scholarly journals Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes

2010 ◽  
Vol 76 (12) ◽  
pp. 3886-3897 ◽  
Author(s):  
Anna Y. Pei ◽  
William E. Oberdorf ◽  
Carlos W. Nossa ◽  
Ankush Agarwal ◽  
Pooja Chokshi ◽  
...  
Keyword(s):  
16S Rrna ◽  
Bacterial Species ◽  
Human Microbiome ◽  
Operational Definition ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Thermoanaerobacter Tengcongensis ◽  
A Genome ◽  
Prokaryotic Genomes

ABSTRACT Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp ). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 ± 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% ± 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2° structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in “Candidatus Protochlamydia amoebophila.” Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.

scholarly journals In-depth analysis of Bacillus anthracis 16S rRNA genes and transcripts reveals intra- and intergenomic diversity and facilitates anthrax detection

2021 ◽  
Author(s):  
Peter Braun ◽  
Fee Zimmermann ◽  
Mathias C Walter ◽  
Sonja Mantel ◽  
Karin Aistleitner ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Close Relative ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Depth Analysis

Analysis of 16S ribosomal RNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ-, in vitro- and in silico-assays to assess the yet unknown 16S-state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing and bioinformatics we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B. anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis genome data-sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy-numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy-numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons.

scholarly journals 16S rRNA Gene-Based Identification of Midgut Bacteria from Field-Caught Anopheles gambiae Sensu Lato and A. funestus Mosquitoes Reveals New Species Related to Known Insect Symbionts

2005 ◽  
Vol 71 (11) ◽  
pp. 7217-7223 ◽  
Author(s):  
Jenny M. Lindh ◽  
Olle Terenius ◽  
Ingrid Faye
Keyword(s):  
16S Rrna ◽  
Anopheles Gambiae ◽  
Bacterial Species ◽  
Anopheles Funestus ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Malaria Vectors ◽  
Rrna Gene ◽  
Species Determination ◽  
Insect Symbionts

ABSTRACT Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the γ-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.

scholarly journals Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

1998 ◽  
Vol 64 (2) ◽  
pp. 795-799 ◽  
Author(s):  
Julian R. Marchesi ◽  
Takuichi Sato ◽  
Andrew J. Weightman ◽  
Tracey A. Martin ◽  
John C. Fry ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Amplification ◽  
Environmental Samples ◽  
Bacterial Species ◽  
Pcr Primers ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Specific Pcr

ABSTRACT We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.

scholarly journals Species-Specific PCR for Identification of Common Contaminant Mollicutes in Cell Culture

2001 ◽  
Vol 67 (7) ◽  
pp. 3195-3200 ◽  
Author(s):  
Fanrong Kong ◽  
Gregory James ◽  
Susanna Gordon ◽  
Anna Zelynski ◽  
Gwendolyn L. Gilbert
Keyword(s):  
16S Rrna ◽  
Cell Cultures ◽  
Bacterial Species ◽  
Intergenic Spacer ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Species Specific

ABSTRACT Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5′ end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5′ end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.

scholarly journals Characterization of the core bacteriobiome in the rhizosphere of greenhouse vegetables: taxonomic diversity and putative functions

2021 ◽  
Author(s):  
Natalia B. Naumova ◽  
Oleg A. Savenkov ◽  
Tatiana Yu. Alikina ◽  
Yury V. Fotev
Keyword(s):  
16S Rrna ◽  
Plant Pathogens ◽  
Bacterial Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bitter Melon ◽  
Soil Substrate ◽  
Benincasa Hispida ◽  
Wax Gourd

The aim of the study. The aim was to profile 16S rRNA gene diversity and to assess functional potential of bacterial assemblages in the rhizosphere of some unconventional vegetables grown in protected greenhouse conditions in West Siberia. Location and time of the study. Novosibirsk, Russia, 2016. Methodology. At the end of the growing season in the middle of September the rhizosphere soil was collected from the plants of wax gourd (Benincasa hispida), bitter melon (Momordica charantia), kiwano (Cucumis metuliferus) and cowpea (Vigna unguiculata) grown on peat-based substrate in a polyethylene-protected greenhouse that has been in operation for more than 40 years. The metagenomic DNA was extracted and amplified with V3-V4 primers for 16S rRNA genes, and the amplicons sequenced with Illumina MiSeq. The obtained OTUs tables were used to predict putative functions by running through the FAPROTAX database. Main results. The rhizosphere bacteriobiome was dominated by Proteobacteria (32±11% of the total number of sequence reads), Acidobacteria (23±7%) and Actinobacteria (18±3%) phyla, together accounting for about three quarters of the rhizosphere bacteriobiome. In total 20 bacterial phyla were found. The rhizosphere bacteriobiome was surprisingly diverse with Shannon index ranging 7.0–7.5. The number of the observed operational taxonomic units (OTUs) per sample was very high, ranging 4,500–4,900, and the potential number of OTUs estimated as 5,100–5,700; all those OTUs were evenly and equitably represented in the bacteriobiome, and dominance indices (Simpson dominance and Berger-Parker) were very low. The main dominant OTU represented Bradyrhizobiaceae family and accounted for just 1% on average. Overall the study identified 27 OTUs belonging to the Bradyrhizobiaceae family, but only four of them were ascribed to nitrogen fixation by FAPROTAX. Function prediction by FAPROTAX also suggested that bacteriobiome had a marked potential for the carbon cycle, denitrification, aromatic compound and plant polymer degradation, but no plant pathogens. The biggest difference in rhizosphere bacteriobiome diversity was observed between the bitter melon and the other three vegetable crops: bitter melon had much increased abundance of Arthrobacter and Sphingomonas as compared with wax gourd, kiwano and cowpea, and increased number of bacterial species associated with aromatic compounds degradation. Conclusion. Based on the finding that the studied rhizosphere bacteriobiomes were very diverse, we conclude that the crops were able to recruit diverse microbiota from the peat-based soil substrate, which, in its turn, means that diverse soil substrate microbiota has been sustained over several decades of the greenhouse operation. All crops apparently shaped distinct bacteriobiomes in their rhizosphere, which ideally should be included into studies of plant-associated bacterial diversity profiles for breeding and sustainable production.

scholarly journals Dictyobacter vulcani sp. nov., belonging to the class Ktedonobacteria, isolated from soil of the Mt Zao volcano

2020 ◽  
Vol 70 (3) ◽  
pp. 1805-1813 ◽  
Author(s):  
Yu Zheng ◽  
Chiung-mei Wang ◽  
Yasuteru Sakai ◽  
Keietsu Abe ◽  
Akira Yokota ◽  
...  
Keyword(s):  
Cell Wall ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
A Genome

An aerobic, Gram-stain-positive, mesophilic Ktedonobacteria strain, W12T, was isolated from soil of the Mt Zao volcano in Miyagi, Japan. Cells were filamentous, non-motile, and grew at 20–37 °C (optimally at 30 °C), at pH 5.0–7.0 (optimally at pH 6.0) and with <2 % (w/v) NaCl on 10-fold diluted Reasoner’s 2A (R2A) medium. Oval-shaped spores were formed on aerial mycelia. Strain W12T hydrolysed microcrystalline cellulose and xylan very weakly, and used d-glucose as its sole carbon source. The major menaquinone was MK-9, and the major cellular fatty acids were C16 : 1 2-OH, iso-C17 : 0, summed feature 9 (10-methyl C16 : 0 and/or iso-C17 : 1ω9c) and anteiso-C17 : 0. Cell-wall sugars were mannose and xylose, and cell-wall amino acids were d-glutamic acid, glycine, l-serine, d-alanine, l-alanine, β-alanine and l-ornithine. Polar lipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid and an unidentified phospholipid. Strain W12T has a genome of 7.42 Mb with 49.7 mol% G+C content. Nine copies of 16S rRNA genes with a maximum dissimilarity of 1.02 % and 13 biosynthetic gene clusters mainly coding for peptide products were predicted in the genome. Phylogenetic analysis based on both 16S rRNA gene and whole genome sequences indicated that strain W12T represents a novel species in the genus Dictyobacter . The most closely related Dictyobacter type strain was Dictyobacter alpinus Uno16T, with 16S rRNA gene sequence similarity and genomic average nucleotide identity of 98.37 % and 80.00 %, respectively. Herein, we propose the name Dictyobacter vulcani sp. nov. for the type strain W12T (=NBRC 113551T=BCRC 81169T) in the bacterial class Ktedonobacteria .

scholarly journals Vaginal Microbiota of Women with Frequent Vulvovaginal Candidiasis

2009 ◽  
Vol 77 (9) ◽  
pp. 4130-4135 ◽  
Author(s):  
Xia Zhou ◽  
Rachel Westman ◽  
Roxana Hickey ◽  
Melanie A. Hansmann ◽  
Colleen Kennedy ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Bacterial Communities ◽  
Bacterial Species ◽  
Vulvovaginal Candidiasis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Novel Bacteria

ABSTRACT Vulvovaginal candidiasis (VVC) is an insidious infection that afflicts a large proportion of women of all ages, and 5 to 8% of affected women experience recurrent VVC (RVVC). The aim of this study was to explore the possible importance of vaginal bacterial communities in reducing the risk of RVVC. The species composition and diversity of microbial communities were evaluated for 42 women with and without frequent VVC based on profiles of terminal restriction fragment polymorphisms of 16S rRNA genes and phylogenetic analysis of cloned 16S rRNA gene sequences from the numerically dominant microbial populations. The data showed that there were no significant differences between the vaginal microbial communities of women in the two groups (likelihood score, 5.948; bootstrap P value, 0.26). Moreover, no novel bacteria were found in the communities of women with frequent VVC. The vaginal communities of most women in both groups (38/42; 90%) were dominated by species of Lactobacillus. The results of this study failed to provide evidence for the existence of altered or unusual vaginal bacterial communities in women who have frequent VVC compared to women who do not have frequent VVC. The findings suggest that commensal vaginal bacterial species may not be able to prevent VVC.

scholarly journals FILOGENI MOLEKULER BAKTERI DARI MEDIA PEMELIHARAAN ROTIFER YANG DIBERI OLAHAN LIMBAH IKAN SEBAGAI SUMBER NUTRISI

2021 ◽  
Vol 9 (1) ◽  
pp. 38
Author(s):  
Herlin S Hubu ◽  
Stenly Wullur ◽  
Veibe Warouw ◽  
Elvy L Ginting ◽  
Robert A Bara ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Local Alignment ◽  
Rrna Gene ◽  
Fish Waste ◽  
Positive Effects ◽  
The 16S Rrna Gene

This study aims to identify and construct molecular phylogeny of an isolate bacteria from culture media of rotifer Brachionus rotudiforis supplied with processed fishery waste feed as nutritional source. The use of fish waste-based food for rotifer showed positive effects on growth and nutrient content of the rotifers. Genomic DNA of the isolate bacteria BRLI- 01 was extracted and the 16S rRNA gene was amplified using primers (8F and 1492F) and further sequenced using Sanger sequence technique. The 16S rRNA gene was analysed using SeqScanner® and MEGA® followed with BLAST (Basic Local Alignment Search Tool) analyses in the NCBI (National Centre for Biotechnology Information). Amplification result of 16S rRNA gene bacteria s NCBI site as a reference for identification and phylogeny of bacterial species. BRLI-01 was successfully cultured on rotifer rearing media. The results of the 16S rRNA gene amplification of the isolate bacteria showed a DNA band with a length of 1400 bp. The BLAST result on the NCBI showed that the isolate bacteria BRLI-01 had a percent identity (98.46%) and is in the same phylogony branching position with Vibrio rotiferianus Keywords: Rotifers, Bacteria, Fish waste, 16S rRNA Genes, Phylogeny identification

scholarly journals Engineering CRISPR/Cas9 to mitigate abundant host contamination for 16S rRNA gene-based amplicon sequencing

Microbiome ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Luyang Song ◽  
Kabin Xie
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput Sequencing ◽  
Bacterial Species ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Plant Microbiota

Abstract Background High-throughput sequencing of bacterial 16S rRNA gene (16S-seq) is a useful and common method for studying bacterial community structures. However, contamination of the 16S rRNA genes from the mitochondrion and plastid hinders the sensitive bacterial 16S-seq in plant microbiota profiling, especially for some plant species such as rice. To date, efficiently mitigating such host contamination without a bias is challenging in 16S rRNA gene-based amplicon sequencing. Results We developed Cas-16S-seq method to reduce abundant host contamination for plant microbiota profiling. This method utilizes the Cas9 nuclease and specific guide RNA (gRNA) to cut 16S rRNA targets during library construction, thereby removing host contamination in 16S-seq. We used rice as an example to validate the feasibility and effectiveness of Cas-16S-seq. We established a bioinformatics pipeline to design gRNAs that specifically target rice 16S rRNA genes without bacterial 16S rRNA off-targets. We compared the effectiveness of Cas-16S-seq with that of the commonly used 16S-seq method for artificially mixed 16S rRNA gene communities, paddy soil, rice root, and phyllosphere samples. The results showed that Cas-16S-seq substantially reduces the fraction of rice 16S rRNA gene sequences from 63.2 to 2.9% in root samples and from 99.4 to 11.6% in phyllosphere samples on average. Consequently, Cas-16S-seq detected more bacterial species than the 16S-seq in plant samples. Importantly, when analyzing soil samples, Cas-16S-seq and 16S-seq showed almost identical bacterial communities, suggesting that Cas-16S-seq with host-specific gRNAs that we designed has no off-target in rice microbiota profiling. Conclusion Our Cas-16S-seq can efficiently remove abundant host contamination without a bias for 16S rRNA gene-based amplicon sequencing, thereby enabling deeper bacterial community profiling with a low cost and high flexibility. Thus, we anticipate that this method would be a useful tool for plant microbiomics.

scholarly journals Amplicon sequence variants artificially split bacterial genomes into separate clusters

2021 ◽  
Author(s):  
Patrick D. Schloss
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Genome ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Genomes ◽  
A Genome ◽  
The 16S Rrna Gene

AbstractAmplicon sequencing variants (ASVs) have been proposed as an alternative to operational taxonomic units (OTUs) for analyzing microbial communities. ASVs have grown in popularity, in part, because of a desire to reflect a more refined level of taxonomy since they do not cluster sequences based on a distance-based threshold. However, ASVs and the use of overly narrow thresholds to identify OTUs increase the risk of splitting a single genome into separate clusters. To assess this risk, I analyzed the intragenomic variation of 16S rRNA genes from the bacterial genomes represented in a rrn copy number database, which contained 20,427 genomes from 5,972 species. As the number of copies of the 16S rRNA gene increased in a genome, the number of ASVs also increased. There was an average of 0.58 ASVs per copy of the 16S rRNA gene for full length 16S rRNA genes. It was necessary to use a distance threshold of 5.25% to cluster full length ASVs from the same genome into a single OTU with 95% confidence for genomes with 7 copies of the 16S rRNA, such as E. coli. This research highlights the risk of splitting a single bacterial genome into separate clusters when ASVs are used to analyze 16S rRNA gene sequence data. Although there is also a risk of clustering ASVs from different species into the same OTU when using broad distance thresholds, those risks are of less concern than artificially splitting a genome into separate ASVs and OTUs.

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