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The area of transcriptomics analysis is among the more established in computational biology, having evolved in both technology and experimental design. Transcriptomics has a strong impetus to develop sophisticated computational methods due to the large amounts of available whole-genome datasets for many species and because of powerful applications in regulatory network reconstruction as well as elucidation and modeling of cellular transcriptional responses. While gene expression microarray data can be noisy and comparisons across experiments challenging, there are a number of sophisticated methods that aid in arriving at statistically and biologically significant conclusions. As such, computational transcriptomics analysis can provide guidance for analysis of results from newer experimental technologies. More recently, search methods have been developed to identify modules of genes, which exhibit coherent expression patterns in only a subset of experimental conditions. The latest advances in these methods allow to integrate multiple data types and datasets, both experimental and computational, within a single statistical framework accounting for data confidence and relevance to specific biological questions. Such frameworks provide a unified environment for the exploration of specific biological hypothesis and for the discovery of coherent data patterns along with the evidence supporting them.

Presenter

Joachimiak, Marcin P.

Funding Source

Environmental Stress Pathway Project (ESPP)

Keywords

Bioinformatics, Transcriptomics

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The overview of changes in protein levels or states in response to a growth condition, stress, mutation or metabolic engineering is invaluable in understanding the physiology of a microbial system. The lipid profile of the cell is similarly a valuable diagnostic of the cellular response and health, especially in context of survival in a fluctuating environment. To obtain comprehensive cellular models, post-transcriptional cell wide surveys at the levels of proteins and lipids are required. Both these fields have been greatly bolstered by the development of high throughput methods using mass spectrometry. Multiple strategies now exist for the identification of proteins, and numerous workflows to quantify protein abundance have also been developed. Cellular profiling such as these allows us to assess the potential of a microbial system for environmental applications such as bioremediation and bio-energy.

Presenter

Mukhopadhyay, Aindrila

Funding Source

Environmental Stress Pathway Project (ESPP), Metabolomics

Keywords

Bioremediation, Functional Genomics, Lipidomics, Proteomics

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Hexavalent chromium is a widespread contaminant found in groundwater. In order to stimulate microbially mediated Cr(VI)-reduction, a poly-lactate compound was injected into Cr(VI)-contaminated aquifers at site 100H at Hanford. Investigation of bacterial community composition using high-density DNA microarray analysis of 16S rRNA gene products revealed a stimulation of Pseudomonas, Desulfovibrio and Geobacter species amongst others. Enrichment of these organisms coincided with continued Cr(VI) depletion. Functional gene-array analysis of DNA from monitoring well indicated high abundance of genes involved in nitrate-reduction, sulfate-reduction, iron-reduction, methanogenesis, chromium tolerance/reduction. Clone-library data revealed Psedomonas was the dominant genus in these samples. Based on above results, we conducted lab investigations to study the dominant anaerobic culturable microbial populations present at this site and their role in Cr(VI)-reduction. Enrichments using defined anaerobic media resulted in isolation of an iron-reducing, a sulfate-reducing and a nitrate-reducing isolate among several others. Preliminary 16S rDNA sequence analysis identified the isolates as Geobacter metallireducens, Pseudomonas stutzeri and Desulfovibrio vulgaris species respectively. The Pseudomonas isolate utilized acetate, lactate, glycerol and pyruvate as alternative carbon sources, and reduced Cr(VI). Anaerobic washed cell suspension of strain HLN reduced almost 95μM Cr(VI) within 4 hr. Further, with 100μM Cr(VI) as sole electron-acceptor, cells grew to 4.05 x 107 /ml over 24 h after an initial lag, demonstrating direct enzymatic Cr(VI) reduction coupled to growth. These results demonstrate that Cr(VI)-immobilization at Hanford 100H site could be mediated by direct microbial metabolism in addition to indirect chemical reduction of Cr(VI) by end-products of microbial activity.

Presenter

Chakraborty, Romy

Funding Source

Environmental Stress Pathway Project (ESPP)

Keywords

Biogeochemistry, Bioremediation, Environmental Genomics, Field Studies, Phenomics, Sulfate Reducers

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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/

Presenter

Shutkin, Amy

Funding Source

Environmental Stress Pathway Project (ESPP)

Keywords

Models

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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.

Presenter

Hillesland, Kristina

Funding Source

Environmental Stress Pathway Project (ESPP)

Keywords

Comparative Genomics, Environmental Genomics, Evolutionary Biology, Extremophiles, Sulfate Reducers

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Adaptive 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.

Presenter

Shapiro, B. Jesse

Funding Source

Environmental Stress Pathway Project (ESPP)

Keywords

Evolutionary Biology, Proteomics

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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.

Presenter

Bender, Kelly S.

Funding Source

Environmental Stress Pathway Project (ESPP), Protein Complex Analysis Project (PCAP)

Keywords

Functional Genomics, Sequencing, Sulfate Reducers

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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.

Presenter

Novichkov, Pavel

Funding Source

Environmental Stress Pathway Project (ESPP)

Keywords

Bioinformatics, Stress Response, Transcriptomics

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One 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.

Presenter

Zhou, Aifen

Funding Source

Environmental Stress Pathway Project (ESPP)

Keywords

Evolutionary Biology, Extremophiles, Functional Genomics, Sequencing, Stress Response, Sulfate Reducers, Transcriptomics

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The 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.

Presenter

Hadi, Masood

Funding Source

Environmental Stress Pathway Project (ESPP), ERSP (formerly known as NABIR)

Keywords

Bioremediation, Environmental Genomics, Field Studies, Functional Genomics, Microarrays, Sequencing, Sulfate Reducers