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HuMiChip: Development of a Functional Gene Array for the Study of Human Microbiomes

Description: Microbiomes play very important roles in terms of nutrition, health and disease by interacting with their hosts. Based on sequence data currently available in public domains, we have developed a functional gene array to monitor both organismal and functional gene profiles of normal microbiota in human and mouse hosts, and such an array is called human and mouse microbiota array, HMM-Chip. First, seed sequences were identified from KEGG databases, and used to construct a seed database (seedDB) containing 136 gene families in 19 metabolic pathways closely related to human and mouse microbiomes. Second, a mother database (motherDB) was constructed with 81 genomes of bacterial strains with 54 from gut and 27 from oral environments, and 16 metagenomes, and used for selection of genes and probe design. Gene prediction was performed by Glimmer3 for bacterial genomes, and by the Metagene program for metagenomes. In total, 228,240 and 801,599 genes were identified for bacterial genomes and metagenomes, respectively. Then the motherDB was searched against the seedDB using the HMMer program, and gene sequences in the motherDB that were highly homologous with seed sequences in the seedDB were used for probe design by the CommOligo software. Different degrees of specific probes, including gene-specific, inclusive and exclusive group-specific probes were selected. All candidate probes were checked against the motherDB and NCBI databases for specificity. Finally, 7,763 probes covering 91.2percent (12,601 out of 13,814) HMMer confirmed sequences from 75 bacterial genomes and 16 metagenomes were selected. This developed HMM-Chip is able to detect the diversity and abundance of functional genes, the gene expression of microbial communities, and potentially, the interactions of microorganisms and their hosts.
Date: May 17, 2010
Creator: Tu, Q.; Deng, Ye; Lin, Lu; Hemme, Chris L.; 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

Functional Ecological Gene Networks to Reveal the Changes Among Microbial Interactions Under Elevated Carbon Dioxide Conditions

Description: Biodiversity and its responses to environmental changes is a central issue in ecology, and for society. Almost all microbial biodiversity researches focus on species richness and abundance but ignore the interactions among different microbial species/populations. However, determining the interactions and their relationships to environmental changes in microbial communities is a grand challenge, primarily due to the lack of information on the network structure among different microbial species/populations. Here, a novel random matrix theory (RMT)-based conceptual framework for identifying functional ecological gene networks (fEGNs) is developed with the high throughput functional gene array hybridization data from the grassland microbial communities in a long-term FACE (Free Air CO2 Enrichment) experiment. Both fEGNs under elevated CO2 (eCO2) and ambient CO2 (aCO2) possessed general characteristics of many complex systems such as scale-free, small-world, modular and hierarchical. However, the topological structure of the fEGNs is distinctly different between eCO2 and aCO2, suggesting that eCO2 dramatically altered the interactions among different microbial functional groups/populations. In addition, the changes in network structure were significantly correlated with soil carbon and nitrogen dynamics, and plant productivity, indicating the potential importance of network interactions in ecosystem functioning. Elucidating network interactions in microbial communities and their responses to environmental changes are fundamentally important for research in microbial ecology, systems microbiology, and global change.
Date: May 17, 2010
Creator: Deng, Ye; Zhou, Jizhong; Luo, Feng; He, Zhili; Tu, Qichao & Zhi, Xiaoyang
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

Effects of experimental warming and clipping on metabolic change of microbial community in a US Great Plains tallgrass prairie

Description: While more and more studies are being conducted on the effects of global warming, little is known regarding the response of metabolic change of whole soil microbial communities to this phenomenon. In this study, functional gene changes at the mRNA level were analyzed by our new developed GeoChip 3.0. Soil samples were taken from a long-term climate warming experiment site, which has been conducted for ~;;8 years at the Kessler Farm Field Laboratory, a 137.6-ha farm located in the Central Redbed Plains, in McClain County, Oklahoma. The experiment uses a paired factorial design with warming as the primary factor nested with clipping as a secondary factor. An infrared heater was used to simulate global warming, and clipping was used to mimic mowing hay. Twelve 2m x 2m plots were divided into six pairs of warmed and control plots. The heater generates a constant output of ~;;100 Watts m-2 to approximately 2 oC increase in soil temperature above the ambient plots, which is at the low range of the projected climate warming by IPCC. Soil whole microbial communities? mRNA was extracted, amplified, labeled and hybridized with our GeoChip 3.0, a functional gene array covering genes involved in N, C, P, and S cycling, metal resistance and contaminant degradation, to examine expressed genes. The results showed that a greater number and higher diversity of genes were expressed under warmed plots compared to control. Detrended correspondence analysis (DCA) of all detected genes showed that the soil microbial communities were clearly altered by warming, with or without clipping. The dissimilarity of the communities based on functional genes was tested and results showed that warming and control communities were significantly different (P&lt;0.05), with or without clipping. Most genes involved in C, N, P and S cycling were expressed at higher levels in warming samples compared ...
Date: May 17, 2010
Creator: Xie, Jianping; Liu, Xinxing; Liu, Xueduan; Nostrand, Joy D. Van; Deng, Ye; Wu, Liyou et al.
Partner: UNT Libraries Government Documents Department

GeoChip-based Analysis of Groundwater Microbial Diversity in Norman Landfill

Description: The Norman Landfill is a closed municipal solid waste landfill located on an alluvium associated with the Canadian River in Norman, Oklahoma. It has operated as a research site since 1994 because it is typical of many closed landfill sites across the U.S. Leachate from the unlined landfill forms a groundwater plume that extends downgradient approximately 250 m from the landfill toward the Canadian River. To investigate the impact of the landfill leachate on the diversity and functional structure of microbial communities, groundwater samples were taken from eight monitoring wells at a depth of 5m, and analyzed using a comprehensive functional gene array covering about 50,000 genes involved in key microbial processes, such as biogeochemical cycling of C, N, P, and S, and bioremediation of organic contaminants and metals. Wells are located within a transect along a presumed flow path with different distances to the center of the leachate plume. Our analyses showed that microbial communities were obviously impacted by the leachate-component from the landfill. The number of genes detected and microbial diversity indices in the center (LF2B) and its closest (MLS35) wells were significantly less than those detected in other more downgradient wells, while no significant changes were observed in the relative abundance (i.e., percentage of each gene category) for most gene categories. However, the microbial community composition or structure of the landfill groundwater did not clearly show a significant correlation with the distance from well LF2B. Burkholderia sp. and Pseudomonas sp. were found to be the dominant microbial populations detected in all wells, while Bradyrhizobium sp. and Ralstonia sp. were dominant populations for seven wells except LF2B. In addition, Mantel test and canonical correspondence analysis (CCA) indicate that pH, sulfate, ammonia nitrogen and dissolved organic carbon (DOC) have significant effects on the microbial community structure. The results suggest ...
Date: May 17, 2010
Creator: Lu, Zhenmei; He, Zhili; Parisi, Victoria; Kang, Sanghoon; Deng, Ye; Nostrand, Joy Van et al.
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

GeoChip 3.0: A High Throughput Tool for Analyzing Microbial Community, Composition, Structure, and Functional Activity

Description: Microarray-based genomic technology has been widely used for microbial community analysis, and it is expected that microarray-based genomic technologies will revolutionize the analysis of microbial community structure, function and dynamics. A new generation of functional gene arrays (GeoChip 3.0) has been developed, with 27,812 probes covering 56,990 gene variants from 292 functional gene families involved in carbon, nitrogen, phosphorus and sulfur cycles, energy metabolism, antibiotic resistance, metal resistance, and organic contaminant degradation. Those probes were derived from 2,744, 140, and 262 species for bacteria, archaea, and fungi, respectively. GeoChip 3.0 has several other distinct features, such as a common oligo reference standard (CORS) for data normalization and comparison, a software package for data management and future updating, and the gyrB gene for phylogenetic analysis. Our computational evaluation of probe specificity indicated that all designed probes had a high specificity to their corresponding targets. Also, experimental analysis with synthesized oligonucleotides and genomic DNAs showed that only 0.0036percent-0.025percent false positive rates were observed, suggesting that the designed probes are highly specific under the experimental conditions examined. In addition, GeoChip 3.0 was applied to analyze soil microbial communities in a multifactor grassland ecosystem in Minnesota, USA, which demonstrated that the structure, composition, and potential activity of soil microbial communities significantly changed with the plant species diversity. All results indicate that GeoChip 3.0 is a high throughput powerful tool for studying microbial community functional structure, and linking microbial communities to ecosystem processes and functioning. To our knowledge, GeoChip 3.0 is the most comprehensive microarrays currently available for studying microbial communities associated with geobiochemical cycling, global climate change, bioenergy, agricuture, land use, ecosystem management, environmental cleanup and restoration, bioreactor systems, and human health.
Date: May 17, 2010
Creator: He, Zhili; Deng, Ye; Nostrand, Joy Van; Tu, Qichao; Xu, Meiying; Hemme, Chris 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

Comparative Metagenomics of Freshwater Microbial Communities

Description: Previous analyses of a microbial metagenome from uranium and nitric-acid contaminated groundwater (FW106) showed significant environmental effects resulting from the rapid introduction of multiple contaminants. Effects include a massive loss of species and strain biodiversity, accumulation of toxin resistant genes in the metagenome and lateral transfer of toxin resistance genes between community members. To better understand these results in an ecological context, a second metagenome from a pristine groundwater system located along the same geological strike was sequenced and analyzed (FW301). It is hypothesized that FW301 approximates the ancestral FW106 community based on phylogenetic profiles and common geological parameters; however, even if is not the case, the datasets still permit comparisons between healthy and stressed groundwater ecosystems. Complex carbohydrate metabolism has been almost entirely lost in the stressed ecosystem. In contrast, the pristine system encodes a wide diversity of complex carbohydrate metabolism systems, suggesting that carbon turnover is very rapid and less leaky in the healthy groundwater system. FW301 encodes many (~;;160+) carbon monoxide dehydrogenase genes while FW106 encodes none. This result suggests that the community is frequently exposed to oxygen from aerated rainwater percolating into the subsurface, with a resulting high rate of carbon metabolism and CO production. When oxygen levels fall, the CO then serves as a major carbon source for the community. FW301 appears to be capable of CO2 fixation via the reductive carboxylase (reverse TCA) cycle and possibly acetogenesis, activities; these activities are lacking in the heterotrophic FW106 system which relies exclusively on respiration of nitrate and/or oxygen for energy production. FW301 encodes a complete set of B12 biosynthesis pathway at high abundance suggesting the use of sodium gradients for energy production in the healthy groundwater community. Overall comparative analysis suggests that the introduction of contaminants is accompanied by a decrease in biodiversity, loss of nutrient ...
Date: May 17, 2010
Creator: Hemme, Chris; Deng, Ye; Tu, Qichao; Fields, Matthew; Gentry, Terry; Wu, Liyou et al.
Partner: UNT Libraries Government Documents Department

Metagenomic Insights into Evolution of a Heavy Metal-Contaminated Groundwater Microbial Community

Description: Understanding adaptation of biological communities to environmental change is a central issue in ecology and evolution. Metagenomic analysis of a stressed groundwater microbial community reveals that prolonged exposure to high concentrations of heavy metals, nitric acid and organic solvents (~;;50 years) have resulted in a massive decrease in species and allelic diversity as well as a significant loss of metabolic diversity. Although the surviving microbial community possesses all metabolic pathways necessary for survival and growth in such an extreme environment, its structure is very simple, primarily composed of clonal denitrifying ?- and ?-proteobacterial populations. The resulting community is over-abundant in key genes conferring resistance to specific stresses including nitrate, heavy metals and acetone. Evolutionary analysis indicates that lateral gene transfer could be a key mechanism in rapidly responding and adapting to environmental contamination. The results presented in this study have important implications in understanding, assessing and predicting the impacts of human-induced activities on microbial communities ranging from human health to agriculture to environmental management, and their responses to environmental changes.
Date: February 15, 2010
Creator: Hemme, Christopher L.; Deng, Ye; Gentry, Terry J.; Fields, Matthew W.; Wu, Liyou; Barua, Soumitra et al.
Partner: UNT Libraries Government Documents Department