scholarly journals Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies

2006 ◽  
Vol 73 (1) ◽  
pp. 278-288 ◽  
Author(s):  
Rebecca J. Case ◽  
Yan Boucher ◽  
Ingela Dahllöf ◽  
Carola Holmström ◽  
W. Ford Doolittle ◽  
...  
Keyword(s):  
16S Rrna ◽  
Molecular Marker ◽  
16S Rrna Gene ◽  
Microbial Ecology ◽  
Gene Tree ◽  
Single Copy ◽  
Rrna Gene ◽  
Phylogenetic Resolution ◽  
The Impact ◽  
The 16S Rrna Gene

ABSTRACT Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such “heterogeneity hot spots” occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

scholarly journals Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor

2017 ◽  
Vol 28 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Anniina Rintala ◽  
Sami Pietilä ◽  
Eveliina Munukka ◽  
Erkki Eerola ◽  
Juha-Pekka Pursiheimo ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Rrna Gene ◽  
Gene Target ◽  
Target Region ◽  
Microbiota Analysis ◽  
The Impact ◽  
The 16S Rrna Gene

The applications of the 16S rRNA gene in microbial ecology: current situation and problems

2015 ◽  
Vol 35 (9) ◽  
Author(s):  
刘驰 LIU Chi ◽  
李家宝 LI Jiabao ◽  
芮俊鹏 RUI Junpeng ◽  
安家兴 AN Jiaxing ◽  
李香真 LI Xiangzhen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Ecology ◽  
Current Situation ◽  
Rrna Gene ◽  
The 16S Rrna Gene

The structure and organization of the 16S ribosomal RNA gene from the archaebacterium Thermoplasma acidophilum

1989 ◽  
Vol 35 (1) ◽  
pp. 124-133 ◽  
Author(s):  
Heesoo K. Ree ◽  
Kaiming Cao ◽  
David L. Thurlow ◽  
Robert A. Zimmermann
Keyword(s):  
Secondary Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Transcription Initiation ◽  
Single Copy ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Transcription Control ◽  
Thermoplasma Acidophilum ◽  
The 16S Rrna Gene

The complete nucleotide sequence of the 16S rRNA gene from Thermoplasma acidophilum, as well as its 5′ and 3′ flanking regions, were determined by the dideoxynucleotide chain termination method. The 16S rRNA gene encodes 1471 nucleotides. The primary and secondary structures of T. acidophilum 16S rRNA both exhibit typical archaebacterial features. The sequence appears to be more closely related to 16S rRNAs of the methanogen–halophile group than to those of the thermoacidophile group. Secondary-structure comparisons generally support this relationship, although there are several examples in which the single-stranded loops in particular helices of T. acidophilum 16S rRNA more strongly resemble their counterparts in the 16S rRNA of Sulfolobus solfataricus, a member of the thermoacidophile group. In contrast to the polycistronic rRNA operons found in most organisms, the three rRNA genes from T. acidophilum occur in only a single copy per genome and appear to be physically unlinked. Consistent with this, the 16S rRNA gene is flanked by putative promoter and terminator sequences that are comparable to the transcription control signals from other archaebacterial genes. The sequence TATATATA, which is very similar to the archaebacterial promoter consensus TTTAT/AATA, is located 18 bases before the probable site of transcription initiation, TGCACAT. There is a potential transcription termination site immediately downstream from the gene that consists of a relatively stable stem and loop structure followed by stretches of Tresidues.Key words: archaebacteria, thermoacidophile, rRNA sequence, rRNA secondary structure, promoter.

scholarly journals Direct Quantification of Campylobacter jejuni and Campylobacter lanienae in Feces of Cattle by Real-Time Quantitative PCR

2004 ◽  
Vol 70 (4) ◽  
pp. 2296-2306 ◽  
Author(s):  
G. Douglas Inglis ◽  
Lisa D. Kalischuk
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Campylobacter Jejuni ◽  
Single Copy ◽  
Mitigation Strategies ◽  
Rrna Gene ◽  
Limit Of Quantification ◽  
Cattle Feces ◽  
The Impact

ABSTRACT Campylobacter species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify Campylobacter jejuni and Campylobacter lanienae in cattle feces. For C. jejuni, the single-copy mapA gene was selected. For C. lanienae, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of C. jejuni quantification, and the limit of quantification was 19 to 25 genome copies (≈3 × 103 CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on C. lanienae by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies (≈250 CFU/g of feces), and there was no discernible difference between the two C. lanienae secondary primer sets evaluated. Detection and quantification of C. jejuni in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect C. lanienae in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of C. lanienae, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.

scholarly journals Resolving microbial microdiversity with high accuracy full length 16S rRNA Illumina sequencing

2014 ◽  
Author(s):  
Catherine Burke ◽  
Aaron E Darling
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Single Molecule ◽  
Illumina Miseq ◽  
Full Length ◽  
Rrna Gene ◽  
Variable Regions ◽  
Phylogenetic Resolution ◽  
Miseq Platform ◽  
The 16S Rrna Gene

We describe a method for sequencing full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform. The resulting sequences have about 100-fold higher accuracy than standard Illumina reads and are chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. We demonstrate that the data provides fine scale phylogenetic resolution not available from Illumina amplicon methods targeting smaller variable regions of the 16S rRNA gene.

scholarly journals Phylogenetic evaluation of the genus Nostoc and description of Nostoc neudorfense sp. nov., from the Czech Republic

2020 ◽  
Vol 70 (4) ◽  
pp. 2740-2749 ◽  
Author(s):  
Prashant Singh ◽  
Jana Šnokhousová ◽  
Aniket Saraf ◽  
Archana Suradkar ◽  
Josef Elster
Keyword(s):  
Czech Republic ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Tree ◽  
Rrna Gene ◽  
Phylogenetic Distance ◽  
Content Type ◽  
Gene Phylogeny ◽  
Link Type ◽  
The 16S Rrna Gene

Cyanobacterial strain ARC8 was isolated from seepage coming into the river Dračice, Františkov, Czech Republic, and was characterized using a polyphasic approach. Strain ARC8 showed a typical Nostoc -like morphology and in-depth morphological characterization indicated that it is a member of the genus Nostoc . Furthermore, in the 16S rRNA gene phylogeny inferred using Bayesian inference, maximum likelihood and neighbour joining methods, strain ARC8 clustered within the Nostoc sensu stricto clade. The phylogenetic distance and the positioning of strain ARC8 also indicated that it is a member of the genus Nostoc . Furthermore, the rbcL gene phylogeny along with the 16S–23S ITS secondary structure analysis also supported the findings from the 16S rRNA gene tree. In accordance with the International Code of Nomenclature for Algae, Fungi and Plants we describe a novel species of Nostoc with the name Nostoc neudorfense sp. nov.

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).

scholarly journals Genetic relatedness within the genus Campylobacter inferred from rpoB sequences

2006 ◽  
Vol 56 (5) ◽  
pp. 937-945 ◽  
Author(s):  
Bożena M. Korczak ◽  
Regina Stieber ◽  
Stefan Emler ◽  
André P. Burnens ◽  
Joachim Frey ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Genetic Relatedness ◽  
Gene Tree ◽  
Rrna Gene ◽  
Human Pathogens ◽  
Gene Encoding ◽  
Type Strains ◽  
The 16S Rrna Gene

The genus Campylobacter comprises 17 species, some of which are important animal and human pathogens. To gain more insight into the genetic relatedness of this genus and to improve the molecular tools available for diagnosis, a universal sequencing approach was established for the gene encoding the beta-subunit of RNA polymerase (rpoB) for the genus Campylobacter. A total of 59 strains, including the type strains of currently recognized species as well as field isolates, were investigated in the study. A primer set specific for Campylobacter species enabled straightforward amplification and sequencing of a 530 bp fragment of the rpoB gene. The 16S rRNA gene sequences of all of the strains were determined in parallel. A good congruence was obtained between 16S rRNA and rpoB gene sequence-based trees within the genus Campylobacter. The branching of the rpoB tree was similar to that of the 16S rRNA gene tree, even though a few discrepancies were observed for certain species. The resolution of the rpoB gene within the genus Campylobacter was generally much higher than that of the 16S rRNA gene sequence, resulting in a clear separation of most species and even some subspecies. The universally applicable amplification and sequencing approach for partial rpoB gene sequence determination provides a powerful tool for DNA sequence-based discrimination of Campylobacter species.

scholarly journals Williamsia faeni sp. nov., an actinomycete isolated from a hay meadow

2010 ◽  
Vol 60 (11) ◽  
pp. 2548-2551 ◽  
Author(s):  
Amanda L. Jones ◽  
Gail D. Payne ◽  
Michael Goodfellow
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Tree ◽  
Taxonomic Status ◽  
Rrna Gene ◽  
Phenotypic Properties ◽  
Phyletic Line ◽  
Type Strains ◽  
The 16S Rrna Gene ◽  
Sequence Similarities

The taxonomic status of an actinomycete isolated from soil collected from a hay meadow was determined using a polyphasic approach. The strain, designated N1350T, had morphological and chemotaxonomic properties consistent with its classification in the genus Williamsia and formed a distinct phyletic line within the clade comprising the type strains of species of the genus Williamsia in the 16S rRNA gene tree. Strain N1350T shared highest 16S rRNA gene sequence similarities with Williamsia marianensis MT8T (98.1 %) and Williamsia muralis MA140-96T (98.3 %). However, strain N1350T was readily distinguished from the type strains of Williamsia species using a combination of phenotypic properties. On the basis of these data, strain N1350T is considered to represent a novel species of the genus Williamsia. The name proposed for this taxon is Williamsia faeni sp. nov., with the type strain N1350T (=DSM 45372T =NCIMB 14575T =NRRL B-24794T).

scholarly journals Effects of Growth Conditions on Expression of Mycobacterial murA and tyrS Genes and Contributions of Their Transcripts to Precursor rRNA Synthesis

1999 ◽  
Vol 181 (15) ◽  
pp. 4617-4627 ◽  
Author(s):  
J. A. Gonzalez-y-Merchand ◽  
M. J. Colston ◽  
R. A. Cox
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Single Copy ◽  
Growth Cycle ◽  
Trna Synthetase ◽  
Rrna Operon ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Rnase Protection ◽  
The 16S Rrna Gene

ABSTRACT All mycobacteria studied to date have an rRNA operon, designatedrrnA, located downstream from a single copy of themurA gene, which encodes an enzyme (EC 2.5.1.7 ) important for peptidoglycan synthesis. The rrnA operon has a promoter, P1(A), located within the coding region of murA, near the 3′ end. Samples of RNA were isolated from Mycobacterium tuberculosis at different stages of the growth cycle and fromMycobacterium smegmatis grown under different conditions. RNase protection assays were used to investigate transcripts of bothmurA and rrnA. Transcription ofmurA was found to continue into the 16S rRNA gene, as ifmurA and rrnA form a hybrid (protein coding-rRNA coding) operon. During the growth of M. tuberculosis, the hybrid operon contributed approximately 2% to total pre-rRNA. Analysis of M. smegmatis RNA revealed that the level of murA RNA depended on the growth rate and that the patterns of expression during the growth cycle were different formurA and rrnA. M. smegmatis has a second rRNA operon, rrnB, located downstream from a single copy of the tyrS gene, encoding tyrosyl-tRNA synthetase. Transcription of tyrS was found to continue into the 16S rRNA gene rrnB. The hybrid tyrS-rrnB operon contributed 0.2 to 0.6% to rrnB transcripts. The pattern of tyrS expression during the growth cycle matched the pattern of rrnB expression, reflecting the essential role of TyrS and rRNA in protein biosynthesis.

Sign in / Sign up

Export Citation Format

Share Document