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320 presentations were found
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Shutkin, Amy Scientific Research Project Management, 06/17/2008, Park Hyatt Philadelphia Hotel | Philadelphia, PA, The Pharmaceutical Strategy Series Fifth Annual Managing Projects and Resources: Strategies, Tools and Tactics for Effective Project Management, [ShutkinESPP2_PM_PhilCHI0608.pdf]The success of a mature scientific research program depends on maintaining an agile Performance Monitoring Project Management structure while implementing more formal risk management oversight practices as proposed new technologies are integrated into core research groups. Project Management offers the stability of a rational and logical process for managing work in both co-located and virtual research institutes. A Department of Energy Genomics:GTL sponsored research project initiated at three national laboratories and seven universities from coast to coast and is comprised of ~75 individuals collaborating within three matrixed core research groups: Applied Environmental Microbiology Core (AEMC), Functional Genomics and Imaging Core (FGIC) and Computational and Systems Biology Core (CSBC). We have found that there is a balance between tight project management to create synergy, focus and continuity to the project and well-tracked individual-investigator-driven initiatives and follow-up that must be struck to maintain creative engagement and productivity of the project team, all while remaining vigilant against scope creep. It has been our experience that frequent and rapid communication at different levels with different media is critical to exploiting the scale and diversity of the team project’s capabilities for this multidisciplinary, multi-institutional collaboration.
To learn more about the Virtual Institute of Microbial Stress and Survival (VIMSS) and the Environmental Stress Pathway Project (ESPP), please visit us online at: http://vimss.lbl.gov/ PresenterShutkin, Amy Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsModels |
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Hemme, C.; Deng, Y.; Gentry, T. J.; Wu, L.; Fields, M. W.; Green-Tringe, S.; Detter, J. C.; Barry, K.; Kyrpides, N.; Watson, D.; Richardson, P.; Hazen, T.C.; Tiedje, J. and Zhou, J. Comparative Metagenomics of Microbial Communities from Pristine and Contaminated Groundwater, 06/04/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [325_CHemmeASM2008.pdf]Microbial community DNA isolated from contaminated groundwater located at the US Dept. of Energy Field Research Center (FRC) in Oak Ridge, TN, was analyzed to determine the effects of chronic exposure to contaminants on microbial community structure. The sample was obtained from a site (FW106) experiencing long-term exposure to high levels of uranium and other heavy metals, nitric acid and organic solvents. Analysis indicates very low species diversity community (~13 OTU) dominated by denitrifying γ- and β-proteobacteria. Metabolic reconstruction of the dominant γ-proteobacterial species revealed adaptations for specific geochemical parameters including the following: denitrification pathways; pathways for degradation of organic compounds such as1,2-dichloroethene, acetone, butanol, methanol and formaldehyde; accumulation of multiple heavy metal efflux systems (czcABC, czcD, cadA-family, mer operon genes, etc.). Analysis indicates that lateral gene transfer is the predominant mechanism of introducing genetic variation in the community, resulting in the lateral acquisition of, for example, an acetone carboxylation pathway and heavy metal efflux systems. The sample was compared to a second groundwater metagenome from a pristine FRC site (FW301) to determine differences between the two communities. In contrast to the low species diversity of FW106, the FW301 is represented by multiple phyla including all 5 classes of proteobacteria, Planctomycetes, Chloriflexi, Actinobacteria, Acidobacteria, Bacteroidetes and Firmicutes. In contrast to the FW106 sample which resulted in significant read assembly, the FW301 sample is composed largely of single reads that do not assemble into contigs (95%). Abundance profiling of geochemical and cytochrome genes between FW106 and FW301 and between FW106 and the acid mine drainage (AMD) metagenome show distinct environmental signatures between the samples. This analysis verified the previous observation of accumulation of heavy metal other toxin efflux mechanisms in FW106 as well as an accumulation of specific c-type cytochromes in FW106 that may be important in heavy metal resistance. PresenterGentry, T. J. Funding SourceEnvironmental Stress Pathway Project (ESPP), ERSP (formerly known as NABIR) KeywordsBiogeochemistry, Environmental Genomics, Extremophiles, Field Studies, Functional Genomics, Metagenomics |
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Stolyar, S.; Pinel, N.; Walker, C. B.; Wall, J.; Stahl, D.A. The physiological role of the cytoplasmic hydrogenases in D. vulgaris Hildenborough, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [SStolyarASM2008.pdf]The Gram-negative Deltaproteobacterium D. vulgaris Hildenborough is able to grow with sulfate, sulfite and thiosulfate as electron acceptors and in their absence via fermentation or syntrophic association with hydrogenotrophic organisms. Despite decades of research, the mechanism of energy generation by D. vulgaris is not well understood. Genome sequence revealed genes for at list six different hydrogenases, four periplasmic and two cytoplasmic. Although some of them have been characterized, their roles in D. vulgaris remain obscure. We have examined the consequences of mutations in two cytoplasmic hydrogenases on respiratory and syntrophic growth: 1) echA (DVU0434), the first gene in the operon for the Ech type NiFe- containing hydrogenase and 2) cool (DVU2288), the third gene in the operon coding for the second cytoplasmic NiFe-containing hydrogenase Coo. Growth rate, cell yield, and metabolite production were characterized for three growth conditions: i) sulfate with lactate or pyruvate, ii) sulfate with acetate and hydrogen, and iii) in syntrophic association with a hydrogenotrophic methanogen. Hydrogen oxidation activities in soluble and membrane fractions of the mutants and a wild type were not significantly different. Although growth rates of both mutants on sulfate with pyruvate or lactate were comparable to the wild type, hydrogen evolution was much greater for the echA mutant. Growth of the echA mutant was severely impaired relative to the wild type or cooL mutant with sulfate and hydrogen/CO2, but this mutation had little affect on syntrophic growth on either lactate or pyruvate. Syntrophic growth of the cooL mutant was severely impaired on lactate but not on pyruvate. Based on these observations we concluded that the main role of the Ech hydrogenase is in hydrogen oxidation. The Coo hydrogenase likely directly coupled the oxidation of lactate to pyruvate by accepting electrons and reducing protons inside the cells. PresenterHillesland, Kristina Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsComparative Genomics, Environmental Genomics, Evolutionary Biology, Extremophiles, Sulfate Reducers |
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Materna, A.C.; Clarke, S.A.; Cruz, C.; Gao, X.; Alm, E.J. Natural Diversity and Experimental Evolution of Environmental Stress Tolerance in Marine Bacteria, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for MicrobiologyGenome sequencing has revealed extensive genetic variation within bacterial species and among co-existing bacteria. Using marine Vibrio strains as a model system, we investigate to what extent observed sequence diversity corresponds to measurable differences in salinity and temperature tolerance phenotypes, two ecologically important factors for this group of organisms. Using directed evolution, we quantify how malleable these phenotypes are with respect to a small number of mutation events. We have designed two-dimensional gradients in 24 cm square dishes containing solid growth medium to monitor temperature and salinity tolerances over a broad range of both factors. Growth patterns indicate the strain-specific minimum and maximum tolerances and interactions between the factors (salinity and temperature). We compared the specific boundaries of growth for multiple strains of Vibrio splendidus and V. alginolyticus. While the obtained profiles differ in their shape and limits, some consistent features appear. Tolerance to increasing salinities correlated positively with temperature tolerance. However, higher salinity constrained the limits of temperature tolerance, so that the maximum salinity tolerance occurred at intermediate temperatures. Similarly, growth at higher temperatures led to a tradeoff, limiting the range of salinity tolerance. Interestingly, at high salinities, low temperatures tended to suspend growth, leaving viable cells that could be regenerated when the temperature gradient was removed, while higher temperatures led to killing. In addition to comparing related environmental isolates, this method was further applied to study differences between parental and evolved strains. Serial application of 106 cells/cm2 to the solid medium gradients enabled selection for spontaneous, more tolerant mutants. In future work, this integrated ecological and experimental approach will be combined with genome re-sequencing to draw connections between genetic diversity and ecologically relevant phenotypes and tradeoffs. PresenterMaterna, A.C. Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsEnvironmental Genomics, Evolutionary Biology, Extremophiles, Models, Sequencing, Stress Response |
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Shapiro, B. Jesse; Alm, Eric J. A Novel Method to Detect Ancient Natural Selection at the Protein Level, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for MicrobiologyAdaptive changes in sequence and structure allow orthologous proteins to adopt novel or altered functions in different species. From sequence data alone, it remains a challenge to determine which proteins have undergone adaptation in different species, and which residues in the protein are responsible for the change. These challenges are exacerbated when comparing proteins from anciently diverged species, where synonymous nucleotide sites are often saturated with substitutions, rendering the often used dN/dS (nonsynonymous:synonymous substitution ratio) metric powerless to detect selected changes.
Here we describe a novel method to identify proteins with an excess of substitutions in conserved amino acid sites, relative to the neutral standard of substitutions in unconserved sites. The method works on the assumption that amino acid substitutions in conserved (slow-evolving) sites is unexpected, and might thus represent adaptive evolution. Conserved (slow-evolving; ‘S’) and unconserved (fast-evolving; ‘F’) site classes are assigned based on the most probable gamma-distributed rate category for each site in a multiple sequence alignment. The number of substitutions per site in each class (S or F) is then estimated, and species with an unexpectedly high S:F ratio (>1) are considered candidate targets of positive selection.
We computed the S:F ratio for ~900 protein families in 30 species of gammaproteobacteria. The distribution of S:F ratios (across all genes) in each branch of the phylogeny was compared to the distribution over all branches, revealing several branches with significantly higher S:F ratios. This suggests that some lineages (e.g. the Buchnera clade of insect endosymbionts) may have relaxed purifying selection on all loci, due to their low effective population size, while others - especially some of the ancestral branches connecting major ecologically-differentiated groups - may actually reflect excess positive selection on that branch. We also found that the set of genes with high S:F within a species or branch is often enriched in specific cellular functions, providing information about possible phenotypic consequences of substitutions in slow sites.
PresenterShapiro, B. Jesse Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsEvolutionary Biology, Proteomics |
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Keller, K. L.; Bender, K. S.; Wall, J. D. The Development of an In-frame Deletion System in Desulfovibrio vulgaris Hildenborough , 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [KKellerASM2008.pdf]In recent years, genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress; however, the current method of deletion construction via marker exchange mutagenesis does not allow for easy selection of multiple sequential gene deletions because of the low number of selectable markers now available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation of D. vulgaris, an in-frame markerless deletion system is being developed based on the upp-encoded uracil phosphoribosyltransferase as an element for a counterselection strategy. In wild-type D. vulgaris, growth is inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene is resistant to 5-FU. The introduction of a plasmid containing the wild-type upp gene expressed constitutively from the aph(5’)-III promoter (the promoter for the kanamycin resistance gene in Tn5) into the upp deletion strain restored sensitivity to 5-FU. This observation is the basis for the establishment of a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion does not contain an antibiotic cassette, multiple gene deletions can be generated in a single strain using this method. To construct such a markerless deletion for the R-subunit (DVU1703) of a type I restriction-modification system, Gateway Technology methods (Invitrogen) are being used. A destination vector containing the constitutively expressed wild-type upp gene has been constructed and is available for generating deletion vectors. Its use is reported here. PresenterKeller, K. L. Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsFunctional Genomics, Sulfate Reducers |
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Bender, Kelly S.; Burns, Andrew S.; Wall, Judy D. Identification of a Small Non-Coding RNA in Desulfovibrio vulgaris , 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [KBenderASM2008.pdf]Identification and analysis of small non-coding RNAs (sRNAs) in D. vulgaris and other metal-reducing bacteria are essential for uncovering novel regulatory mechanisms involved in processes critical to the DOE such as stress response, environmental adaptation, and contaminant remediation. Previous work by the ‘Environmental Stress Pathway Project’ has enhanced our systems knowledge of D. vulgaris via valuable transcriptional and proteomic profiles, however regulation by sRNAs could not be detected in those experiments. In an effort to identify sRNAs in D. vulgaris, a strategy for cloning total RNA ranging in size from 20-200 nt was employed. Following addition of directional aptamer sequences, cDNAs were produced and cloned for sequencing. Sequence analysis of a small portion of the resulting cDNA library yielded two identical ~65 nt sRNA clones (Dv-sRNA2) possessing complementary sequence to the RBS of open reading frame (ORF) DVU0678. While DVU0678 is adjacent to the Dv-sRNA2 gene, the ORF is transcribed from the opposite chromosomal strand. Northern analysis specialized for sRNAs verified the expression of Dv-sRNA2 as an individual transcript under anaerobic lactate/sulfate growth (LS4D medium). These data suggest that when Dv-sRNA2 is transcribed, translation of DVU0678 will be inhibited. DVU0678 has been annotated to encode a putative 34 amino acid protein unique to D. vulgaris strains Hildenborough and DP4, hampering our abilities to discern the role of DVU0678 in the cell. Further sequence analysis of the Dv-sRNA DNA locus by ‘PromScan’ identified a putative sigma54-recognition site (97% probability) 43 nt upstream of the predicted sRNA transcriptional start site and therefore suggests that Dv-sRNA2 may be member of the sigma54 regulon. A perfect stem-loop terminator was also identified 26 nt downstream of the Dv-sRNA2 DNA sequence. Current analysis is underway to ascertain the expression profile for this sRNA as well as the effect over-expression has on the physiology and transcriptional response of D. vulgaris under multiple environmental conditions. PresenterBender, Kelly S. Funding SourceEnvironmental Stress Pathway Project (ESPP), Protein Complex Analysis Project (PCAP) KeywordsFunctional Genomics, Sequencing, Sulfate Reducers |
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Chivian, Dylan; Dehal, Paramvir S.; Arkin, Adam P. MicroCOSM: Phylogenetic Classification of Metagenomic Data Using Microbial Clade-oriented Sequence Markers Microbial Clade-oriented Sequence Markers, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [298_DChivian.pdf]Metagenomics projects that seek to elucidate the population structure of microbial communities are faced with the related computational challenges of classifying the sequences obtained and quantifying which organisms are present within a sample. Individually low-proportion species usually make up a large fraction of microbial communities, complicating their detection using traditional phylogenetic marker approaches. Such species usually don't yield sufficient read depth to assemble into longer sequences, leaving fragments that rarely contain traditional markers such as the small subunit (SSU) rRNA gene. BLAST-based approaches for analysis of metagenomic sequences [1] compensate for this rarity of traditional markers, but may be confounded by genes that are subject to horizontal transfer or duplication. Another approach instead makes use only of reliable non-transferred single-copy genes [2] to classify and quantify the organisms present within a sample, but the application has so far been limited to the use of a fairly small set of universal genes found in all organisms. In this work, we have extended the latter approach, boosting the set of reliable marker genes from only about 30-40 universal genes to several hundred by identifying sets of single-copy genes that are not subject to inter-clade horizontal transfer through investigation of finished bacterial and archaeal genomes. These clade-oriented sequence markers allow for a method, which we have named “MicroCOSM”, that greatly increases the probability that a marker will be found in any given sequence and therefore offers improved coverage for phylogenetic classification and quantification of microbial types in an environmental sample. This approach will aid our understanding of the microbial community population structure at contaminated field sites for the VIMSS/ESPP2 project. PresenterChivian, Dylan Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsEnvironmental Genomics, Metagenomics, Sequencing |
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Novichkov, P., Cipriano, Michael J.; Kazakov, Alexei E.; Ravcheev, Dmitry; Arkin, Adam P.; Gelfand, Mikhail S.; Dubchak, Inna The analysis and expansion of regulatory binding site data in a wide range of bacteria using a semi-automatic system - RegTransBase, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [PNovichkovASM2008.pdf]RegTransBase, a database describing regulatory interactions in prokaryotes, has been developed as a component of the MicrobesOnline/RegTransBase framework successfully used for interpretation of microbial stress response and metal reduction pathways. It is manually curated and based on published scientific literature. RegTransBase describes a large number of regulatory interactions and contains experimental data which investigates regulation with known elements. It is available at http://regtransbase.lbl.gov. Currently, the database content is derived from more than 4000 relevant articles describing over 9000 experiments in relation to 155 microbes. It contains data on the regulation of ~14000 genes and evidence for ~7500 interactions with ~850 regulators.
RegTransBase additionally provides an expertly curated library of alignments of known transcription factor binding sites covering a wide range of bacterial species. Each alignment contains information as to the transcription factor which binds the DNA sequence, the exact location of the binding site on a published genome, and links to published articles.
RegTransBase builds upon these alignments by containing a set of computational modules for the comparative analysis of regulons among related organisms. These modules guide a user through the appropriate steps of transferring known or high confidence regulatory binding site results to other microbial organisms, allowing them to study many organisms at one time, while warning of analysis possibly producing low confidence results, and providing them with sound default parameters.
There is an increasingly tight coupling of RegTransBase with MicrobesOnline in reporting cis-regulatory sites and regulatory interactions, and integrating RegTransBase searches into MicrobesOnLine cart functions.
PresenterNovichkov, Pavel Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsBioinformatics, Stress Response, Transcriptomics |
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Joachimiak, Marcin P.; Tuglus, Cathy; van der Laan, Mark; Arkin, Adam P. Statistical search for coherence in functional genomics data, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [MJoachimiakASM2008.pdf]Functional genomics confronts researchers with a deluge of new functional genomic experiments and technologies aimed at understanding biological function on the genome scale. For example, the Genomes to Life (GTL) Environmental Stress Pathway Project generates gene expression, gene knockout, proteomic, metabolomic, and protein-protein interaction data. How to rationally construct biological interpretations and determine their significance based on many instances of multiple data types?
Biclustering of gene expression data is a partitioning of the data that reveals modules of genes and experiments. These modules serve to analyze gene-gene associations and reconstruct regulatory networks. As datasets and data types proliferate it has become advantageous to: a) utilize multiple data types simultaneously, b) determine confidence from combined data, and c) systematically form hypothesis from multiple types of evidence. A recent method, CMONKEY, searches for co-regulated genes using simulated annealing and a Markov chain bicluster model with multi-parametric logistic regression for module membership based on gene expression, association networks, and sequence motifs.
We have developed a random-walk algorithm to search for biological modules that maximize a summary criterion. The main novelty of the algorithm lies in modeling three common data types: gene-experiment, gene-gene, and gene-feature. The summary criterion is computed from a weighted linear combination of correlation measures. Module membership gene-features is determined via a cross-validated R2 of association sub-criterion from data-adaptive polynomial spline fitting. An empirical null distribution provides significance scores for the mean-squared error of sub-criteria. The algorithm identifies multiple potentially overlapping global maxima, each with distinct contributions from specific sub-criteria and datasets.
To benchmark module discovery we evaluate this and related methods using a highly annotated functional genomic compendium as well as simulated datasets with virtual modules. We also present preliminary findings for Saccharomyces cerevisiae and select prokaryotes.
PresenterJoachimiak, Marcin P. Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsBioinformatics, Functional Genomics, Models, Sequencing |
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Zhou, Aifen; He, Zhili; Joachimiak, Marcin P.; Dehal, Paramvir S.; Arkin, Adam P.; Hillesland, Kristina; Stahl, David; Wall, Judy; Hazen, Terry C.; Zhou, Jizhong The dynamics and genetic adaptation to salt stress in experimental evolution of Desulfovibrio vulgaris Hildenborough, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for MicrobiologyOne of the greatest challenges in biology is to understand how genotype and environment interact to determine the phenotype and fitness of an organism. With the recent advances in genome sequencing and high-throughput genomic technologies, it becomes capable to link sub-cellular molecular/metabolic processes with the population-level processes, functions and evolution. One of our goals of the new proposal is to bring the environmental microbe, D. vulgaris Hildenborough to the model organism status (Aim 1). This study particularly intends to mimic the environmental conditions (salt stress) to address the evolution of DvH under such conditions in the lab. Such a study is expected to generate different DvH strains, and allows us to identify multiple beneficial mutations for salt adaptation. Therefore, this study directly links stress responses to the evolution of DvH, and will provide information for our integrative understanding of gene function, regulation, networking and evolution of DvH. To determine the long-term evolutionary responses, diversifications and adaptation of DvH to environmental stresses, the control and evolved cell lines (6 lines each) were obtained from a single DvH colony directly from the original glycerol stock. LS4D was used as standard culture medium for the control lines. Evolved lines were cultured on LS4D + 100 mM NaCl. Cells were kept at 37oC and transferred every 48 hrs with one to one hundred dilutions. The cells from every 100 generations were archived. The results demonstrated that the adaptation of DvH to salt stress was a dynamical process. The enhanced salt tolerance to higher salt (LS4D + 250 mM NaCl) of evolved lines was observed at 300 generations; and this phenomenon became more and more obvious with the increase of generations. Compared to the ancestor and paralleled control lines, both the growth rate and final biomass of the evolved lines were higher. The de-adaptation experiment on 1000 generation evolved lines provided the evidence that the phenotype was due to the genetic change instead of physiological adaptation. The gene expression profile of the 1000 generation evolved lines showed that some poly-cistronic operons such as hmcF-E-D-C-B-A (functional genes), rrf2-rrf1 (regulatory genes), LysA-2-LysX (functional genes) and DVU3290-3291-3292 (glutamate synthase) were significantly up-regulated compared to the control lines. Next, de-adaptation experiment need to be done on different generation samples to confirm that the beneficial genetic mutations are stable and genome sequencing will be performed to reveal the possible genetic mutations. PresenterZhou, Aifen Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsEvolutionary Biology, Extremophiles, Functional Genomics, Sequencing, Stress Response, Sulfate Reducers, Transcriptomics |
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Waldron, P. J.; Wu, L.; Van Nostrand, J. D.; Watson, D. B.; He, Z.; Schadt, C.W.; Hazen, T. C.; Zhou, J. Z. Functional gene array-based analysis of microbial community structure in a gradient of nitrate and heavy metal-contaminated groundwaters, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [PWaldronASM2008.pdf]Six groundwater monitoring wells from the Field Research Center, site of the U.S. DOE Environmental Remediation Science Program (ERSP) at the Oak Ridge Reservation, Oak Ridge, TN, were selected to compose a gradient of pH (3.25 – 7.11), nitrate (1.2 – 41,790 mg/l) and heavy metal contamination (0 – 500 mg/l U; 0 – 39896 mg/l Tc). To determine the functional populations of bacteria present within the gradient, DNA was extracted from groundwater and analyzed with a functional gene array containing 2,006 gene probes for the detection of genes involved in metal-resistance, sulfate-reduction, contaminant degradation and carbon and nitrogen cycling. The signal intensities for each probe were used to measure community diversity and were correlated to the geochemical profile of each well. Diversity decreased in relation to the level of contamination within each well, and each community exhibited a different distribution of genes. Heatmaps of metal resistance genes and nirK and nirS genes indicate that highly contaminated wells had lower gene diversity, but greater signal intensity for detected genes. Wells with the highest sulfate concentrations had the greatest diversity and signal intensity for dsrAB genes. A greater number of carbon fixation genes (cbbL, cbbM) were detected than fermentation genes (FTHFS) in all wells. A variety of organic contaminant degradation genes were detected. Results of Mantel tests and canonical correspondence analysis indicate that nitrate, sulfate, pH, uranium and technetium have a significant (p = 0.05) effect on bacterial community structure. This study provides an overall picture of bacterial community structure in contaminated environments across many different functional genes and shows that diversity can vary widely in relation to the degree of contamination. PresenterWaldron, P. J. Funding SourceEnvironmental Stress Pathway Project (ESPP), ERSP (formerly known as NABIR) KeywordsBiogeochemistry, Bioremediation, Environmental Genomics, Extremophiles, Field Studies, Functional Genomics, Microarrays, Sulfate Reducers |
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Hadi, Masood; Chakraborty, Romy; Light, Yooli; Meagher, Robert; Hazen, Terry C.; Singh, Anup cleaning up of legacy waste , 06/02/2008, Boston, MA, 108th General Meeting of the American Society for MicrobiologyThe current plan for cleaning up of legacy waste at several sites is expected to cost over 300 million dollars over 70 years. Alternative techniques that are cost effective are an active area of investigation. In situ bioremediation using indigenous microorganisms is one of the many techniques that has shown promising results in recent years. Although Indigenous bacteria at the site can degrade/sequester a wide range of hazardous compounds however, the bacterial population and decontamination efficiency can be affected by high concentrations of toxic compounds (e.g. heavy metals etc). Furthermore, current molecular techniques do not exist to identify the predominant contributor specie and the mechanism of detoxification/sequestration.
Recently a hydrogen release compound (HRC) stimulation field test was preformed to investigate bioreduction of Cr(VI) to Cr (III) at Hanford (site 100H). Post injection of the HRC, the Cr(VI) levels drastically decreased. Microarray analysis identified four key bacterial species that could be the major contributors to this bioreduction process. Based on the microarray results, several anaerobic enrichments were initiated in defined media, and 3 strains were isolated that were very closely related to the Desulfovibrio vulgaris, Geobacter metallireducens and Pseeudomonas stutzeri spp respectively. When individually tested in the lab, all the isolates were capable of Cr(VI) reduction. However to elucidate the contribution of these organisms to the Cr(VI) reduction process in-situ, we attempted a mesocosm study and monitored the stoichiometery of bacterial population and the expression of several key enzymes reported to be involved in Cr(VI) reduction using digital PCR as a function of the Cr(IV) depletion. This is a novel technique not commonly used for bioremediation studies. We discuss our results and present a preliminary model for Cr(IV) bioreduction in this complex environment. We also outline some future detection strategies for similar studies.
PresenterHadi, Masood Funding SourceEnvironmental Stress Pathway Project (ESPP), ERSP (formerly known as NABIR) KeywordsBioremediation, Environmental Genomics, Field Studies, Functional Genomics, Microarrays, Sequencing, Sulfate Reducers |
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Fortney, Julian; Chakraborty,Romy; Joachimiak, Marcin; Borglin, Sharon; Arkin, Adam P.; Hazen, Terry C. Investigation of stress response in the metal reducing organism Geobacter metallireducens, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for MicrobiologyAs part of the ongoing project on determination of stress response pathways in metal and radionuclide reducing bacteria, it is imperative to compare and contrast effects of environmental stressors on bacteria that are found to co-exist in several DOE-contaminated sites. In this study we have optimized growth kinetics of Geobacter metallireducens strain GS15 (GM) to monitor response to stressors NaCl and NO3 through several analyses including Fe(III) reduction, total cell protein production, transcriptomics, direct cell counts, and phospholipid fatty acid analysis (PLFA). Studying responses of GS15 to these stressors will provide a greater understanding of bioremediation under stress conditions. Growth of GM was optimized in defined LS4D medium at 30°C containing 10mM acetate and 10mM Fe-NTA in place of lactate and sulfate. In this medium, cell numbers of 4.3x107 are achieved in midlog phase of growth while 4.5mM of the initial Fe (III) is reduced. The minimal inhibitory concentration of NaCl of this organism in this media was determined to be 100mM, and that of NO3 was determined to be ___mM. The MICs were determined by following Fe(II) concentration and cell counts. To examine salt stress response of GM in detail, 100mM NaCl was amended to a midlog phase culture. At this time, 4.5mM Fe(III) was reduced and there were 3.5X107 cells/ml. After 4 hours of salt stress, the stressed cultures had reduced 7.1 mM Fe(III) and contained 5.23x107 cells/ml, which is significantly less than that of control cultures where 9.0mM Fe(III) was reduced and 9.53x107 cells/ml were present. PLFA analysis of stressed cells was performed and compared to non stressed cells which showed shifts in relative amounts of saturated and unsaturated fatty acids in response to stress. PresenterFortney, Julian Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsBioremediation, Extremophiles, Stress Response, Transcriptomics |
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Elias, Dwayne A.; Kucken, Amy M.; Brown, Steven D.; Drake, Meghan M.; Fagan, Lisa A.; Chakraborty, Romy; Brandt, Craig C.; Podar, Mircea; Hazen, Terry C.; Wall, Judy D.; Palumbo, Anthony V. Environmental Parameters and the Genes Involved in Mercury Methylation in the Pleomorphic Desulfovibrio africanus, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for MicrobiologyThe sulfate-reducing bacterium Desulfovibrio africanus ATCC 19997 has been shown to methylate mercury (Hg), a process that increases Hg toxicity. The goals of this work are to determine optimal growth conditions for this strain, assess the rate and extent of Hg methylation, and develop a genetic system for the identification of the genes involved in methylation. Initial Hg methylation analysis indicated that cultures spiked with mercuric nitrate generated 1 ng/L of methyl-Hg after several days. For optimal growth, media, NaCl concentrations, and pHs have been tested. A complex medium with yeast extract, 0.5% (w/v) NaCl, lactate as the carbon and electron donor, and sulfate as the electron acceptor yields 1x109 cells/ml in 48 hours. Microscopic observations revealed that the organism changed from a “lemon-shaped” cell in the lag phase to long, slender multi-flagellated rods in the exponential phase, and back to “lemon-shaped” cells in the transition to stationary phase. This phenomenon has been seen in the fresh-water strain Desulfovibrio africanus ATCC 19996 as well as the newly described Desulfovibrio strain PCS. Metabolic capacity and genetic accessibility of the different morphological types is being explored in strain 19997. Preliminary growth studies toward a genetic system have thus far shown antibiotic resistance to only kanamycin and ampicillin of seven antibiotics tested. We are currently determining transformation parameters. Proof-of-principle testing has targeted three candidate loci in D. africanus ATCC19997 for potential roles in Hg methylation of which orthologs were either not found in non-methylating Desulfovibrio spp. or did not share synteny with non-methlyators. These are a hypothetical gene, a SAM dependent methyltransferase, and a cobalamin-dependent methionine synthase I gene. Hence, heterologous expression of each of these genes in the non Hg-methlyating D. vulgaris Hildenborough will allow for a functional analysis of these proteins, and combined with their deletion in D. africanus may reveal the mechanisms by which methyl Hg is generated in the environment. PresenterElias, Dwayne A. Funding SourceEnvironmental Stress Pathway Project (ESPP), Protein Complex Analysis Project (PCAP) KeywordsEnvironmental Genomics, Extremophiles, Functional Genomics, Sequencing, Sulfate Reducers |
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Wu, Cindy H.; Chou, Joyce; Fujita, Yoshiko; Bill, Markus; Brodie, Eoin L.; Andersen, Gary L.; Hazen, Terry C.; Conrad, Mark S. Microbial Community Stimulated by Triethyl Phosphate for Vadose Zone Sequestration of Strontium-90 and Uranium, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for MicrobiologySignificant quantities of metals and radionuclides are contained in thick unsaturated zones at several contaminated sites in the western US. In many cases, this contamination has migrated to underlying groundwater, sometimes decades after being released into the subsurface. Because of the prohibitive costs associated with physically removing the contamination, an attractive remedy to this problem is to develop methods for long-term in situ stabilization of the contamination in the vadose zone. Triethyl phosphate (TEP) gas has been used to enhance biodegradation of organic pollutants in aquifers and vadose zones, presumably by alleviating phosphate limitations. We hypothesize that microbially-mediated TEP hydrolysis generating inorganic phosphate could also be used to promote metal and radionuclide sequestration in situ. Phosphate minerals are effective scavengers of contaminants such as Sr-90 and U. However, it is unclear which bacteria are capable of metabolizing TEP. A high-density microarray (PhyloChip) has been used to characterize the microbial community composition in TEP-enriched slurries. Preliminary results had indicated a 30% increase in inorganic phosphate production and 20% lower TEP concentration in slurries without heat sterilization. Microarray analysis reveals that Proteobacteria taxa are enriched by TEP, especially Methylophilaceae, Comamonadacea, Burkholderiaceae, and unclassified Rhizobiales. Future work includes 1) microarray analysis of metabolically active microbes through extraction and hybridization of rRNA, 2) evaluation of isotopic methods for monitoring microbial utilization of TEP, and 3) demonstration of radionuclide and metal sequestration induced by microbial metabolism of TEP. PresenterWu, Cindy H. Funding SourceEnvironmental Stress Pathway Project (ESPP), ERSP (formerly known as NABIR) KeywordsBiogeochemistry, Bioremediation, Environmental Genomics, Microarrays |
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Sercu, Bram; Wu, Cindy H.; Van De Werfhorst, Laurie C.; Brodie, Eoin L.; Hazen, Terry C.; Andersen, Gary L.; Holden, Patricia A. High Density Microarray as an Additional Tool for Microbial Source Tracking in a California Urban Creek, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for MicrobiologyHigh levels of fecal bacteriological contamination in coastal waters are often attributed to nearby human development. Tracking inland sources of fecal bacteria can be a significant challenge. To address this in Santa Barbara, CA, a multi-phase study has been conducted in an urban coastal creek that discharges into an impacted beach frequently posted with warnings. Culture-based IDEXX assays, quantitative PCR specific for human-specific Bacteroides, TRFLP and a high density microarray were used to determine human fecal contamination during dry weather at several locations during 3 consecutive days. Hierarchical clustering using microarray data indicated different degrees of relatedness of the microbial communities of water samples and separately collected sewage samples, and about half of the samples showed relatedness to sewage. When only fecal taxa (taxa most abundant in sewage samples) were considered, grouping according to location was stronger, and relatedness to sewage remained similar. Fecal OTUs belonged to families such as Bacteroidaceae, Lachnospiracae, Enterococcaceae, and Streptococcacea. Quantitative PCR identified a few samples containing elevated concentrations of the human-specific Bacteroides marker. Those samples also contained more fecal taxa, indicating agreement between qPCR and microarray results. However, the microarray results suggested human fecal pollution in additional samples, where no human-specific Bacteroides markers were found. Finally, the absence of important water-related pathogens (e.g. Campylobacter jejuni), and the presence of other pathogens (e.g. Helicobacter pylori) was shown. In conclusion, the microarray metagenomics approach appeared a valuable addition to the DNA toolbox for detection of human fecal waste in urban creek water samples. The results obtained in this study will guide further development of a microarray designed specifically for microbial water quality purposes. PresenterSercu, Bram Funding SourceEnvironmental Stress Pathway Project (ESPP), ERSP (formerly known as NABIR) KeywordsBiogeochemistry, Bioremediation, Field Studies, Microarrays |
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Kang, Sanghoon; Gough, Heidi L.; Van Nostrand, Joy; He, Zhili; Wu, Liyou; Stahl, David A.; Hazen, Terry C.; Zhou, Jizhong Microbial communities at the metal contaminated lake sediment, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology Microbes are known for their versatility under different and even certain extreme environmental conditions. The versatility is of particular interest in many applications including bioremediation. Lake DePue has been subjected to metal contamination over the last 80 years by adjacent zinc smelting activities. Sediments were collected in triplicate from five areas of the lake. These areas were previously identified as having varied metals contamination levels, and are located along a transect from near a creek inlet to the main body of the lake. GeoChip II, with over 10,000 microbial functional genes, was used to investigate metal impact on the microbial diversity and community structure. Three microbial communities with functional genes supposedly relevant to the metal contaminated condition were analyzed (S cycle, metal resistance and reduction, and C cycle genes). Geographic location of samples in relation to the contamination source was important factor in shaping microbial communities. Community structures at two sites closest to the contamination source (Sites 1 and 2), measured by NMDS, hierarchical clustering and k-means clustering, were clustered together in all three communities. Interestingly, same two sampling sites showed the greatest diversity (Shannon’s H’, Simpson’s –lnD and Fisher’s α). Proximity of sampling locations alone could not explain community variability as samples from Site 4, located between Sites 3 and 5, were more similar to samples from Sites 1 and 2. Multivariate correlation analysis by both Mantel test and Procrustes test revealed very significant correlation among three communities (P < 0.001). Spatial structures were fairly distinctive among three microbial communities in that smaller scale patch size in C cycle community and larger scale patch size in metal-related community were observed while virtually no patchiness was observed in S cycle community by multivariate correlogram analysis. In conclusion, three microbial communities relevant to metal contamination were rather similar in their composition and diversity while their spatial structure was quite distinctive. PresenterKang, Sanghoon Funding SourceEnvironmental Stress Pathway Project (ESPP), ERSP (formerly known as NABIR) KeywordsBiogeochemistry, Bioremediation, Environmental Genomics, Extremophiles, Field Studies, Functional Genomics, Microarrays, Stress Response |
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Huang, Katherine; Price, Morgan; Alm, Eric; Dehal, Paramvir and Arkin, Adam P. Large Lineage-Specific Expansions Are Driven By Mobile Genetic Elements, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [KHuangASM2008.pdf]Lineage-specifi c expansion (LSE) plays a vital role in how prokaryotes gain new gene functions and adapt to their environments. To uncover the mechanisms behind LSE, we identify genes that arise from LSE by constructing phylogenetic trees of protein families across 400+ bacterial genomes. We found that LSE genes tend to cluster on the chromosomes and form hyper LSE regions. Such regions could not be explained solely by operon duplication. The locations of these hyper LSE regions are often remarkably conserved among closely related strains, even though the gene content may not be conserved. Furthermore, these hyper LSE regions frequently overlap with clusters of mobile genetic elements
(MGE) and strain-specifi c genomic islands. We hypothesize that the majority of large strain-specifi c gene duplications are mediated by MGE and are concentrated in regions prone to site-speci fic MGE-driven recombinations. And the same regions for the same reason are more susceptible to phage
integration and to inter-genomic information exchange. PresenterHuang, Katherine Funding SourceEnvironmental Stress Pathway Project (ESPP) KeywordsBioinformatics, Comparative Genomics, Evolutionary Biology |
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Zane, Grant M.; Yen, Huei-Che; Wall, Judy D. Complementation with and without hypothetical protein DVU0851 in a qmoABC, hypothetical Protein (DVU0851) Deletion Mutation of Desulfovibrio vulgaris Hildenborough, 06/02/2008, Boston, MA, 108th General Meeting of the American Society for Microbiology, [GZaneASM2008.pdf]Deletion of the qmoABC and a hypothetical protein (HP) (DVU0848-51) in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough resulted in an inability to reduce sulfate. No suppressor mutations appeared in cultures of the deletion incubated in the presence of sulfate. Curiously, the D(qmoABC, HP) mutant was also unable to ferment pyruvate. Respiration of sulfate and fermentation of pyruvate was restored by supplying the qmoABC, HP genes. In order to verify if the hypothetical protein at the end of the operon contributes to the ability of this organism to reduce sulfate and ferment pyruvate, two complemented strains were constructed: both with the qmoABC genes, one with and one without the hypothetical protein. These two strains are compared for their ability to grow on sulfate as the sole electron-donor. Latest results suggest an integral role of the hypothetical protein in sulfate reduction.
PresenterWall, Judy D. Funding SourceEnvironmental Stress Pathway Project (ESPP), Protein Complex Analysis Project (PCAP) KeywordsComparative Genomics, Functional Genomics, Metabolomics, Sequencing, Sulfate Reducers |
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