scholarly journals Analysis of Bacterial Communities in the Rhizosphere of Chrysanthemum via Denaturing Gradient Gel Electrophoresis of PCR-Amplified 16S rRNA as Well as DNA Fragments Coding for 16S rRNA

2001 ◽  
Vol 67 (1) ◽  
pp. 172-178 ◽  
Author(s):  
Bernadette M. Duineveld ◽  
George A. Kowalchuk ◽  
Anneke Keijzer ◽  
Jan Dirk van Elsas ◽  
Johannes A. van Veen
Keyword(s):  
16S Rrna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bulk Soil ◽  
Root Tip ◽  
Rt Pcr ◽  
Root Tips ◽  
Bacterial Populations ◽  
Gradient Gel Electrophoresis ◽  
Species Specific

ABSTRACT The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such asPseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root.

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.

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.

Molecular Analysis of Spoilage-Related Bacteria in Pasteurized Milk during Refrigeration by PCR and Denaturing Gradient Gel Electrophoresis

2009 ◽  
Vol 72 (3) ◽  
pp. 572-577 ◽  
Author(s):  
HONGFEI HE ◽  
JIN DONG ◽  
CHIN NYEAN LEE ◽  
YONG LI
Keyword(s):  
Shelf Life ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Plate Count ◽  
Pasteurized Milk ◽  
Milk Samples ◽  
Gradient Gel Electrophoresis ◽  
Aerobic Plate Count ◽  
Species Specific

Bacterial diversity in fluid milk products has been extensively studied in order to improve milk quality. Here, we illustrate the utility of viable counts and PCR–denaturing gradient gel electrophoresis (DGGE) for monitoring the microbial spoilage of pasteurized milk during shelf life. Five pasteurized milk samples stored at 4°C were examined at 10 and 5 days before expiration and on the expiration day. With bacterial DNA extracted directly from the samples, PCR-DGGE analysis indicated that Pseudomonas became dominant in four samples. Meanwhile, the aerobic plate count of these four samples exceeded the regulatory limit of 20,000 CFU/ml at 5 days before expiration, and the rapid psychrotrophic count markedly surpassed the aerobic plate count on the expiration day. Streptococcus and Buttiauxella spp. were detected in several samples. Sequence analysis of DGGE fragments revealed high diversity among Pseudomonas spp. in the milk samples. P. putida and P. migulae grew to high numbers during refrigerated storage. Further identification of Pseudomonas at the species level was facilitated by PCR and multiplex PCR using species-specific primers; consequently, P. fluorescens and P. fragi were observed. These results highlight an important role of Pseudomonas in the shelf life of pasteurized milk.

scholarly journals Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

2003 ◽  
Vol 69 (11) ◽  
pp. 6801-6807 ◽  
Author(s):  
Isabel Lopez ◽  
Fernanda Ruiz-Larrea ◽  
Luca Cocolin ◽  
Erica Orr ◽  
Trevor Phister ◽  
...  
Keyword(s):  
Lactic Acid ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Pcr Primers ◽  
Fungal Species ◽  
Bacterial Populations ◽  
Gradient Gel Electrophoresis ◽  
Primer Sets

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

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