scholarly journals Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

2001 ◽  
Vol 67 (1) ◽  
pp. 469-472 ◽  
Author(s):  
Arjen G. C. L. Speksnijder ◽  
George A. Kowalchuk ◽  
Sander De Jong ◽  
Elizabeth Kline ◽  
John R. Stephen ◽  
...  
Keyword(s):  
16S Rdna ◽  
Sequence Variation ◽  
Hot Spot ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Pcr Cloning ◽  
Clone Libraries ◽  
High Degree ◽  
Cloning Experiment ◽  
Rdna Clone

ABSTRACT A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.

scholarly journals Resuscitation of eleven-year VBNC Citrobacter

2008 ◽  
Vol 6 (4) ◽  
pp. 565-568 ◽  
Author(s):  
Amel Dhiaf ◽  
Amina Bakhrouf ◽  
Karl-Paul Witzel
Keyword(s):  
Growth Factors ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Reference Strain ◽  
Rrna Gene ◽  
Citrobacter Freundii ◽  
16S Rrna Gene Sequences ◽  
High Degree ◽  
Degree Of Similarity ◽  
The 16S Rrna Gene

Citrobacter freundii strain WA1 was stressed by incubation in seawater microcosms for eleven years. After two years of starvation, no culturable strain was observed. Incubation of samples in nutrient-rich broth medium not supplemented with growth factors, however, allowed resuscitation of VBNC cells so that subsequent plating yielded observable colonies for significantly extended periods of time. Recovery of VBNC Citrobacter freundii was obtained by incubation in nutrient broth even after eleven years of starvation. To see whether the samples contained the same strain of Citrobacter freundii inoculated 11 years ago. The complete 16S rRNA gene was PCR amplified and sequenced from initial, stressed and revived strains of Citrobacter freundii strain WA1.The 16S rRNA gene sequences from eleven-year stressed strains were homologous with a high degree of similarity to the GenBank reference strain and were identical to each other.

scholarly journals Change in Bacterial Community Structure during In Situ Biostimulation of Subsurface Sediment Cocontaminated with Uranium and Nitrate

2004 ◽  
Vol 70 (8) ◽  
pp. 4911-4920 ◽  
Author(s):  
Nadia N. North ◽  
Sherry L. Dollhopf ◽  
Lainie Petrie ◽  
Jonathan D. Istok ◽  
David L. Balkwill ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Quantitative Pcr ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Clone Libraries ◽  
Subsurface Sediments

ABSTRACT Previous studies have demonstrated that metal-reducing microorganisms can effectively promote the precipitation and removal of uranium from contaminated groundwater. Microbial communities were stimulated in the acidic subsurface by pH neutralization and addition of an electron donor to wells. In single-well push-pull tests at a number of treated sites, nitrate, Fe(III), and uranium were extensively reduced and electron donors (glucose, ethanol) were consumed. Examination of sediment chemistry in cores sampled immediately adjacent to treated wells 3.5 months after treatment revealed that sediment pH increased substantially (by 1 to 2 pH units) while nitrate was largely depleted. A large diversity of 16S rRNA gene sequences were retrieved from subsurface sediments, including species from the α, β, δ, and γ subdivisions of the class Proteobacteria, as well as low- and high-G+C gram-positive species. Following in situ biostimulation of microbial communities within contaminated sediments, sequences related to previously cultured metal-reducing δ-Proteobacteria increased from 5% to nearly 40% of the clone libraries. Quantitative PCR revealed that Geobacter-type 16S rRNA gene sequences increased in biostimulated sediments by 1 to 2 orders of magnitude at two of the four sites tested. Evidence from the quantitative PCR analysis corroborated information obtained from 16S rRNA gene clone libraries, indicating that members of the δ-Proteobacteria subdivision, including Anaeromyxobacter dehalogenans-related and Geobacter-related sequences, are important metal-reducing organisms in acidic subsurface sediments. This study provides the first cultivation-independent analysis of the change in metal-reducing microbial communities in subsurface sediments during an in situ bioremediation experiment.

scholarly journals Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries

2006 ◽  
Vol 55 (1) ◽  
pp. 101-107 ◽  
Author(s):  
Daniel Saito ◽  
Renato de Toledo Leonardo ◽  
Jorge Luiz Mazza Rodrigues ◽  
Siu Mui Tsai ◽  
José Francisco Höfling ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rdna ◽  
Significant Proportion ◽  
Oral Bacteria ◽  
Clone Libraries ◽  
Root Canals ◽  
Gram Positive Bacteria ◽  
Identification Of Bacteria ◽  
Endodontic Infections ◽  
Rdna Clone

A significant proportion of oral bacteria are unable to undergo cultivation by existing techniques. In this regard, the microbiota from root canals still requires complementary characterization. The present study aimed at the identification of bacteria by sequence analysis of 16S rDNA clone libraries from seven endodontically infected teeth. Samples were collected from the root canals, subjected to the PCR with universal 16S rDNA primers, cloned and partially sequenced. Clones were clustered into groups of closely related sequences (phylotypes) and identification to the species level was performed by comparative analysis with the GenBank, EMBL and DDBJ databases, according to a 98 % minimum identity. All samples were positive for bacteria and the number of phylotypes detected per subject varied from two to 14. The majority of taxa (65·2 %) belonged to the phylum Firmicutes of the Gram-positive bacteria, followed by Proteobacteria (10·9 %), Spirochaetes (4·3 %), Bacteroidetes (6·5 %), Actinobacteria (2·2 %) and Deferribacteres (2·2 %). A total of 46 distinct taxonomic units was identified. Four clones with low similarity to sequences previously deposited in the databases were sequenced to nearly full extent and were classified taxonomically as novel representatives of the order Clostridiales, including a putative novel species of Mogibacterium. The identification of novel phylotypes associated with endodontic infections suggests that the endodontium may still harbour a relevant proportion of uncharacterized taxa.

scholarly journals Cryptic haplotypes of "Candidatus Liberibacter africanus"

2015 ◽  
Author(s):  
Warrick Nelson ◽  
Sandrine Eveillard ◽  
Marie-Pierre Dubrana ◽  
Joseph Bové
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rdna ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Rdna Sequences ◽  
New Information ◽  
Candidatus Liberibacter ◽  
Citrus Leaves

“Candidatus Liberibacter africanus” (Laf) has long been recognised as a causal agent of the devastating citrus disease huanglongbing (HLB) or citrus greening. This species is currently restricted to Africa, the Arabian Peninsula and some Indian Ocean islands and vectored by the African citrus psyllid, Trioza erytreae. Blotchy mottle on citrus leaves is characteristic of the disease. Somewhat similar symptoms in the Rutaceous tree Calodendrum capensis (Cape Chestnut) resulted in the discovery of Laf outside commercial citrus crops in South Africa. This was classed as a subspecies of Laf (capensis, hence LafC). In subsequent surveys of both commercial citrus crops and Calodendrum, both natural and ornamental specimens, LafC was not found in the citrus crop, nor has Laf been found in C. capensis. HLB was reported from Madagascar in 1968 but no sequences from this source have so far been published. Until fairly recently, only the reference 16S rRNA gene sequences of Laf (L22533) and LafC (AF137368) had been deposited in GenBank. Both of these reference sequences contain a number of unresolved nucleotides. Resolving these nucleotide positions by aligning against more recently available sequences, it becomes evident that these unresolved positions represent one percentage point difference in similarity between Laf and LafC. The originally reported 97.4% similarity is therefore incorrect based on this new information. Recalculating the similarity on the full length 16S rDNA sequence results in 99.54% similarity, a value too high to justify a subspecies status. LafC should therefore be reduced to that of a haplotype of Laf. Further, the six 16S rRNA gene sequences currently available in GenBank identified as the species Laf separate into 2 haplotype groups. The 3 haplotypes of Laf are therefore LafA designated as the first accession sequenced (L22533), LafC for the former capensis subspecies and to recognise the prior use of this term, and LafB for the third haplotype not previously recognised. Thus the cryptic presence of 3 haplotypes is revealed by this review of the Laf 16S rDNA sequences.

ARDRA and Identification of Intestinal Aerobic Bacteria from 4th Instar Larvae of Dendrolimu. kikuchii

2012 ◽  
Vol 518-523 ◽  
pp. 5523-5527
Author(s):  
Wu Xian Zhang ◽  
Jin Hua Wang ◽  
You He Sun ◽  
Biao Li ◽  
Zhi Xiong
Keyword(s):  
Genetic Diversity ◽  
Bacillus Subtilis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rdna ◽  
Enzyme Digestion ◽  
Aerobic Bacteria ◽  
Rrna Gene ◽  
Accession Number ◽  
16S Rrna Gene Sequences

Genetic diversity of 11 intestinal aerobic bacteria isolated from Dendrolimu. kikuchii was analysed, using PCR and ARDRA which used enzyme digestion of cloned 16S rRNA gene sequences. The results showed that 11 strains could be divided into 6 groups on 84% similarity level, it indicated that the intestinal aerobic bacteria genetic diversity was abundant. Sequencing the 6 representative strains’ 16S rDNA and submitting to GenBank, the accession number being JQ308104 to JQ308109 respectively. The 6 strains belonged to Klebsiella sp., Lysinibacillus sp., Brevibacillus sp., Bacillus subtilis, Gamma Proteobacterium and Brevibacillus limnophilus.

scholarly journals Estimation of the Relative Abundance of DifferentBacteroides and Prevotella Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences

1998 ◽  
Vol 64 (10) ◽  
pp. 3683-3689 ◽  
Author(s):  
Jacqueline Wood ◽  
Karen P. Scott ◽  
Gorazd Avguštin ◽  
C. James Newbold ◽  
Harry J. Flint
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rdna ◽  
Restriction Enzymes ◽  
Pcr Primers ◽  
Gut Contents ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Bacterial Populations

ABSTRACT We describe an approach for determining the genetic composition ofBacteroides and Prevotellapopulations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides andPrevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution ofBacteroides and Prevotellasequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA.Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotellastrains, together accounted for between 20 and 86% of the total amplified Bacteroides andPrevotella rDNA in these samples. The most abundantBacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundantBacteroides and Prevotella groups in the rumen are underrepresented among cultured rumenPrevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.

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