scholarly journals Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in Compost and Composted Materials

1999 ◽  
Vol 65 (2) ◽  
pp. 396-403 ◽  
Author(s):  
George A. Kowalchuk ◽  
Zinaida S. Naoumenko ◽  
Piet J. L. Derikx ◽  
Andreas Felske ◽  
John R. Stephen ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Ammonia Oxidizer ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrogen Transformations ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Specific Pcr ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
Almost All

ABSTRACT Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteriain a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms.

Nitrifier and denitrifier molecular operational taxonomic unit compositions from sites of a freshwater estuary of Chesapeake Bay

2009 ◽  
Vol 55 (3) ◽  
pp. 333-346 ◽  
Author(s):  
Caroline S. Fortunato ◽  
David B. Carlini ◽  
Evan Ewers ◽  
Karen L. Bushaw-Newton
Keyword(s):  
Organic Matter ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Organic Matter Content ◽  
Sequence Divergence ◽  
Operational Taxonomic Unit ◽  
Matter Content ◽  
Ammonia Oxidizing Bacteria ◽  
Overlying Water ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis

Temporal and spatial changes in the molecular operational taxonomic unit (OTU) compositions of bacteria harboring genes for nitrification and denitrification were assessed using denaturing gradient gel electrophoresis (DGGE), clone-based DNA sequencing of selected PCR products, and analyses of ammonium and organic matter concentrations. Sediment, overlying water, and pore-water samples were taken from different vegetated sites of Jug Bay National Estuarine Research Reserve, Maryland, during spring, summer, and fall 2006. OTU richness and the diversities of nitrifiers and denitrifiers were assessed by the presence of bands on DGGE gels, both ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were seasonally dependent. AOB OTU richness was highest in the summer when NOB richness was decreased, whereas NOB richness was highest in the spring when AOB richness was decreased. The OTU diversities of nitrifiers did not correlate with ammonium concentrations, organic matter concentrations, or the presence of vegetation. The OTU diversities of denitrifiers possessing either the nirK or nosZ genes were not seasonally dependent but were positively correlated with organic matter content (p = 0.0015, r2 = 0.27; p < 0.0001, r2 = 0.39, respectively). Additionally, the presence of vegetation significantly enhanced nosZ species richness (Wilcoxon/Kruskal–Wallis test, p < 0.008), but this trend was not seen for nirK OTU richness. Banding patterns for nirK OTUs were more similar within sites for each season compared with any of the other genes. Over all seasons, nirK OTU richness was highest and AOB and nosZ OTU richness were lowest (Wilcoxon/Kruskal–Wallis test, p < 0.0001). High levels of sequence divergence among cloned nirK PCR products indicate a broad diversity of nirK homologs in this freshwater estuary.

scholarly journals Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

2001 ◽  
Vol 67 (2) ◽  
pp. 504-513 ◽  
Author(s):  
Reetta M. Satokari ◽  
Elaine E. Vaughan ◽  
Antoon D. L. Akkermans ◽  
Maria Saarela ◽  
Willem M. de Vos
Keyword(s):  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Methodological Approach ◽  
Common Species ◽  
Bifidobacterium Adolescentis ◽  
Specific Pcr ◽  
Rdna Sequences ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis

ABSTRACT We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE).Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

scholarly journals Molecular Profiling of the Clostridium leptum Subgroup in Human Fecal Microflora by PCR-Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

2006 ◽  
Vol 72 (8) ◽  
pp. 5232-5238 ◽  
Author(s):  
Jian Shen ◽  
Baorang Zhang ◽  
Guifang Wei ◽  
Xiaoyan Pang ◽  
Hua Wei ◽  
...  
Keyword(s):  
Clone Library ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Clone Library Analysis ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
Universal Bacterial Primer ◽  
Group Specific Primer

ABSTRACT A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, ∼99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.

scholarly journals amoAGene Abundances and Nitrification Potential Rates Suggest that Benthic Ammonia-Oxidizing Bacteria and Not Archaea Dominate N Cycling in the Colne Estuary, United Kingdom

2014 ◽  
Vol 81 (1) ◽  
pp. 159-165 ◽  
Author(s):  
Jialin Li ◽  
David B. Nedwell ◽  
Jessica Beddow ◽  
Alex J. Dumbrell ◽  
Boyd A. McKew ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Dry Weight ◽  
Estuarine Sediments ◽  
Ammonia Oxidizer ◽  
Ammonia Oxidizing Bacteria ◽  
Content Type ◽  
Gene Abundance ◽  
Nitrification Potential ◽  
Gradient Gel Electrophoresis ◽  
Colne Estuary

ABSTRACTNitrification, mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), is important in global nitrogen cycling. In estuaries where gradients of salinity and ammonia concentrations occur, there may be differential selections for ammonia-oxidizer populations. The aim of this study was to examine the activity, abundance, and diversity of AOA and AOB in surface oxic sediments of a highly nutrified estuary that exhibits gradients of salinity and ammonium. AOB and AOA communities were investigated by measuring ammonia monooxygenase (amoA) gene abundance and nitrification potentials both spatially and temporally. Nitrification potentials differed along the estuary and over time, with the greatest nitrification potentials occurring mid-estuary (8.2 μmol N grams dry weight [gdw]−1day−1in June, increasing to 37.4 μmol N gdw−1day−1in January). At the estuary head, the nitrification potential was 4.3 μmol N gdw−1day−1in June, increasing to 11.7 μmol N gdw−1day−1in January. At the estuary head and mouth, nitrification potentials fluctuated throughout the year. AOBamoAgene abundances were significantly greater (by 100-fold) than those of AOA both spatially and temporally.Nitrosomonasspp. were detected along the estuary by denaturing gradient gel electrophoresis (DGGE) band sequence analysis. In conclusion, AOB dominated over AOA in the estuarine sediments, with the ratio of AOB/AOAamoAgene abundance increasing from the upper (freshwater) to lower (marine) regions of the Colne estuary. These findings suggest that in this nutrified estuary, AOB (possiblyNitrosomonasspp.) were of major significance in nitrification.

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 of Transcripts of Nitrite Reductase Genes (nirK and nirS) in Rhizospheres of Grain Legumes

2005 ◽  
Vol 71 (4) ◽  
pp. 2001-2007 ◽  
Author(s):  
Shilpi Sharma ◽  
Manish Kumar Aneja ◽  
Jochen Mayer ◽  
Jean Charles Munch ◽  
Michael Schloter
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Lupinus Albus ◽  
Grain Legumes ◽  
Rt Pcr ◽  
Insert Size ◽  
Reverse Transcription Pcr ◽  
Distinct Cluster ◽  
Nitrite Reductases ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis

ABSTRACT Transcription of the nirK and nirS genes coding for dissimilatory bacterial nitrite reductases was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from rhizosphere samples of three economically important grain legumes at maturity: Vicia faba, Lupinus albus, and Pisum sativum. The nirK gene and transcripts could be detected in all the rhizosphere samples. In contrast, nirS could not be detected. Sampling variations were analyzed by comparing denaturing gradient gel electrophoresis profiles derived from nirK RT-PCR products. High similarity was observed between the replicates, and so one representative product per legume was cloned. Clones with the correct insert size were screened by restriction fragment length polymorphism by using the restriction enzyme MspI. The clones could be distributed into 12 different patterns. Patterns 1, 3, 4, 5, and 7 were common in clone libraries of the three rhizosphere types under study. Patterns 2, 9, 10, and 11 were absent from Pisum rhizospheres, while patterns 6, 8, and 12 were absent from the Vicia library. Pattern 1, which was the most dominant in the Vicia and Lupinus libraries, constituted about 25% of all clones. The Lupinus library had clones representing all 12 patterns, indicating it to be the most diverse among the three. Clones representative of each pattern were sequenced. All patterns grouped together forming a distinct cluster, which was divergent from previously described nirK sequences in the database. The study revealed a hitherto unknown diversity of denitrifiers in legume rhizospheres. A plant-dependent rhizosphere effect on the transcripts of a gene was evident.

scholarly journals Influence of Effluent Irrigation on Community Composition and Function of Ammonia-Oxidizing Bacteria in Soil

2001 ◽  
Vol 67 (8) ◽  
pp. 3426-3433 ◽  
Author(s):  
Tamar Oved ◽  
Avi Shaviv ◽  
Tal Goldrath ◽  
Raphi T. Mandelbaum ◽  
Dror Minz
Keyword(s):  
Community Composition ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Ammonium Concentration ◽  
Ammonia Oxidizing Bacteria ◽  
Gene Encoding ◽  
Α Subunit ◽  
Irrigation Period ◽  
Effluent Irrigation ◽  
Gradient Gel Electrophoresis ◽  
And Function

ABSTRACT The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the α-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein.

Seasonal variation in communities of ammonia-oxidizing bacteria based on polymerase chain reaction – denaturing gradient gel electrophoresis in a biofilm reactor for drinking water pretreatment

2008 ◽  
Vol 54 (5) ◽  
pp. 358-365 ◽  
Author(s):  
Xiao-Wen Zhang ◽  
Ying-Ying Qin ◽  
Hong-Qiang Ren ◽  
Dao-Tang Li ◽  
Hong Yang
Keyword(s):  
Drinking Water ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Biofilm Reactor ◽  
Ammonia Oxidizer ◽  
Ammonia Oxidizing Bacteria ◽  
Clone Libraries ◽  
Community Shift ◽  
Water Pretreatment ◽  
Gradient Gel Electrophoresis

The diversity and variation of total and active ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment were characterized by clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA and its gene during a whole year. Sequences obtained from clone libraries affiliated with the Nitrosomonas oligotropha lineage and the Nitrosomonas communis lineage. An uncultured subgroup of Nitrosomonas communis lineage was also detected. Seasonal variations in both total and active ammonia-oxidizing bacteria communities were observed in the DGGE profiles, but an RNA-based analysis reflected more obvious dynamic changes in ammonia-oxidizer community than a DNA-based approach. Statistical study based on canonical correspondence analysis showed that a community shift of active ammonia oxidizers was significantly influenced by temperature and pH, but no significant correlation was found between environmental variables and total ammonia-oxidizer community shift.

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.

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