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Title: Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA)

Abstract

A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis {Delta}fur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwatermore » system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion.« less

Authors:
 [1];  [2];  [3];  [4];  [2];  [4]
  1. University of Oklahoma
  2. ORNL
  3. Texas A&M University
  4. University of Oklahoma, Norman
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
931177
DOE Contract Number:
DE-AC05-00OR22725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Applied and Environmental Microbiology; Journal Volume: 73; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; ABUNDANCE; AMPLIFICATION; BIOREACTORS; COMMUNITIES; DENITRIFICATION; DETECTION; EVALUATION; FUNCTIONALS; GENES; MIXTURES; NUCLEOTIDES; RNA; STRAINS

Citation Formats

Gao, Haichun, Yang, Zamin Koo, Gentry, Terry, Wu, Liyou, Schadt, Christopher Warren, and Zhou, Jizhong. Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA). United States: N. p., 2007. Web. doi:10.1128/AEM.01771-06.
Gao, Haichun, Yang, Zamin Koo, Gentry, Terry, Wu, Liyou, Schadt, Christopher Warren, & Zhou, Jizhong. Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA). United States. doi:10.1128/AEM.01771-06.
Gao, Haichun, Yang, Zamin Koo, Gentry, Terry, Wu, Liyou, Schadt, Christopher Warren, and Zhou, Jizhong. Mon . "Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA)". United States. doi:10.1128/AEM.01771-06.
@article{osti_931177,
title = {Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA)},
author = {Gao, Haichun and Yang, Zamin Koo and Gentry, Terry and Wu, Liyou and Schadt, Christopher Warren and Zhou, Jizhong},
abstractNote = {A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis {Delta}fur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion.},
doi = {10.1128/AEM.01771-06},
journal = {Applied and Environmental Microbiology},
number = 2,
volume = 73,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • Microarray technology provides the opportunity to identifythousands of microbial genes or populations simultaneously, but lowmicrobial biomass often prevents application of this technology to manynatural microbial communities. We developed a whole-community genomeamplification-assisted microarray detection approach basedon multipledisplacement amplification. The representativeness of amplification wasevaluatedusing several types of microarrays and quantitative indexes.Representative detection of individual genes or genomes was obtained with1 to 100 ng DNA from individual or mixed genomes of equal or unequalabundance and with 1 to 500 ng community DNAs from groundwater. Lowerconcentrations of DNA (as low as 10 fg) could be detected, but the lowertemplate concentrations affected the representativeness ofmore » amplification.Robustquantitative detection was also observed by significant linearrelationships between signal intensities and initial DNA concentrationsranging from (i) 0.04 to 125 ng (r2 = 0.65 to 0.99) for DNA from purecultures as detected by whole-genome open reading frame arrays, (ii) 0.1to 1,000 ng (r2 = 0.91) for genomic DNA using community genome arrays,and (iii) 0.01 to 250 ng (r2 = 0.96 to 0.98) for community DNAs fromethanol amended groundwater using 50-mer functional gene arrays. Thismethod allowed us to investigate the oligotrophic microbial communitiesin groundwater contaminated with uranium and other metals. The resultsindicated that microorganisms containing genes involved in contaminantdegradation and immobilization are present in these communities, thattheir spatial distribution is heterogeneous, and that microbial diversityis greatly reduced in the highly contaminated environment.« less
  • A 15 h ozonation was performed on bioremediated soil to remove recalcitrant residual oil. To monitor the survival of indigenous microorganisms in the soil during in-situ chemical oxidation(ISCO) culturing and a functional genearray, GeoChip, was used to examine the functional genes and structure of the microbial community during ozonation (0h, 2h, 4h, 6h, 10hand15h). Breakthrough ozonation decreased the population of cultivable heterotrophic bacteria by about 3 orders of magnitude. The total functional gene abundance and diversity decreased during ozonation, as the number of functional genes was reduced by 48percent after 15 h. However, functional genes were evenly distributed during ozonationmore » as judged by the Shannon-Weaver Evenness index. A sharp decrease in gene number was observed in the first 6 h of ozonation followed by a slower decrease in the next 9 h, which was consistent with microbial populations measured by a culture based method. Functional genes involved in carbon, nitrogen, phosphors and sulfur cycling, metal resistance and organic remediation were detected in all samples. Though the pattern of gene categories detected was similar for all time points, hierarchica lcluster of all functional genes and major functional categories all showed a time-serial pattern. Bacteria, archaea and fungi decreased by 96.1percent, 95.1percent and 91.3percent, respectively, after 15 h ozonation. Delta proteobacteria, which were reduced by 94.3percent, showed the highest resistance to ozonation while Actinobacteria, reduced by 96.3percent, showed the lowest resistance. Microorganisms similar to Rhodothermus, Obesumbacterium, Staphylothermus, Gluconobacter, and Enterococcus were dominant at all time points. Functional genes related to petroleum degradation decreased 1~;;2 orders of magnitude. Most of the key functional genes were still detected after ozonation, allowing a rapid recovery of the microbial community after ozonation. While ozone had a large impact on the indigenous soil microorganisms, a fraction of the key functional gene-containing microorganisms survived during ozonation and kept the community functional.« less