Polymerase Chain Reaction-denaturing Gradient Gel Electrophoresis Analysis Research Articles

Bacterial communities in iron mottles in the plow pan layer in a Japanese rice field: Estimation using PCR-DGGE and sequencing analyses

Bacterial communities in iron (Fe) mottles in the plow pan layer in a Japanese rice field were estimated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis targeting 16S rDNA genes. The DGGE band patterns indicate that distinct bacterial communities with lower diversity inhabit Fe mottles compared with the reference soil matrix. Many of the DGGE bands of the Fe mottles that were sequenced (12 of 29 DGGE bands) were closely related to bacteria involved with Fe oxidation and reduction: Siderooxidans ghiorsii, Azoarcus sp., Azovibrio sp., Dechloromonas sp., Acidimicrobium ferrooxidans, Geobacter psychrophilus, Clostridium sp., Desulfovibrio sp. and Desulfonatronum cooperativum. The results suggest that specific bacteria inhabit Fe mottles and may play a role in the Fe cycle.

PCR-DGGE Analysis of Nematode Diversity in Cu-Contaminated Soil

A wheat pot experiment was conducted under greenhouse conditions to assess the effect of copper contamination on soil nematode diversity by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method and morphological analysis. The soil was treated with CuSO 4·5H 2O at the following concentrations: 0, 50, 100, 200, 400, and 800 mg kg −1 dry soil, and the soil samples were collected at wheat jointing and ripening stages. Nematode diversity index ( H′) from morphological analysis showed no difference between the control and the treated samples in either of the sampling dates. At the wheat ripening stage, nematode diversity obtained by the PCR-DGGE method decreased noticeably in the Cu800 treatment in comparison with the control. With optimization of the method of nematode DNA extraction, PCR-DGGE could give more information on nematode genera, and the intensity of the bands could reflect the abundance of nematode genera in the assemblage. The PCR-DGGE method proved promising in distinguishing nematode diversity in heavy metal contaminated soil.

PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium

Bacteria attached to the rumen epithelium (or epimural community) are not well characterised and their role in rumen functioning is not totally understood. There is just one published report of a clone library from one cow that suggests that this epimural community differs from the bacteria associated with the rumen digestive contents. However, this time-consuming approach is not adapted for examining microbial population changes in groups of animals. In in vivo studies, when samples from several animals have to be analysed simultaneously, a simpler technique has to be used. In this study, a genetic fingerprinting technique, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterise the structure of the bacterial population attached to the rumen epithelium. This community was compared with that present in the solid and liquid phases of rumen content under two contrasting diets. Rumen samples were obtained from four forage-fed and four high-concentrate-fed (80 : 20, wheat grain : hay) 5-month-old lambs. After slaughter, samples from five epithelial sites and the solid and liquid digesta phases were taken for DNA extraction and analysis. Bacterial communities were profiled by PCR-DGGE using bacterial-specific 16S rDNA primers. Analysis of the fingerprint revealed that the epithelial community differed from those of rumen content in both diets. As expected, the nature of the feed influenced the bacterial communities from the solid and liquid rumen phases but no diet effect was observed in the rumen epithelial profiles suggesting a strong host effect on this bacterial population. Additionally, no differences were observed among the five epithelial sampling sites taken from each animal. The profile of the bacterial population attached to the rumen epithelium presented a high inter-animal variation, whether this difference has an influence in the function of this community remains to be determined.

Bacterial Activity, Community Structure, and Centimeter-Scale Spatial Heterogeneity in Contaminated Soil

In an anthropogenically disturbed soil (88% sand, 8% silt, 4% clay), 150-mg samples were studied to examine the fine-scale relationship of bacterial activity and community structure to heavy metal contaminants. The soils had been contaminated for over 40 years with aromatic solvents, Pb, and Cr. Samples from distances of <1, 5, 15, and 50 cm over a depth range of 40-90 cm underwent a sequential analysis to determine metabolic potential (from 14C glucose mineralization), bacterial community structure [using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)], and total extractable Pb and Cr levels. Metabolic potential varied by as much as 10,000-fold in samples <1 cm apart; log-log plots of metal concentration and microbial metabolic potential showed no correlation with each other. Overall, metal concentrations ranged from 9 to 29,000 mg kg(-1) for Pb and from 3 to 8500 mg kg(-1) for Cr with small zones of high contamination present. All regions exhibited variable metal concentrations, with some soil samples having 30-fold differences in metal concentration in sites <1 cm apart. Geostatistical analysis revealed a strong spatial dependence for all three parameters tested (metabolic activity, Pb, and Cr levels) with a range up to 30 cm. Kriging maps showed that in zones of high metal, the corresponding metabolic activity was low suggesting that metals negatively impacted the microbial community. PCR-DGGE analysis revealed that diverse communities were present in the soils with a random distribution of phylotypes throughout the sampling zones. These results suggest the presence of spatially isolated microbial communities within the soil profile.

Stereospecific effect of hexachlorocyclohexane on activity and structure of soil methanotrophic communities

In the past decades, large amounts of non-insecticidal hexachlorocyclohexane (HCH) isomers (alpha-, beta-, delta- and epsilon-HCH) have been dumped as side-products of the insecticide gamma-HCH (lindane). This study investigates the effect of HCH isomers on methane oxidation, an important soil function performed by methanotrophic bacteria. Both activity and structure of the methanotrophic community were assessed, using methane oxidation assays and PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) respectively. Methane oxidation assays with historically polluted soils revealed that on the long-term methane oxidation was inhibited by HCH pollution. PCR-DGGE and diversity analysis based on Lorenz curves showed that the type I methanotrophic community was less evenly distributed in historically HCH-polluted soils compared with less polluted reference soils. Short-term experiments with methane-enriched consortia further demonstrated that only gamma- and delta-isomers inhibited methane oxidation. Type I methanotrophs of methane-enriched microbial consortia that received gamma- or delta-HCH evolved towards higher species richness. Apparently, for historically HCH-polluted soils, a narrow community remained after long-term exposure while in case of short-term exposures, methane-enriched consortia were converted into less active, but richer communities when they were stressed by the presence of gamma- or delta-HCH. This work demonstrates the importance of incorporating all isomers and possible other side-products in risk assessment studies of persistent organic pollutants and the use of structural analysis of type I methanotrophic communities as evaluating tool.

Succession and phylogenetic profile of eukaryotic communities in the composting process of rice straw estimated by PCR-DGGE analysis

The succession and phylogenetic profile of eukaryotic communities during the composting process of rice straw (RS) were studied by applying polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by sequencing of 18S rDNA. Principal component analysis and cluster analysis of the DGGE band patterns of eukaryotic communities resulted in exactly the same grouping as found with phospholipid fatty acid (PLFA) analysis (Cahyani et al. in Soil Sci Plant Nutr 48:735, 2002) and by the DGGE pattern analysis of the bacterial communities (Cahyani et al. in Soil Sci Plant Nutr 49:619, 2003) for the same samples, namely the communities characterizing the pre-composting stage (initial RS materials), and thermophilic, middle, and curing stages of the compost. Different eukaryotic members characterized the respective stages as follows: fungi (Ascomycota) for the initial RS materials, protozoans (Apicomplexa) as well as the fungi (Ascomycota) of the initial RS materials for the thermophilic stage, fungi (Ascomycota and Basidiomycota), protozoans (Opalozoa, Ciliophora and Leptomyxida), nematodes and stramenopiles for the middle stage, and fungi (Ascomycota, Zygomycota and Oomycota), algae (Haptophyceae and Chrysophyceae), and nematodes for the curing stage, respectively. Temperature, moisture content, and substrates available seemed to play a key role in determining the composition of eukaryotic members present at the respective stages of the composting process of RS.

Effect of long-term herbicide applications on the bacterial community structure and function in an agricultural soil

Little is known about the chronic effect of herbicides on the soil microbial community, with most studies focusing on acute impacts. In this study, we investigated the effect of 20 years of atrazine and metolachlor application on the community structure, abundance and function of bacterial groups in the bulk soil of a maize monoculture. Group-specific PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) of 16S rRNA genes was used to characterize the composition of the microbial community. Primers specific for the entire bacterial group, as well as for the Acidobacterium group, the actinomycetes, the ammonium oxidizers, and the Type I and II methanotrophs were used in the PCR-DGGE analysis. Cluster analysis of the DGGE patterns obtained with the different primer sets differentiated between the herbicide-treated and the non-treated control soil. In particular the methanotrophic community differed, with three bands (phylotypes) being absent (or showing a lower intensity) in the DGGE patterns of the herbicide-treated soils compared to the patterns of the control soil. The differences in the methanotrophic community structure were not reflected in the methane oxidation capacity, which was similar for the two treatments. The prevalence of methanotrophs as evaluated with real-time PCR analysis also did not differ between the herbicide-treated and non-treated soil. Our results indicate that the long-term use of the herbicides atrazine and metolachlor resulted in an altered soil community structure, in particular for the methanotrophic bacteria. These observed changes did not cause a decreased community function (methane oxidation), probably because the total abundance of the methanotrophs in the soil system was preserved.

Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis

The use of Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technique in identifying the microorganisms present in commercial probiotic yoghurts and lyophilised products was evaluated. Two reference ladders were assembled constituted by PCR-amplified V2–V3 regions of 16S rDNA from bacterial species generally used as probiotics. Identification was achieved comparing the PCR-DGGE patterns obtained from the analysed products with the ladder bands. Bands from members of the same species showed the same migration distance in denaturing gel, hence supporting the identificative value of the method. The validity of the technique was also proven confirming the PCR-DGGE identification results by sequence data analysis and by species-specific PCR. General congruence between microorganisms declared on the label and those revealed by PCR-DGGE was found for probiotic yoghurts. Conversely, some discrepancies were observed for probiotic lyophilised preparations, i.e. the incorrect identification of some Bifidobacterium and Bacillus species and the presence of not declared microorganisms. PCR-DGGE turned out to be an appropriate culture-independent approach for a rapid detection of the predominant species in mixed probiotic cultures.

Molecular Ecological Analysis of Dietary and Antibiotic-Induced Alterations of the Mouse Intestinal Microbiota

A cultivation-independent approach, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterize changes in fecal bacterial populations resulting from consumption of a low residue diet or oral administration of a broad-spectrum antibiotic. C57BL/6NHsd mice were weaned to either a standard nonpurified diet (LC-diet) or a low residue diet (LR-diet) and at 17 wk of age were randomly assigned to receive drinking water with or without 25 ppm cefoxitin for 14 d. On d 1, 2, 7 and 14, microbial DNA was extracted from feces, and the V3 region of the 16S rDNA gene was amplified by PCR and analyzed by DGGE. The diversity of fecal microbial populations, assessed using Shannon's index (H'), which incorporates species richness (number of species, or in this case, PCR-DGGE bands) and evenness (the relative distribution of species), was not affected by cefoxitin. However, use of Sorenson's pairwise similarity coefficient (C(s)), an index that measures the species in common between different habitats, indicated that the species composition of fecal bacterial communities was altered by cefoxitin in mice fed either diet. Dietary effects on fecal microbial communities were more pronounced, with greater H' values (P < 0.05) in mice fed the LR-diet (1.9 +/- 0.1) compared with the LC-diet (1.6 +/- 0.1). The C(s) values were also greater (P < 0.05) in fecal bacterial populations from mice fed the LR-diet (C(s) = 69.8 +/- 2.0%) compared with mice fed the LC-diet (C(s) = 50.1 +/- 3.8%), indicating greater homogeneity of fecal bacterial communities in mice fed the LR-diet. These results demonstrate the utility of cultivation-independent PCR-DGGE analysis combined with measurements of ecological diversity for monitoring diet- and antibiotic-induced alterations of the complex intestinal microbial ecosystem.

Competitive PCR–DGGE analysis of bacterial mixtures: an internal standard and an appraisal of template enumeration accuracy

Analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments from environmental samples by denaturing gradients of chemicals or heat [denaturing gradient gel electrophoresis (DGGE) and thermal gradient gel electrophoresis (TGGE)] within polyacrylamide gels is a popular tool in microbial ecology. Difficulties in acceptance of the technique and interpretation of the results remain, due to its qualitative nature. In this study we have addressed this problem by the construction and evaluation of a quantitative standard for incorporation into test DNA samples. The standard was based on a naturally occurring 16S rRNA gene carried by the X-endosymbiont of the psyllid Anomoneura mori, a γ-proteobacterium. This sequence is the most AT-rich 16S rDNA gene recovered from any cultured organism or environmental sample described to date, and a specifically amplified rDNA fragment denatured under exceptionally low stringency denaturing conditions. The native sequence was modified to incorporate perfect matches to the PCR primers used. The efficiency of amplification of this standard in comparison to a range of 16S rDNA sequences and the errors involved in enumerating template molecules under a range of PCR conditions are demonstrated and quantified. Tests indicated that highly accurate counts of released target molecules from a range of bacterial cells could be achieved in both laboratory mixtures and compost.