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

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

Applied and Environmental Microbiology ◽

10.1128/aem.00516-06 ◽

2006 ◽

Vol 72 (9) ◽

pp. 5877-5883 ◽

Cited By ~ 94

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.

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Determination of Bifidobacterium and Lactobacillus in breast milk of healthy women by digital PCR

Beneficial Microbes ◽

10.3920/bm2015.0195 ◽

2016 ◽

Vol 7 (4) ◽

pp. 559-569 ◽

Cited By ~ 8

Author(s):

L. Qian ◽

H. Song ◽

W. Cai

Keyword(s):

Breast Milk ◽

16S Rrna ◽

16S Rrna Gene ◽

Digital Pcr ◽

16S Rrna Genes ◽

Rrna Genes ◽

Gene Copy ◽

Rrna Gene ◽

Qrt Pcr ◽

Gene Copies

Breast milk is one of the most important sources of postnatal microbes. Quantitative real-time polymerase chain reaction (qRT-PCR) is currently used for the quantitative analysis of bacterial 16S rRNA genes in breast milk. However, this method relies on the use of standard curves and is imprecise when quantitating target DNA of low abundance. In contrast, droplet digital PCR (DD-PCR) provides an absolute quantitation without the need for calibration curves. A comparison between DD-PCR and qRT-PCR was conducted for the quantitation of Bifidobacterium and Lactobacillus 16S RNA genes in human breast milk, and the impacts of selected maternal factors were studied on the composition of these two bacteria in breast milk. From this study, DD-PCR reported between 0-34,460 16S rRNA gene copies of Bifidobacterium genera and between 1,108-634,000 16S rRNA gene copies of Lactobacillus genera in 1 ml breast milk. The 16S rRNA gene copy number of Lactobacillus genera was much greater than that of Bifidobacterium genera in breast milk. DD-PCR showed a 10-fold lower limit of quantitation as compared to qRT-PCR. A higher correlation and agreement was observed between qRT-PCR and DD-PCR in Lactobacillus quantitation as compared to Bifidobacterium quantitation. Based on our DD-PCR quantitation, a low abundance of Bifidobacterium bacteria in breast milk was correlated to higher pre-pregnancy body mass index (BMI). However, no significant difference was observed for these two bacteria in breast milk between mothers who had vaginal deliveries and caesarean deliveries. This study suggests that DD-PCR is a better tool to quantitate the bacterial load of breast milk compared to the conventional qRT-PCR method. The number of breast milk Bifidobacterium bacteria is influenced by maternal pre-pregnancy BMI.

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Isolation and Characterization of “Dehalococcoides” sp. Strain MB, Which Dechlorinates Tetrachloroethene to trans-1,2-Dichloroethene

Applied and Environmental Microbiology ◽

10.1128/aem.00767-09 ◽

2009 ◽

Vol 75 (18) ◽

pp. 5910-5918 ◽

Cited By ~ 74

Author(s):

Dan Cheng ◽

Jianzhong He

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Anaerobic Respiration ◽

Rrna Gene ◽

Reductive Dehalogenase ◽

Isolation And Characterization ◽

Dehalococcoides Ethenogenes ◽

Pure Cultures ◽

Microbial Group ◽

Rdase Genes

ABSTRACT In an attempt to understand the microorganisms involved in the generation of trans-1,2-dichloroethene (trans-DCE), pure-culture “Dehalococcoides” sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate cis-DCE as the predominant product, Dehalococcoides sp. strain MB reductively dechlorinates PCE to trans-DCE and cis-DCE at a ratio of 7.3 (±0.4):1. It utilizes H2 as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 μm in diameter and ∼150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with Dehalococcoides ethenogenes strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of trans-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group, Dehalococcoides species.

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Gastrointestinal Segments Influenced Fermentation End-Products, Microbiota and Microbial Abundances in Goats

10.21203/rs.3.rs-359515/v1 ◽

2021 ◽

Author(s):

Tsegay Gebremariam ◽

Zhiliang Tan

Keyword(s):

Fatty Acids ◽

Microbial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

18S Rrna ◽

18S Rrna Gene ◽

Rrna Gene ◽

Gene Copies ◽

End Products ◽

The Impact

Abstract Purpose: Carbohydrate diets altered fermentation end-products and microbial community in the gastrointestinal tracts (GIT) of goats. Gastrointestinal contents used to determine the impact of carbohydrate feeds on fermentation end-products and microbial community in goats.Methodology: in the study goats were assigned to one of the two treatments corn meal (CM) or Corn gluten (CG) in a randomized block design (400 g/kg DM each). Goats were slaughtered, GIT liquids were used to determine dissolved gasses, fatty acids and microbial community.Results: Goats fed CG increased molar acetate (P < 0.05), lowered butyrate and propionate in the fore and hindgut comparing to those goats received CM. Goats received CM had higher (P < 0.05) dH2 while lowered dH2S in the fore and hindgut than those goats fed with CG treatment. The fore and hindgut had higher (P < 0.01) 16S rRNA gene copies of bacteria, protozoa, methanogens and 18S rRNA gene copies fungi than in the ileum and cecum. Goats fed CG diet had higher (P < 0.05)16S rRNA gene copies of bacteria, protozoa, methanogens, and 18S rRNA gene copies of fungi than those goats fed with CM diet. Conclusion fore and hindguts improved dissolved gasses, fatty acids and microbial community comparing with in the ileum and cecum. Goats fed CM had improved the Methanobacterials order and Methanobrevibacter genus as compared with those goats fed CG. The study suggested that hindgut segments have a reasonable contribution as foregut to methane emissions from goats.

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Development of a Real-Time PCR Assay for Detection and Quantification of Rhizobium leguminosarum Bacteria and Discrimination between Different Biovars in Zinc-Contaminated Soil

Applied and Environmental Microbiology ◽

10.1128/aem.02232-10 ◽

2011 ◽

Vol 77 (13) ◽

pp. 4626-4633 ◽

Cited By ~ 13

Author(s):

Catriona A. Macdonald ◽

Ian M. Clark ◽

Penny R. Hirsch ◽

Fang-Jie Zhao ◽

Steve P. McGrath

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Rhizobium Leguminosarum ◽

Gene Copy Number ◽

Gene Copy ◽

Most Probable Number ◽

Rrna Gene ◽

Probable Number ◽

Content Type ◽

Gene Copies

ABSTRACTPrimers were designed to target 16S rRNA andnodDgenes ofRhizobium leguminosarumfrom DNA extracted from two different soil types contaminated with Zn applied in sewage sludge. Numbers of rhizobia estimated using 16S rRNA gene copy number showed higher abundance than those estimated by bothnodDand the most-probable-number (MPN) enumeration method using a plant trap host. Both 16S rRNA gene copies and the MPN rhizobia declined with increased levels of Zn contamination, as did the abundance of the functional genenodD, providing compelling evidence of a toxic effect of Zn onR. leguminosarumpopulations in the soil. Regression analysis suggested the total Zn concentration in soil as a better predictor of rhizobial numbers than both NH4NO3-extractable and soil solution Zn.R. leguminosarumbv. viciaenodDgene copies were generally less sensitive to Zn thanR. leguminosarumbv. trifoliinodD.The latter were generally below detection limits at Zn levels of >250 mg kg−1. Although there were differences in the actual numbers estimated by each approach, the response to Zn was broadly similar across all methods. These differences were likely to result from the fact that the molecular approaches assess the potential for nodulation while the MPN approach assesses actual nodulation. The results demonstrate that the use of targeted gene probes for assessing environmental perturbations of indigenous soil rhizobial populations may be more sensitive than the conventional plant bioassay and MPN methods.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02199-07 ◽

2008 ◽

Vol 74 (9) ◽

pp. 2728-2739 ◽

Cited By ~ 117

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.

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The diversity of active microbial groups in an activated sludge process treating painting process wastewater

E3S Web of Conferences ◽

10.1051/e3sconf/202014801002 ◽

2020 ◽

Vol 148 ◽

pp. 01002

Author(s):

Herto Dwi Ariesyady ◽

Mentari Rizki Mayanda ◽

Tsukasa Ito

Keyword(s):

Wastewater Treatment ◽

Microbial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Activated Sludge ◽

Biological Treatment ◽

Rrna Gene ◽

Activated Sludge Process ◽

Painting Process ◽

Process Wastewater

Activated sludge process is one of the wastewater treatment method that is applied for many wastewater types including painting process wastewater of automotive industry. This wastewater is well-known to have high heavy metals concentration which could deteriorate water environment if appropriate performance of the wastewater treatment could not be achieved. In this study, we monitored microbial community diversity in a Painting Biological Treatment (PBT) system. We applied a combination of cultivation and genotypic biological methods based on 16S rRNA gene sequence analysis to identify the diversity of active microbial community. The results showed that active microbes that could grow in this activated sludge system were dominated by Gram-negative bacteria. Based on 16S rRNA gene sequencing analysis, it was revealed that their microbial diversity has close association with Bacterium strain E286, Isosphaera pallida, Lycinibacillus fusiformis, Microbacterium sp., Orchobactrum sp., Pseudomonas guariconensis, Pseudomonas sp. strain MR84, Pseudomonas sp. MC 54, Serpens sp., Stenotrophomonas acidaminiphila, and Xylella fastidiosa with similarity of 86 – 99%. This findings reflects that microbial community in a Painting Biological Treatment (PBT) system using activated sludge process could adapt with xenobiotics in the wastewater and has a wide range of diversity indicating a complex metabolism mechanism in the treatment process.

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Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella

Microbiology ◽

10.1099/mic.0.038224-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 2080-2091 ◽

Cited By ~ 28

Author(s):

Anne-Laure Michon ◽

Fabien Aujoulat ◽

Laurent Roudière ◽

Olivier Soulier ◽

Isabelle Zorgniotti ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Diversity ◽

Natural Populations ◽

Variable Region ◽

Housekeeping Gene ◽

Single Copy ◽

Population Based ◽

Rrna Gene ◽

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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