scholarly journals Viable microbes in ice: application of molecular assays to McMurdo Dry Valley lake ice communities

2010 ◽  
Vol 22 (5) ◽  
pp. 470-476 ◽  
Author(s):  
Markus Dieser ◽  
Andreas Nocker ◽  
John C. Priscu ◽  
Christine M. Foreman
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Extreme Environments ◽  
Membrane Integrity ◽  
Pcr Amplification ◽  
Ice Cores ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Dry Valleys ◽  
Dry Valley ◽  
Gradient Gel Electrophoresis

AbstractThe permanent ice covers of the McMurdo Dry Valley lakes, Antarctica, are colonized by a diverse microbial assemblage. We collected ice cores from Lakes Fryxell, Hoare and Bonney. Propidium monoazide (PMA) was used in combination with quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE) to examine membrane integrity of prokaryotes in these extreme environments. PMA selectively penetrates cells with compromised membranes and modifies their DNA resulting in the suppression of PCR amplification. Our results based on analysis of 16S rRNA genes demonstrate that despite the hostile conditions of the Dry Valleys, the permanent ice covers of the lakes support a ‘potentially viable’ microbial community. The level of membrane integrity, as well as diversity, was higher in samples where sediment was entrapped in the ice cover. Pronounced differences in the fraction of cells with intact and compromised cell membranes were found for Lake Fryxell and east lobe of Lake Bonney, both expressed in differences in DGGE banding patterns and qPCR signal reductions. Limitations in the ability to distinguish between intact or compromised cells occurred in samples from Lake Hoare and west lobe of Lake Bonney due to low DNA template concentrations recovered from the samples.

Changes in the structure and diversity of bacterial communities during the process of adaptation to organic wastewater

2010 ◽  
Vol 56 (4) ◽  
pp. 352-355 ◽  
Author(s):  
Junmin Li ◽  
Zexin Jin ◽  
Binbin Yu
Keyword(s):  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Similarity Index ◽  
Pcr Amplification ◽  
Genotypic Diversity ◽  
Diversity Indices ◽  
Inhibitory Effect ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gradient Gel Electrophoresis

To explore changes in the structure and diversity of activated sludge-derived microbial communities during adaptation to gradual increases in the concentration of wastewater, RAPD–PCR and the combination of PCR amplification of 16S rRNA genes with denaturing gradient gel electrophoresis (DGGE) analysis were used. In bacterial communities exposed to 0%, 5%, 10%, 20%, or 40% wastewater, there were 27, 25, 18, 17 and 16 bands, respectively, based on DGGE data, while there were 69, 83, 97, 86, and 88 bands, respectively, based on RAPD data. The community similarity index among bacterial communities during the process of adaptation to different concentrations of wastewater was different based on DGGE and RAPD data. Based on DGGE and RAPD profiles, the Shannon–Weiner and Simpson’s diversity indices decreased sharply upon exposure to 10% wastewater, indicating that 10% wastewater might be a critical point at which the growth of bacteria could be significantly inhibited and the genotypic diversity could change. This indicated that changes in structure and diversity might have an inhibitory effect on the toxicity of organic matter and that selection and adaptation could play important roles in the changes.

scholarly journals Suicide Polymerase Endonuclease Restriction, a Novel Technique for Enhancing PCR Amplification of Minor DNA Templates

2005 ◽  
Vol 71 (8) ◽  
pp. 4721-4727 ◽  
Author(s):  
Stefan J. Green ◽  
Dror Minz
Keyword(s):  
16S Rrna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Dna Templates ◽  
Target Dna ◽  
Gradient Gel Electrophoresis ◽  
Pcr Method

ABSTRACT PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.

scholarly journals Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions

2000 ◽  
Vol 66 (10) ◽  
pp. 4372-4377 ◽  
Author(s):  
Bo Normander ◽  
Jim I. Prosser
Keyword(s):  
Community Composition ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Community Composition ◽  
Pcr Amplification ◽  
Molecular Techniques ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Populations ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis

ABSTRACT An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.

scholarly journals Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology

2007 ◽  
Vol 73 (16) ◽  
pp. 5111-5117 ◽  
Author(s):  
Andreas Nocker ◽  
Priscilla Sossa-Fernandez ◽  
Mark D. Burr ◽  
Anne K. Camper
Keyword(s):  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Intact Cell ◽  
Treatment Plant ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Propidium Monoazide ◽  
End Point ◽  
Gradient Gel Electrophoresis

ABSTRACT One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.

scholarly journals Phylogenetic and Morphological Diversity of Cyanobacteria in Soil Desert Crusts from the Colorado Plateau

2001 ◽  
Vol 67 (4) ◽  
pp. 1902-1910 ◽  
Author(s):  
Ferran Garcia-Pichel ◽  
Alejandro López-Cortés ◽  
Ulrich Nübel
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Colorado Plateau ◽  
Morphological Diversity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Filamentous Cyanobacteria ◽  
Phylogenetic Cluster ◽  
Field Samples ◽  
Gradient Gel Electrophoresis ◽  
Well Differentiated

ABSTRACT We compared the community structures of cyanobacteria in four biological desert crusts from Utah's Colorado Plateau developing on different substrata. We analyzed natural samples, cultures, and cyanobacterial filaments or colonies retrieved by micromanipulation from field samples using microscopy, denaturing gradient gel electrophoresis, and sequencing of 16S rRNA genes. While microscopic analyses apparently underestimated the biodiversity of thin filamentous cyanobacteria, molecular analyses failed to retrieve signals for otherwise conspicuous heterocystous cyanobacteria with thick sheaths. The diversity found in desert crusts was underrepresented in currently available nucleotide sequence databases, and several novel phylogenetic clusters could be identified. Morphotypes fitting the description of Microcoleus vaginatus Gomont, dominant in most samples, corresponded to a tight phylogenetic cluster of probable cosmopolitan distribution, which was well differentiated from other cyanobacteria traditionally classified within the same genus. A new, diverse phylogenetic cluster, named “Xeronema,” grouped a series of thin filamentousPhormidium-like cyanobacteria. These were also ubiquitous in our samples and probably correspond to various botanicalPhormidium and Schizothrix spp., but they are phylogenetically distant from thin filamentous cyanobacteria from other environments. Significant differences in community structure were found among soil types, indicating that soil characteristics may select for specific cyanobacteria. Gypsum crusts were most deviant from the rest, while sandy, silt, and shale crusts were relatively more similar among themselves.

Perchlorate removal using two component biodegradable carriers in particle-fixed biofilm reactor

2014 ◽  
Vol 49 (3) ◽  
pp. 234-244
Author(s):  
Fang He ◽  
Fusheng Li ◽  
Haihong Zhou ◽  
Lingling Niu ◽  
Liguo Wang
Keyword(s):  
Carbon Source ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Polymer Particle ◽  
Biofilm Reactor ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Biofilm Reactors ◽  
Gradient Gel Electrophoresis ◽  
Perchlorate Removal ◽  
Fixed Biofilm

In this research, biocompounds designed out of two polymers having different degradability was investigated for use as the sole carbon source and biofilm carrier to remove perchlorate in particle-fixed biofilm reactors. Both laboratory batch and column experiments were conducted with perchlorate contaminated groundwater. Batch experiments demonstrated clearly that ClO4– was removed from the aqueous phase readily and the degradation rate constants (k) changed in the range of 0.23–0.37 mg/L h as ClO4– concentration increased from 2 to 8 mg/L. Simultaneous perchlorate and nitrate degradation occurred in the polymer bioreactor. Effluent concentrations of perchlorate varied positively with temperature and fitted the Arrhenius equation expression as k=k20•100.0316(t–20) over the range of 13–30 °C. No perchlorate was detected in the effluent of polymer columns after 20 days’ startup. Complete perchlorate removal was observed at a hydraulic loading rate doubled to 1.8 mL/min. Images prove the concept of the pore and filament structure within the biocompounds, which provide both a heterotrophic biofilm and carbon source. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes indicated that formerly reported perchlorate-reducing bacteria were present in the polymer particle-fixed biofilm reactors.

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.

scholarly journals Effects of Lactococcus lactis on Composition of Intestinal Microbiota: Role of Nisin

2006 ◽  
Vol 72 (1) ◽  
pp. 239-244 ◽  
Author(s):  
Nete Bernbom ◽  
Tine Rask Licht ◽  
Carl-Henrik Brogren ◽  
Birthe Jelle ◽  
Anette H. Johansen ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Intestinal Microbiota ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Fecal Microbiota ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gradient Gel Electrophoresis ◽  
Intact Molecule ◽  
Human Flora

ABSTRACT This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.

scholarly journals Comparison of Different Denaturing Gradient Gel Electrophoresis Primer Sets for the Study of Marine Bacterioplankton Communities

2007 ◽  
Vol 73 (18) ◽  
pp. 5962-5967 ◽  
Author(s):  
Olga Sánchez ◽  
Josep M. Gasol ◽  
Ramon Massana ◽  
Jordi Mas ◽  
Carlos Pedrós-Alió
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Coastal System ◽  
Marine Bacterioplankton ◽  
Gradient Gel Electrophoresis ◽  
Primer Sets ◽  
Bacterial Groups ◽  
Plankton Communities

ABSTRACT An annual seasonal cycle of composition of a bacterioplankton community in an oligotrophic coastal system was studied by denaturing gradient gel electrophoresis (DGGE) using five different primer sets. Analysis of DGGE fingerprints showed that primer set 357fGC-907rM grouped samples according to seasons. Additionally, we used the set of 16S rRNA genes archived in the RDPII database to check the percentage of perfect matches of each primer for the most abundant bacterial groups inhabiting coastal plankton communities. Overall, primer set 357fGC-907rM was the most suitable for the routine use of PCR-DGGE analyses in this environment.

scholarly journals Identification of Bacteria Potentially Responsible for Oxic and Anoxic Sulfide Oxidation in Biofilters of a Recirculating Mariculture System

2005 ◽  
Vol 71 (10) ◽  
pp. 6134-6141 ◽  
Author(s):  
Eddie Cytryn ◽  
Jaap van Rijn ◽  
Andreas Schramm ◽  
Armin Gieseke ◽  
Dirk de Beer ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Sulfide Oxidation ◽  
16S Rrna Genes ◽  
Microbial Consortia ◽  
Rrna Genes ◽  
Marine Aquaculture ◽  
Dgge Analysis ◽  
Dna And Rna ◽  
Gradient Gel Electrophoresis

ABSTRACT Bacteria presumably involved in oxygen- or nitrate-dependent sulfide oxidation in the biofilters of a recirculating marine aquaculture system were identified using a new application of reverse transcription-PCR denaturing gradient gel electrophoresis (DGGE) analysis termed differential-transcription (DT)-DGGE. Biofilter samples were incubated in various concentrations of sulfide or thiosulfate (0 to 5 mM) with either oxygen or nitrate as the sole electron acceptor. Before and after short-term incubations (10 to 20 h), total DNA and RNA were extracted, and a 550-bp fragment of the 16S rRNA genes was PCR amplified either directly or after reverse transcription. DGGE analysis of DNA showed no significant change of the original microbial consortia upon incubation. In contrast, DGGE of cDNA revealed several phylotypes whose relative band intensities markedly increased or decreased in response to certain incubation conditions, indicating enhanced or suppressed rRNA transcription and thus implying metabolic activity under these conditions. Specifically, species of the gammaproteobacterial genus Thiomicrospira and phylotypes related to symbiotic sulfide oxidizers could be linked to oxygen-dependent sulfide oxidation, while members of the Rhodobacteraceae (genera Roseobacter, Rhodobacter, and Rhodobium) were putatively active in anoxic, nitrate-dependent sulfide oxidation. For all these organisms, the physiology of their closest cultured relatives matches their DT-DGGE-inferred function. In addition, higher band intensities following exposure to 5 mM sulfide and nitrate were observed for Thauera-, Hydrogenophaga-, and Dethiosulfovibrio-like phylotypes. For these genera, nitrate-dependent sulfide oxidation has not been documented previously and therefore DT-DGGE might indicate a higher relative tolerance to high sulfide concentrations than that of other community members. We anticipate that DT-DGGE will be of general use in tracing functionally equivalent yet phylogenetically diverse microbial populations in nature.

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