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Geochip: A high throughput genomic tool for linking community structure to functions

Description: GeoChip is a comprehensive functional gene array that targets key functional genes involved in the geochemical cycling of N, C, and P, sulfate reduction, metal resistance and reduction, and contaminant degradation. Studies have shown the GeoChip to be a sensitive, specific, and high-throughput tool for microbial community analysis that has the power to link geochemical processes with microbial community structure. However, several challenges remain regarding the development and applications of microarrays for microbial community analysis.
Date: January 30, 2009
Creator: Van Nostrand, Joy D.; Liang, Yuting; He, Zhili; Li, Guanghe & Zhou, Jizhong
Partner: UNT Libraries Government Documents Department

GeoChips for Analysis of Microbial Functional Communities

Description: Functional gene arrays (FGA) are microarrays that contain probes for genes encoding proteins or enzymes involved in functions of interest and allow for the study of thousands of genes at one time. The most comprehensive FGA to date is the GeoChip, which contains ~;;24,000 probes for ~;;10,000 genes involved in the geochemical cycling of C, N, P, and S, as well as genes involved in metal resistance and reduction and contaminant degradation. This chapter details the methods necessary for GeoChip analysis. Methods covered include preparation of DNA (whole community genome amplification and labeling), array setup (prehybridization steps), hybridization (sample and hybridization buffers), and post hybridization steps (slide washing and array scanning).
Date: September 30, 2008
Creator: Van Nostrand, Joy D.; Wu, Liyou; He, Zhili & Zhou, Jizhong
Partner: UNT Libraries Government Documents Department

Design and analysis of mismatch probes for long oligonucleotide microarrays

Description: Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.
Date: August 15, 2008
Creator: Deng, Ye; He, Zhili; Van Nostrand, Joy D. & Zhou, Jizhong
Partner: UNT Libraries Government Documents Department

Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

Description: Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.
Date: May 17, 2010
Creator: Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy & Zhou, Jizhong
Partner: UNT Libraries Government Documents Department

Alternations of Structure and Functional Activity of Below Ground Microbial Communities at Elevated Atmospheric Carbon Dioxide

Description: The global atmospheric concentration of CO2 has increased by more than 30percent since the industrial revolution. Although the stimulating effects of elevated CO2 (eCO2) on plant growth and primary productivity have been well studied, its influences on belowground microbial communities are poorly understood and controversial. In this study, we showed a significant change in the structure and functional potential of soil microbial communities at eCO2 in a grassland ecosystem, the BioCON (Biodiversity, CO2 and Nitrogen) experimental site (http://www.biocon.umn.edu/) using a comprehensive functional gene array, GeoChip 3.0, which contains about 28,0000 probes and covers approximately 57,000 gene variants from 292 functional gene families involved in carbon, nitrogen, phosphorus and sulfur cycles as well as other functional processes. GeoChip data indicated that the functional structure of microbial communities was markedly different between ambient CO2 (aCO2) and eCO2 by detrended correspondence analysis (DCA) of all 5001 detected functional gene probes although no significant differences were detected in the overall microbial diversity. A further analysis of 1503 detected functional genes involved in C, N, P, and S cycles showed that a considerable portion (39percent) of them were only detected under either aCO2 (14percent) or eCO2 (25percent), indicating that the functional characteristics of the microbial community were significantly altered by eCO2. Also, for those shared genes (61percent) detected, some significantly (p<0.05) changed their abundance at eCO2. Especially, genes involved in labile C degradation, such as amyA, egl, and ara for starch, cellulose, and hemicelluloses, respectively, C fixation (e.g., rbcL, pcc/acc), N fixation (nifH), and phosphorus utilization (ppx) were significantly increased under eCO2, while those involved in decomposing recalcitrant C, such as glx, lip, and mnp for lignin degradation remained unchanged. This study provides insights into our understanding of belowground microbial communities and their feedbacks to terrestrial ecosystems at eCO2.
Date: May 17, 2010
Creator: He, Zhili; Xu, Meiying; Deng, Ye; Kang, Sanghoon; Wu, Liyou; Van Nostrand, Joy D. et al.
Partner: UNT Libraries Government Documents Department

Prokaryotic diversity, distribution, and insights into their role in biogeochemical cycling in marine basalts

Description: We used molecular techniques to analyze basalts of varying ages that were collected from the East Pacific Rise, 9 oN, from the rift axis of the Juan de Fuca Ridge, and from neighboring seamounts. Cluster analysis of 16S rDNA Terminal Restriction Fragment Polymorphism data revealed that basalt endoliths are distinct from seawater and that communities clustered, to some degree, based on the age of the host rock. This age-based clustering suggests that alteration processes may affect community structure. Cloning and sequencing of bacterial and archaeal 16S rRNA genes revealed twelve different phyla and sub-phyla associated with basalts. These include the Gemmatimonadetes, Nitrospirae, the candidate phylum SBR1093 in the c, andin the Archaea Marine Benthic Group B, none of which have been previously reported in basalts. We delineated novel ocean crust clades in the gamma-Proteobacteria, Planctomycetes, and Actinobacteria that are composed entirely of basalt associated microflora, and may represent basalt ecotypes. Finally, microarray analysis of functional genes in basalt revealed that genes coding for previously unreported processes such as carbon fixation, methane-oxidation, methanogenesis, and nitrogen fixation are present, suggesting that basalts harbor previously unrecognized metabolic diversity. These novel processes could exert a profound influence on ocean chemistry.
Date: September 30, 2008
Creator: Mason, Olivia U.; Di Meo-Savoie, Carol A.; Van Nostrand, Joy D.; Zhou, Jizhong; Fisk, Martin R. & Giovannoni, Stephen J.
Partner: UNT Libraries Government Documents Department

Taxa-area Relationship (TAR) of Microbial Functional Genes with Long-TGerm Fertilization

Description: Diversity and spatial patterns in plant and animal communities are well documented as a positive-power law of a taxa-area relationship (TAR). At present little is known whether this also applies to soil microbial communities and whether long-term fertilization has an influence on the underlying microbial diversity. To test the effects of long-term fertilization on above-ground botanical diversity and below-ground microbial diversity, a nested sampling approach on Park Grass plots (12d&amp; 11/2c) of Rothamsted Reseach in United Kingdom, both at ~;; pH 5 but with plant diversities of between 42 and 13 respectively were used. GeoChip 3.0, covering approximately 57, 000 gene sequences of 292 gene families involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance, and organic contaminant degradation, was used to determine the gene area relationships for both functional and phylogenetic groups and the relationship to plant diversity. Our analysis indicated that the microbial communities were separated by different plant diversity based on DCA. The soil microbial diversity was in accord with plant diversity. Soil microbial community exhibited different z value with different plant diversity, z = 0.0449 with higher plant diversity and z = 0.0583 with lower plant diversity (P&lt; 0.0001). These results suggest that the turnover in space of microorganisms may be higher with long-term fertilization.
Date: May 17, 2010
Creator: Liang, Yuting; Wu, Liyou; Clark, Ian; Xue, Kai; Van Nostrand, Joy D.; Deng, Ye et al.
Partner: UNT Libraries Government Documents Department

Microarray-based analysis of survival of soil microbial community during ozonation

Description: 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 ozonation 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 ...
Date: May 17, 2010
Creator: Wang, Jian; Van Nostrand, Joy D.; He, Zhili; Wu, Liyou; Deng, Ye; Zhang, Xu et al.
Partner: UNT Libraries Government Documents Department

Effects of Nitrate Exposure on the Functional Structure of a Microbial Community in a Uranium-contaminated Aquifer

Description: Increasing nitrogen deposition, increasing atmospheric CO2, and decreasing biodiversity are three main environmental changes occurring on a global scale. The BioCON (Biodiversity, CO2, and Nitrogen) ecological experiment site at the University of Minnesota's Cedar Creek Ecosystem Science Reserve started in 1997, to better understand how these changes would affect soil systems. To understand how increasing nitrogen deposition affects the microbial community diversity, heterogeneity, and functional structure impact soil microbial communities, 12 samples were collected from the BioCON plots in which nitrogenous fertilizer was added to simulate the effect of increasing nitrogen deposition and 12 samples from without added fertilizer. DNA from the 24 samples was extracted using a freeze-grind protocol, amplified, labeled with a fluorescent dye, and then hybridized to GeoChip, a functional gene array containing probes for genes involved in N, S and C cycling, metal resistance and organic contaminant degradation. Detrended correspondence analysis (DCA) of all genes detected was performed to analyze microbial community patterns. The first two axes accounted for 23.5percent of the total variation. The samples fell into two major groups: fertilized and non-fertilized, suggesting that nitrogenous fertilizer had a significant impact on soil microbial community structure and diversity. The functional gene numbers detected in fertilized samples was less that detected in non-fertilizer samples. Functional genes involving in the N cycling were mainly discussed.
Date: May 17, 2010
Creator: Van Nostrand, Joy; Waldron, P.; Wu, W.; Zhou, B.; Wu, Liyou; Deng, Ye et al.
Partner: UNT Libraries Government Documents Department

Microbial community changes during sustained Cr(VI) reduction at the 100H site in Hanford, WA

Description: Hexavalent Chromium is a widespread contaminant found in soil, sediment, and groundwater. In order to stimulate microbially-mediated reduction of Cr(VI), a poly-lactate compound (HRC) was injected into the Chromium-contaminated aquifer at the Hanford (WA) 100H site in 2004. Cr(VI) concentrations rapidly declined to below the detection limit and remained so for more than three years after injection. Based on the results of the bacterial community composition using high-density DNA 16S rRNA gene microarrays, we observed the community to transition through denitrifying, ironreducing and sulfate-reducing populations. As a result, we specifically focused isolation efforts on three bacterial species that were significant components of the community. Positive enrichments in defined anaerobic media resulted in the isolation of an iron-reducing Geobacter metallireducens-like isolate, a sulfate-reducing Desulfovibrio vukgaris-like strain and a nitrate-reducing Pseudomonas stutzeri-like isolate among several others. All of these isolates were capable of reducing Cr(VI) anoxically and have been submitted for genome sequencing to JGI. To further characterize the microbial, and geochemical mechanisms associated with in situ Cr(VI) reduction at the site, additional HRC was injected in 2008. The goal was to restimulate the indigenous microbial community and to regenerate the reducing conditions necessary for continued Cr(VI) bio-immobilization in the groundwater. Analysis of the microbial populations post-injection revealed that they recovered to a similar density as after the first injection in 2004. In this study, we present the results from our investigation into microbially-mediated Cr(VI) reduction at Hanford, and a comparison of the microbial community development following two HRC injections four years apart.
Date: May 17, 2010
Creator: Chakraborty, Romy; Brodie, Eoin L; Faybishenko, Boris; Piceno, Yvette M; Tom, Lauren; Choudhuri, Swati et al.
Partner: UNT Libraries Government Documents Department