scholarly journals Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes

2006 ◽  
Vol 72 (1) ◽  
pp. 428-436 ◽  
Author(s):  
Ariel Grostern ◽  
Elizabeth A. Edwards
Keyword(s):  
16S Rrna ◽  
Reductive Dechlorination ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Anaerobic Bacteria ◽  
Organic Substrate ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Real Time Quantitative Pcr ◽  
Gradient Gel Electrophoresis

ABSTRACT Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.

scholarly journals Development and Application of a Selective PCR-Denaturing Gradient Gel Electrophoresis Approach To Detect a Recently Cultivated Bacillus Group Predominant in Soil

2004 ◽  
Vol 70 (10) ◽  
pp. 5801-5809 ◽  
Author(s):  
Vesela A. Tzeneva ◽  
Youguo Li ◽  
Andreas D. M. Felske ◽  
Willem M. de Vos ◽  
Antoon D. L. Akkermans ◽  
...  
Keyword(s):  
16S Rrna ◽  
The Netherlands ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Physiological State ◽  
Soil Samples ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis

ABSTRACT The worldwide presence of a hitherto-nondescribed group of predominant soil microorganisms related to Bacillus benzoevorans was analyzed after development of two sets of selective primers targeting 16S rRNA genes in combination with denaturing gradient gel electrophoresis (DGGE). The high abundance and cultivability of at least some of these microorganisms makes them an appropriate subject for studies on their biogeographical dissemination and diversity. Since cultivability can vary significantly with the physiological state and even between closely related strains, we developed a culture-independent 16S rRNA gene-targeted DGGE fingerprinting protocol for the detection of these bacteria from soil samples. The composition of the B. benzoevorans relatives in the soil samples from The Netherlands, Bulgaria, Russia, Pakistan, and Portugal showed remarkable differences between the different countries. Differences in the DGGE profiles of these communities in archived soil samples from the Dutch Wieringermeer polder were observed over time during which a shift from anaerobic to aerobic and from saline to freshwater conditions occurred. To complement the molecular methods, we additionally cultivated B. benzoevorans-related strains from all of the soil samples. The highest number of B. benzoevorans relatives was found in the soils from the northern part of The Netherlands. The present study contributes to our knowledge of the diversity and abundance of this interesting group of microbes in soils throughout the world.

scholarly journals Evaluations of Different Hypervariable Regions of Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient Gel Electrophoresis

2007 ◽  
Vol 74 (3) ◽  
pp. 889-893 ◽  
Author(s):  
Zhongtang Yu ◽  
Rubén García-González ◽  
Floyd L. Schanbacher ◽  
Mark Morrison
Keyword(s):  
16S Rrna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
V Region

ABSTRACT Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.

scholarly journals Diversity and Distribution in Hypersaline Microbial Mats of Bacteria Related to Chloroflexusspp

2001 ◽  
Vol 67 (9) ◽  
pp. 4365-4371 ◽  
Author(s):  
Ulrich Nübel ◽  
Mary M. Bateson ◽  
Michael T. Madigan ◽  
Michael Kühl ◽  
David M. Ward
Keyword(s):  
16S Rrna ◽  
Microbial Mats ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Phylogenetic Analyses ◽  
Pcr Primers ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis ◽  
Hypersaline Microbial Mats

ABSTRACT Filamentous bacteria containing bacteriochlorophylls cand a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related toChloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of “green nonsulfur bacteria.” PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related toChloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.

scholarly journals Sequential Reductive Dechlorination of meta-Chlorinated Polychlorinated Biphenyl Congeners in Sediment Microcosms by Two Different Chloroflexi Phylotypes

2005 ◽  
Vol 71 (12) ◽  
pp. 8085-8090 ◽  
Author(s):  
Sonja K. Fagervold ◽  
Joy E. M. Watts ◽  
Harold D. May ◽  
Kevin R. Sowers
Keyword(s):  
16S Rrna ◽  
Reductive Dechlorination ◽  
Polychlorinated Biphenyl ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Individual Species ◽  
Rrna Gene ◽  
Probable Number

ABSTRACT Three species within a deeply branching cluster of the Chloroflexi are the only microorganisms currently known to anaerobically transform polychlorinated biphenyls (PCBs) by the mechanism of reductive dechlorination. A selective PCR primer set was designed that amplifies the 16S rRNA genes of a monophyletic group within the Chloroflexi including Dehalococcoides spp. and the o-17/DF-1 group. Assays for both qualitative and quantitative analyses by denaturing gradient gel electrophoresis and most probable number-PCR, respectively, were developed to assess sediment microcosm enrichments that reductively dechlorinated PCBs 101 (2,2′,4,5,5′-CB) and 132 (2,2′,3,3′,4,6′-CB). PCB 101 was reductively dechlorinated at the para-flanked meta position to PCB 49 (2,2′,4,5′-CB) by phylotype DEH10, which belongs to the Dehalococcoides group. This same species reductively dechlorinated the para- and ortho-flanked meta-chlorine of PCB 132 to PCB 91 (2,2′,3′,4,6′-CB). However, another phylotype designated SF1, which is more closely related to the o-17/DF-1 group, was responsible for the subsequent dechlorination of PCB 91 to PCB 51 (2,2′,4,6′-CB). Using the selective primer set, an increase in 16S rRNA gene copies was observed only with actively dechlorinating cultures, indicating that PCB-dechlorinating activities by both phylotype DEH10 and SF1 were linked to growth. The results suggest that individual species within the Chloroflexi exhibit a limited range of congener specificities and that a relatively diverse community of species within a deeply branching group of Chloroflexi with complementary congener specificities is likely required for the reductive dechlorination of different PCBs congeners in the environment.

scholarly journals Unique Kinetic Properties of Phenol-Degrading Variovorax Strains Responsible for Efficient Trichloroethylene Degradation in a Chemostat Enrichment Culture

2005 ◽  
Vol 71 (2) ◽  
pp. 904-911 ◽  
Author(s):  
Hiroyuki Futamata ◽  
Yayoi Nagano ◽  
Kazuya Watanabe ◽  
Akira Hiraishi
Keyword(s):  
16S Rrna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Competitive Inhibition ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Kinetic Properties ◽  
Rrna Gene ◽  
High Affinity ◽  
Degrading Bacteria ◽  
Tce Degradation

ABSTRACT A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities (H. Futamata, S. Harayama, and K. Watanabe, Appl. Environ. Microbiol. 67:4671-4677, 2001). To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter−1 day−1) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter−1 day−1 (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.

Genetic Diversity of the Biofilm CoveringMontacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA†

1998 ◽  
Vol 64 (9) ◽  
pp. 3464-3472 ◽  
Author(s):  
David C. Gillan ◽  
Arjen G. C. L. Speksnijder ◽  
Gabriel Zwart ◽  
Chantal De Ridder
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
Dna Extraction ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gradient Gel Electrophoresis ◽  
Biofilm Bacteria ◽  
Sequence Types

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas,Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of theCytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.

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 Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State

2008 ◽  
Vol 74 (15) ◽  
pp. 4877-4888 ◽  
Author(s):  
Pedro A. Dimitriu ◽  
Holly C. Pinkart ◽  
Brent M. Peyton ◽  
Melanie R. Mormile
Keyword(s):  
16S Rrna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Community Composition ◽  
Soda Lake ◽  
Community Diversity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Probable Number

ABSTRACT The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.

scholarly journals Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

1999 ◽  
Vol 65 (2) ◽  
pp. 422-430 ◽  
Author(s):  
Ulrich Nübel ◽  
Ferran Garcia-Pichel ◽  
Michael Kühl ◽  
Gerard Muyzer
Keyword(s):  
16S Rrna ◽  
Microbial Mats ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Morphological Diversity ◽  
Numerical Approach ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Phototrophic Microorganisms

ABSTRACT We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.

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