scholarly journals Quantifying Genes and Transcripts To Assess the In Situ Physiology of “Dehalococcoides” spp. in a Trichloroethene-Contaminated Groundwater Site

2008 ◽  
Vol 74 (9) ◽  
pp. 2728-2739 ◽  
Author(s):  
Patrick K. H. Lee ◽  
Tamzen W. Macbeth ◽  
Kent S. Sorenson ◽  
Rula A. Deeb ◽  
Lisa Alvarez-Cohen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Field Samples ◽  
Rdase Genes ◽  
Gene Copy Numbers ◽  
The 16S Rrna Gene

ABSTRACT Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of “Dehalococcoides” spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among Dehalococcoides cells. The Dehalococcoides cells containing the tceA gene consistently lagged behind other Dehalococcoides cells in population numbers and made up less than 5% of the total Dehalococcoides population, whereas the vcrA- and bvcA-containing cells represented the dominant fractions. Quantification of transcripts in groundwater samples verified that the 16S rRNA gene and the bvcA and vcrA genes were consistently highly expressed in all samples examined, while the tceA transcripts were detected inconsistently, suggesting a less active physiological state of the cells with this gene. The production of vinyl chloride and ethene toward the end of treatment supported the physiological activity of the bvcA- and vcrA-carrying cells. A clone library of the expressed RDase genes in field samples produced with degenerate primers revealed the expression of two putative RDase genes that were not previously monitored with RT-qPCR. The level of abundance of one of the putative RDase genes (FtL-RDase-1638) identified in the cDNA clone library tracked closely in field samples with abundance of the bvcA gene, suggesting that the FtL-RDase-1638 gene was likely colocated in genomes containing the bvcA gene. Overall, results from this study demonstrate that quantification of biomarker dynamics at field sites can provide useful information about the in situ physiology of Dehalococcoides strains and their associated activity.

scholarly journals High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts

2021 ◽  
Vol 7 (9) ◽  
Author(s):  
Žana Kapustina ◽  
Justina Medžiūnė ◽  
Gediminas Alzbutas ◽  
Irmantas Rokaitis ◽  
Karolis Matjošaitis ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Sequencing ◽  
Gene Copy ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Copy Numbers ◽  
Rrna Sequencing ◽  
Metagenome Sequencing ◽  
Gene Copy Numbers

Sequence-based characterization of bacterial communities has long been a hostage of limitations of both 16S rRNA gene and whole metagenome sequencing. Neither approach is universally applicable, and the main efforts to resolve constraints have been devoted to improvement of computational prediction tools. Here, we present semi-targeted 16S rRNA sequencing (st16S-seq), a method designed for sequencing V1–V2 regions of the 16S rRNA gene along with the genomic locus upstream of the gene. By in silico analysis of 13 570 bacterial genome assemblies, we show that genome-linked 16S rRNA sequencing is superior to individual hypervariable regions or full-length gene sequences in terms of classification accuracy and identification of gene copy numbers. Using mock communities and soil samples we experimentally validate st16S-seq and benchmark it against the established microbial classification techniques. We show that st16S-seq delivers accurate estimation of 16S rRNA gene copy numbers, enables taxonomic resolution at the species level and closely approximates community structures obtainable by whole metagenome sequencing.

scholarly journals Correcting for 16S rRNA gene copy numbers in microbiome surveys remains an unsolved problem

Microbiome ◽  
2018 ◽  
Vol 6 (1) ◽  
Author(s):  
Stilianos Louca ◽  
Michael Doebeli ◽  
Laura Wegener Parfrey
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Unsolved Problem ◽  
Gene Copy ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Gene Copy Numbers

scholarly journals Quantification of Burkholderia coxL Genes in Hawaiian Volcanic Deposits

2010 ◽  
Vol 76 (7) ◽  
pp. 2212-2217 ◽  
Author(s):  
C. F. Weber ◽  
G. M. King
Keyword(s):  
Carbon Monoxide ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Large Subunit ◽  
Dry Weight ◽  
Gene Copy ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Volcanic Deposits ◽  
Gene Copy Numbers

ABSTRACT Isolation of multiple carbon monoxide (CO)-oxidizing Burkholderia strains and detection by culture-independent approaches suggest that Burkholderia may be an important component of CO-oxidizing communities in Hawaiian volcanic deposits. The absolute and relative abundance of the bacteria in these communities remains unknown, however. In this study, a quantitative PCR (Q-PCR) approach has been developed to enumerate Burkholderia coxL genes (large subunit of carbon monoxide dehydrogenase). This represents the first attempt to enumerate coxL genes from CO oxidizers in environmental samples. coxL copy numbers have been determined for samples from three sites representing a vegetation gradient on a 1959 volcanic deposit that included unvegetated cinders (bare), edges of vegetated sites (edge), and sites within tree stands (canopy). Q-PCR has also been used to estimate copy numbers of Betaproteobacteria 16S rRNA gene copy numbers and total Bacteria 16S rRNA. coxL genes could not be detected in the bare site (detection limit, ≥4.7 � 103 copies per reaction) but average 1.0 � 108 � 2.4 � 107 and 8.6 � 108 � 7.6 �107 copies g−1 (dry weight) in edge and canopy sites, respectively, which differ statistically (P = 0.0007). Average Burkholderia coxL gene copy numbers, expressed as a percentage of total Bacteria 16S rRNA gene copy numbers, are 6.2 and 0.7% for the edge and canopy sites, respectively. Although the percentage of Burkholderia coxL is lower in the canopy site, significantly greater gene copy numbers demonstrate that absolute abundance of coxL increases in vegetated sites and contributes to the expansion of CO oxidizer communities during biological succession on volcanic deposits.

scholarly journals Gene copy normalization of the 16S rRNA gene cannot outweigh the methodological biases of sequencing

2019 ◽  
Author(s):  
Robert Starke ◽  
Daniel Morais
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Rrna Gene ◽  
16S Sequencing ◽  
Copy Numbers ◽  
Α Diversity ◽  
Golden Standard ◽  
The 16S Rrna Gene ◽  
Mock Communities

The 16S rRNA gene is the golden standard target of sequencing to uncover the composition of bacterial communities but the presence of multiple copies of the gene makes gene copy normalization (GCN) inevitable. Even though GCN resulted in abundances closer to the metagenome, it should be validated by communities with known composition as both amplicon and shotgun sequencing are prone to methodological biases. Here we compared the composition of three mock communities to the composition derived from 16S sequencing without and with GCN. In all of them, the 16S composition was different from the mock community and GCN improved the picture only in the community with the lowest Shannon diversity. Albeit with low abundance, half of the identified genera were not present in the mock communities. Our approach provides empirical evidence to the methodological biases introduced by sequencing that was only counteracted by GCN in the case of low α-diversity, potentially due to the small number of bacterial taxa with known gene copy numbers. We thus cannot recommend the use of GCN moving forward and it is questionable whether a complete catalogue of 16S rRNA copy numbers can outweigh the methodological biases of sequencing.

scholarly journals Archaeal Abundance across a pH Gradient in an Arable Soil and Its Relationship to Bacterial and Fungal Growth Rates

2012 ◽  
Vol 78 (16) ◽  
pp. 5906-5911 ◽  
Author(s):  
Per Bengtson ◽  
Anna E. Sterngren ◽  
Johannes Rousk
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Soil Ph ◽  
Fungal Growth ◽  
Ph Gradient ◽  
Gene Copy ◽  
Rrna Gene ◽  
Copy Numbers ◽  
Ph Range ◽  
Gene Copy Numbers

ABSTRACTSoil pH is one of the most influential factors for the composition of bacterial and fungal communities, but the influence of soil pH on the distribution and composition of soil archaeal communities has yet to be systematically addressed. The primary aim of this study was to determine how total archaeal abundance (quantitative PCR [qPCR]-based estimates of 16S rRNA gene copy numbers) is related to soil pH across a pH gradient (pH 4.0 to 8.3). Secondarily, we wanted to assess how archaeal abundance related to bacterial and fungal growth rates across the same pH gradient. We identified two distinct and opposite effects of pH on the archaeal abundance. In the lowest pH range (pH 4.0 to 4.7), the abundance of archaea did not seem to correspond to pH. Above this pH range, there was a sharp, almost 4-fold decrease in archaeal abundance, reaching a minimum at pH 5.1 to 5.2. The low abundance of archaeal 16S rRNA gene copy numbers at this pH range then sharply increased almost 150-fold with pH, resulting in an increase in the ratio between archaeal and bacterial copy numbers from a minimum of 0.002 to more than 0.07 at pH 8. The nonuniform archaeal response to pH could reflect variation in the archaeal community composition along the gradient, with some archaea adapted to acidic conditions and others to neutral to slightly alkaline conditions. This suggestion is reinforced by observations of contrasting outcomes of the (competitive) interactions between archaea, bacteria, and fungi toward the lower and higher ends of the examined pH gradient.

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 Discrimination of Multiple Dehalococcoides Strains in a Trichloroethene Enrichment by Quantification of Their Reductive Dehalogenase Genes

2006 ◽  
Vol 72 (9) ◽  
pp. 5877-5883 ◽  
Author(s):  
Victor F. Holmes ◽  
Jianzhong He ◽  
Patrick K. H. Lee ◽  
Lisa Alvarez-Cohen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Rrna Gene ◽  
Sequence Matching ◽  
Relative Copy Number ◽  
Reductive Dehalogenase ◽  
Copy Numbers ◽  
Reductase Gene ◽  
Reductive Dehalogenase Genes

ABSTRACT While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.

scholarly journals Quantification of the Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example

2008 ◽  
Vol 74 (5) ◽  
pp. 1660-1663 ◽  
Author(s):  
D. Bru ◽  
F. Martin-Laurent ◽  
L. Philippot
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Gene Copy ◽  
Rrna Gene ◽  
Template Dna ◽  
Single Primer ◽  
The 16S Rrna Gene ◽  
Extension Sequence

ABSTRACT We investigated the effects of internal primer-template mismatches on the efficiency of PCR amplification using the 16S rRNA gene as the model template DNA. We observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.

scholarly journals Correlation of Dehalococcoides 16S rRNA and Chloroethene-Reductive Dehalogenase Genes with Geochemical Conditions in Chloroethene-Contaminated Groundwater

2009 ◽  
Vol 76 (3) ◽  
pp. 843-850 ◽  
Author(s):  
Bas van der Zaan ◽  
Fredericke Hannes ◽  
Nanne Hoekstra ◽  
Huub Rijnaarts ◽  
Willem M. de Vos ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gene Copy ◽  
Rrna Gene ◽  
Chlorinated Ethene ◽  
Monitoring Wells ◽  
Geochemical Conditions ◽  
Copy Numbers ◽  
Gene Copies ◽  
Reductase Gene ◽  
Gene Copy Numbers

ABSTRACT Quantitative analysis of genes that code for Dehalococcoides 16S rRNA and chloroethene-reductive dehalogenases TceA, VcrA, and BvcA was done on groundwater sampled from 150 monitoring wells spread over 11 chlorinated ethene polluted European locations. Redundancy analysis was used to relate molecular data to geochemical conditions. Dehalococcoides 16S rRNA- and vinyl chloride (VC)-reductase genes were present at all tested locations in concentrations up to 106 gene copies per ml of groundwater. However, differences between and also within locations were observed. Variation in Dehalococcoides 16S rRNA gene copy numbers were most strongly correlated to dissolved organic carbon concentration in groundwater and to conditions appropriate for biodegradation of chlorinated ethenes (U.S. Environmental Protection Agency score). In contrast, vcrA gene copy numbers correlated most significantly to VC and chlorinated ethene concentrations. Interestingly, bvcA and especially tceA were more correlated with oxidizing conditions. In groundwater microcosms, dechlorination of 1 mM VC was correlated to an increase of vcrA and/or bvcA gene copies by 2 to 4 orders of magnitude. Interestingly, in 34% of the monitoring wells and in 40% of the active microcosms, the amount of individual VC-reductase gene copies exceeded that of Dehalococcoides 16S rRNA gene copies. It is concluded that the geographical distribution of the genes was not homogeneous, depending on the geochemical conditions, whereby tceA and bvcA correlated to more oxidized conditions than Dehalococcoides 16S rRNA and vcrA. Because the variation in VC-reductase gene numbers was not directly correlated to variation in Dehalococcoides spp., VC-reductase genes are better monitoring parameters for VC dechlorination capacity than Dehalococcoides spp.

scholarly journals Monitoring Abundance and Expression of “Dehalococcoides” Species Chloroethene-Reductive Dehalogenases in a Tetrachloroethene-Dechlorinating Flow Column

2008 ◽  
Vol 74 (18) ◽  
pp. 5695-5703 ◽  
Author(s):  
Sebastian Behrens ◽  
Mohammad F. Azizian ◽  
Paul J. McMurdie ◽  
Andrew Sabalowsky ◽  
Mark E. Dolan ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Reductive Dehalogenation ◽  
Gene Copy ◽  
Rrna Gene ◽  
Reductive Dehalogenase ◽  
Gene Copies ◽  
Donor And Acceptor ◽  
Rdase Genes

ABSTRACT We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified “Dehalococcoides” species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes (pceA, tceA, vcrA, and bvcA) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 ± 0.6) × 106 (mean ± standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA, and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.

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