scholarly journals Combining Culture-Dependent and -Independent Methodologies for Estimation of Richness of Estuarine Bacterioplankton Consuming Riverine Dissolved Organic Matter

2003 ◽  
Vol 69 (6) ◽  
pp. 3607-3616 ◽  
Author(s):  
Veljo Kisand ◽  
Johan Wikner
Keyword(s):  
Organic Matter ◽  
Dissolved Organic Matter ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Experimental System ◽  
Community Diversity ◽  
Rrna Gene ◽  
Solid Media ◽  
Gradient Gel Electrophoresis ◽  
Culture Dependent

ABSTRACT Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated β-proteobacteria to be RDOM consumers.

scholarly journals Phylogenetic Diversity and Specificity of Bacteria Closely Associated with Alexandrium spp. and Other Phytoplankton

2005 ◽  
Vol 71 (7) ◽  
pp. 3483-3494 ◽  
Author(s):  
Suresh Jasti ◽  
Michael E. Sieracki ◽  
Nicole J. Poulton ◽  
Michael W. Giewat ◽  
Juliette N. Rooney-Varga
Keyword(s):  
Phylogenetic Diversity ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Gulf Of Maine ◽  
Harmful Algal Bloom ◽  
Bacterial Species ◽  
Geographic Origin ◽  
Rrna Gene ◽  
Phytoplankton Communities ◽  
Gradient Gel Electrophoresis ◽  
Phytoplankton Cell

ABSTRACT While several studies have suggested that bacterium-phytoplankton interactions have the potential to dramatically influence harmful algal bloom dynamics, little is known about how bacteria and phytoplankton communities interact at the species composition level. The objective of the current study was to determine whether there are specific associations between diverse phytoplankton and the bacteria that co-occur with them. We determined the phylogenetic diversity of bacterial assemblages associated with 10 Alexandrium strains and representatives of the major taxonomic groups of phytoplankton in the Gulf of Maine. For this analysis we chose xenic phytoplankton cultures that (i) represented a broad taxonomic range, (ii) represented a broad geographic range for Alexandrium spp. isolates, (iii) grew under similar cultivation conditions, (iv) had a minimal length of time since the original isolation, and (v) had been isolated from a vegetative phytoplankton cell. 16S rRNA gene fragments of most Bacteria were amplified from DNA extracted from cultures and were analyzed by denaturing gradient gel electrophoresis and sequencing. A greater number of bacterial species were shared by different Alexandrium cultures, regardless of the geographic origin, than by Alexandrium species and nontoxic phytoplankton from the Gulf of Maine. In particular, members of the Roseobacter clade showed a higher degree of association with Alexandrium than with other bacterial groups, and many sequences matched sequences reported to be associated with other toxic dinoflagellates. These results provide evidence for specificity in bacterium-phytoplankton associations.

Comparative Analysis of Bacterial Diversity for Cutlassfish (Trichiurus haumela) during Cold Storage by the Culture-Dependent and Culture-Independent Methods

2012 ◽  
Vol 535-537 ◽  
pp. 1046-1053 ◽  
Author(s):  
Wei Qing Lan ◽  
Jing Xie
Keyword(s):  
16S Rrna ◽  
Pseudomonas Fluorescens ◽  
Cold Storage ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Region Gene ◽  
Gradient Gel Electrophoresis ◽  
Dominant Bacteria ◽  
V3 Region ◽  
Culture Dependent

The methods of culture-dependent and denaturing gradient gel electrophoresis (DGGE) based on the sequence of 16S rRNA V3 region gene were described to comparatively characterize the microbial population and community structure of cutlassfish (Trichiurus haumela) under the cold storage. The results showed that 13 kinds of bacteria were identified by the traditional culture-dependent methods, the dominant bacteria belonged to Shewanella putrefaciens and Pseudomonas fluorescens. To determine the community profiles of the samples on variable V3 region, the bacteria of 16S rRNA gene were amplified by PCR and 11 distinct PCR products were separated by DGGE fingerprinting technology. From the sequence analysis, Psychrobacter sp. was found to be the predominant bacteria in the initial stage of the storage. The proportion of Shewanella sp., Pseudomonas sp. increased gradually with the extension of storage time, and they took the place of Psychrobacter sp. to be the dominant bacteria. Thereinto, both Pseudomonas fluorescens and Vibrio sp. took high proportions in the process of storage due to the deterioration of cutlassfish (Trichiurus haumela).

scholarly journals Characterization of a Highly Enriched Microbial Consortium Reductively Dechlorinating 2,3-Dichlorophenol and 2,4,6-Trichlorophenol and the Corresponding cprA Genes from River Sediment

2016 ◽  
Vol 65 (3) ◽  
pp. 341-352 ◽  
Author(s):  
Wael S. El-Sayed
Keyword(s):  
Electron Donor ◽  
Reductive Dechlorination ◽  
River Sediment ◽  
Microbial Consortium ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis ◽  
Dominant Bacteria

Anaerobic reductive dechlorination of 2,3-dichlorophenol (2,3DCP) and 2,4,6-trichlorophenol (2,4,6TCP) was investigated in microcosms from River Nile sediment. A stable sediment-free anaerobic microbial consortium reductively dechlorinating 2,3DCP and 2,4,6TCP was established. Defined sediment-free cultures showing stable dechlorination were restricted to ortho chlorine when enriched with hydrogen as the electron donor, acetate as the carbon source, and either 2,3-DCP or 2,4,6-TCP as electron acceptors. When acetate, formate, or pyruvate were used as electron donors, dechlorination activity was lost. Only lactate can replace dihydrogen as an electron donor. However, the dechlorination potential was decreased after successive transfers. To reveal chlororespiring species, the microbial community structure of chlorophenol-reductive dechlorinating enrichment cultures was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Eight dominant bacteria were detected in the dechlorinating microcosms including members of the genera Citrobacter, Geobacter, Pseudomonas, Desulfitobacterium, Desulfovibrio, and Clostridium. Highly enriched dechlorinating cultures were dominated by four bacterial species belonging to the genera Pseudomonas, Desulfitobacterium, and Clostridium. Desulfitobacterium represented the major fraction in DGGE profiles indicating its importance in dechlorination activity, which was further confirmed by its absence resulting in complete loss of dechlorination. Reductive dechlorination was confirmed by the stoichiometric dechlorination of 2,3DCP and 2,4,6TCP to metabolites with less chloride groups and by the detection of chlorophenol RD cprA gene fragments in dechlorinating cultures. PCR amplified cprA gene fragments were cloned and sequenced and found to cluster with the cprA/pceA type genes of Dehalobacter restrictus.

scholarly journals Soil and Rhizosphere Bacterial Diversity in Maize Agro- Ecosystem

2017 ◽  
Vol 6 (3) ◽  
pp. 35 ◽  
Author(s):  
Maria Teresa Federici Rodriguez ◽  
Natalia Bajsa Valverde ◽  
Paula Lagurara ◽  
Santiago Revale ◽  
Jackson Antonio Marcondes de Souza ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Management Practices ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Community Diversity ◽  
Rrna Gene ◽  
Phenological Stages ◽  
Maize Production ◽  
Maize Cultivars ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis

Management practices used in maize production have an impact on soil agro- ecosystems where different microbial communities coexist. Soil inhabiting bacteria are numerous and diverse, but we know very little about their ecological distribution. Here we analyzed the bacterial community diversity in the rhizosphere of two transgenic maize cultivars, in agricultural soil before sowing and in non-cultivated soil in an experimental site in the south region of Uruguay. We followed two culture-independent methods: DGGE (denaturing gradient gel electrophoresis) and 454-pyrosequencing of 16S rRNA gene amplicon. Through pyrosequencing, the three environments analyzed presented differences in terms of bacterial composition. However, no differences were found in the relative abundance of the ten most represented phyla in the rhizosphere of the two cultivars at different phenological stages. We found significant differences of Bacteroidetes, Gemmatimonadetes, Planctomycetes, Proteobacteria and Verrucomicrobia phyla when comparing agricultural and non-cultivated soils, as well as a significant enrichment of members of the phylum Gemmatimonadetes in all rhizosphere samples compared to soil. Through DGGE analysis we evidenced that maize rhizosphere bacterial communities changed at different phenological stages in both cultivars. We also provided baseline information about bacterial specific taxa within maize agro- ecosystem for further evaluation of possible rhizosphere bacterial community shifts of genetically modified maize cultivars under different management practices.

scholarly journals Biodiversity in Oscypek, a Traditional Polish Cheese, Determined by Culture-Dependent and -Independent Approaches

2012 ◽  
Vol 78 (6) ◽  
pp. 1890-1898 ◽  
Author(s):  
Ángel Alegría ◽  
Pawel Szczesny ◽  
Baltasar Mayo ◽  
Jacek Bardowski ◽  
Magdalena Kowalczyk
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Content Type ◽  
Protected Designation Of Origin ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis ◽  
Microbial Groups ◽  
The Tatra Mountains ◽  
Culture Dependent

ABSTRACTOscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g.,Lactococcus,Lactobacillus,Leuconostoc,Streptococcus, andEnterococcus, identified by all three methods, other, subdominant bacteria belonging to the familiesBifidobacteriaceaeandMoraxellaceae(mostlyEnhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.

Effects of Elevated Carbon Dioxide on Soil Bacterial Community Structure

2014 ◽  
Vol 1010-1012 ◽  
pp. 422-428
Author(s):  
Chun Rong Li ◽  
Wen Ke Wang ◽  
Hong Zhang Deng ◽  
Xiao Hong Zhao ◽  
Feng Han ◽  
...  
Keyword(s):  
Soil Bacteria ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Brucella Melitensis ◽  
Bacterial Species ◽  
Control Area ◽  
Community Diversity ◽  
High Concentration ◽  
Bacteria Diversity ◽  
Gradient Gel Electrophoresis ◽  
Dominant Bacteria

The soil bacteria diversity in different concentrations of CO2was investigated in the simulation test area, by using PCR (Polymerase Chain Reaction) -DGGE (Denaturing Gradient Gel Electrophoresis) and 16Sr-DNA library technology. The results showed that the bacteria diversity coefficient (P) got down to 0.8710, 0.8710 and 0.7742 from 0.9032 (from control area) when the concentration of CO2in soil reached 20000ppm, 40000ppm, 60000ppmrespectively. With the increasing concentration of CO2in soil, the abundance of the original low-density bacteria such asAsticcacaulis excentricus, etc. increased, while the abundance ofunclassified_Rhizobialesreduced significantly. Dominant bacteria such asBrucella melitensisetc. had the higher homology. It can be revealed that high concentration of CO2had a significant impact on the soil bacteria community diversity, while a weak influence on main bacterial species. Azotobacter was sensitive to the increasing of the CO2concentration. Great reducing of their abundance had an adverse effect on nitrogen-fixing capability of soil.

scholarly journals Analysis of Vaginal Lactobacilli from Healthy and Infected Brazilian Women

2008 ◽  
Vol 74 (14) ◽  
pp. 4539-4542 ◽  
Author(s):  
Rafael C. R. Martinez ◽  
Sílvio A. Franceschini ◽  
Maristela C. Patta ◽  
Silvana M. Quintana ◽  
Álvaro C. Nunes ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Restriction Analysis ◽  
Rrna Gene ◽  
Vaginal Lactobacilli ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis ◽  
Gene Restriction ◽  
Culture Dependent

ABSTRACT Culture-dependent PCR-amplified rRNA gene restriction analysis and culture-independent (PCR-denaturing gradient gel electrophoresis) methodologies were used to examine vaginal lactobacilli from Brazilian women who were healthy or had been diagnosed with vulvovaginal candidiasis (VVC) or bacterial vaginosis. Only Lactobacillus crispatus was detected accordingly by both methods, and H2O2-producing lactobacilli were not associated with protection against VVC.

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 Differences in Microbial Activity and Microbial Populations of Peat Associated with Suppression of Damping-Off Disease Caused by Pythium sylvaticum

2006 ◽  
Vol 72 (10) ◽  
pp. 6452-6460 ◽  
Author(s):  
Paul J. Hunter ◽  
Geoff M. Petch ◽  
Leo A. Calvo-Bado ◽  
Tim R. Pettitt ◽  
Nick R. Parsons ◽  
...  
Keyword(s):  
Microbial Activity ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Small Subunit ◽  
Disease Suppression ◽  
Rrna Gene ◽  
Damping Off ◽  
Small Subunit Rrna ◽  
Pythium Sylvaticum ◽  
Gradient Gel Electrophoresis ◽  
Microbial Groups

ABSTRACT The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.

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