scholarly journals Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

2013 ◽  
Vol 79 (24) ◽  
pp. 7598-7609 ◽  
Author(s):  
Jer-Horng Wu ◽  
Hui-Ping Chuang ◽  
Mao-Hsuan Hsu ◽  
Wei-Yu Chen
Keyword(s):  
Anaerobic Digestion ◽  
16S Rrna ◽  
Industrial Wastewater ◽  
Anaerobic Treatment ◽  
Primer Extension ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Oligonucleotide Primer ◽  
Content Type ◽  
Relative Abundances

ABSTRACTIn this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using anArchaea-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within theMethanobacteriales,Methanomicrobiales, andMethanosarcinalesand displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of theMethanosaeta,Methanolinea, andMethanomethylovorans, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products.

scholarly journals Relative Abundance of Bacteroides spp. in Stools and Wastewaters as Determined by Hierarchical Oligonucleotide Primer Extension

2008 ◽  
Vol 74 (9) ◽  
pp. 2882-2893 ◽  
Author(s):  
Pei-Ying Hong ◽  
Jer-Horng Wu ◽  
Wen-Tso Liu
Keyword(s):  
16S Rrna ◽  
Fecal Microbiota ◽  
Primer Extension ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Oligonucleotide Primer ◽  
Healthy Human ◽  
Dna Templates ◽  
Human Adults ◽  
Relative Abundances

ABSTRACT A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), was used to determine the relative abundances of predominant Bacteroides spp. present in fecal microbiota and wastewaters. For this analysis, genomic DNA in feces of healthy human adults, bovines, and swine and in wastewaters was extracted and total bacterial 16S rRNA genes were PCR amplified and used as the DNA templates for HOPE. Nineteen oligonucleotide primers were designed to detect 14 Bacteroides spp. at different hierarchical levels (domain, order, cluster, and species) and were arranged into and used in six multiplex HOPE reaction mixtures. Results showed that species like B. vulgatus, B. thetaiotaomicron, B. caccae, B. uniformis, B. fragilis, B. eggerthii, and B. massiliensis could be individually detected in human feces at abundances corresponding to as little as 0.1% of PCR-amplified 16S rRNA genes. Minor species like B. pyogenes, B. salyersiae, and B. nordii were detected only collectively using a primer that targeted the B. fragilis subgroup (corresponding to ∼0.2% of PCR-amplified 16S rRNA genes). Furthermore, Bac303-related targets (i.e., most Bacteroidales) were observed to account for 28 to 44% of PCR-amplified 16S rRNA genes from human fecal microbiota, and their abundances were higher than those detected in the bovine and swine fecal microbiota and in wastewaters by factors of five and two, respectively. These results were comparable to those obtained by quantitative PCR and to those reported previously from studies using whole-cell fluorescence hybridization and 16S rRNA clone library methods, supporting the conclusion that HOPE can be a sensitive, specific, and rapid method to determine the relative abundances of Bacteroides spp. predominant in fecal samples.

scholarly journals Pyrosequencing ofmcrAand Archaeal 16S rRNA Genes Reveals Diversity and Substrate Preferences of Methanogen Communities in Anaerobic Digesters

2014 ◽  
Vol 81 (2) ◽  
pp. 604-613 ◽  
Author(s):  
David Wilkins ◽  
Xiao-Ying Lu ◽  
Zhiyong Shen ◽  
Jiapeng Chen ◽  
Patrick K. H. Lee
Keyword(s):  
Anaerobic Digestion ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Methanogenic Archaea ◽  
16S Rrna Gene Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
The 16S Rrna Gene

ABSTRACTMethanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes.Methanobacteriales,Methanomicrobiales, andMethanosarcinaleswere detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of themcrAgenes suggested that these digesters were dominated by acetoclastic methanogens, particularlyMethanosarcinales, while the other digesters were dominated by hydrogenotrophicMethanomicrobiales. The proposed euryarchaeotal orderMethanomassiliicoccalesand the uncultured WSA2 group were detected with the 16S rRNA gene, and potentialmcrAgenes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using themcrAgene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance ofmcrAtranscripts in digesters treating sludge and wastewater samples, supporting themcrAgene as a biomarker for methane yield.

scholarly journals Hierarchical Oligonucleotide Primer Extension as a Time- and Cost-Effective Approach for Quantitative Determination of Bifidobacterium spp. in Infant Feces

2009 ◽  
Vol 75 (8) ◽  
pp. 2573-2576 ◽  
Author(s):  
Pei-Ying Hong ◽  
Gaik Chin Yap ◽  
Bee Wah Lee ◽  
Kaw Yan Chua ◽  
Wen-Tso Liu
Keyword(s):  
Quantitative Determination ◽  
Bifidobacterium Longum ◽  
Cost Effective ◽  
Primer Extension ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Oligonucleotide Primer ◽  
Effective Manner ◽  
Relative Abundances

ABSTRACT The Bifidobacterium spp. present in 10 infant fecal samples (4 from infants with eczema and 6 from healthy infants) were quantified with both hierarchical oligonucleotide primer extension (HOPE) and fluorescence in situ hybridization-flow cytometry. The relative abundances of Bifidobacterium longum and B. catenulatum with respect to the total bifidobacteria had a poor correlation (ρ, <0.600; P value, >0.208), presumably due to differences in primer specificity and the level of hybridization stringency of both methods. In contrast, the relative abundances of organisms of the genus Bifidobacterium against the total amplified 16S rRNA genes and those of B. adolescentis, B. bifidum, and B. breve against the genus Bifidobacterium exhibited a good statistical correlation (ρ, >0.783; P value, <0.066). This good comparability supports HOPE as a method to achieve high-throughput quantitative determination of bacterial targets in a time- and cost-effective manner.

scholarly journals Temporal Variability of Coastal Planctomycetes Clades at Kabeltonne Station, North Sea

2011 ◽  
Vol 77 (14) ◽  
pp. 5009-5017 ◽  
Author(s):  
Ilaria Pizzetti ◽  
Bernhard M. Fuchs ◽  
Gunnar Gerdts ◽  
Antje Wichels ◽  
Karen H. Wiltshire ◽  
...  
Keyword(s):  
16S Rrna ◽  
North Sea ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Group A ◽  
Related Group ◽  
Group B ◽  
Card Fish ◽  
Group D

ABSTRACTMembers of the bacterial phylumPlanctomycetesare reported in marine water samples worldwide, but quantitative information is scarce. Here we investigated the phylogenetic diversity, abundance, and distribution ofPlanctomycetesin surface waters off the German North Sea island Helgoland during different seasons by 16S rRNA gene analysis and catalyzed reporter deposition fluorescencein situhybridization (CARD-FISH). GenerallyPlanctomycetesare more abundant in samples collected in summer and autumn than in samples collected in winter and spring. Statistical analysis revealed thatPlanctomycetesabundance was correlated to theCentralesdiatom bloom in spring 2007. The analysis of size-fractionated seawater samples and of macroaggregates showed that ∼90% of thePlanctomycetesreside in the >3-μm size fraction. Comparative sequence analysis of 184 almost full-length 16S rRNA genes revealed three dominant clades. The clades, namedPlanctomyces-related group A, unculturedPlanctomycetesgroup B, andPirellula-related group D, were monitored by CARD-FISH using newly developed oligonucleotide probes. All three clades showed recurrent abundance patterns during two annual sampling campaigns. UnculturedPlanctomycetesgroup B was most abundant in autumn samples, whilePlanctomyces-related group A was present in high numbers only during late autumn and winter. The levels ofPirellula-related group D were more constant throughout the year, with elevated counts in summer. Our analyses suggest that the seasonal succession of thePlanctomycetesis correlated with algal blooms. We hypothesize that the niche partitioning of the different clades might be caused by their algal substrates.

scholarly journals Mucilaginibacter koreensis sp. nov., isolated from leaf mould

2014 ◽  
Vol 64 (Pt_7) ◽  
pp. 2274-2279 ◽  
Author(s):  
Cheol Su Park ◽  
Kyudong Han ◽  
Tae-Young Ahn
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
Sequence Similarity ◽  
Optimal Growth ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Strictly Aerobic ◽  
Content Type ◽  
Link Type

A Gram-staining-negative, strictly aerobic, rod-shaped, pale-pink pigmented bacterial strain, designated TF8T, was isolated from leaf mould in Cheonan, Republic of Korea. Its taxonomic position was determined through a polyphasic approach. Optimal growth occurred on R2A agar without NaCl supplementation, at 25–28 °C and at pH 6.0–7.0. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain TF8T belongs to the genus Mucilaginibacter in the family Sphingobacteriaceae . The sequence similarity between 16S rRNA genes of strain TF8T and the type strains of other species of the genus Mucilaginibacter ranged from 92.1 to 94.7 %. The closest relatives of strain TF8T were Mucilaginibacter lutimaris BR-3T (94.7 %), M. soli R9-65T (94.5 %), M. litoreus BR-18T (94.5 %), M. rigui WPCB133T (94.0 %) and M. daejeonensis Jip 10T (93.8 %). The major isoprenoid quinone was MK-7 and the major cellular fatty acids were iso-C15 : 0 (33.0 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 24.8 %) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 13.0 %). The major polar lipids of TF8T were phosphatidylethanolamine and three unidentified aminophospholipids. The G+C content of the genomic DNA was 46.2 mol%. On the basis of the data presented here, strain TF8T is considered to represent a novel species of the genus Mucilaginibacter , for which the name Mucilaginibacter koreensis sp. nov. is proposed. The type strain is TF8T ( = KACC 17468T = JCM 19323T).

scholarly journals Active Ammonia Oxidizers in an Acidic Soil Are Phylogenetically Closely Related to Neutrophilic Archaeon

2013 ◽  
Vol 80 (5) ◽  
pp. 1684-1691 ◽  
Author(s):  
Baozhan Wang ◽  
Yan Zheng ◽  
Rong Huang ◽  
Xue Zhou ◽  
Dongmei Wang ◽  
...  
Keyword(s):  
16S Rrna ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Acidic Soil ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Evidence ◽  
Ammonia Oxidizing Bacteria ◽  
Content Type ◽  
Metabolic Versatility

ABSTRACTAll cultivated ammonia-oxidizing archaea (AOA) within theNitrososphaeracluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence ofNitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth ofNitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis ofamoAgenes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the “heavy” DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that13CO2assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both13C-labeledamoAand 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strainsNitrososphaera viennensisEN76 and JG1 within theNitrososphaeracluster. Our results provide strong evidence for the adaptive growth ofNitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated.

scholarly journals Wastewater Irrigation Increases the Abundance of Potentially Harmful Gammaproteobacteria in Soils in Mezquital Valley, Mexico

2014 ◽  
Vol 80 (17) ◽  
pp. 5282-5291 ◽  
Author(s):  
Melanie Broszat ◽  
Heiko Nacke ◽  
Ronja Blasi ◽  
Christina Siebe ◽  
Johannes Huebner ◽  
...  
Keyword(s):  
16S Rrna ◽  
Rainy Season ◽  
Soil Microbial Communities ◽  
Wastewater Irrigation ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Resistance Determinants ◽  
Oxacillin Resistance ◽  
Irrigated Soils

ABSTRACTWastewater contains large amounts of pharmaceuticals, pathogens, and antimicrobial resistance determinants. Only a little is known about the dissemination of resistance determinants and changes in soil microbial communities affected by wastewater irrigation. Community DNAs from Mezquital Valley soils under irrigation with untreated wastewater for 0 to 100 years were analyzed by quantitative real-time PCR for the presence ofsulgenes, encoding resistance to sulfonamides. Amplicon sequencing of bacterial 16S rRNA genes from community DNAs from soils irrigated for 0, 8, 10, 85, and 100 years was performed and revealed a 14% increase of the relative abundance ofProteobacteriain rainy season soils and a 26.7% increase in dry season soils for soils irrigated for 100 years with wastewater. In particular,Gammaproteobacteria, including potential pathogens, such asPseudomonas,Stenotrophomonas, andAcinetobacterspp., were found in wastewater-irrigated fields. 16S rRNA gene sequencing of 96 isolates from soils irrigated with wastewater for 100 years (48 from dry and 48 from rainy season soils) revealed that 46% were affiliated with theGammaproteobacteria(mainly potentially pathogenicStenotrophomonasstrains) and 50% with theBacilli, whereas all 96 isolates from rain-fed soils (48 from dry and 48 from rainy season soils) were affiliated with theBacilli. Up to six types of antibiotic resistance were found in isolates from wastewater-irrigated soils; sulfamethoxazole resistance was the most abundant (33.3% of the isolates), followed by oxacillin resistance (21.9% of the isolates). In summary, we detected an increase of potentially harmful bacteria and a larger incidence of resistance determinants in wastewater-irrigated soils, which might result in health risks for farm workers and consumers of wastewater-irrigated crops.

scholarly journals Detection of Putatively Thermophilic Anaerobic Methanotrophs in Diffuse Hydrothermal Vent Fluids

2012 ◽  
Vol 79 (3) ◽  
pp. 915-923 ◽  
Author(s):  
Alexander Y. Merkel ◽  
Julie A. Huber ◽  
Nikolay A. Chernyh ◽  
Elizaveta A. Bonch-Osmolovskaya ◽  
Alexander V. Lebedinsky
Keyword(s):  
16S Rrna ◽  
Deep Sea ◽  
Hydrothermal Vents ◽  
Optimal Growth ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Anaerobic Oxidation Of Methane ◽  
Phylogenetic Groups ◽  
Content Type

ABSTRACTThe anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivatedEuryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (PGC) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between thePGCand optimal growth temperatures (Topt) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested bothin silicoand in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.

scholarly journals Diversity and Partitioning of Bacterial Populations within the Accessory Nidamental Gland of the Squid Euprymna scolopes

2012 ◽  
Vol 78 (12) ◽  
pp. 4200-4208 ◽  
Author(s):  
Andrew J. Collins ◽  
Brenna A. LaBarre ◽  
Brian S. Wong Won ◽  
Monica V. Shah ◽  
Steven Heng ◽  
...  
Keyword(s):  
16S Rrna ◽  
Bacterial Consortium ◽  
16S Rrna Genes ◽  
Microbial Consortia ◽  
Rrna Genes ◽  
Euprymna Scolopes ◽  
Sequencing Data ◽  
Content Type ◽  
Exact Function ◽  
Nidamental Gland

ABSTRACTMicrobial consortia confer important benefits to animal and plant hosts, and model associations are necessary to examine these types of host/microbe interactions. The accessory nidamental gland (ANG) is a female reproductive organ found among cephalopod mollusks that contains a consortium of bacteria, the exact function of which is unknown. To begin to understand the role of this organ, the bacterial consortium was characterized in the Hawaiian bobtail squid,Euprymna scolopes, a well-studied model organism for symbiosis research. Transmission electron microscopy (TEM) analysis of the ANG revealed dense bacterial assemblages of rod- and coccus-shaped cells segregated by morphology into separate, epithelium-lined tubules. The host epithelium was morphologically heterogeneous, containing ciliated and nonciliated cells with various brush border thicknesses. Hemocytes of the host's innate immune system were also found in close proximity to the bacteria within the tubules. A census of 16S rRNA genes suggested thatRhodobacterales, Rhizobiales, andVerrucomicrobiabacteria were prevalent, with members of the genusPhaeobacterdominating the consortium. Analysis of 454-shotgun sequencing data confirmed the presence of members of these taxa and revealed members of a fourth,Flavobacteriaof theBacteroidetesphylum. 16S rRNA fluorescentin situhybridization (FISH) revealed that many ANG tubules were dominated by members of specific taxa, namely,Rhodobacterales,Verrucomicrobia, orCytophaga-Flavobacteria-Bacteroidetes, suggesting symbiont partitioning to specific host tubules. In addition, FISH revealed that bacteria, includingPhaeobacterspecies from the ANG, are likely deposited into the jelly coat of freshly laid eggs. This report establishes the ANG of the invertebrateE. scolopesas a model to examine interactions between a bacterial consortium and its host.

scholarly journals Mycoplasma anserisalpingitidis sp. nov., isolated from European domestic geese (Anser anser domesticus) with reproductive pathology

2020 ◽  
Vol 70 (4) ◽  
pp. 2369-2381 ◽  
Author(s):  
Dmitriy V. Volokhov ◽  
Dénes Grózner ◽  
Miklós Gyuranecz ◽  
Naola Ferguson-Noel ◽  
Yamei Gao ◽  
...  
Keyword(s):  
16S Rrna ◽  
Type Strain ◽  
Type Species ◽  
Novel Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type

In 1983, Mycoplasma sp. strain 1220 was isolated in Hungary from the phallus lymph of a gander with phallus inflammation. Between 1983 and 2017, Mycoplasma sp. 1220 was also identified and isolated from the respiratory tract, liver, ovary, testis, peritoneum and cloaca of diseased geese in several countries. Seventeen studied strains produced acid from glucose and fructose but did not hydrolyse arginine or urea, and all grew under aerobic, microaerophilic and anaerobic conditions at 35 to 37 ˚C in either SP4 or pleuropneumonia-like organism medium supplemented with glucose and serum. Colonies on agar showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. Molecular characterization included analysis of the following genetic loci: 16S rRNA, 23S rRNA, 16S–23S rRNA ITS, rpoB, rpoC, rpoD, uvrA, parC, topA, dnaE, fusA and pyk. The genome was sequenced for type strain 1220T. The 16S rRNA gene sequences of studied strains of Mycoplasma sp. 1220 shared 99.02–99.19 % nucleotide similarity with M. anatis strains but demonstrated ≤95.00–96.70 % nucleotide similarity to the 16S rRNA genes of other species of the genus Mycoplasma . Phylogenetic, average nucleotide and amino acid identity analyses revealed that the novel species was most closely related to Mycoplasma anatis . Based on the genetic data, we propose a novel species of the genus Mycoplasma , for which the name Mycoplasma anserisalpingitidis sp. nov. is proposed with the type strain 1220T (=ATCC BAA-2147T=NCTC 13513T=DSM 23982T). The G+C content is 26.70 mol%, genome size is 959110 bp.

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