scholarly journals Molecular Characterization of Functional and Phylogenetic Genes from Natural Populations of Methanotrophs in Lake Sediments

1999 ◽  
Vol 65 (11) ◽  
pp. 5066-5074 ◽  
Author(s):  
Andria M. Costello ◽  
Mary E. Lidstrom
Keyword(s):  
16S Rrna ◽  
Phylogenetic Analyses ◽  
Natural Populations ◽  
Restriction Enzymes ◽  
Clone Libraries ◽  
Operational Taxonomic Units ◽  
Methane Oxidizing Bacteria ◽  
Lake Washington ◽  
Pcr Products ◽  
Total Community

ABSTRACT 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 enzymesMspI, 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 generaMethylomonas, Methylosinus, andMethylocystis 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.

scholarly journals Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades

2004 ◽  
Vol 70 (11) ◽  
pp. 6559-6568 ◽  
Author(s):  
Hector Castro ◽  
Andrew Ogram ◽  
K. R. Reddy
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Conservation ◽  
Phylogenetic Analyses ◽  
Florida Everglades ◽  
Rrna Gene ◽  
Conservation Area ◽  
Clone Libraries ◽  
Phylogenetic Characterization ◽  
Acetoclastic Methanogen

ABSTRACT Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.

Quantitatively evaluating mistaken clone assignments by RFLP analysis of 16S rRNA genes: a case study

2008 ◽  
Vol 54 (6) ◽  
pp. 479-482 ◽  
Author(s):  
Han-Bo Zhang ◽  
Chan-Wen Xu ◽  
Miao-Miao Wang ◽  
Tao Li ◽  
Zhi-Wei Zhao
Keyword(s):  
16S Rrna ◽  
Sequence Similarity ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Taxonomic Rank ◽  
Operational Taxonomic Units

We quantitatively evaluated the errors of clone assignment based on the restriction fragment length polymorphism (RFLP) pattern of 16S rRNA genes. Eighty clones were randomly selected from a 16S rRNA gene library and were categorized into 35 operational taxonomic units (OTU) based on their indistinguishable enzyme restriction patterns of 3 tetrameric restriction enzymes RsaI, BsuRI, and HinfI. All of these clones were then sequenced and were reassigned into 36–53 OTUs using the DOTUR program when sequence similarities of 95%–100% were used. The number of the identically assigned clones ranged from 53 to 61 and the percentage varied from 66.3% to 76.3%. The Shannon–Weaver index for the bacterial community observed by RFLP analysis was 2.75, equal to that estimated by DOTUR at a 97% sequence similarity. Compared with clones assigned with the DOTUR program at a 97% sequence similarity, only 61 clones (76.3%) were correctly assigned by RFLP analysis. Six clones (7.5%) were assigned mistakenly at the phylum level, and the positions of 13 clones (16.2%) were phylogenetically different at a lower taxonomic rank.

scholarly journals Improved Assessment of Denitrifying, N2-Fixing, and Total-Community Bacteria by Terminal Restriction Fragment Length Polymorphism Analysis Using Multiple Restriction Enzymes

2005 ◽  
Vol 71 (4) ◽  
pp. 2026-2035 ◽  
Author(s):  
Christopher Rösch ◽  
Hermann Bothe
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dominant Role ◽  
Restriction Enzymes ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
Data Content ◽  
Total Community ◽  
Set Up ◽  
The 16S Rrna Gene

ABSTRACT A database of terminal restriction fragments (tRFs) of the 16S rRNA gene was set up utilizing 13 restriction enzymes and 17,327 GenBank sequences. A computer program, termed TReFID, was developed to allow identification of any of these 17,327 sequences by means of polygons generated from the specific tRFs of each bacterium. The TReFID program complements and exceeds in its data content the Web-based phylogenetic assignment tool recently described by A. D. Kent, D. J. Smith, B. J. Benson, and E. W. Triplett (Appl. Environ. Microb. 69:6768-6766, 2003). The method to identify bacteria is different, as is the region of the 16S rRNA gene employed in the present program. For the present communication the software of the tRF profiles has also been extended to allow screening for genes coding for N2 fixation (nifH) and denitrification (nosZ) in any bacterium or environmental sample. A number of controls were performed to test the reliability of the TReFID program. Furthermore, the TReFID program has been shown to permit the analysis of the bacterial population structure of bacteria by means of their 16S rRNA, nifH, and nosZ gene content in an environmental habitat, as exemplified for a sample from a forest soil. The use of the TReFID program reveals that noncultured denitrifying and dinitrogen-fixing bacteria might play a more dominant role in soils than believed hitherto.

Meloidogyne lopezi n. sp. (Nematoda: Meloidogynidae), a new root-knot nematode associated with coffee (Coffea arabica L.) in Costa Rica, its diagnosis and phylogenetic relationship with other coffee-parasitising Meloidogyne species

Nematology ◽  
2014 ◽  
Vol 16 (6) ◽  
pp. 643-661 ◽  
Author(s):  
Danny A. Humphreys-Pereira ◽  
Danny A. Humphreys-Pereira ◽  
Lorena Flores-Chaves ◽  
Danny A. Humphreys-Pereira ◽  
Lorena Flores-Chaves ◽  
...  
Keyword(s):  
Costa Rica ◽  
16S Rrna ◽  
Coffea Arabica ◽  
Phylogenetic Analyses ◽  
Mitochondrial Gene ◽  
Restriction Enzymes ◽  
Nematode Species ◽  
Male Body ◽  
Root Knot Nematode ◽  
Pcr Rflp

Coffee (Coffea arabica L. cv. Catuai) seedlings with abundant small root galls caused by an unknown root-knot nematode were found in southern Costa Rica. Morphology, esterase and malate dehydrogenase isozyme phenotypes and DNA markers differentiated this nematode from known Meloidogyne spp. A new species, M. lopezi n. sp., with common name Costa Rican root-knot nematode, is suggested. Meloidogyne lopezi n. sp. is distinguished from other coffee-associated Meloidogyne spp. by size of female lips and stylet, male body length and stylet and second-stage juvenile body and tail morphology. The region of the mitochondrial genome between COII and 16S rRNA showed a unique amplicon size of 1370 bp, and digestions with restriction enzymes HinfI, AluI, DraI and DraIII revealed characteristic PCR-RFLP patterns that differed from the tropical root-knot nematode species M. arabicida, M. incognita, M. izalcoensis, M. javanica and M. paranaensis. Characterisation of the protein-coding map-1 gene and phylogenetic analyses suggested that M. lopezi n. sp. might reproduce by mitotic parthenogenesis. Phylogenies estimated using Bayesian analyses based on the region between the COII and 16S rRNA mitochondrial genes, as well as the 18S and 28S ribosomal nuclear genes, indicated that M. lopezi n. sp. is closely related to other tropical Meloidogyne spp. that infect coffee, especially M. arabicida, M. izalcoensis and M. paranaensis from Central and South America. Isozyme analyses and PCR-RFLP of the COII-16S rRNA mitochondrial gene region enable a clear diagnostic differentiation between these species.

scholarly journals Development and Evaluation of a PCR-Based Assay for Detection of Haemobartonella felis in Cats and Differentiation of H. felis from Related Bacteria by Restriction Fragment Length Polymorphism Analysis

1998 ◽  
Vol 36 (2) ◽  
pp. 462-466 ◽  
Author(s):  
Joanne B. Messick ◽  
Linda M. Berent ◽  
Sandra K. Cooper
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Data ◽  
Mycoplasma Genitalium ◽  
Restriction Enzymes ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
Doxycycline Treatment ◽  
Pcr Products ◽  
The 16S Rrna Gene

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set ofH. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA ofEperythrozoon suis, Mycoplasma genitalium, andBartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI andMnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felisinfection in cats.

scholarly journals Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

2004 ◽  
Vol 70 (4) ◽  
pp. 2245-2253 ◽  
Author(s):  
K. Nanba ◽  
G. M. King ◽  
K. Dunfield
Keyword(s):  
Nucleotide Diversity ◽  
Microbial Mats ◽  
Phylogenetic Analyses ◽  
Large Subunit ◽  
Hydrogen Uptake ◽  
Clone Libraries ◽  
Bisphosphate Carboxylase ◽  
Gene Coding ◽  
Volcanic Deposits ◽  
Pcr Products

ABSTRACT A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.

scholarly journals Molecular Comparison of Bacterial Communities within Iron-Containing Flocculent Mats Associated with Submarine Volcanoes along the Kermadec Arc

2008 ◽  
Vol 75 (6) ◽  
pp. 1650-1657 ◽  
Author(s):  
Tyler W. Hodges ◽  
Julie B. Olson
Keyword(s):  
Bacterial Communities ◽  
Fragment Length Polymorphism ◽  
Phylogenetic Analyses ◽  
Clone Libraries ◽  
Site Specific ◽  
Operational Taxonomic Units ◽  
Molecular Comparison ◽  
Flocculent Material ◽  
Hydrothermal Environments

ABSTRACT Iron oxide sheaths and filaments are commonly found in hydrothermal environments and have been shown to have a biogenic origin. These structures were seen in the flocculent material associated with two submarine volcanoes along the Kermadec Arc north of New Zealand. Molecular characterization of the bacterial communities associated with the flocculent samples indicated that no known Fe-oxidizing bacteria dominated the recovered clone libraries. However, clones related to the recently described Fe-oxidizing bacterium Mariprofundus ferrooxydans were obtained from both the iron-containing flocculent (Fe-floc) and sediment samples, and peaks corresponding to Mariprofundus ferrooxydans, as well as the related clones, were observed in several of our terminal restriction fragment length polymorphism profiles. A large group of epsilonproteobacterial sequences, for which there is no cultured representative, dominated clones from the Fe-floc libraries and were less prevalent in the sediment sample. Phylogenetic analyses indicated that several operational taxonomic units appeared to be site specific, and statistical analyses of the clone libraries found that all samples were significantly different from each other. Thus, the bacterial communities in the Fe-floc samples were not more closely related to each other than to the sediment communities.

scholarly journals Improvement of the Representation of Bifidobacteria in Fecal Microbiota Metagenomic Libraries by Application of the cpn60 Universal Primer Cocktail

2010 ◽  
Vol 76 (13) ◽  
pp. 4550-4552 ◽  
Author(s):  
Janet E. Hill ◽  
W. M. Ursla Fernando ◽  
Gordon A. Zello ◽  
Robert T. Tyler ◽  
Wendy J. Dahl ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Fecal Microbiota ◽  
Universal Primer ◽  
Clone Libraries ◽  
Metagenomic Libraries ◽  
Large Numbers ◽  
Pcr Products

ABSTRACT Actinobacteria, particularly bifidobacteria, are widely observed to be underrepresented in metagenomic studies of microbial communities. We have compared human fecal microbiota clone libraries based on 16S rRNA and cpn60 PCR products. Taxonomic profiles were similar except that the cpn60 libraries contained large numbers of bifidobacterial sequences.

scholarly journals Comparative Analysis of the Methanogen Diversity in Horse and Pony by UsingmcrAGene and Archaeal 16S rRNA Gene Clone Libraries

Archaea ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Khin-Ohnmar Lwin ◽  
Hiroki Matsui
Keyword(s):  
Comparative Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Clone Libraries ◽  
Operational Taxonomic Units ◽  
Gene 16S Rrna ◽  
Methanogen Population ◽  
Methyl Coenzyme M Reductase ◽  
16S Rrna Clone Libraries

Comparative analysis of methanogen compositions in the feces of horse and pony was carried out by constructing theα-subunit of methyl coenzyme-M reductase (mcrA) gene and 16S ribosomal RNA gene (16S rRNA) clone libraries. ThemcrAclone library analysis indicated that Methanomicrobiales was predominant in both horse and pony. Furthermore, most of the clones of the 16S rRNA gene library showed that Methanomicrobiales was also predominant in horse and pony, but the LIBSHUFF analysis showed that the horse and pony libraries were significantly different (P<0.05). Most of operational taxonomic units (OTUs) showed low similarity to the identified methanogens in both themcrAand the 16S rRNA clone libraries. The results suggest that horse and pony harbor unidentified and novel methanogens in their hindgut. The methanogen population was higher in horse than in pony; however, the anaerobic fungal population was similar in horse and pony. The methanogen diversity was different between two breeds ofEquus caballus.

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