNA genes of the three most different samples have demonstrated that these genes were identical and display 99.9% homology with the sequence of Phyllobacterium trifolii 16S rRNA gene.     

Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region

A rapid and reliable PCR-based method for distinguishing closely related species within two groups of lactobacilli is described. Primers complementary to species-specific sequences in the 16S/23S rDNA spacer regions were designed after sequencing and sequence comparison of the spacer regions of 32 strains. The strains belong to two groups of closely related Lactobacillus species; one composed of Lactobacillus curvatus, Lactobacillus graminis and Lactobacillus sake, the other of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum. PCR assays with the designed primers and subsequent agarose gel analysis of the amplified fragments allowed the same species identification as the DNA/DNA hybridization procedure.     

Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria.

We have conducted a preliminary phylogenetic survey of ammonia-oxidizing beta-proteobacteria, using 16S rRNA gene libraries prepared by selective PCR and DNA from acid and neutral soils and polluted and nonpolluted marine sediments. Enrichment cultures were established from samples and analyzed by PCR. Analysis of 111 partial sequences of c. 300 bases revealed that the environmental sequences formed seven clusters, four of which are novel, within the phylogenetic radiation defined by cultured autotrophic ammonia oxidizers. Longer sequences from 13 cluster representatives support their phylogenetic positions relative to cultured taxa. These data suggest that known taxa may not be representative of the ammonia-oxidizing beta-proteobacteria in our samples. Our data provide further evidence that molecular and culture-based enrichment methods can select for different community members. Most enrichments contained novel Nitrosomonas-like sequences whereas novel Nitrosospira-like sequences were more common from gene libraries of soils and marine sediments. This is the first evidence for the occurrence of Nitrosospira-like strains in marine samples. Clear differences between the sequences of soil and marine sediment libraries were detected. Comparison of 16S rRNA sequences from polluted and nonpolluted sediments provided no strong evidence that the community composition was determined by the degree of pollution. Soil clone sequences fell into four clusters, each containing sequences from acid and neutral soils in varying proportions. Our data suggest that some related strains may be present in both samples, but further work is needed to resolve whether there is selection due to pH for particular sequence types.     

Comparison of genotypic and phylogenetic relationships of environmental Enterococcus isolates by BOX-PCR typing and 16S rRNA gene sequencing

Environmental Enterococcus spp. were compared by BOX-PCR genotyping and 16S rRNA gene sequencing to clarify the predictive relationship of BOX-PCR fingerprints to species designation. BOX-PCR and 16S rRNA gene relationships agreed for 77% of strains. BOX-PCR provided superior intraspecies discrimination but incorrectly identified some strains to the species level and divided some species into multiple groups.     

Profiling of the bacteria responsible for pyogenic liver abscess by 16S rRNA gene pyrosequencing

Pyogenic liver abscess (PLA) is a severe disease with considerable mortality and is often polymicrobial. Understanding the pathogens that cause PLA is the basis for PLA treatment. Here, we profiled the bacterial composition in PLA fluid by pyrosequencing the 16S ribosomal RNA (rRNA) gene based on next-generation sequencing (NGS) technology to identify etiological agents of PLA and to provide information of their 16S rRNA sequences for application to DNA-based techniques in the hospital. Twenty patients with PLA who underwent percutaneous catheter drainage, abscess culture, and blood culture for isolates were included. Genomic DNAs from abscess fluids were subjected to polymerase chain reaction and pyrosequencing of the 16S rRNA gene with a 454 GS Junior System. The abscess and blood cultures were positive in nine (45%) and four (20%) patients, respectively. Pyrosequencing of 16S rRNA gene showed that 90% of the PLA fluid samples contained single or multiple genera of known bacteria such as Klebsiella, Fusobacterium, Streptococcus, Bacteroides, Prevotella, Peptostreptococcus, unassigned Enterobacteriaceae, and Dialister. Klebsiella was predominantly found in the PLA fluid samples. All samples that carried unassigned bacteria had 26.8% reads on average. We demonstrated that the occurrence of PLA was associated with eight known bacterial genera as well as unassigned bacteria and that 16S rRNA gene sequencing was more useful than conventional culture methods for accurate identification of bacterial pathogens from PLA.     

A renaissance for the pioneering 16S rRNA gene

Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the past quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata, and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.     

Rapid identification of Enterococcus italicus by PCR with primers targeted to 16S rRNA gene

AIMS: To develop a species-specific PCR assay with primers targeted to 16S rRNA gene for the identification of Enterococcus italicus, a new species of Enterococcus, involved in the production of Italian cheeses. METHODS AND RESULTS: The type strain of E. italicus (DSM 15952(T) - 16S rRNA gene accession no. AJ582753) and other strains of the species were subjected to a rapid identification by PCR using primer pairs located within the 16S rRNA gene. A species-specific PCR product of approximately 323 bp was obtained after amplification of all E. italicus strains tested. The specificity of the primers was validated with representatives of the most closely related genera and species and a number of other bacterial species. In addition, the technique enabled the recognition of E. italicus from cheeses. CONCLUSIONS: The protocol was highly efficient and sensitive, enabling the identification of E. italicus from cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific PCR offers a reliable and rapid alternative to conventional phenotypic methods for the identification of E. italicus within the heterogeneous genus Enterococcus.     

Phylogeny of spore-forming lactic acid bacteria based on 16S rRNA gene sequences

Abstract The phylogeny of spore-forming lactic acid bacteria was investigated on the basis of 16S rRNA gene sequences. Sixteen strains were separated into three lines of descent; one consisted of 14 strains assigned to Sporolactobacillus spp. and Bacillus spp., and the other two each consisted of " Sporolactobacillus dextrus " and Bacillus coagulans . Strains of all the first lineage but one composed a cluster of similarity values of 97.2% and higher, and were represented by the type of S. inulinus . The cluster was further separated into five subclusters, four catalase negative and one positive. The definition of the genus Sporolactobacillus should be amended to accomodate catalase positive strains. Spore-forming lactic acid bacteria originated at different phylogenetic positions, and would have evolved convergently in the area of Bacillus .     

Characterization and identification of compost bacteria based on 16S rRNA gene sequencing

The aim of this study was to isolate and characterize bacteria from the compost of fruit and vegetable waste (FVW) for plant growth-promoting (PGP) activities and investigate the pro-active influence of bacterial isolates on wheat growth. Fourteen bacterial strains (RHC-1 to RHC-14) were isolated and purified in tryptic soya agar (TSA). In addition to being biochemically characterized, these bacterial strains were also tested for their PGP traits, such as phosphate (P)-solubilization, nifH gene amplification, indole-3-acetic acid (IAA) quantification and the production of ammonia, oxidase and catalase. Based on 16S rRNA gene sequencing, these bacterial strains were identified as belonging to species of Bacillus, Lysinibacillus, Lysobacter, Staphylococcus, Enterobacter, Pseudomonas and Serratia. All bacterial strains solubilized tri-calcium phosphate and produced IAA. Two bacterial strains RHC-8 (Enterobacter sp.) and RHC-13 (Pseudomonas sp.) solubilized the maximum amount of tri-calcium phosphate, i.e. 486 and 464 μg/ml, respectively. P-solubilization was associated with a significant drop in the pH of the broth culture from an initial pH of 7 to pH 4.43. In addition to P-solubilization and IAA production, six bacterial strains also carried the nifH gene and were further evaluated for their effect on wheat (Triticum aestivum) growth under controlled conditions. All six bacterial strains enhanced wheat growth as compared to uninoculated control plants. Two of the bacterial strains, RHC-8 and RHC-13, identified as Enterobacter aerogenes and Pseudomonas brenneri, respectively, were assessed as potential PGP rhizobacteria due to exhibiting characteristics of four or more PGP traits and enhancing wheat growth though their specific mechanism of action.     

16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria

Aims

Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.

Methods and Results

Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1–V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture.

Conclusions

Sequencing the V1–V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare.

Significance and Impact of the Study

We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.     

16S rRNA gene phylogeny and tfdA gene analysis of 2,4-D-degrading bacteria isolated in China

Twenty-two 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial isolates were collected from agricultural soils at three sites in China. Sequence analysis of the 16S rRNA genes indicated that the isolates were phylogenetically grouped into four categories: Ochrobactrum anthropi, in the Alpha- class of the phylum Proteobacteria (3 out of 22 isolates), Cupriavidus sp., of the Betaproteobacteria (3 out of 22), Pseudomonas sp. and Stenotrophomonas sp., which are Gammaproteobacteria (7 out of 22), and Bacillus sp., of the phylum Firmicutes (9 out of 22). Primers were designed to amplify the conserved domain of tfdA, which is known to be involved in the degradation of 2,4-D. Results showed that the tfdA genes of all 22 strains were most similar to that of Cupriavidus necator JMP134, which belongs to the 2,4-D/α-ketoglutarate dioxygenase TfdA protein family, indicating that the JMP134-type tfdA gene is likely to be almost universal among the 2,4-D-degrading bacteria isolated from China. Degradation abilities of these 22 strains were investigated in assays using 2,4-D as the sole source of carbon and energy. Thirteen strains degraded >60 % of the available 2,4-D (500 mg l^?1) over a 1-week incubation period, while a further nine Bacillus sp. strains degraded 50–81 % of the available 2,4-D. None of these nine strains degraded other selected herbicides, such as mecoprop, 2-methyl-4-chlorophenoxyacetic acid, quizalofop, and fluroxypyr. This is the first report of 2,4-D-degradation by Bacilli.     

Isolation of endophytic bacteria from arboreal species of the Amazon and identification by sequencing of the 16S rRNA encoding gene

Endophytic bacteria from three arboreal species native to the Amazon (Carapa guianenses, Ceiba pentandra, and Swietenia macrophylla), were isolated and identified, through partial sequencing of the 16S rRNA encoding gene. From these, 16 isolates were obtained, although, when compared to sequences deposited in GenBank, only seven had produced identifiable fragments. Bacillus, Pantoea and two non-culturable samples were identified. Results obtained through sequence analysis revealed low genetic diversity across the isolates, even when analyzing different species and plant structures. This is the first report concerning the isolation and identification of endophytic bacteria in these plant species.     

Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene

To allow rapid identification of bacteria in pure cultures and blood culture bottles, an assay was developed which is based on real-time amplification and sequencing of bacterial 16 S rRNA genes. In principle, this assay allows identification of bacteria from pure cultures within 6.5 h, and from blood cultures within approximately 7 h.