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Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. (1997)

by W T Liu, T L Marsh, H Cheng, L J Forney
Venue:Applied and Environmental Microbiology,
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Counting the uncountable: statistical approaches to estimating microbial diversity

by Jennifer B. Hughes, Jessica J. Hellmann, Taylor H. Ricketts, Brendan J. M. Bohannan, Jennifer B. Hughes, Jessica J. Hellmann, Taylor H. Ricketts, Brendan J. M. Bohannan - Appl. Environ , 2001
"... This article cites 52 articles, 16 of which can be accessed free ..."
Abstract - Cited by 110 (0 self) - Add to MetaCart
This article cites 52 articles, 16 of which can be accessed free
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...will often refer to species as the measured unit of diversity, but our discussion can be applied to any operational taxonomic units (OTUs), such as the number of unique terminal restriction fragments =-=(35)-=- or number of 16S ribosomal DNA (rDNA) sequence similarity groups (41). Finally, we are concerned here with estimating richness and do not address how this diversity is related to functional diversity...

Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning

by John Dunbar, Shannon Takala, Susan M. Barns, Jody A. Davis, Cheryl, R. Kuske - Appl. Environ. Microbiol , 1999
"... Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community d ..."
Abstract - Cited by 75 (1 self) - Add to MetaCart
Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6 % based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities
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...to compare the composition, richness, and structure of microbial communities. Such methods include denaturing gradient gel electrophoresis (13, 19, 23, 37, 49), terminal restriction fragment analysis =-=(4, 6, 28)-=-, and 16S rDNA cloning (2, 8, 15, 38). Like cultivation, amplification of rDNA can distort the apparent structure of a community as a result of biases in cellular rDNA copy number (11), DNA extraction...

A new approach to utilize PCR-singlestrand-conformation polymorphism for 16S rRNA gene-based microbial community analysis

by Frank Schwieger, Christoph, C. Tebbe - Appl. Environ , 1998
"... Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S ..."
Abstract - Cited by 65 (1 self) - Add to MetaCart
Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5 � end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach
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...yzed by electrophoresis, since the sizes of the products are almost identical for all eubacteria. To detect sequence variations, PCR products can be analyzed after restriction endonuclease digestions =-=(23, 26, 27)-=- or without digestions directly on denaturing gradient gel matrices. Such gradients can be generated chemically by increasing urea and formamide concentrations (denaturing gradient gel electrophoresis...

2000): A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. - Appl Environ Microbiol 66

by Anne E. Bernhard, Katharine, G. Field
"... Our purpose was to develop a rapid, inexpensive method of diagnosing the source of fecal pollution in water. In previous research, we identified Bacteroides-Prevotella ribosomal DNA (rDNA) PCR markers based on analysis. These markers length heterogeneity PCR and terminal restriction fragment length ..."
Abstract - Cited by 60 (4 self) - Add to MetaCart
Our purpose was to develop a rapid, inexpensive method of diagnosing the source of fecal pollution in water. In previous research, we identified Bacteroides-Prevotella ribosomal DNA (rDNA) PCR markers based on analysis. These markers length heterogeneity PCR and terminal restriction fragment length polymorphism distinguish cow from human feces. Here, we recovered 16S rDNA clones from natural waters that were close phylogenetic relatives of the markers. From the sequence data, we designed specific PCR primers that discriminate human and ruminant sources of fecal contamination. The inability to identify the source of fecal contamination is partly to blame for the persistent problem of fecal pollution in coastal and inland waters. Although methods exist to quantify fecal pollution, none quickly and accurately identifies the an-imal source. Antibiotic resistance patterns of fecal streptococci (8, 16, 17) and Escherichia coli ribosomal DNA (rDNA) track-ing (14; D. Akre and J. Wilcox, Northwest Algal Symp. Pacific Estuarine Res. Soc. Joint Meet., 1998) have recently emerged as potentially useful, but labor-intensive, solutions to
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...st-specific Bacteroides-Prevotella 16S rDNA markers for humans and cows by screening fecal DNAs by length heterogeneity PCR (LH-PCR) (15) or terminal restriction fragment length polymorphism (T-RFLP) =-=(11)-=- analysis (2). Cloning and sequencing experiments revealed that each marker comprised multiple sequences forming host-specific gene clusters. Here, we have identified additional clones, recovered from...

Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities

by Madeline M. Fisher, Eric, W. Triplett - Appl. Environ , 1999
"... An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid esti-mation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA o ..."
Abstract - Cited by 52 (0 self) - Add to MetaCart
An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid esti-mation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplifi-cation products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and
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...to as automated RISA (ARISA). This method is similar to the recently published terminal restriction fragment length polymorphism and length heterogeneity analysis by PCR community analysis techniques =-=(13, 24)-=-. In the automated approach, the initial steps of DNA extraction and PCR amplification are the same as in RISA, except that PCR is conducted with a fluorescencetagged oligonucleotide primer. The elect...

Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities

by Christopher B. Blackwood, Terry Marsh, Sang-hoon Kim, Eldor A. Paul - Applied and Environmental Microbiology , 2003
"... Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profi ..."
Abstract - Cited by 46 (2 self) - Add to MetaCart
Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) perform-ing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward’s method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward’s method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger dis-tance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all
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...bosomal intergenic spacer analysis (2), single-strand conformation polymorphism (25), amplified ribosomal DNA restriction analysis (19), and terminal restriction fragment length polymorphism (T-RFLP) =-=(3, 16)-=-. These methods do not reveal diversity per se unless the community is very simple, since only a fraction of the species indicated by DNA rehybridization rates or sequence analysis of a clone library ...

Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments

by Andria M. Costello, Mary, E. Lidstrom - Appl. Environ , 1999
"... The 16S rRNA and pmoA genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes ..."
Abstract - Cited by 41 (5 self) - Add to MetaCart
The 16S rRNA and pmoA genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes MspI, HaeIII, and HhaI. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the genera Methylomonas, Methylosinus, and Methylocystis were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs, pmoA. Phylogenetic analyses of both the 16S rRNA and pmoA gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.
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...-Methylocystis This study A189gc GGNGACTGGGACTTCTGG pmoA 16 mb661 CCGGMGCAACGTCYTTACC pmoA This study VOL. 65, 1999 METHANOTROPHS IN LAKE SEDIMENTS 5067 environmental clone libraries by RFLP analysis =-=(10, 21, 27, 30, 34, 36)-=-. Common restriction fragments obtained from such analyses that distinguish between taxonomic groups are known as operational taxonomic units (OTUs) (27). Identification of OTUs for methanotrophs woul...

Distribution of archaea in a black smoker chimney structure

by Ken Takai, Tetsushi Komatsu, Fumio Inagaki, Updated Information, Ken Takai, Tetsushi Komatsu, Fumio Inagaki, Koki Horikoshi - Appl Environ Microbiol , 2001
"... These include: This article cites 47 articles, 30 of which can be accessed free at: ..."
Abstract - Cited by 40 (5 self) - Add to MetaCart
These include: This article cites 47 articles, 30 of which can be accessed free at:
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...2, pISA48, pISA16, and pMC1A11) was used as a standard (52). T-RFLP analysis of archaeal rDNA. In order to rapidly identify archaeal sequences in the subsamples, T-RFLP analysis of rDNA was performed =-=(34)-=-. Archaeal rDNA was amplified by PCR using LA Taq polymerase (TaKaRa, Kyoto, Japan). The oligonucleotide primers used were Arch21F and Arch958RFAM (13). Reaction mixtures were prepared in which the co...

Microbial population structures in soil particle size fractions of a long-term fertilizer field experiment

by Angela Sessitsch, Alexandra Weilharter, Martin H. Gerzabek, Holger Kirchmann, Ellen Kandeler - Appl. Environ. Microbiol
"... Soil structure depends on the association between mineral soil particles (sand, silt, and clay) and organic matter, in which aggregates of different size and stability are formed. Although the chemistry of organic materials, total microbial biomass, and different enzyme activities in different soil ..."
Abstract - Cited by 39 (3 self) - Add to MetaCart
Soil structure depends on the association between mineral soil particles (sand, silt, and clay) and organic matter, in which aggregates of different size and stability are formed. Although the chemistry of organic materials, total microbial biomass, and different enzyme activities in different soil particle size fractions have been well studied, little information is available on the structure of microbial populations in microhabitats. In this study, topsoil samples of different fertilizer treatments of a long-term field experiment were analyzed. Size
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... has become a frequently employed phylogenetic marker to describe microbial diversity in soils without the need of cultivation (10, 14, 49). Terminal restriction length polymorphism (T-RFLP) analysis =-=(7, 11, 38, 47, 50)-=- and cloning and sequencing of 16S rRNA genes were applied to analyze bacterial diversity. Data on the nutrient turnover and soil organic matter fractions and changes in physical soil properties and e...

Neutrophilic Fe-oxidizing bacteria are abundant at the Loihi Seamount hydrothermal vents and play a major role in Fe oxide deposition

by David Emerson, Craig L. Moyer - Appl. Environ , 2002
"... A number of hydrothermal vent sites exist on the summit of the Loihi Seamount, a shield volcano that is part of the Hawaiian archipelago. The vents are 1,100 to 1,325 m below the surface and range in temperature from slightly above ambient (10°C) to high temperature (167°C). The vent fluid is charac ..."
Abstract - Cited by 36 (9 self) - Add to MetaCart
A number of hydrothermal vent sites exist on the summit of the Loihi Seamount, a shield volcano that is part of the Hawaiian archipelago. The vents are 1,100 to 1,325 m below the surface and range in temperature from slightly above ambient (10°C) to high temperature (167°C). The vent fluid is characterized by high concentrations of CO 2 (up to 17 mM) and Fe(II) (up to 268 �M), but there is a general paucity of H 2S. Most of the vents are surrounded by microbial mats that have a gelatinous texture and are heavily encrusted with rust-colored Fe oxides. Visually, the Fe oxides appeared homogeneous. However, light microscopy revealed that the oxides had different morphologies, which fell into three classes: (i) sheaths, (ii) twisted or irregular filaments, and (iii) amorphous oxides. A morphological analysis of eight different samples indicated that the amorphous oxides were overall the most abundant; however, five sites had>50 % sheaths and filamentous oxides. These latter morphologies are most likely the direct result of microbial deposition. Direct cell counts revealed that all of the oxides had abundant microbial populations associated with them, from 6.9 � 10 7 to 5.3 � 10 8 cells per ml of mat material. At most sites, end point dilution series for lithotrophic Fe oxidizers were successful out to dilutions of 10 �6 and 10 �7. A pure culture was obtained from a 10 �7 dilution tube; this strain, JV-1, was an obligate, microaerophilic Fe oxidizer that grew at 25 to 30°C. A non-cultivation-based molecular approach with terminal-restriction fragment length polymorphism also indicated the common presence of
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...NA from specific groups of taxa. PCR products are then digested with tetrameric restriction enzymes, and the fluorescently labeled terminal restriction fragments are precisely measured and quantified =-=(27)-=-. Samples of the Fe mat were subjected to extraction and isolation of total genomic DNA followed by multitemplate PCR (30) with an annealing temperature of 56°C and a total reaction volume of 50 �l. A...

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