Six species of nontuberculous mycobacteria carry non-identical 16S rRNA gene copies

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5–2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

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Heterogeneity between 16S ribosomal RNA gene copies borne by one Desulfitobacterium strain is caused by different 100-200 bp insertions in the 5´ region

Canadian Journal of Microbiology ◽

10.1139/w06-111 ◽

2007 ◽

Vol 53 (1) ◽

pp. 116-128 ◽

Cited By ~ 13

Author(s):

Richard Villemur ◽

Philippe Constant ◽

Annie Gauthier ◽

Martine Shareck ◽

Réjean Beaudet

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

16S Rrna Gene ◽

Ribosomal Rna ◽

16S Ribosomal Rna ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Gene Copies

Strains of Desulfitobacterium hafniense, such as strains PCP-1, DP7, TCE1, and TCP-A, have unusual long 16S ribosomal RNA (rRNA) genes due to an insertion of approximately 100 bp in the 5' region. In this report, we analyzed the 16S rRNA genes of different Desulfitobacterium strains to determine if such an insertion is a common feature of desulfitobacteria. We amplified this region by polymerase chain reaction (PCR) from eight Desulfitobacterium strains (D. hafniense strains PCP-1, DP7, TCP-A, TCE1, and DCB-2; D. dehalogenans; D. chlororespirans; and Desulfitobacterium sp. PCE1) and resolved each PCR product by denaturing gradient gel electrophoresis (DGGE). All strains had from two to seven DGGE- migrating bands, suggesting heterogeneity in their 16S rRNA gene copies. For each strain, the 5' region of the 16S rRNA genes was amplified and a clone library was derived. Clones corresponding to most PCR–DGGE migration bands were isolated. Sequencing of representative clones revealed that the heterogeneity was generated by insertions of 100–200 bp. An insertion was found in at least one copy of the 16S rRNA gene in all examined strains. In total, we found eight different types of insertions (INS1–INS8) that varied from 123 to 193 nt in length. Two-dimensional structural analyses of transcribed sequences predicted that all insertions would form an energetically stable loop. Reverse transcriptase – PCR experiments revealed that most of the observed insertions in the Desulfitobacterium strains were excised from the mature 16S rRNA transcripts. Insertions were not commonly found in bacterial 16S rRNA genes, and having a different insertion in several 16S rRNA gene copies borne by a single bacterial species was rarely observed. The function of these insertions is not known, but their occurrence can have an important impact in deriving 16S rRNA oligonucleotidic fluorescence in situ hybridization probes, as these insertions can be excised from 16S rRNA transcripts.Key words: Desulfitobacterium, 16S ribosomal RNA genes, heterogeneity, gene insertions, fluorescence in situ hybridization.

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Bacterial DNAemia is associated with serum zonulin levels in older subjects

10.1101/2020.04.10.035519 ◽

2020 ◽

Author(s):

Giorgio Gargari ◽

Valentina Taverniti ◽

Cristian Del Bo’ ◽

Stefano Bernardi ◽

Cristina Andres-Lacueva ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Serum Levels ◽

Gene Profiling ◽

Rrna Gene ◽

Bacterial Dna ◽

Blood Samples ◽

Gene Copies ◽

Older Subjects ◽

Pcr Targeting

AbstractThe increased presence of bacteria in blood is a plausible contributing factor in the development and progression of aging-associated diseases. In this context, we performed the quantification and the taxonomic profiling of the bacterial DNA in blood samples collected from a group of forty-three older subjects enrolled in a nursing home. Quantitative PCR targeting the 16S rRNA gene revealed that all the older volunteers contained detectable amounts of bacterial DNA in their blood. The total amount of 16S rRNA gene copies varied considerably between subjects. Correlation analyses revealed that the bacterial DNAemia (expressed as concentration of 16S rRNA gene copies in blood) significantly correlated with the serum levels of zonulin, an emerging marker of intestinal permeability. This result was confirmed by the analysis of a second set of blood samples collected after approximately four months from the same subjects. Analyses of 16S rRNA gene profiling revealed that most of the bacterial DNA detected in blood was ascribable to the phylum Proteobacteria with a predominance of Pseudomonadaceae and Enterobacteriaceae. Several control samples were also analyzed to assess the influence exerted by contaminant bacterial DNA potentially originating from reagents and materials. The date reported here suggest that para-cellular permeability of epithelial (and potentially also endothelial) cell layers may play an important role in bacterial migration into the bloodstream. Bacterial DNAemia is likely to impact on several aspects of host physiology and could underpin the development and prognosis of various diseases in older subjects.

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Six species of non-tuberculosis mycobacteria carry non-identical 16S rRNA gene copies

10.1101/302836 ◽

2018 ◽

Author(s):

Keita Takeda ◽

Kinuyo Chikamatsu ◽

Yuriko Igarashi ◽

Yuta Morishige ◽

Yoshiro Murase ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Rrna Gene ◽

Target Sequence ◽

Nucleotide Polymorphisms ◽

16S Rrna Gene Sequences ◽

Cloning And Sequencing ◽

Single Nucleotide ◽

Gene Copies ◽

The 16S Rrna Gene

AbstractNon-tuberculosis mycobacteria (NTM) can carry two or more 16S rRNA gene copies that are, in some instances, non-identical. In this study, we used a combined cloning and sequencing approach to analyze the 16S rRNA gene sequences of six NTM species,Mycobacterium cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, andM. xenopi. The approach facilitated the identification of two distinct gene copies in each species. The twoM. cosmeticumgenes had a single nucleotide difference, whereas two nucleotide polymorphisms were identified inM. hodleri, M. flavescens, andM. xenopi. M. pallenshad a difference in four nucleotides andM. crocinumin 23. Hence, we showed that the six NTM species possess at least two non-identical 16S rRNA gene copies.ImportanceThe presence of multiple 16S rRNA gene copies with nucleotide polymorphisms represents a challenge for species identification using 16S rRNA as the target sequence. Our analysis was focused on six NTM species,M. cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, andM. xenopi. As a result, we generated the full-length sequences of two non-identical 16S rRNA copies for each NTM species. The data will be helpful for the sequence analysis of specimens or other samples.

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Corn oil supplementation enhances hydrogen use for biohydrogenation, inhibits methanogenesis, and alters fermentation pathways and the microbial community in the rumen of goats

Journal of Animal Science ◽

10.1093/jas/skz352 ◽

2019 ◽

Vol 97 (12) ◽

pp. 4999-5008 ◽

Cited By ~ 1

Author(s):

Xiu Min Zhang ◽

Rodolfo F Medrano ◽

Min Wang ◽

Karen A Beauchemin ◽

Zhi Yuan Ma ◽

...

Keyword(s):

Fatty Acids ◽

16S Rrna ◽

16S Rrna Gene ◽

Relative Abundance ◽

Unsaturated Fatty Acids ◽

Corn Oil ◽

Nutrient Digestibility ◽

Rrna Gene ◽

Ch4 Emissions ◽

Gene Copies

Abstract Enteric methane (CH4) emissions are not only an important source of greenhouse gases but also a loss of dietary energy in livestock. Corn oil (CO) is rich in unsaturated fatty acid with >50% PUFA, which may enhance ruminal biohydrogenation of unsaturated fatty acids, leading to changes in ruminal H2 metabolism and methanogenesis. The objective of this study was to investigate the effect of CO supplementation of a diet on CH4 emissions, nutrient digestibility, ruminal dissolved gases, fermentation, and microbiota in goats. Six female goats were used in a crossover design with two dietary treatments, which included control and CO supplementation (30 g/kg DM basis). CO supplementation did not alter total-tract organic matter digestibility or populations of predominant ruminal fibrolytic microorganisms (protozoa, fungi, Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes), but reduced enteric CH4 emissions (g/kg DMI, −15.1%, P = 0.003). CO supplementation decreased ruminal dissolved hydrogen (dH2, P < 0.001) and dissolved CH4 (P < 0.001) concentrations, proportions of total unsaturated fatty acids (P < 0.001) and propionate (P = 0.015), and increased proportions of total SFAs (P < 0.001) and acetate (P < 0.001), and acetate to propionate ratio (P = 0.038) in rumen fluid. CO supplementation decreased relative abundance of family Bacteroidales_BS11_gut_group (P = 0.032), increased relative abundance of family Rikenellaceae (P = 0.021) and Lachnospiraceae (P = 0.025), and tended to increase relative abundance of genus Butyrivibrio_2 (P = 0.06). Relative abundance (P = 0.09) and 16S rRNA gene copies (P = 0.043) of order Methanomicrobiales, and relative abundance of genus Methanomicrobium (P = 0.09) also decreased with CO supplementation, but relative abundance (P = 0.012) and 16S rRNA gene copies (P = 0.08) of genus Methanobrevibacter increased. In summary, CO supplementation increased rumen biohydrogenatation by facilitating growth of biohydrogenating bacteria of family Lachnospiraceae and genus Butyrivibrio_2 and may have enhanced reductive acetogenesis by facilitating growth of family Lachnospiraceae. In conclusion, dietary supplementation of CO led to a shift of fermentation pathways that enhanced acetate production and decreased rumen dH2 concentration and CH4 emissions.

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Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types

10.1101/2020.10.21.348508 ◽

2020 ◽

Author(s):

Michael E.C. Abundo ◽

John M. Ngunjiri ◽

Kara J.M. Taylor ◽

Hana Ji ◽

Amir Ghorbani ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Extraction ◽

Extraction Process ◽

Production Performance ◽

Rrna Gene ◽

Sample Type ◽

Alpha And Beta Diversity ◽

Gene Copies ◽

Diversity Metrics

AbstractCharacterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. Furthermore, between-kit differences in alpha and beta diversity metrics were statistically indistinguishable. Therefore, both kits perform similarly with regard to 16S rRNA gene-based poultry microbiome analysis.

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Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types

PLoS ONE ◽

10.1371/journal.pone.0241732 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0241732

Author(s):

Michael E. C. Abundo ◽

John M. Ngunjiri ◽

Kara J. M. Taylor ◽

Hana Ji ◽

Amir Ghorbani ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Extraction ◽

Production Performance ◽

Rrna Gene ◽

Sequence Variant ◽

Sample Type ◽

Alpha And Beta Diversity ◽

Gene Copies ◽

Diversity Metrics

Characterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant bacterial taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. However, a few differentially abundant shared taxa were observed at family and genus levels. Furthermore, between-kit differences in alpha and beta diversity metrics at the amplicon sequence variant level were statistically indistinguishable. Therefore, both kits perform similarly in terms of 16S rRNA gene-based poultry microbiome analysis for the sample types analyzed in this study.

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Blood bacterial DNA, intestinal adenoma and colorectal cancer

10.1101/2021.07.22.21260498 ◽

2021 ◽

Author(s):

Massimiliano Mutignani ◽

Roberto Penagini ◽

Giorgio Gargari ◽

Simone Guglielmetti ◽

Marcello Cintolo ◽

...

Keyword(s):

Colorectal Cancer ◽

Colon Cancer ◽

16S Rrna ◽

16S Rrna Gene ◽

Healthy Subjects ◽

Bacterial Load ◽

Rrna Gene ◽

Bacterial Dna ◽

Gene Copies ◽

Intestinal Adenoma

Objective We aimed to investigate the relation of blood bacterial DNA load and profiling with intestinal adenoma (IA) and colorectal cancer (CRC) patients. Design We performed 16S rRNA gene analysis of blood from 100 incident histologically confirmed CRC cases, 100 IA and 100 healthy subjects, matched to cases by centre, sex and age. Bacterial load was analysed using multiple conditional logistic regression. Differences in terms of abundance of bacteria between groups were estimated through analysis based on negative binomial distribution normalization. Random Forest was applied to predict the group assignment. Results We found an overrepresentation of blood 16S rRNA gene copies in colon cancer as compared to tumor-free controls (IA and healthy subjects). The odds ratio of colon cancer for the highest versus the lowest three quintiles of gene copies was 2.62. (95% confidence interval=1.22-5.65). No difference was found for rectal cancer and IA. For high 16S rRNA, community diversity was higher in colon cancers than controls. CRC cases had an enrichment of Peptostreptococcaceae and Acetobacteriaceae and a reduced abundance of Bacteroidaceae, Lachnospiraceae, and Ruminococcaceae. Identified variables predicted CRC from control and IA patients with an accuracy of 0.70. Conclusion Colon cancer patients had a higher DNA bacterial load and a different bacterial profiling as compared to healthy subjects, IA and rectal cancers, indicating a higher passage of bacteria from gastrointestinal tract to bloodstream. Further studies are needed to confirm this result and exploit it to conceive new non-invasive techniques for an early diagnosis of CRC.

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Quantification of 16S rRNA Gene Copies Using ddPCR (EvaGreen-based assay: 338F-805R) v3 (protocols.io.bpy6mpze)

protocols.io ◽

10.17504/protocols.io.bpy6mpze ◽

2020 ◽

Author(s):

Roey Angel ◽

Eva Petrova

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Rrna Gene ◽

Gene Copies

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Detection and Quantification of Geobacter lovleyi Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites

Applied and Environmental Microbiology ◽

10.1128/aem.01218-07 ◽

2007 ◽

Vol 73 (21) ◽

pp. 6898-6904 ◽

Cited By ~ 36

Author(s):

Benjamin K. Amos ◽

Youlboong Sung ◽

Kelly E. Fletcher ◽

Terry J. Gentry ◽

Wei-Min Wu ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Nested Pcr ◽

Rrna Gene ◽

Chlorinated Ethene ◽

Oak Ridge ◽

Chlorinated Solvent ◽

Gene Copies ◽

Groundwater Samples ◽

Template Dna

ABSTRACTGeobacter lovleyistrain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducingGeobactergroup capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely relatedGeobacterisolates, including its closest cultured relative,G. thiogenes. Detection limits for direct and nested PCR were approximately 1 ï¿½ 106and 1 ï¿½ 10416S rRNA gene copies per μl of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per μl of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ fromG. thiogeneswhen analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 ï¿½ 107ï¿½ 0.1 ï¿½ 107per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. TheG. lovleyistrain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.

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