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

2006 ◽  
Vol 72 (8) ◽  
pp. 5232-5238 ◽  
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.

scholarly journals Evaluations of Different Hypervariable Regions of Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient Gel Electrophoresis

2007 ◽  
Vol 74 (3) ◽  
pp. 889-893 ◽  
Author(s):  
Zhongtang Yu ◽  
Rubén García-González ◽  
Floyd L. Schanbacher ◽  
Mark Morrison
Keyword(s):  
16S Rrna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
V Region

ABSTRACT Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

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.

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

2001 ◽  
Vol 67 (2) ◽  
pp. 504-513 ◽  
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.

scholarly journals Evidence for Vertical Transmission of Bacterial Symbionts from Adult to Embryo in the Caribbean Sponge Svenzea zeai

2009 ◽  
Vol 75 (19) ◽  
pp. 6147-6156 ◽  
Author(s):  
On On Lee ◽  
Pui Yi Chui ◽  
Yue Him Wong ◽  
Joseph R. Pawlik ◽  
Pei-Yuan Qian
Keyword(s):  
Water Column ◽  
Clone Library ◽  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Vertical Transfer ◽  
Clone Library Analysis ◽  
Gradient Gel Electrophoresis ◽  
The Caribbean ◽  
Surrounding Seawater

ABSTRACT The Caribbean reef sponge Svenzea zeai was previously found to contain substantial quantities of unicellular photosynthetic and autotrophic microbes in its tissues, but the identities of these symbionts and their method of transfer from adult to progeny are largely unknown. In this study, both a 16S rRNA gene-based fingerprinting technique (denaturing gradient gel electrophoresis [DGGE]) and clone library analysis were applied to compare the bacterial communities associated with adults and embryos of S. zeai to test the hypothesis of vertical transfer across generations. In addition, the same techniques were applied to the bacterial community from the seawater adjacent to adult sponges to test the hypothesis that water column bacteria could be transferred horizontally as sponge symbionts. Results of both DGGE and clone library analysis support the vertical transfer hypothesis in that the bacterial communities associated with sponge adults and embryos were highly similar to each other but completely different from those in the surrounding seawater. Sequencing of prominent DGGE bands and of clones from the libraries revealed that the bacterial communities associated with the sponge, whether adult or embryo, consisted of a large proportion of bacteria in the phyla Chloroflexi and Acidobacteria, while most of the sequences recovered from the community in the adjacent water column belonged to the class Alphaproteobacteria. Altogether, 21 monophyletic sequence clusters, comprising sequences from both sponge adults and embryos but not from the seawater, were identified. More than half of the sponge-derived sequences fell into these clusters. Comparison of sequences recovered in this study with those deposited in GenBank revealed that more than 75% of S. zeai-derived sequences were closely related to sequences derived from other sponge species, but none of the sequences recovered from the seawater column overlapped with those from adults or embryos of S. zeai. In conclusion, there is strong evidence that a dominant proportion of sponge-specific bacteria present in the tissues of S. zeai are maintained through vertical transfer during embryogenesis rather than through acquisition from the environment (horizontal transfer).

scholarly journals Impact of Trichloroethylene Exposure on the Microbial Diversity and Protein Expression in Anaerobic Granular Biomass at 37°C and 15°C

Archaea ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Alma Siggins ◽  
Anne-Marie Enright ◽  
Florence Abram ◽  
Catherine Botting ◽  
Vincent O'Flaherty
Keyword(s):  
Community Structure ◽  
Bacterial Community ◽  
Clone Library ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Glyoxylate Cycle ◽  
Bacterial Community Composition ◽  
Rrna Gene ◽  
Clone Library Analysis ◽  
Granular Biomass

Granular biomass from a laboratory-scale anaerobic bioreactor trial was analysed to identify changes in microbial community structure and function in response to temperature and trichloroethylene (TCE). Two bioreactors were operated at 37°C, while two were operated at 15°C. At the time of sampling, one of each temperature pair of bioreactors was exposed to process failure-inducing concentrations of TCE (60 mg L−1) while the other served as a TCE-free control. Bacterial community structure was investigated using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis. Temperature was identified as an important factor for bacterial community composition, while minor differences were associated with trichloroethylene supplementation. Proteobacteria was the dominant phylum in all bioreactors, while clone library analysis revealed a higher proportion of Bacteroidetes-, Chloroflexi-, and Firmicutes-like clones at 15°C than at 37°C. Comparative metaproteomics in the presence and absence of TCE was carried out by two-dimensional gel electrophoresis (2-DGE), and 28 protein spots were identified, with putative functions related to cellular processes, including methanogenesis, glycolysis, the glyoxylate cycle, and the methyl malonyl pathway. A good agreement between metaproteomic species assignment and phylogenetic information was observed, with 10 of the identified proteins associated with members of the phylum Proteobacteria.

scholarly journals MOLECULAR IDENTIFICATION OF YEAST OF THE PULQUE BY PCR-DGGE, A TRADITIONAL MEXICANBEVERAGE

2015 ◽  
Vol 3 (3) ◽  
pp. 1-15
Author(s):  
Marcial-Quino J. ◽  
Garcia-Ocón B. ◽  
Mendoza-Espinoza J.A. ◽  
Gómez-Manzo S. ◽  
Sierra-Palacios E
Keyword(s):  
Gel Electrophoresis ◽  
Fermentation Process ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Beverage Samples ◽  
D2 Domain ◽  
Gradient Gel Electrophoresis ◽  
26S Rrna

Currently it is well known that yeasts play an essential role in the production of different beverages. In this paper, were identified some of the yeasts involved in the fermentation process of the pulque, a Mexican traditional beverage. Samples were collected from different regions of Mexico and yeasts were detected directly from samples without cultivation. Identifying the yeasts was obtained using amplification the D1/D2 domain of the 26S rRNA gene and Denaturing Gradient Gel Electrophoresis (DGGE). The results of DGGE showed different profiles of bands in each of the analyzed samples, indicating the presence of several species of yeast, which was also confirmed by sequencing of the bands corresponding to the domain D1/D2, succeeded in identifying five species of yeasts. The results obtained in this work demonstrated that the technique used for identification of yeasts of pulque was efficient. Besides, the optimization of this method could also allow rapid identification of yeasts and help understand the role of these in the fermentation process of this beverage, as well as the isolation of strains of interest for biotechnological purposes such as production of ethanol or metabolites with nutraceutical activity.

scholarly journals Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages

2001 ◽  
Vol 67 (11) ◽  
pp. 5113-5121 ◽  
Author(s):  
Luca Cocolin ◽  
Marisa Manzano ◽  
Carlo Cantoni ◽  
Giuseppe Comi
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Dynamic Changes ◽  
Good Tool ◽  
Reverse Transcription Pcr ◽  
Dna And Rna ◽  
Fermented Sausages ◽  
Brochothrix Thermosphacta ◽  
Gradient Gel Electrophoresis

ABSTRACT In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from the sausages and subjected to PCR and reverse transcription-PCR, and the amplicons obtained were analyzed by DGGE. Lactic acid bacteria (LAB) were present together with other organisms, mainly members of the family Micrococcaceae and meat contaminants, such as Brochothrix thermosphacta andEnterococcus sp., during the first 3 days of fermentation. After 3 days, LAB represented the main population, which was responsible for the acidification and proteolysis that determined the characteristic organoleptic profile of the Friuli Venezia Giulia fermented sausages. The PCR-DGGE protocol for studying sausage fermentation proved to be a good tool for monitoring the process in real time, and it makes technological adjustments possible when they are required.

Study of microbial community of brewery-treating granular sludge by denaturing gradient gel electrophoresis of 16S rRNA gene

2001 ◽  
Vol 43 (1) ◽  
pp. 77-82 ◽  
Author(s):  
O.-C. Chan ◽  
W.-T. Liu ◽  
H. H. Fang
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Granular Sludge ◽  
Upflow Anaerobic Sludge Blanket ◽  
Anaerobic Sludge ◽  
Rrna Gene ◽  
Related Sequence ◽  
Gradient Gel Electrophoresis

The microbial community structure of granular sludge from an upflow anaerobic sludge blanket (UASB) reactor treating brewery effluent was studied by denaturing gradient gel electrophoresis (DGGE). Twelve major bands were observed in the DGGE fingerprint for the Bacteria domain and four bands for the Archaea domain. Of the bacterial bands observed, six were successfully purified and sequenced. Among them, three were related to the gram-positive low G+C group, one to the Delta subclass of the Proteobacteria, one to the Gamma subclass, and one to the Cytophaga group with no close related sequence. The 16S rRNA sequences of the four archaeal bands were closely associated with Methanosaeta concilii and Methanobacterium formicum.

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