scholarly journals Postinoculation Protozoan Establishment and Association Patterns of Methanogenic Archaea in the Ovine Rumen

2007 ◽  
Vol 73 (14) ◽  
pp. 4609-4618 ◽  
Author(s):  
Samuel Ohene-Adjei ◽  
Ronald M. Teather ◽  
Michael Ivan ◽  
Robert J. Forster
Keyword(s):  
Phylogenetic Analysis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Methanogenic Archaea ◽  
Type A ◽  
Negative Control ◽  
Rrna Gene ◽  
Association Patterns ◽  
Clone Libraries ◽  
Symbiotic Relationships ◽  
The Impact

ABSTRACT Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.

scholarly journals Differences in the Rumen Methanogen Populations of Lactating Jersey and Holstein Dairy Cows under the Same Diet Regimen

2011 ◽  
Vol 77 (16) ◽  
pp. 5682-5687 ◽  
Author(s):  
Erin E. King ◽  
Rachel P. Smith ◽  
Benoit St-Pierre ◽  
André-Denis G. Wright
Keyword(s):  
Methanogenic Archaea ◽  
Milk Composition ◽  
Species Level ◽  
Rrna Gene ◽  
Content Type ◽  
Sequence Identity ◽  
Diet Regimen ◽  
Pcr Products ◽  
The Impact ◽  
Rumen Methanogen

ABSTRACTIn the dairy cattle industry, Holstein and Jersey are the breeds most commonly used for production. They differ in performance by various traits, such as body size, milk production, and milk composition. With increased concerns about the impact of agriculture on climate change, potential differences in other traits, such as methane emission, also need to be characterized further. Since methane is produced in the rumen by methanogenic archaea, we investigated whether the population structure of methanogen communities would differ between Holsteins and Jerseys. Breed-specific rumen methanogen 16S rRNA gene clone libraries were constructed from pooled PCR products obtained from lactating Holstein and Jersey cows, generating 180 and 185 clones, respectively. The combined 365 sequences were assigned to 55 species-level operational taxonomic units (OTUs). Twenty OTUs, representing 85% of the combined library sequences, were common to both breeds, while 23 OTUs (36 sequences) were found only in the Holstein library and 12 OTUs (18 sequences) were found only in the Jersey library, highlighting increased diversity in the Holstein library. Other differences included the observation that sequences with species-like sequence identity toMethanobrevibacter milleraewere represented more highly in the Jersey breed, whileMethanosphaera-related sequences and novel uncultured methanogen clones were more frequent in the Holstein library. In contrast, OTU sequences with species-level sequence identity toMethanobrevibacter ruminantiumwere represented similarly in both libraries. Since the sampled animals were from a single herd consisting of two breeds which were fed the same diet and maintained under the same environmental conditions, the differences we observed may be due to differences in host breed genetics.

scholarly journals Molecular Analysis of Surfactant-Driven Microbial Population Shifts in Hydrocarbon-Contaminated Soil

2000 ◽  
Vol 66 (7) ◽  
pp. 2959-2964 ◽  
Author(s):  
Gregory M. Colores ◽  
Richard E. Macur ◽  
David M. Ward ◽  
William P. Inskeep
Keyword(s):  
Gel Electrophoresis ◽  
Microbial Population ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Microbial Populations ◽  
Alcohol Ethoxylate ◽  
Rrna Gene ◽  
Mineralization Kinetics ◽  
Gradient Gel Electrophoresis ◽  
The Impact ◽  
Parallel Cultivation

ABSTRACT We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC′) (determined to be 13 mg g−1). Addition of the surfactant at a concentration below the CMC′ (2 mg g−1) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC′ (10 mg g−1), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC′ (40 mg g−1) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulatedRhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenespopulations at elevated surfactant levels. Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas andAlcaligenes populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization.

scholarly journals Impact of Consumption of Oligosaccharide-Containing Biscuits on the Fecal Microbiota of Humans

2004 ◽  
Vol 70 (4) ◽  
pp. 2129-2136 ◽  
Author(s):  
Gerald W. Tannock ◽  
Karen Munro ◽  
Rodrigo Bibiloni ◽  
Mary A. Simon ◽  
Patrick Hargreaves ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Internal Standard ◽  
Fecal Microbiota ◽  
Rrna Gene ◽  
Galactosidase Activity ◽  
Bifidobacterium Adolescentis ◽  
S1 Nuclease ◽  
The Impact

ABSTRACT Human subjects consumed biscuits containing either galacto-oligosaccharides or fructo-oligosaccharides in a double-blinded, crossover study. The impact of supplementing the diet with three biscuits per day on the fecal microbiota was evaluated by selective culture of particular bacterial groups, measurement of β-galactosidase activity, and nucleic acid-based analytical methods (PCR-denaturing gradient gel electrophoresis [PCR-DGGE] and fluorescent in situ hybridization). The composition of the bifidobacterial populations was monitored at the level of species (PCR-DGGE) and strains (pulsed-field gel electrophoresis of DNA digests), and representative cultures were tested quantitatively for their ability to use galacto-oligosaccharides. Technical improvements to DGGE analysis of the microbiota were made by the use of an internal standard that allowed valid comparisons of fragment staining intensities to be made between profiles, the use of S1 nuclease digestion to remove single-stranded DNA to facilitate cloning of DNA sequences cut from gels, and the extraction of RNA to be used as the template in reverse transcription-PCR-DGGE. RNA-DGGE profiles were markedly different (Dice's similarity coefficient, 58.5%) from those generated by DNA-DGGE. Neither the sizes of the bacterial populations nor the DNA-DGGE profiles of the microbiota were altered by the consumption of the biscuits, but the RNA-DGGE profiles were altered by the detection or increased staining intensity of 16S rRNA gene sequences originating from Bifidobacterium adolescentis and/or Colinsella aerofaciens in the feces of 11 of 15 subjects. β-Galactosidase activity was elevated in the feces of some subjects as a result of biscuit consumption. Subjects differed in the ability of the bifidobacterial strains harbored in their feces to use galacto-oligosaccharides. Our observations suggest that a phylogenetic approach to analysis of the gut ecosystem may not always be optimal and that a more physiological (biochemical) method might be more informative.

Higher seasonal variation of actinobacterial communities than spatial heterogeneity in the surface sediments of Taihu Lake, China

2013 ◽  
Vol 59 (5) ◽  
pp. 353-358 ◽  
Author(s):  
Jianjun Wang ◽  
Yong Zhang ◽  
Zhengkui Li ◽  
Ji Shen
Keyword(s):  
Seasonal Variation ◽  
Spatial Heterogeneity ◽  
Lake Sediments ◽  
Surface Sediments ◽  
Taihu Lake ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Detrended Correspondence Analysis ◽  
Rrna Gene ◽  
Clone Libraries ◽  
Principal Coordinates

Much more attention has been paid to the actinobacterial community in soils or water columns of aquatic habitats. However, there are few studies on their composition and diversity in lake sediments. Here, we used denaturing gradient gel electrophoresis and clone libraries of partial 16S rRNA gene to study the spatial variations of actinobacterial communities across 4 seasons in the surface sediments of the shallow, subtropical Taihu Lake. Cluster analysis based on fingerprints showed clear spatiotemporal variations of actinobacterial communities and higher seasonal variation than spatial heterogeneity. Based on clone libraries, this pattern was supported by the principal coordinates analysis in the phylogenetic context and by detrended correspondence analysis on the operational taxonomic unit table. Additionally, phylogenetic analysis showed that the putative freshwater-specific actinobacterial lineages (e.g., acI) were also detected in the lake sediments, which suggests that these subclusters may also adapt to the sediment environments. Summarily, our results suggested that actinobacterial communities of the surface sediments were more affected by seasonal variation than spatial heterogeneity in the intrahabitat of Taihu Lake.

scholarly journals Analysis of Bacterial Communities in Soil by Use of Denaturing Gradient Gel Electrophoresis and Clone Libraries, as Influenced by Different Reverse Primers

2008 ◽  
Vol 74 (9) ◽  
pp. 2717-2727 ◽  
Author(s):  
Jolanda K. Brons ◽  
Jan Dirk van Elsas
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Soil Analysis ◽  
Rrna Gene ◽  
Sequence Information ◽  
Soil Bacterial Diversity ◽  
Clone Libraries ◽  
Gradient Gel Electrophoresis ◽  
Candidate Division ◽  
Forward Primer

ABSTRACT To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel reverse primers was tested with the canonical forward primer as to the DGGE fingerprints obtained from grassland soil. Analysis of these DGGE profiles by GelCompar showed that they all fell into two main clusters separated by a G/A alteration at position 14 in the reverse primer used. To assess differences between the dominant bacteria amplified, we then produced four (100-membered) 16S rRNA gene clone libraries by using reverse primers with either an A or a G at position 14, designated R1401-1a, R1401-1b, R1401-2a, and R1401-2b. Subsequent sequence analysis revealed that, on the basis of the about 410-bp sequence information, all four primers amplified similar, as well as different (including novel), bacterial groups from soil. Most of the clones fell into two main phyla, Firmicutes and Proteobacteria. Within Firmicutes, the majority of the clones belonged to the genus Bacillus. Within Proteobacteria, the majority of the clones fell into the alpha or gamma subgroup whereas a few were delta and beta proteobacteria. The other phyla found were Actinobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Gemmatimonadetes, Chlorobi, Bacteroidetes, Chlamydiae, candidate division TM7, Ferribacter, Cyanobacteria, and Deinococcus. Statistical analysis of the data revealed that reverse primers R1401-1b and R1401-1a both produced libraries with the highest diversities yet amplified different types. Their concomitant use is recommended.

scholarly journals Identification and Removal of Potential Contaminants in 16S rRNA Gene Sequence Datasets from Low Microbial Biomass Samples: An Example from the Mosquito Gut

2020 ◽  
Author(s):  
Sebastián Diaz ◽  
Juan Sebastián Escobar ◽  
Frank William Avila
Keyword(s):  
Microbial Biomass ◽  
16S Rrna ◽  
Gut Microbiota ◽  
Relative Abundance ◽  
Vector Competence ◽  
Negative Control ◽  
Rrna Gene ◽  
Physiological Processes ◽  
Physiological Relevance ◽  
The Impact

Abstract Background: The bacterial gut microbiota of the female mosquito influences numerous physiological processes, including vector competence. As a low-microbial-biomass ecosystem, mosquito gut tissue is prone to contamination from the laboratory environment and from reagents commonly used to dissect and/or isolate DNA from gut tissue. In this report, we analyze five 16S rRNA datasets, including new data obtained by us, to gain insight into the impact of potential contaminating sequences on the composition, diversity, and structure of the mosquito gut microbial community. Results: We present a clustering-free approach that, based on the relative abundance of amplicon sequence variants (ASVs) in gut and negative control samples , allowed for the identification of candidate contaminating sequences. Some of these sequences belong to bacterial taxa previously identified as common contaminants in metagenomic studies; they have also been identified as part of the mosquito core gut microbiota, with putative physiological relevance for the host. By using different relative abundance cutoffs, we show that contaminating sequences have a significant impact on gut microbiota diversity and structure.Conclusions: The approach presented here allows the identification and removal of purported contaminating sequences in datasets obtained from low-microbial biomass samples. While it was exemplified with the analysis of gut microbiota from mosquitos, it can easily extend to other datasets dealing with similar technical artifacts.

scholarly journals Culture-Independent Characterization of Archaeal Biodiversity in Swine Confinement Building Bioaerosols

2009 ◽  
Vol 75 (17) ◽  
pp. 5445-5450 ◽  
Author(s):  
Benjamin Nehm� ◽  
Yan Gilbert ◽  
Val�rie L�tourneau ◽  
Robert J. Forster ◽  
Marc Veillette ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Molecular Ecology ◽  
Pcr Amplification ◽  
Methanogenic Archaea ◽  
Archaeal Community ◽  
Pig Manure ◽  
Rrna Gene ◽  
Swine Confinement

ABSTRACT It was previously demonstrated that microbial communities of pig manure were composed of both bacteria and archaea. Recent studies have shown that bacteria are aerosolized from pig manure, but none have ever focused on the airborne archaeal burden. We sought here to develop and apply molecular ecology approaches to thoroughly characterize airborne archaea from swine confinement buildings (SCBs). Eight swine operations were visited, twice in winter and once during summer. Institute of Occupational Medicine cassettes loaded with 25-mm gelatin filters were used to capture the inhalable microbial biomass. The total genomic DNA was extracted and used as a template for PCR amplification of the archaeal 16S rRNA gene. High concentrations of archaea were found in SCB bioaerosols, being as high as 108 16S rRNA gene copies per cubic meter of air. Construction and sequencing of 16S rRNA gene libraries revealed that all sequences were closely related to methanogenic archaea, such as Methanosphaera stadtmanae (94.7% of the archaeal biodiversity). Archaeal community profiles were compared by 16S rRNA gene denaturing gradient gel electrophoresis. This analysis showed similar fingerprints in each SCB and confirmed the predominance of methanogenic archaea in the bioaerosols. This study sheds new light on the nature of bioaerosols in SCBs and suggests that archaea are also aerosolized from pig manure.

scholarly journals Microbiota Dynamics and Diversity at Different Stages of Industrial Processing of Cocoa Beans into Cocoa Powder

2012 ◽  
Vol 78 (8) ◽  
pp. 2904-2913 ◽  
Author(s):  
Lídia J. R. Lima ◽  
Vera van der Velpen ◽  
Judith Wolkers-Rooijackers ◽  
Henri J. Kamphuis ◽  
Marcel H. Zwietering ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Cocoa Beans ◽  
Community Size ◽  
Cocoa Powder ◽  
Content Type ◽  
Industrial Processing ◽  
Microbiological Methods ◽  
Gradient Gel Electrophoresis ◽  
The Impact

ABSTRACTWe sampled a cocoa powder production line to investigate the impact of processing on the microbial community size and diversity at different stages. Classical microbiological methods were combined with 16S rRNA gene PCR-denaturing gradient gel electrophoresis, coupled with clone library construction, to analyze the samples. Aerobic thermoresistant spores (ThrS) (100°C; 10 min) were also isolated and characterized (identity, genetic diversity, and spore heat resistance), in view of their relevance to the quality of downstream heat-treated cocoa-flavored drinks. In the nibs (broken, shelled cocoa beans), average levels of total aerobic microorganisms (TAM) (4.4 to 5.6 log CFU/g) and aerobic total spores (TS) (80°C; 10 min; 4.3 to 5.5 log CFU/g) were significantly reduced (P< 0.05) as a result of alkalizing, while fungi (4.2 to 4.4 log CFU/g) andEnterobacteriaceae(1.7 to 2.8 log CFU/g) were inactivated to levels below the detection limit, remaining undetectable throughout processing. Roasting further decreased the levels of TAM and TS, but they increased slightly during subsequent processing. Molecular characterization of bacterial communities based on enriched cocoa samples revealed a predominance of members of theBacillaceae,Pseudomonadaceae, andEnterococcaceae. Eleven species of ThrS were found, butBacillus licheniformisand theBacillus subtiliscomplex were prominent and revealed great genetic heterogeneity. We concluded that the microbiota of cocoa powder resulted from microorganisms that could have been initially present in the nibs, as well as microorganisms that originated during processing.B. subtiliscomplex members, particularlyB. subtilissubsp.subtilis, formed the most heat-resistant spores. Their occurrence in cocoa powder needs to be considered to ensure the stability of derived products, such as ultrahigh-temperature-treated chocolate drinks.

scholarly journals Multilocus sequence analysis of the family Halomonadaceae

2012 ◽  
Vol 62 (Pt_3) ◽  
pp. 520-538 ◽  
Author(s):  
Rafael R. de la Haba ◽  
M. Carmen Márquez ◽  
R. Thane Papke ◽  
Antonio Ventosa
Keyword(s):  
Phylogenetic Analysis ◽  
Sequence Analysis ◽  
16S Rrna ◽  
Halophilic Bacteria ◽  
Multilocus Sequence Analysis ◽  
23S Rrna ◽  
Rrna Gene ◽  
The Family ◽  
Micro Organisms ◽  
The Impact

Multilocus sequence analysis (MLSA) protocols have been developed for species circumscription for many taxa. However, at present, no studies based on MLSA have been performed within any moderately halophilic bacterial group. To test the usefulness of MLSA with these kinds of micro-organisms, the family Halomonadaceae, which includes mainly halophilic bacteria, was chosen as a model. This family comprises ten genera with validly published names and 85 species of environmental, biotechnological and clinical interest. In some cases, the phylogenetic relationships between members of this family, based on 16S rRNA gene sequence comparisons, are not clear and a deep phylogenetic analysis using several housekeeping genes seemed appropriate. Here, MLSA was applied using the 16S rRNA, 23S rRNA, atpA, gyrB, rpoD and secA genes for species of the family Halomonadaceae. Phylogenetic trees based on the individual and concatenated gene sequences revealed that the family Halomonadaceae formed a monophyletic group of micro-organisms within the order Oceanospirillales. With the exception of the genera Halomonas and Modicisalibacter, all other genera within this family were phylogenetically coherent. Five of the six studied genes (16S rRNA, 23S rRNA, gyrB, rpoD and secA) showed a consistent evolutionary history. However, the results obtained with the atpA gene were different; thus, this gene may not be considered useful as an individual gene phylogenetic marker within this family. The phylogenetic methods produced variable results, with those generated from the maximum-likelihood and neighbour-joining algorithms being more similar than those obtained by maximum-parsimony methods. Horizontal gene transfer (HGT) plays an important evolutionary role in the family Halomonadaceae; however, the impact of recombination events in the phylogenetic analysis was minimized by concatenating the six loci, which agreed with the current taxonomic scheme for this family. Finally, the findings of this study also indicated that the 16S rRNA, gyrB and rpoD genes were the most suitable genes for future taxonomic studies using MLSA within the family Halomonadaceae.

Sign in / Sign up

Export Citation Format

Share Document