scholarly journals Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples

2012 ◽  
Vol 78 (12) ◽  
pp. 4481-4489 ◽  
Author(s):  
Robert Brankatschk ◽  
Natacha Bodenhausen ◽  
Josef Zeyer ◽  
Helmut Bürgmann
Keyword(s):  
Microbial Community ◽  
Quantitative Pcr ◽  
Environmental Samples ◽  
Absolute Quantification ◽  
Geobacter Sulfurreducens ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gene Copy ◽  
Nostoc Commune ◽  
Content Type

ABSTRACTReal-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting thenifHand 16S rRNA genes. TheEvalues of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in theirEvalues, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences inEand was derived from the ΔΔCTmethod with correction forE, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures fromGeobacter sulfurreducens(DSM 12127) andNostoc commune(Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in theirEvalues. While the SC method deviated from the expectednifHgene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences inEbetween the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.

scholarly journals Defining the Core Citrus Leaf- and Root-Associated Microbiota: Factors Associated with Community Structure and Implications for Managing Huanglongbing (Citrus Greening) Disease

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Ryan A. Blaustein ◽  
Graciela L. Lorca ◽  
Julie L. Meyer ◽  
Claudio F. Gonzalez ◽  
Max Teplitski
Keyword(s):  
Community Structure ◽  
Microbial Community ◽  
Microbial Communities ◽  
Illumina Sequencing ◽  
Symptom Severity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
The Core ◽  
Symptom Progression

ABSTRACTStable associations between plants and microbes are critical to promoting host health and productivity. The objective of this work was to test the hypothesis that restructuring of the core microbiota may be associated with the progression of huanglongbing (HLB), the devastating citrus disease caused byLiberibacter asiaticus,Liberibacter americanus, andLiberibacter africanus. The microbial communities of leaves (n= 94) and roots (n= 79) from citrus trees that varied by HLB symptom severity, cultivar, location, and season/time were characterized with Illumina sequencing of 16S rRNA genes. The taxonomically rich communities contained abundant core members (i.e., detected in at least 95% of the respective leaf or root samples), some overrepresented site-specific members, and a diverse community of low-abundance variable taxa. The composition and diversity of the leaf and root microbiota were strongly associated with HLB symptom severity and location; there was also an association with host cultivar. The relative abundance ofLiberibacterspp. among leaf microbiota positively correlated with HLB symptom severity and negatively correlated with alpha diversity, suggesting that community diversity decreases as symptoms progress. Network analysis of the microbial community time series identified a mutually exclusive relationship betweenLiberibacterspp. and members of theBurkholderiaceae,Micromonosporaceae, andXanthomonadaceae. This work confirmed several previously described plant disease-associated bacteria, as well as identified new potential implications for biological control. Our findings advance the understanding of (i) plant microbiota selection across multiple variables and (ii) changes in (core) community structure that may be a precondition to disease establishment and/or may be associated with symptom progression.IMPORTANCEThis study provides a comprehensive overview of the core microbial community within the microbiomes of plant hosts that vary in extent of disease symptom progression. With 16S Illumina sequencing analyses, we not only confirmed previously described bacterial associations with plant health (e.g., potentially beneficial bacteria) but also identified new associations and potential interactions between certain bacteria and an economically important phytopathogen. The importance of core taxa within broader plant-associated microbial communities is discussed.

scholarly journals Microbial Community Response during the Iron Fertilization Experiment LOHAFEX

2012 ◽  
Vol 78 (24) ◽  
pp. 8803-8812 ◽  
Author(s):  
Stefan Thiele ◽  
Bernhard M. Fuchs ◽  
Nagappa Ramaiah ◽  
Rudolf Amann
Keyword(s):  
Microbial Community ◽  
Total Cell ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Thymidine Uptake ◽  
Phytoplankton Blooms ◽  
Minor Response ◽  
Content Type ◽  
Cell Numbers ◽  
Iron Fertilization

ABSTRACTIron fertilization experiments in high-nutrient, low-chlorophyll areas are known to induce phytoplankton blooms. However, little is known about the response of the microbial community upon iron fertilization. As part of the LOHAFEX experiment in the southern Atlantic Ocean,BacteriaandArchaeawere monitored within and outside an induced bloom, dominated byPhaeocystis-like nanoplankton, during the 38 days of the experiment. The microbial production increased 1.6-fold (thymidine uptake) and 2.1-fold (leucine uptake), while total cell numbers increased only slightly over the course of the experiment. 454 tag pyrosequencing of partial 16S rRNA genes and catalyzed reporter deposition fluorescencein situhybridization (CARD FISH) showed that the composition and abundance of the bacterial and archaeal community in the iron-fertilized water body were remarkably constant without development of typical bloom-related succession patterns. Members of groups usually found in phytoplankton blooms, such asRoseobacterandGammaproteobacteria, showed no response or only a minor response to the bloom. However, sequence numbers and total cell numbers of the SAR11 and SAR86 clades increased slightly but significantly toward the end of the experiment. It seems that although microbial productivity was enhanced within the fertilized area, a succession-like response of the microbial community upon the algal bloom was averted by highly effective grazing. Only small-celled members like the SAR11 and SAR86 clades could possibly escape the grazing pressure, explaining a net increase of those clades in numbers.

scholarly journals Soil Microbial Community Responses to Additions of Organic Carbon Substrates and Heavy Metals (Pb and Cr)

2005 ◽  
Vol 71 (12) ◽  
pp. 7679-7689 ◽  
Author(s):  
Cindy H. Nakatsu ◽  
Nadia Carmosini ◽  
Brett Baldwin ◽  
Federico Beasley ◽  
Peter Kourtev ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Microbial Community ◽  
Organic Carbon ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Carbon Sources ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gene Copy ◽  
Organic Substrates ◽  
Gradient Gel Electrophoresis

ABSTRACT Microcosm experiments were conducted with soils contaminated with heavy metals (Pb and Cr) and aromatic hydrocarbons to determine the effects of each upon microbial community structure and function. Organic substrates were added as a driving force for change in the microbial community. Glucose represented an energy source used by a broad variety of bacteria, whereas fewer soil species were expected to use xylene. The metal amendments were chosen to inhibit the acute rate of organic mineralization by either 50% or 90%, and lower mineralization rates persisted over the entire 31-day incubation period. Significant biomass increases were abolished when metals were added in addition to organic carbon. The addition of organic carbon alone had the most significant impact on community composition and led to the proliferation of a few dominant phylotypes, as detected by PCR-denaturing gradient gel electrophoresis of bacterial 16S rRNA genes. However, the community-wide effects of heavy metal addition differed between the two carbon sources. For glucose, either Pb or Cr produced large changes and replacement with new phylotypes. In contrast, many phylotypes selected by xylene treatment were retained when either metal was added. Members of the Actinomycetales were very prevalent in microcosms with xylene and Cr(VI); gene copy numbers of biphenyl dioxygenase and phenol hydroxylase (but not other oxygenases) were elevated in these microcosms, as determined by real-time PCR. Much lower metal concentrations were needed to inhibit the catabolism of xylene than of glucose. Cr(VI) appeared to be reduced during the 31-day incubations, but in the case of glucose there was substantial microbial activity when much of the Cr(VI) remained. In the case of xylene, this was less clear.

scholarly journals Correlation between Composition of the Bacterial Community and Concentration of Volatile Fatty Acids in the Rumen during the Transition Period and Ketosis in Dairy Cows

2012 ◽  
Vol 78 (7) ◽  
pp. 2386-2392 ◽  
Author(s):  
Xiaoxu Wang ◽  
Xiaobing Li ◽  
Chenxu Zhao ◽  
Pan Hu ◽  
Hui Chen ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Quantitative Pcr ◽  
Volatile Fatty Acids ◽  
Transition Period ◽  
Negative Energy ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Selenomonas Ruminantium ◽  
Megasphaera Elsdenii

ABSTRACTThe transition period is a severe challenge to dairy cows. Glucose supply cannot meet demand and body fat is mobilized, potentially leading to negative energy balance (NEB), ketosis, or fatty liver. Propionate produces glucose by gluconeogenesis, which depends heavily on the number and species of microbes. In the present study, we analyzed the rumen microbiome composition of cows in the transition period, cows with ketosis, and nonperinatal cows by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes and quantitative PCR. TRFLP analysis indicated that the quantity ofVeillonellaceaeorganisms was reduced and that ofStreptococcaceaeorganisms was increased in rumen samples from the transition period and ketosis groups, with the number ofLactobacillaceaeorganisms increased after calving. Quantitative PCR data suggested that the numbers of the main propionate-producing microbes,Megasphaera elsdeniiandSelenomonas ruminantium, were decreased, while numbers of the main lactate-producing bacterium,Streptococcus bovis, were increased in the rumen of cows from the transition period and ketosis groups, with the number ofLactobacillussp. organisms increased after calving. Volatile fatty acid (VFA) and glucose concentrations were decreased, but the lactic acid concentration was increased, in rumen samples from the transition period and ketosis groups. Our results indicate that the VFA concentration is significantly related to the numbers ofSelenomonas ruminantiumandMegasphaera elsdeniiorganisms in the rumen.

scholarly journals Revisiting the Dilution Procedure Used To Manipulate Microbial Biodiversity in Terrestrial Systems

2015 ◽  
Vol 81 (13) ◽  
pp. 4246-4252 ◽  
Author(s):  
Yan Yan ◽  
Eiko E. Kuramae ◽  
Peter G. L. Klinkhamer ◽  
Johannes A. van Veen
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Terrestrial Ecosystems ◽  
Diversity Indices ◽  
Soil Samples ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Dilution Method ◽  
Soil Suspension ◽  
Content Type

ABSTRACTIt is hard to assess experimentally the importance of microbial diversity in soil for the functioning of terrestrial ecosystems. An approach that is often used to make such assessment is the so-called dilution method. This method is based on the assumption that the biodiversity of the microbial community is reduced after dilution of a soil suspension and that the reduced diversity persists after incubation of more or less diluted inocula in soil. However, little is known about how the communities develop in soil after inoculation. In this study, serial dilutions of a soil suspension were made and reinoculated into the original soil previously sterilized by gamma irradiation. We determined the structure of the microbial communities in the suspensions and in the inoculated soils using 454-pyrosequencing of 16S rRNA genes. Upon dilution, several diversity indices showed that, indeed, the diversity of the bacterial communities in the suspensions decreased dramatically, withProteobacteriaas the dominant phylum of bacteria detected in all dilutions. The structure of the microbial community was changed considerably in soil, withProteobacteria,Bacteroidetes, andVerrucomicrobiaas the dominant groups in most diluted samples, indicating the importance of soil-related mechanisms operating in the assembly of the communities. We found unique operational taxonomic units (OTUs) even in the highest dilution in both the suspensions and the incubated soil samples. We conclude that the dilution approach reduces the diversity of microbial communities in soil samples but that it does not allow accurate predictions of the community assemblage during incubation of (diluted) suspensions in soil.

scholarly journals Effect of Sodium Bisulfite Injection on the Microbial Community Composition in a Brackish-Water-Transporting Pipeline

2011 ◽  
Vol 77 (19) ◽  
pp. 6908-6917 ◽  
Author(s):  
Hyung Soo Park ◽  
Indranil Chatterjee ◽  
Xiaoli Dong ◽  
Sheng-Hung Wang ◽  
Christoph W. Sensen ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Microbial Community Composition ◽  
Sodium Bisulfite ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Iron Corrosion ◽  
Injection Point ◽  
Content Type ◽  
Sulfate Reducing

ABSTRACTPipelines transporting brackish subsurface water, used in the production of bitumen by steam-assisted gravity drainage, are subject to frequent corrosion failures despite the addition of the oxygen scavenger sodium bisulfite (SBS). Pyrosequencing of 16S rRNA genes was used to determine the microbial community composition for planktonic samples of transported water and for sessile samples of pipe-associated solids (PAS) scraped from pipeline cutouts representing corrosion failures. These were obtained from upstream (PAS-616P) and downstream (PAS-821TP and PAS-821LP, collected under rapid-flow and stagnant conditions, respectively) of the SBS injection point. Most transported water samples had a large fraction (1.8% to 97% of pyrosequencing reads) ofPseudomonasnot found in sessile pipe samples. The sessile population of PAS-616P had methanogens (Methanobacteriaceae) as the main (56%) community component, whereasDeltaproteobacteriaof the generaDesulfomicrobiumandDesulfocapsawere not detected. In contrast, PAS-821TP and PAS-821LP had lower fractions (41% and 0.6%) ofMethanobacteriaceaearchaea but increased fractions of sulfate-reducingDesulfomicrobium(18% and 48%) and of bisulfite-disproportionatingDesulfocapsa(35% and 22%) bacteria. Hence, SBS injection strongly changed the sessile microbial community populations. X-ray diffraction analysis of pipeline scale indicated that iron carbonate was present both upstream and downstream, whereas iron sulfide and sulfur were found only downstream of the SBS injection point, suggesting a contribution of the bisulfite-disproportionating and sulfate-reducing bacteria in the scale to iron corrosion. Incubation of iron coupons with pipeline waters indicated iron corrosion coupled to the formation of methane. Hence, both methanogenic and sulfidogenic microbial communities contributed to corrosion of pipelines transporting these brackish waters.

scholarly journals Deterministic Assembly of Complex Bacterial Communities in Guts of Germ-Free Cockroaches

2015 ◽  
Vol 82 (4) ◽  
pp. 1256-1263 ◽  
Author(s):  
Aram Mikaelyan ◽  
Claire L. Thompson ◽  
Markus J. Hofer ◽  
Andreas Brune
Keyword(s):  
Microbial Community ◽  
Gut Microbiota ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Experimental Proof ◽  
Content Type ◽  
Germ Free ◽  
Microbe Interactions ◽  
Host Microbe Interactions ◽  
Axenic Conditions

ABSTRACTThe gut microbiota of termites plays important roles in the symbiotic digestion of lignocellulose. However, the factors shaping the microbial community structure remain poorly understood. Because termites cannot be raised under axenic conditions, we established the closely related cockroachShelfordella lateralisas a germ-free model to study microbial community assembly and host-microbe interactions. In this study, we determined the composition of the bacterial assemblages in cockroaches inoculated with the gut microbiota of termites and mice using pyrosequencing analysis of their 16S rRNA genes. Although the composition of the xenobiotic communities was influenced by the lineages present in the foreign inocula, their structure resembled that of conventional cockroaches. Bacterial taxa abundant in conventional cockroaches but rare in the foreign inocula, such asDysgonomonasandParabacteroidesspp., were selectively enriched in the xenobiotic communities. Donor-specific taxa, such as endomicrobia or spirochete lineages restricted to the gut microbiota of termites, however, either were unable to colonize germ-free cockroaches or formed only small populations. The exposure of xenobiotic cockroaches to conventional adults restored their normal microbiota, which indicated that autochthonous lineages outcompete foreign ones. Our results provide experimental proof that the assembly of a complex gut microbiota in insects is deterministic.

scholarly journals Pyrosequencing Analysis of Bacterial Diversity in 14 Wastewater Treatment Systems in China

2012 ◽  
Vol 78 (19) ◽  
pp. 7042-7047 ◽  
Author(s):  
Xiaohui Wang ◽  
Man Hu ◽  
Yu Xia ◽  
Xianghua Wen ◽  
Kun Ding
Keyword(s):  
Wastewater Treatment ◽  
Microbial Community ◽  
Bacterial Community ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Microbial Populations ◽  
Operational Parameters ◽  
Geographic Locations ◽  
Content Type ◽  
Wastewater Treatment Systems

ABSTRACTTo determine if there is a core microbial community in the microbial populations of different wastewater treatment plants (WWTPs) and to investigate the effects of wastewater characteristics, operational parameters, and geographic locations on microbial communities, activated sludge samples were collected from 14 wastewater treatment systems located in 4 cities in China. High-throughput pyrosequencing was used to examine the 16S rRNA genes of bacteria in the wastewater treatment systems. Our results showed that there were 60 genera of bacterial populations commonly shared by all 14 samples, includingFerruginibacter,Prosthecobacter,Zoogloea, Subdivision 3 generaincertae sedis, Gp4, Gp6, etc., indicating that there is a core microbial community in the microbial populations of WWTPs at different geographic locations. The canonical correspondence analysis (CCA) results showed that the bacterial community variance correlated most strongly with water temperature, conductivity, pH, and dissolved oxygen (DO) content. Variance partitioning analyses suggested that wastewater characteristics had the greatest contribution to the bacterial community variance, explaining 25.7% of the variance of bacterial communities independently, followed by operational parameters (23.9%) and geographic location (14.7%). Results of this study provided insights into the bacterial community structure and diversity in geographically distributed WWTPs and discerned the relationships between bacterial community and environmental variables in WWTPs.

scholarly journals Defining the Functional Potential and Active Community Members of a Sediment Microbial Community in a High-Arctic Hypersaline Subzero Spring

2013 ◽  
Vol 79 (12) ◽  
pp. 3637-3648 ◽  
Author(s):  
Chih-Ying Lay ◽  
Nadia C. S. Mykytczuk ◽  
Étienne Yergeau ◽  
Guillaume Lamarche-Gagnon ◽  
Charles W. Greer ◽  
...  
Keyword(s):  
Microbial Community ◽  
Sulfur Metabolism ◽  
Close Association ◽  
High Arctic ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Metagenomic Data ◽  
Metabolic Potential ◽  
Data Set ◽  
Content Type

ABSTRACTThe Lost Hammer (LH) Spring is the coldest and saltiest terrestrial spring discovered to date and is characterized by perennial discharges at subzero temperatures (−5°C), hypersalinity (salinity, 24%), and reducing (≈−165 mV), microoxic, and oligotrophic conditions. It is rich in sulfates (10.0%, wt/wt), dissolved H2S/sulfides (up to 25 ppm), ammonia (≈381 μM), and methane (11.1 g day−1). To determine its total functional and genetic potential and to identify its active microbial components, we performed metagenomic analyses of the LH Spring outlet microbial community and pyrosequencing analyses of the cDNA of its 16S rRNA genes. Reads related toCyanobacteria(19.7%),Bacteroidetes(13.3%), andProteobacteria(6.6%) represented the dominant phyla identified among the classified sequences. Reconstruction of the enzyme pathways responsible for bacterial nitrification/denitrification/ammonification and sulfate reduction appeared nearly complete in the metagenomic data set. In the cDNA profile of the LH Spring active community, ammonia oxidizers (Thaumarchaeota), denitrifiers (Pseudomonasspp.), sulfate reducers (Desulfobulbusspp.), and other sulfur oxidizers (Thermoprotei) were present, highlighting their involvement in nitrogen and sulfur cycling. Stress response genes for adapting to cold, osmotic stress, and oxidative stress were also abundant in the metagenome. Comparison of the composition of the functional community of the LH Spring to metagenomes from other saline/subzero environments revealed a close association between the LH Spring and another Canadian high-Arctic permafrost environment, particularly in genes related to sulfur metabolism and dormancy. Overall, this study provides insights into the metabolic potential and the active microbial populations that exist in this hypersaline cryoenvironment and contributes to our understanding of microbial ecology in extreme environments.

scholarly journals Establishment of Bacterial Herbicide Degraders in a Rapid Sand Filter for Bioremediation of Phenoxypropionate-Polluted Groundwater

2015 ◽  
Vol 82 (3) ◽  
pp. 878-887 ◽  
Author(s):  
Louise Feld ◽  
Tue Kjærgaard Nielsen ◽  
Lars Hestbjerg Hansen ◽  
Jens Aamand ◽  
Christian Nyrop Albers
Keyword(s):  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gene Copy ◽  
Most Probable Number ◽  
Steady Increase ◽  
Propanoic Acid ◽  
Sand Filter ◽  
Probable Number ◽  
Content Type ◽  
Pcr Targeting

ABSTRACTIn this study, we investigated the establishment of natural bacterial degraders in a sand filter treating groundwater contaminated with the phenoxypropionate herbicides (RS)-2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) and (RS)-2-(2,4-dichlorophenoxy)propanoic acid (DCPP) and the associated impurity/catabolite 4-chlorophenoxypropanoic acid (4-CPP). A pilot facility was set up in a contaminated landfill site. Anaerobic groundwater was pumped up and passed through an aeration basin and subsequently through a rapid sand filter, which is characterized by a short residence time of the water in the filter. For 3 months, the degradation of DCPP, MCPP, and 4-CPP in the sand filter increased to 15 to 30% of the inlet concentration. A significant selection for natural bacterial herbicide degraders also occurred in the sand filter. Using a most-probable-number (MPN) method, we found a steady increase in the number of culturable phenoxypropionate degraders, reaching approximately 5 × 105degraders per g sand by the end of the study. Using a quantitative PCR targeting the two phenoxypropionate degradation genes,rdpAandsdpA, encoding stereospecific dioxygenases, a parallel increase was observed, but with the gene copy numbers being about 2 to 3 log units higher than the MPN. In general, thesdpAgene was more abundant than therdpAgene, and the establishment of a significant population of bacteria harboringsdpAoccurred faster than the establishment of anrdpAgene-carrying population. The identities of the specific herbicide degraders in the sand filter were assessed by Illumina MiSeq sequencing of 16S rRNA genes from sand filter samples and from selected MPN plate wells. We propose a list of potential degrader bacteria involved in herbicide degradation, including representatives belonging to theComamonadaceaeandSphingomonadales.

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