scholarly journals Toxic and Nontoxic Microcystis Colonies in Natural Populations Can Be Differentiated on the Basis of rRNA Gene Internal Transcribed Spacer Diversity

2004 ◽  
Vol 70 (7) ◽  
pp. 3979-3987 ◽  
Author(s):  
Ingmar Janse ◽  
W. Edwin A. Kardinaal ◽  
Marion Meima ◽  
Jutta Fastner ◽  
Petra M. Visser ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Natural Populations ◽  
Genotypic Diversity ◽  
Toxin Production ◽  
Its Sequences ◽  
Rrna Gene ◽  
Flight Mass Spectrometry ◽  
Gradient Gel Electrophoresis ◽  
Natural Communities

ABSTRACT Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.

scholarly journals Diversity of Bacteria Associated with Natural Aphid Populations

2003 ◽  
Vol 69 (12) ◽  
pp. 7216-7223 ◽  
Author(s):  
S. Haynes ◽  
A. C. Darby ◽  
T. J. Daniell ◽  
G. Webster ◽  
F. J. F. van Veen ◽  
...  
Keyword(s):  
Acyrthosiphon Pisum ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Natural Populations ◽  
Geographical Location ◽  
Laboratory Culture ◽  
Aphid Species ◽  
Aphis Fabae ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis ◽  
Aphid Populations

ABSTRACT The bacterial communities of aphids were investigated by terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments generated by PCR with general eubacterial primers. By both methods, theγ -proteobacterium Buchnera was detected in laboratory cultures of six parthenogenetic lines of the pea aphid Acyrthosiphon pisum and one line of the black bean aphid Aphis fabae, and one or more of four previously described bacterial taxa were also detected in all aphid lines except one of A. pisum. These latter bacteria, collectively known as secondary symbionts or accessory bacteria, comprised three taxa of γ-proteobacteria (R-type [PASS], T-type [PABS], and U-type [PAUS]) and a rickettsia (S-type [PAR]). Complementary analysis of aphids from natural populations of four aphid species (A. pisum [n= 74], Amphorophora rubi [n= 109], Aphis sarothamni [n= 42], and Microlophium carnosum [n= 101]) from a single geographical location revealed Buchnera and up to three taxa of accessory bacteria, but no other bacterial taxa, in each aphid. The prevalence of accessory bacterial taxa varied significantly among aphid species but not with the sampling month (between June and August 2000). These results indicate that the accessory bacterial taxa are distributed across multiple aphid species, although with variable prevalence, and that laboratory culture does not generally result in a shift in the bacterial community in aphids. Both the transmission patterns of the accessory bacteria between individual aphids and their impact on aphid fitness are suggested to influence the prevalence of accessory bacterial taxa in natural aphid populations.

scholarly journals High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis

2003 ◽  
Vol 69 (11) ◽  
pp. 6634-6643 ◽  
Author(s):  
Ingmar Janse ◽  
Marion Meima ◽  
W. Edwin A. Kardinaal ◽  
Gabriel Zwart
Keyword(s):  
High Resolution ◽  
Gel Electrophoresis ◽  
Internal Transcribed Spacer ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Advantages And Disadvantages ◽  
Field Samples ◽  
Gradient Gel Electrophoresis

ABSTRACT For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3′ end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.

scholarly journals sxtA4+ and sxtA4- Genotypes Occur Together within Natural Pyrodinium bahamense Sub-Populations from the Western Atlantic

2021 ◽  
Vol 9 (6) ◽  
pp. 1128
Author(s):  
Kathleen Cusick ◽  
Gabriel Duran
Keyword(s):  
Molecular Mechanisms ◽  
Harmful Algal Bloom ◽  
Single Cells ◽  
Natural Populations ◽  
Indian River Lagoon ◽  
Toxin Production ◽  
Genotype Frequency ◽  
Rrna Gene ◽  
Western Atlantic ◽  
Pyrodinium Bahamense

Saxitoxin (STX) is a secondary metabolite and potent neurotoxin produced by several genera of harmful algal bloom (HAB) marine dinoflagellates. The basis for variability in STX production within natural bloom populations is undefined as both toxic and non-toxic strains (of the same species) have been isolated from the same geographic locations. Pyrodinium bahamense is a STX-producing bioluminescent dinoflagellate that blooms along the east coast of Florida as well as the bioluminescent bays in Puerto Rico (PR), though no toxicity reports exist for PR populations. The core genes in the dinoflagellate STX biosynthetic pathway have been identified, and the sxtA4 gene is essential for toxin production. Using sxtA4 as a molecular proxy for the genetic capacity of STX production, we examined sxtA4+ and sxtA4- genotype frequency at the single cell level in P. bahamense populations from different locations in the Indian River Lagoon (IRL), FL, and Mosquito Bay (MB), a bioluminescent bay in PR. Multiplex PCR was performed on individual cells with Pyrodinium-specific primers targeting the 18S rRNA gene and sxtA4. The results reveal that within discrete natural populations of P. bahamense, both sxtA4+ and sxtA4- genotypes occur, and the sxtA4+ genotype dominates. In the IRL, the frequency of the sxtA4+ genotype ranged from ca. 80–100%. In MB, sxtA4+ genotype frequency ranged from ca 40–66%. To assess the extent of sxtA4 variation within individual cells, sxtA4 amplicons from single cells representative of the different sampling sites were cloned and sequenced. Overall, two variants were consistently obtained, one of which is likely a pseudogene based on alignment with cDNA sequences. These are the first data demonstrating the existence of both genotypes in natural P. bahamense sub-populations, as well as sxtA4 presence in P. bahamense from PR. These results provide insights on underlying genetic factors influencing the potential for toxin variability among natural sub-populations of HAB species and highlight the need to study the genetic diversity within HAB sub-populations at a fine level in order to identify the molecular mechanisms driving HAB evolution.

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.

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.

scholarly journals Molecular Analysis of Surfactant-Driven Microbial Population Shifts in Hydrocarbon-Contaminated Soil

2000 ◽  
Vol 66 (7) ◽  
pp. 2959-2964 ◽  
Author(s):  
Gregory M. Colores ◽  
Richard E. Macur ◽  
David M. Ward ◽  
William P. Inskeep
Keyword(s):  
Gel Electrophoresis ◽  
Microbial Population ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Microbial Populations ◽  
Alcohol Ethoxylate ◽  
Rrna Gene ◽  
Mineralization Kinetics ◽  
Gradient Gel Electrophoresis ◽  
The Impact ◽  
Parallel Cultivation

ABSTRACT We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC′) (determined to be 13 mg g−1). Addition of the surfactant at a concentration below the CMC′ (2 mg g−1) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC′ (10 mg g−1), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC′ (40 mg g−1) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulatedRhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenespopulations at elevated surfactant levels. Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas andAlcaligenes populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization.

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