Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

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Relationship between wood-inhabiting fungi determined by molecular analysis (denaturing gradient gel electrophoresis) and quality of decaying logs

Canadian Journal of Forest Research ◽

10.1139/x10-176 ◽

2010 ◽

Vol 40 (12) ◽

pp. 2384-2397 ◽

Cited By ~ 41

Author(s):

Tiina Rajala ◽

Mikko Peltoniemi ◽

Taina Pennanen ◽

Raisa Mäkipää

Keyword(s):

Tree Species ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Lignin Content ◽

Fruit Body ◽

Fungal Communities ◽

Important Species ◽

European Aspen ◽

Gradient Gel Electrophoresis ◽

Decaying Wood

We investigated the fungal communities inhabiting decaying logs in a seminatural boreal forest stand in relation to host tree species, stage of decay, density, diameter, moisture, C to N ratio, Klason lignin content, and water- and ethanol-soluble extractives. Communities were profiled using denaturing gradient gel electrophoresis fingerprinting of the rDNA ITS1 region coupled with sequencing of fungal DNA extracted directly from the wood. In addition, polypore fruit bodies were inventoried. Logs from different tree species had different fungal communities and different physicochemical properties (e.g., C to N ratio, density, ethanol extractives, and diameter). Ascomycetes comprised a larger portion of communities inhabiting deciduous birch ( Betula spp.) and European aspen ( Populus tremula L.) logs compared with those living on coniferous Norway spruce ( Picea abies (L.) Karst.) and Scots pine ( Pinus sylvestris L.). A relationship between mycelial community structure and density of decaying spruce logs suggested a succession of fungi with mass loss of wood. The fruit body inventory underestimated fungal diversity in comparison with the culture-free denaturing gradient gel electrophoresis analysis that also detected inconspicuous but important species inhabiting decaying wood.

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Methanotrophic diversity in high arctic wetlands on the islands of Svalbard (Norway) - denaturing gradient gel electrophoresis analysis of soil DNA and enrichment cultures

Canadian Journal of Microbiology ◽

10.1139/w03-080 ◽

2003 ◽

Vol 49 (10) ◽

pp. 602-612 ◽

Cited By ~ 47

Author(s):

Ingvild Wartiainen ◽

Anne Grethe Hestnes ◽

Mette M Svenning

Keyword(s):

Methane Emission ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

The Arctic ◽

Rrna Gene ◽

Climatic Warming ◽

Soil Dna ◽

Enrichment Cultures ◽

Arctic Soil ◽

Gradient Gel Electrophoresis

The methanotrophic community in arctic soil from the islands of Svalbard, Norway (78°N) was analysed by combining group-specific PCR with PCR of the highly variable V3 region of the 16S rRNA gene and then by denaturing gradient gel electrophoresis (DGGE). Selected bands were sequenced for identification. The analyses were performed with DNA extracted directly from soil and from enrichment cultures at 10 and 20 °C. The two genera Methylobacter and Methylosinus were found in all localities studied. The DGGE band patterns were simple, and DNA fragments with single base differences were separated. The arctic tundra is a potential source of extensive methane emission due to climatic warming because of its large reservoirs of stored organic carbon. Higher temperatures due to climatic warming can cause increased methane production, and the abundance and activity of methane-oxidizing bacteria in the arctic soil may be important regulators for methane emission to the atmosphere.Key words: methanotrophic diversity, Svalbard, arctic wetland, denaturing gradient gel electrophoresis.

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Isolation and purification of microbial community DNA from soil naturally enriched for chitin

Biologia ◽

10.2478/s11756-012-0059-0 ◽

2012 ◽

Vol 67 (4) ◽

Author(s):

Pullabhotla Sarma ◽

Vadlamudi Srinivas ◽

Kondreddy Anil ◽

Appa Podile

Keyword(s):

16S Rdna ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Sodium Borate ◽

Metagenomic Dna ◽

Soil Dna ◽

Isolation And Purification ◽

Gradient Gel Electrophoresis ◽

Chitinase Genes ◽

Sodium Borate Buffer

AbstractWe made an attempt to isolate and purify metagenomic DNA from chitin enriched soil. In this communication we report a modified direct lysis method for soil DNA extraction including initial pre-lysis washing of sample, followed by a rapid polyvinylpyrrolidone-agarose-based purification and electroelution of DNA using Gene-capsule™ assembly. Rapidity was achieved using low molarity conducting media (sodium-borate buffer) for electrophoresis by reducing run time for both the gel electrophoresis and electroelution. Extracted DNA was sufficiently pure and of high quality, evidenced by amplification of 16S rDNA and chitinase genes by PCR. Metagenomic nature of the DNA was confirmed by running V3 (16S rDNA) region amplicons using denaturing gradient gel electrophoresis. This method requires 30 min for purification, and less than 2 h for complete execution of protocol and becomes the first report on the isolation of metagenomic DNA from soil naturally enriched for chitin.

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Characterization of bacterial and fungal communities in composted biosolids over a 2 year period using denaturing gradient gel electrophoresis

Canadian Journal of Microbiology ◽

10.1139/w08-152 ◽

2009 ◽

Vol 55 (4) ◽

pp. 375-387 ◽

Cited By ~ 22

Author(s):

Amy Novinscak ◽

Nadine J. DeCoste ◽

Céline Surette ◽

Martin Filion

Keyword(s):

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Species ◽

Fungal Species ◽

Fungal Communities ◽

Whole Process ◽

Agricultural Applications ◽

Gradient Gel Electrophoresis ◽

Microbial Groups ◽

Key Microorganisms

Composting is a microbial process that converts organic waste into a nutrient-rich end product used in horticultural and agricultural applications. The diversity and long-term succession of microorganisms found in composted biosolids has been less characterized than other composts. In this study, bacterial and fungal communities found in composted biosolids aging from 1 to 24 months were studied using denaturing gradient gel electrophoresis (DGGE) and sequencing. The results revealed high levels of diversity, where 53 bacterial species belonging to 10 phyla and 21 fungal species belonging to 4 phyla were identified. Significant differences were observed when comparing the bacterial DGGE patterns of young compost samples, whereas no differences were observed in samples over 8 months. For fungal patterns, no significant differences were observed during the first 4 months of composting, but the diversity then significantly shifted until 24 months. The results indicate that patterns of bacterial species vary during the first few months of composting, whereas fungal patterns generally vary throughout the whole process, except during early stages. The description of the main microbial groups found in composted biosolids could find various applications, including the discovery of biotechnologically relevant microorganisms and the development of novel markers allowing quantitative monitoring of key microorganisms.

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Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.504-513.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 504-513 ◽

Cited By ~ 322

Author(s):

Reetta M. Satokari ◽

Elaine E. Vaughan ◽

Antoon D. L. Akkermans ◽

Maria Saarela ◽

Willem M. de Vos

Keyword(s):

16S Rdna ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Methodological Approach ◽

Common Species ◽

Bifidobacterium Adolescentis ◽

Specific Pcr ◽

Rdna Sequences ◽

Pcr Products ◽

Gradient Gel Electrophoresis

ABSTRACT We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE).Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

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Molecular Profiling of the Clostridium leptum Subgroup in Human Fecal Microflora by PCR-Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.00151-06 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5232-5238 ◽

Cited By ~ 37

Author(s):

Jian Shen ◽

Baorang Zhang ◽

Guifang Wei ◽

Xiaoyan Pang ◽

Hua Wei ◽

...

Keyword(s):

Clone Library ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Rrna Gene ◽

Specific Pcr ◽

Clone Library Analysis ◽

Pcr Products ◽

Gradient Gel Electrophoresis ◽

Universal Bacterial Primer ◽

Group Specific Primer

ABSTRACT A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, ∼99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.

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