Clover proliferation phytoplasma: ‘Candidatus Phytoplasma trifolii’

2004 ◽  
Vol 54 (4) ◽  
pp. 1349-1353 ◽  
Author(s):  
Chuji Hiruki ◽  
Keri Wang
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Reference Strain ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Beet Leafhopper ◽  
Signature Sequences ◽  
Elm Yellows ◽  
The 16S Rrna Gene

Clover proliferation phytoplasma (CPR) is designated as the reference strain for the CP phylogenetic group or subclade, on the basis of molecular analyses of genomic DNA, the 16S rRNA gene and the 16S–23S spacer region. Other strains related to CPR include alfalfa witches'-broom (AWB), brinjal little leaf (BLL), beet leafhopper-transmitted virescence (BLTV), Illinois elm yellows (ILEY), potato witches'-broom (PWB), potato yellows (PY), tomato big bud in California (TBBc) and phytoplasmas from Fragaria multicipita (FM). Phylogenetic analysis of the 16S rRNA gene sequences of BLL, CPR, FM and ILEY, together with sequences from 16 other phytoplasmas that belong to the ash yellows (AshY), jujube witches'-broom (JWB) and elm yellows (EY) groups that were available in GenBank, produced a tree on which these phytoplasmas clearly clustered as a discrete group. Three subgroups have been classified on the basis of sequence homology and the collective RFLP patterns of amplified 16S rRNA genes. AWB, BLTV, PWB and TBBc are assigned to taxonomic subgroup CP-A, FM belongs to subgroup CP-B and BLL and ILEY are assigned to subgroup CP-C. Genetic heterogeneity between different isolates of AWB, CPR and PWB has been observed from heteroduplex mobility assay analysis of amplified 16S rRNA genes and the 16S–23S spacer region. Two unique signature sequences that can be utilized to distinguish the CP group from others were present. On the basis of unique properties of the DNA from clover proliferation phytoplasma, the name ‘Candidatus Phytoplasma trifolii’ is proposed for the CP group.

Overestimation of Streptococcus mutans prevalence by nested PCR detection of the 16S rRNA gene

2006 ◽  
Vol 55 (1) ◽  
pp. 109-113 ◽  
Author(s):  
Ali Al-Ahmad ◽  
Thorsten Mathias Auschill ◽  
Gabriele Braun ◽  
Elmar Hellwig ◽  
Nicole Birgit Arweiler
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Streptococcus Mutans ◽  
Nested Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Direct Pcr ◽  
Specific Primers ◽  
The 16S Rrna Gene

This study was carried out in order to compare two PCR-based methods in the detection of Streptococcus mutans. The first PCR method was based on primers for the 16S rRNA gene and the second method was based on specific primers that targeted the glucosyltransferase gene (gtfB). Each PCR was performed with eight different streptococci from the viridans group, five other streptococci and 17 different non-streptococcal bacterial strains. Direct use of the S. mutans 16S rRNA gene-specific primers revealed that Streptococcus gordonii and Streptococcus infantis were also detected. After amplifying the 16S rRNA gene with universal primers and subsequently performing nested PCR, the S. mutans-specific nested primers based on the 16S rRNA gene detected all tested streptococci. There was no cross-reaction of the gtfB primers after direct PCR. Our results indicate that direct PCR and nested PCR based on 16S rRNA genes can reveal false-positive results for oral streptococci and lead to an overestimation of the prevalence of S. mutans with regards to its role as the most prevalent causative agent of dental caries.

scholarly journals Reassessment of the phylogenetic relationships of Thiomonas cuprina

2007 ◽  
Vol 57 (11) ◽  
pp. 2720-2724 ◽  
Author(s):  
Donovan P. Kelly ◽  
Yoshihito Uchino ◽  
Harald Huber ◽  
Ricardo Amils ◽  
Ann P. Wood
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Significant Similarity ◽  
23S Rrna Gene ◽  
The 16S Rrna Gene

The published sequence of the 16S rRNA gene of Thiomonas cuprina strain Hö5 (=DSM 5495T) (GenBank accession no. U67162) was found to be erroneous. The 16S rRNA genes from the type strain held by the DSMZ since 1990 (DSM 5495T =NBRC 102145T) and strain Hö5 maintained frozen in the Universität Regensburg for 23 years (=NBRC 102094) were sequenced and found to be identical, but to show no significant similarity to the U67162 sequence. This also casts some doubt on the previously published 5S and 23S rRNA gene sequences (GenBank accession nos U67171 and X75567). The correct 16S rRNA gene sequence showed 99.8 % identity to those from Thiomonas delicata NBRC 14566T and ‘Thiomonas arsenivorans’ DSM 16361. The properties of these three species are re-evaluated, and emended descriptions are provided for the genus Thiomonas and the species Thiomonas cuprina.

scholarly journals Horizontal Transfer of Segments of the 16S rRNA Genesbetween Species of the Streptococcus anginosusGroup

2003 ◽  
Vol 185 (24) ◽  
pp. 7241-7246 ◽  
Author(s):  
Leo M. Schouls ◽  
Corrie S. Schot ◽  
Jan A. Jacobs
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Horizontal Transfer ◽  
Blot Hybridization ◽  
Reverse Line Blot ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Streptococcus Anginosus ◽  
The 16S Rrna Gene

ABSTRACT The nature in variation of the 16S rRNA gene of members of the Streptococcus anginosus group was investigated by hybridization and DNA sequencing. A collection of 708 strains was analyzed by reverse line blot hybridization. This revealed the presence of distinct reaction patterns representing 11 different hybridization groups. The 16S rRNA genes of two strains of each hybridization group were sequenced to near-completion, and the sequence data confirmed the reverse line blot hybridization results. Closer inspection of the sequences revealed mosaic-like structures, strongly suggesting horizontal transfer of segments of the 16S rRNA gene between different species belonging to the Streptococcus anginosus group. Southern blot hybridization further showed that within a single strain all copies of the 16S rRNA gene had the same composition, indicating that the apparent mosaic structures were not PCR-induced artifacts. These findings indicate that the highly conserved rRNA genes are also subject to recombination and that these events may be fixed in the population. Such recombination may lead to the construction of incorrect phylogenetic trees based on the 16S rRNA genes.

scholarly journals Comparative Genetic Diversity of the narG, nosZ, and 16S rRNA Genes in Fluorescent Pseudomonads

2003 ◽  
Vol 69 (2) ◽  
pp. 1004-1012 ◽  
Author(s):  
Sandrine Delorme ◽  
Laurent Philippot ◽  
Veronique Edel-Hermann ◽  
Chrystel Deulvot ◽  
Christophe Mougel ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pairwise Comparison ◽  
Fluorescent Pseudomonads ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Good Correspondence ◽  
The 16S Rrna Gene

ABSTRACT The diversity of the membrane-bound nitrate reductase (narG) and nitrous oxide reductase (nosZ) genes in fluorescent pseudomonads isolated from soil and rhizosphere environments was characterized together with that of the 16S rRNA gene by a PCR-restriction fragment length polymorphism assay. Fragments of 1,008 bp and 1,433 bp were amplified via PCR with primers specific for the narG and nosZ genes, respectively. The presence of the narG and nosZ genes in the bacterial strains was confirmed by hybridization of the genomic DNA and the PCR products with the corresponding probes. The ability of the strains to either reduce nitrate or totally dissimilate nitrogen was assessed. Overall, there was a good correspondence between the reductase activities and the presence of the corresponding genes. Distribution in the different ribotypes of strains harboring both the narG and nosZ genes and of strains missing both genes suggests that these two groups of strains had different evolutionary histories. Both dissimilatory genes showed high polymorphism, with similarity indexes (Jaccard) of between 0.04 and 0.8, whereas those of the 16S rRNA gene only varied from 0.77 to 0.99. No correlation between the similarity indexes of 16S rRNA and dissimilatory genes was seen, suggesting that the evolution rates of ribosomal and functional genes differ. Pairwise comparison of similarity indexes of the narG and nosZ genes led to the delineation of two types of strains. Within the first type, the similarity indexes of both genes varied in the same range, suggesting that these two genes have followed a similar evolution. Within the second type of strain, the range of variations was higher for the nosZ than for the narG gene, suggesting that these genes have had a different evolutionary rate.

scholarly journals Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline

2002 ◽  
Vol 184 (8) ◽  
pp. 2131-2140 ◽  
Author(s):  
Catharine A. Trieber ◽  
Diane E. Taylor
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Tetracycline Resistance ◽  
Clinical Isolates ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Nucleotide Substitutions ◽  
The 16S Rrna Gene

ABSTRACT Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 μg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 μg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.

scholarly journals Pyrosequencing ofmcrAand Archaeal 16S rRNA Genes Reveals Diversity and Substrate Preferences of Methanogen Communities in Anaerobic Digesters

2014 ◽  
Vol 81 (2) ◽  
pp. 604-613 ◽  
Author(s):  
David Wilkins ◽  
Xiao-Ying Lu ◽  
Zhiyong Shen ◽  
Jiapeng Chen ◽  
Patrick K. H. Lee
Keyword(s):  
Anaerobic Digestion ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Methanogenic Archaea ◽  
16S Rrna Gene Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
The 16S Rrna Gene

ABSTRACTMethanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes.Methanobacteriales,Methanomicrobiales, andMethanosarcinaleswere detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of themcrAgenes suggested that these digesters were dominated by acetoclastic methanogens, particularlyMethanosarcinales, while the other digesters were dominated by hydrogenotrophicMethanomicrobiales. The proposed euryarchaeotal orderMethanomassiliicoccalesand the uncultured WSA2 group were detected with the 16S rRNA gene, and potentialmcrAgenes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using themcrAgene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance ofmcrAtranscripts in digesters treating sludge and wastewater samples, supporting themcrAgene as a biomarker for methane yield.

scholarly journals Taxonomic note: speciation within the operational group Bacillus amyloliquefaciens based on comparative phylogenies of housekeeping genes

2020 ◽  
pp. 19-26
Author(s):  
Mohamad Syazwan Ngalimat ◽  
Suriana Sabri
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Analyses ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ratio Test ◽  
Group B ◽  
The 16S Rrna Gene ◽  
Operational Group

Many of the publically available Bacillus 16S rRNA genes and genomes in the NCBI database are inconsistently assigned as B. amyloliquefaciens. The highly conserved nature of the 16S rRNA gene makes it fail to differentiate species within the operational group B. amyloliquefaciens. Here, comparative phylogenies of the complete 16S rRNA, gyrB, rpoB, trpB, recA, and cheA nucleotide sequences of bacterial strains within the operational group were analyzed. As the result, the gyrB, rpoB, and trpB phylogenetic analyses showed stable topology that comprised three monophyletic clades: (i) B. amyloliquefaciens; (ii) B. siamensis; and (iii) B. velezensis. Phylogenies derived by comparison of the gyrB, rpoB, trpB, recA, and cheA with the 16S rRNA gene-derived phylogeny was significant as evaluated by the likelihood ratio test. The trpB, rpoB, and trpB gene-derived phylogenies provide a tool for speciation within the operational group B. amyloliquefaciens.

scholarly journals Application of the stochastic labeling methods with random-sequence barcodes for simultaneous quantification and sequencing of environmental 16S rRNA genes

2016 ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Fumio Inagaki
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Copy Number ◽  
Random Sequence ◽  
Digital Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
The 16S Rrna Gene

AbstractNext-generation sequencing (NGS) is a powerful tool for analyzing environmental DNA and provides the comprehensive molecular view of microbial communities. For obtaining the copy number of particular sequences in the NGS library, however, additional quantitative analysis as quantitative PCR (qPCR) or digital PCR (dPCR) is required. Furthermore, number of sequences in a sequence library does not always reflect the original copy number of a target gene because of biases caused by PCR amplification, making it difficult to convert the proportion of particular sequences in the NGS library to the copy number using the mass of input DNA. To address this issue, we applied stochastic labeling approach with random-tag sequences and developed a NGS-based quantification protocol, which enables simultaneous sequencing and quantification of the targeted DNA. This quantitative sequencing (qSeq) is initiated from single-primer extension (SPE) using a primer with random tag adjacent to the 5’ end of target-specific sequence. During SPE, each DNA molecule is stochastically labeled with the random tag. Subsequently, first-round PCR is conducted, specifically targeting the SPE product, followed by second-round PCR to index for NGS. The number of random tags is only determined during the SPE step and is therefore not affected by the two rounds of PCR that may introduce amplification biases. In the case of 16S rRNA genes, after NGS sequencing and taxonomic classification, the absolute number of target phylotypes 16S rRNA gene can be estimated by Poisson statistics by counting random tags incorporated at the end of sequence. To test the feasibility of this approach, the 16S rRNA gene of Sulfolobus tokodaii was subjected to qSeq, which resulted in accurate quantification of 5.0 × 103to 5.0 × 104copies of the 16S rRNA gene. Furthermore, qSeq was applied to mock microbial communities and environmental samples, and the results were comparable to those obtained using digital PCR and relative abundance based on a standard sequence library. We demonstrated that the qSeq protocol proposed here is advantageous for providing less-biased absolute copy numbers of each target DNA with NGS sequencing at one time. By this new experiment scheme in microbial ecology, microbial community compositions can be explored in more quantitative manner, thus expanding our knowledge of microbial ecosystems in natural environments.

scholarly journals Molecular identification of bacteria isolated from culture medium of rotifer fed on fishery waste diet

2020 ◽  
Vol 21 (6) ◽  
Author(s):  
Stenly Wullur ◽  
HATOPAN NAPITUPULU ◽  
LETHA LOISE WANTANIA ◽  
ELVY LIKE GINTING ◽  
VEIBE WAROUW ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Identification ◽  
Culture Medium ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Seawater Salinity ◽  
Identification Of Bacteria ◽  
The 16S Rrna Gene

Abstract. Wullur S, Napitupulu H, Wantania LL, Ginting EL, Warouw V, Tallei TE, Rumengan IFM. 2020. Molecular identification of bacteria isolated from culture medium of rotifer fed on fishery waste diet. Biodiversitas 21: 2735-2740. The aim of this study was 16S-rRNA sequences based molecular identification of bacteria isolated from culture medium of rotifer fed with fishery waste diet (FWD). We cultured rotifer Brachionus rotundiformis in sterilized seawater (salinity 25 ppt) using FWD, following the procedure in Patent No. P00201609066. Bacteria from the culture were collected, homogenized, diluted 10 to 1000 fold, spread on agar plates and incubated at 370C for 24 to 48 hours. Representative colonies of the bacteria according to their morphologies were isolated for further characterization. Genomic DNA of the isolates were extracted, and the 16S rRNA gene of the isolates were amplified. Polymerase Chain Reaction (PCR) product of each isolate was sequenced and queried against the NCBI GenBank database. Six different isolates based on size, color, elevation, margin, and colony were observed during 24-48 hours incubation at 370C. The 16S rRNA genes of the six isolates were successfully amplified and produced DNA band at 1300-1500 bp, with quality value equal to or greater than 20 (QV20+) of each entire sequence around 941-1253 bases. Basic Local Alignment Search Tool (BLAST) queries in the NCBI GenBank and EzBioCloud database using the 16S-rRNA gene sequences showed that the six isolates belong to four different genera, i.e: Bacillus, Staphylococcus, Vibrio, and Alteromonas.

scholarly journals ‘Candidatus Phytoplasma stylosanthis’, a novel taxon with a diverse host range in Australia, characterised using multilocus sequence analysis of 16S rRNA, secA, tuf, and rp genes

2020 ◽  
Author(s):  
Bianca Rodrigues Jardim ◽  
Wycliff M. Kinoti ◽  
Lucy T. T. Tran-Nguyen ◽  
Cherie Gambley ◽  
Brendan Rodoni ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
In Silico ◽  
Phylogenetic Trees ◽  
Gene Tree ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pattern Similarity ◽  
The 16S Rrna Gene

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra, C. papaya, S. officinarum and S. tuberosum were compared and share 99.93–100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other ‘Candidatus Phytoplasma’ species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with ‘Ca. Phytoplasma luffae’ (16SrVIII-A), with which it has 97.17–97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using iPhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of ‘Ca. Phytoplasma luffae’. The unique in silico RFLP patterns were confirmed in vitro. Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein (rp) gene operon (rps19-rpl22-rps3), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon ‘Ca. Phytoplasma stylosanthis’ is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

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