scholarly journals Characterization of a Novel Spirochete Associated with the Hydrothermal Vent Polychaete Annelid, Alvinella pompejana

2001 ◽  
Vol 67 (1) ◽  
pp. 110-117 ◽  
Author(s):  
Barbara J. Campbell ◽  
S. Craig Cary
Keyword(s):  
High Temperature ◽  
Hydrothermal Vent ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Dorsal Surface ◽  
Specific Primers ◽  
Gradient Gel Electrophoresis ◽  
Polychaetous Annelid ◽  
Alvinella Pompejana

ABSTRACT A highly integrated, morphologically diverse bacterial community is associated with the dorsal surface of Alvinella pompejana, a polychaetous annelid that inhabits active high-temperature deep-sea hydrothermal vent sites along the East Pacific Rise (EPR). Analysis of a previously prepared bacterial 16S ribosomal DNA (rDNA) library identified a spirochete most closely related to an endosymbiont of the oligochete Olavius loisae. This spirochete phylotype (spirochete A) comprised only 2.2% of the 16S rDNA clone library but appeared to be much more dominant when the same sample was analyzed by denaturing gradient gel electrophoresis (DGGE) and the terminal restriction fragment length polymorphism procedure (12 to 18%). PCR amplification of the community with spirochete-specific primers used in conjunction with DGGE analysis identified two spirochete phylotypes. The first spirochete was identical to spirochete A but was present in only one A. pompejana specimen. The second spirochete (spirochete B) was 84.5% similar to spirochete A and, more interestingly, was present in the epibiont communities of all of theA. pompejana specimens sampled throughout the geographic range of the worm (13°N to 32°S along the EPR). The sequence variation of the spirochete B phylotype was less than 3% for the range of A. pompejana specimens tested, suggesting that a single spirochete species was present in the A. pompejanaepibiotic community. Additional analysis of the environments surrounding the worm revealed that spirochetes are a ubiquitous component of high-temperature vents and may play an important role in this unique ecosystem.

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 Detection and Characterization of αβ-T-Cell Clonality by Denaturing Gradient Gel Electrophoresis (DGGE)

BioTechniques ◽  
1998 ◽  
Vol 25 (2) ◽  
pp. 244-250 ◽  
Author(s):  
P. thor Straten ◽  
A. Barfoed ◽  
T. Seremet ◽  
I. Saeterdal ◽  
J. Zeuthen ◽  
...  
Keyword(s):  
T Cell ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Gradient Gel Electrophoresis ◽  
T Cell Clonality ◽  
Cell Clonality

scholarly journals Application of Sequence-Dependent Electrophoresis Fingerprinting in Exploring Biodiversity and Population Dynamics of Human Intestinal Microbiota: What Can Be Revealed?

2008 ◽  
Vol 2008 ◽  
pp. 1-26 ◽  
Author(s):  
Geert Huys ◽  
Tom Vanhoutte ◽  
Peter Vandamme
Keyword(s):  
Population Dynamics ◽  
Intestinal Microbiota ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Therapeutic Interventions ◽  
Rrna Gene ◽  
Sequence Dependent ◽  
Human Intestinal Microbiota ◽  
Gradient Gel Electrophoresis ◽  
Community Fingerprinting

Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated.

scholarly journals Phylogenetic Composition of Arctic Ocean Archaeal Assemblages and Comparison with Antarctic Assemblages

2004 ◽  
Vol 70 (2) ◽  
pp. 781-789 ◽  
Author(s):  
Nasreen Bano ◽  
Shomari Ruffin ◽  
Briana Ransom ◽  
James T. Hollibaugh
Keyword(s):  
Arctic Ocean ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
The Arctic ◽  
Rrna Genes ◽  
Specific Primers ◽  
Group I ◽  
The Arctic Ocean ◽  
Gradient Gel Electrophoresis ◽  
Phylogenetic Composition

ABSTRACT Archaea assemblages from the Arctic Ocean and Antarctic waters were compared by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified using the Archaea-specific primers 344f and 517r. Inspection of the DGGE fingerprints of 33 samples from the Arctic Ocean (from SCICEX submarine cruises in 1995, 1996, and 1997) and 7 Antarctic samples from Gerlache Strait and Dallman Bay revealed that the richness of Archaea assemblages was greater in samples from deep water than in those from the upper water column in both polar oceans. DGGE banding patterns suggested that most of the Archaea ribotypes were common to both the Arctic Ocean and the Antarctic Ocean. However, some of the Euryarchaeota ribotypes were unique to each system. Cluster analysis of DGGE fingerprints revealed no seasonal variation but supported depth-related differences in the composition of the Arctic Ocean Archaea assemblage. The phylogenetic composition of the Archaea assemblage was determined by cloning and then sequencing amplicons obtained from the Archaea-specific primers 21f and 958r. Sequences of 198 clones from nine samples covering three seasons and all depths grouped with marine group I Crenarchaeota (111 clones), marine group II Euryarchaeota (86 clones), and group IV Euryarchaeota (1 clone). A sequence obtained only from a DGGE band was similar to those of the marine group III Euryarchaeota.

Model plants for studying the interaction between Methylobacterium mesophilicum and Xylella fastidiosa

2006 ◽  
Vol 52 (5) ◽  
pp. 419-426 ◽  
Author(s):  
Fernando D Andreote ◽  
Paulo T Lacava ◽  
Cláudia S Gai ◽  
Welington L Araújo ◽  
Walter Maccheroni, Jr. ◽  
...  
Keyword(s):  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Xylella Fastidiosa ◽  
Specific Primers ◽  
Gradient Gel Electrophoresis ◽  
Polymerase Chain ◽  
Methylobacterium Mesophilicum ◽  
Biofilm Structure ◽  
Nicotiana Clevelandii

Over the last few years, endophytic bacterial communities associated with citrus have been studied as key components interacting with Xylella fastidiosa. In this study, we investigated the possible interaction between the citrus endophyte Methylobacterium mesophilicum SR1.6/6 and X. fastidiosa in model plants such as Catharanthus roseus (Madagaskar periwinkle) and Nicotiana clevelandii (Clevelands tobacco). The aim of this study was to establish the fate of M. mesophilicum SR1.6/6 after inoculation of C. roseus and N. clevelandii plants, using PCR–DGGE (polymerase chain reaction – denaturing gradient gel electrophoresis) and plating techniques. Shifts in the indigenous endophytic bacterial communities were observed in plants inoculated with strain SR1.6/6, using specific primers targeting α- and β-Proteobacteria. Cells of strain SR1.6/6 were observed in a biofilm structure on the root and hypocotyl surfaces of in vitro seedlings inoculated with M. mesophilicum SR1.6/6. This emphasizes the importance of these tissues as main points of entrance for this organism. The results showed that C. roseus and N. clevelandii could be used as model plants to study the interaction between M. mesophilicum and X. fastidiosa.Key words: endophytic, Methylobacterium, model plants, DGGE.

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