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108 publications were found
| 1 |
Gaucher, Sara P.; Redding, Alyssa M.; Mukhopadhyay, Aindrila; Keasling, Jay D. and Singh, Anup K. (2008) Post-Translational Modifications of Desulfovibrio vulgaris Hildenborough Sulfate Reduction Pathway Proteins. J. Proteome Res., : [View the Publication]closeAbstractRecent developments in shotgun proteomics have enabled high-throughput studies of a variety of microorganisms at a proteome level and provide experimental validation for predicted open reading frames in the corresponding genome. More importantly, advances in mass spectrometric data analysis now allow mining of large proteomics data sets for the presence of post-translational modifications (PTMs). Although PTMs are a critical aspect of cellular activity, such information eludes cell-wide studies conducted at the transcript level. Here, we analyze several mass spectrometric data sets acquired using two-dimensional liquid chromatography tandem mass spectrometry, 2D-LC/MS/MS, for the sulfate reducing bacterium, Desulfovibrio vulgaris Hildenborough. Our searches of the raw spectra led us to discover several post-translationally modified peptides in D. vulgaris. Of these, several peptides containing a lysine with a +42 Da modification were found reproducibly across all data sets. Both acetylation and trimethylation have the same nominal +42 Da mass, and are therefore candidates for this modification. Several spectra were identified having markers for trimethylation, while one is consistent with an acetylation. Surprisingly, these modified peptides predominantly mapped to proteins involved in sulfate respiration. Other highly expressed proteins in D. vulgaris, such as enzymes involved in electron transport and other central metabolic processes, did not contain this modification. Decoy database searches were used to control for random spectrum/sequence matches. Additional validation for these modifications was provided by alternate workflows, for example, two-dimensional gel electrophoresis followed by mass spectrometry analysis of the dissimilatory sulfite reductase γ-subunit (DsrC) protein. MS data for DsrC in this alternate workflow also contained the +42 Da modification at the same loci. Furthermore, the DsrC homologue in another sulfate reducing bacterium, Desulfovibrio desulfuricans G20, also showed similar +42 Da modifications in the same pathway. Here, we discuss our methods and implications of potential trimethylation in the D. vulgaris sulfate reduction pathway. Funding SourceESPP, Metabolomics KeywordsFunctional Genomics, Metabolomics, Proteomics, Sulfate Reducers |
| 2 |
Gough, Heidi L.; Dahl,Amy L.; Nolan,Melissa A.; Gaillard, Jean-Francois
and Stahl, David A. (2008) Metal impacts on microbial biomass in the anoxic sediments of a contaminated lake. J. Geophys. Res., 113(2):G02017 [View the Publication]closeAbstractLittle is known about the long-term impacts of metal contamination on the microbiota of anoxic lake sediments. In this study, we examined microbial biomass and metals arsenic, cadmium, chromium, copper, iron, lead, manganese, and zinc) in the sediments of Lake DePue, a backwater lake located near a former zinc smelter. Sediment core
samples were examined using two independent measures for microbial biomass
(total microscopic counts and total phospholipid phosphate concentrations) and for various
fractions of each metal (pore water extracts, sequential extractions, and total extracts of
all studied metals and zinc speciation by X-ray absorption fine structure). Zinc
concentrations were up to 1000 times higher than reported for sediments in the adjacent Illinois River and ranged from 21,400 mg/kg near the source to 1,680 mg/kg near the river. However, solid metal fractions were not well correlated with pore water
concentrations and were not good predictors of biomass concentrations. Instead, biomass,
which varied among sites by as much as 2 times, was inversely correlated with
concentrations of pore water zinc and arsenic as established by multiple linear regression.
Monitoring of other parameters known to naturally influence biomass in sediments (e.g., organic carbon concentrations, nitrogen concentrations, pH, sediment texture, and macrophytes) revealed no differences that could explain observed biomass trends. This study provides strong support for control of microbial abundance by pore water metal concentrations in contaminated freshwater sediments. Funding SourceESPP KeywordsBiogeochemistry, Extremophiles, Field Studies |
| 3 |
He, Zhili and Zhou, Jizhong (2008) Empirical evaluation of a new method for calculating signal to noise ratio (SNR) for microarray data analysis. Applied and Environmental Microbiology, 74(10):2957-2966 [View the Publication]closeAbstractSignal-to-noise-ratio (SNR) thresholds for microarray data analysis were experimentally determined with an oligonucleotide array that contained perfect match (PM) and mismatch (MM) probes based upon four genes from Shewanella oneidensis MR-1. A new SNR calculation, called signal to both standard deviations ratio (SSDR) was developed, and evaluated along with other two methods, signal to standard deviation ratio (SSR), and signal to background ratio (SBR). At a low stringency, the thresholds of SSR, SBR, and SSDR were 2.5, 1.60 and 0.80 with oligonucleotide and PCR amplicon as target templates, and 2.0, 1.60 and 0.70 with genomic DNA as target templates. Slightly higher thresholds were obtained at the high stringency condition. The thresholds of SSR and SSDR decreased with an increase in the complexity of targets (e.g. target types), and the presence of background DNA, and a decrease in the composition of targets, while SBR remained unchanged under all situations. The lowest percentage of false positives (FP) and false negatives (FN) was observed with the SSDR calculation method, suggesting that it may be a better SNR calculation for more accurate determination of SNR thresholds. Positive spots identified by SNR thresholds were verified by the Student t-test, and consistent results were observed. This study provides general guidance for users to select appropriate SNR thresholds for different samples under different hybridization conditions. Funding SourceESPP, ERSP KeywordsComparative Genomics, Microarrays, Transcriptomics |
| 4 |
Hwang, C., Wu, W.-M., Gentry, T.J., Carley, J., Corbin, G.A., Carroll, S.L., Watson, D.B., Jardine, P.M., Zhou, J.-Z., Criddle, C.S. and Fields, M.W. (2008) Bacterial Community Succession During in situ Uranium Bioremediation: Spatial Similarities Along Controlled Flow Paths. (In Press)closeAbstractBacterial community succession was investigated in a field-scale subsurface reactor formed by a series of wells that received weekly ethanol additions to re-circulating groundwater. Ethanol additions stimulated denitrification, metal reduction, sulfate reduction, and U(VI) reduction to sparingly soluble U(IV). Clone libraries of SSU rRNA gene sequences from groundwater samples enabled tracking of spatial and temporal changes over a 1.5 y period. Analyses showed that the communities changed in a manner consistent with geochemical variations that occurred along temporal and spatial scales. Canonical correspondence analysis revealed that the levels of nitrate, uranium, sulfide, sulfate, and ethanol strongly correlated with particular bacterial populations. As sulfate and U(VI) levels declined, sequences representative of sulfate-reducers and metal-reducers were detected at high levels. Ultimately, sequences associated with sulfate-reducing populations predominated, and sulfate levels declined as U(VI) remained at low levels. When engineering controls were compared to the population variation via canonical ordination, changes could be related to dissolved oxygen control and ethanol addition. The data also indicated that the indigenous populations responded differently to stimulation for bio-reduction; however, the two bio-stimulated communities became more similar after different transitions in an idiosyncratic manner. The strong associations between particular environmental variables and certain populations provide insight into the establishment of practical and successful remediation strategies in radionuclide-contaminated environments with respect to engineering controls and microbial ecology. Funding SourceESPP, ERSP KeywordsBioremediation, Environmental Genomics, Extremophiles, Field Studies |
| 5 |
Klonowska, A.; Clark, M.E.; Thieman, S.B.; Giles, B.J.; Wall, J.D.; Fields, M.W. (2008) Hexavalent chromium reduction in Desulfovibrio vulgaris Hildenborough causes transitory inhibition of sulfate reduction and cell growth. Applied Microbiology and Biotechnology, : [View the Publication]closeAbstractAbstract Desulfovibrio vulgaris Hildenborough is a well-studied sulfate reducer that can reduce heavy metals and radionuclides [e.g., Cr(VI) and U(VI)]. Cultures grown in a defined medium had a lag period of approximately 30 h when exposed to 0.05 mM Cr(VI). Substrate analyses revealed that although Cr(VI) was reduced within the first 5 h, growth was not observed for an additional 20 h. The growth lag could be explained by a decline in cell viability; however, during this time small amounts of lactate were still utilized without sulfate reduction or acetate formation. Approximately 40 h after Cr exposure (0.05 mM), sulfate reduction occurred concurrently with the accumulation of acetate. Similar amounts of hydrogen were produced by Cr exposed cells compared to control cells, and lactate was not converted to glycogen during non-growth conditions. D.
vulgaris cells treated with a reducing agent and then exposed to Cr(VI) still experienced a growth lag, but the addition of ascorbate at the time of Cr(VI) addition prevented the lag period. In addition, cells grown on pyruvate displayed more tolerance to Cr(VI) compared to lactate-grown cells. These results indicated that D. vulgaris utilized lactate during Cr(VI) exposure without the reduction of sulfate or production of acetate, and that ascorbate and pyruvate could protect D. vulgaris cells from Cr(VI)/Cr (III) toxicity. Funding SourceESPP, ERSP KeywordsBioremediation, Environmental Genomics, Metabolism, Stress Response, Sulfate Reducers |
| 6 |
Pereira, Patrícia M.; He, Qiang; Xavier, António V. ; Zhou, Jizhong; Pereira, Inês A. C. and Louro, Ricardo O. (2008) Transcriptional response of Desulfovibrio vulgaris Hildenborough to oxidative stress mimicking environmental conditions. Archives of Microbiology, 189(5):451-461 [View the Publication]closeAbstractSulfate-reducing bacteria (SRB) are anaerobes readily found in oxic–anoxic interfaces. Multiple defense pathways against oxidative conditions were identified in these organisms and proposed to be differentially expressed under different concentrations of oxygen, contributing to their ability to survive oxic conditions. In this study, Desulfovibrio vulgaris Hildenborough cells were exposed to the highest concentration of oxygen that SRB are likely to encounter in natural habitats, and the global transcriptomic response was determined. Three hundred and seven genes were responsive, with cellular roles in energy metabolism, protein fate, cell envelope and regulatory functions, including multiple genes encoding heat shock proteins, peptidases and proteins with heat shock promoters. Of the oxygen reducing mechanisms of D. vulgaris only the periplasmic hydrogen-dependent mechanism was up-regulated, involving the [NiFeSe] hydrogenase, formate dehydrogenase(s) and the Hmc membrane complex. The oxidative defense response concentrated on damage repair by metal-free enzymes. These data, together with the down-regulation of the ferric uptake regulator operon, which restricts the availability of iron, and the lack of response of the peroxide-sensing regulator operon, suggest that a major effect of this oxygen stress is the inactivation and/or degradation of multiple metalloproteins present in D. vulgaris as a consequence of oxidative damage to their metal clusters. Funding SourceESPP KeywordsEnvironmental Genomics, Extremophiles, Functional Genomics, Stress Response, Sulfate Reducers, Transcriptomics |
| 7 |
Shapiro, B. Jesse; Alm, Eric J. (2008) Comparing Patterns of Natural Selection across Species Using Selective Signatures. PLoS Genetics, 4(2):e23 [View the Publication]closeAbstractComparing gene expression profiles over many different conditions has led to insights that were not obvious from single experiments. In the same way, comparing patterns of natural selection across a set of ecologically distinct
species may extend what can be learned from individual genome-wide surveys. Toward this end, we show how variation in protein evolutionary rates, after correcting for genome-wide effects such as mutation rate and demographic factors, can be used to estimate the level and types of natural selection acting on genes across different species. We identify unusually rapidly and slowly evolving genes, relative to empirically derived genome-wide and gene family-specific background rates for 744 core protein families in 30 c-proteobacterial species. We describe the pattern of fast or slow evolution across species as the ‘‘selective signature’’ of a gene. Selective signatures represent a profile of selection across species that is predictive of gene function: pairs of genes with correlated selective signatures are more likely to share the same cellular function, and genes in the same pathway can evolve in concert. For example, glycolysis and phenylalanine metabolism genes evolve rapidly in Idiomarina loihiensis, mirroring an ecological shift in carbon source from sugars to amino acids. In a broader context, our results suggest that the genomic landscape is organized into functional modules even at the level of natural selection, and thus it may be easier than expected to understand the complex evolutionary pressures on a cell. Funding SourceESPP KeywordsEnvironmental Genomics, Evolutionary Biology, Models |
| 8 |
Stolyar S., He, Q., Joachimiak, M.P., He, Z., Yang, Z., Borglin, S. E., Huang, K., Joyner, D., Alm, E., Hazen, T.C. Zhou, J., Wall, J.D., Arkin, A.P. and Stahl, D.A. (2008) Response of Desulfovibrio vulgaris to Alkaline Stress. J. Bacteriol. , 189(24):8944-8952 [View the Publication]closeAbstractThe response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of D. vulgaris to increased pH is generally similar to that of Escherichia coli but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in E. coli appear to be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli were up-regulated in D. vulgaris; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As in E. coli, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a D. vulgaris Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580). Funding SourceESPP KeywordsMicroarrays, Stress Response, Sulfate Reducers, Transcriptomics |
| 9 |
Wall, J.D., Arkin, A.P., Balci, N.C. and Rapp-Giles, B. (2008) Genetics and genomics of sulfate respiration in Desulfovibrio. Springer-Verlag, Berlin, Heidelberg, Microbial Sulfur Metabolism(1):1-12 [View the Publication]closeAbstractBacteria that have evolved to use sulfate as a terminal electron acceptor must commit to spending energy for sulfate activation before there is a return on the investment allowing net energy gain. How sulfate is used and how electron flow
is controlled have provided challenging topics for research for many years. Having the complete genome sequences of several of these bacteria is a monumental step in the elucidation of these questions. This information has provided the tools for determining the quantity of transcripts for genes under defined growth conditions, not just the relative changes in transcripts in two growth conditions. A comparison of the hybridization signal of messenger RNA with that of genomic DNA with oligonucleotide microarrays of all open reading frames reveals the differences in steady-state levels of transcripts for each gene. Growth of Desulfovibrio vulgaris Hildenborough on defined medium with lactate as a carbon and reductant source and with sulfate as the electron acceptor has been examined by this procedure for levels of gene expression. Relative functional importance was inferred from the levels of gene transcription, in spite of the recognized limitations of this interpretation. Not surprisingly, genes encoding established functions for sulfate reduction were highly expressed. However, the high molecular mass c-type cytochrome genes thought to
encode a most important transmembrane electron conduit for sulfate reduction were expressed at quite low levels. Funding SourceESPP KeywordsComparative Genomics, Microarrays, Sequencing, Sulfate Reducers, Transcriptomics |
| 10 |
Zhou, Jizhong; Kang, Sanghoon; Schadt, Christopher W. and Garten, Jr., Charles T. (2008) Spatial scaling of functional gene diversity. PNAS, 105(22):7768-7773 [View the Publication]closeAbstractUnderstanding the spatial patterns of organisms and the underlying
mechanisms shaping biotic communities is a central goal in community ecology. One of the most well documented spatial patterns in plant and animal communities is the positive-power law relationship between species (or taxa) richness and area. Such taxa–area relationships (TARs) are one of the principal generalizations in ecology, and are fundamental to our understanding of the
distribution of global biodiversity. However, TARs remain elusive in microbial communities, especially in soil habitats, because of inadequate sampling methodologies. Here, we describe TARs as gene–area relationships (GARs), at a whole-community level, across various microbial functional and phylogenetic groups in a forest soil, using a comprehensive functional gene array with
>24,000 probes. Our analysis indicated that the forest soil microbial community exhibited a relatively flat gene–area relationship (slope z 0.0624), but the z values varied considerably across different functional and phylogenetic groups (z 0.0475–0.0959). However, the z values are several times lower than those commonly observed in plants and animals. These results suggest that the turnover in space of microorganisms may be, in general, lower than that of plants and animals.
Key words: canonical correspondence analysis | GeoChip | taxa–area relationship Funding SourceERSP KeywordsBiogeochemistry, Environmental Genomics, Functional Genomics |
| 11 |
Bender, K.S., Yen, H.-C., Hemme, C.L., Yang, Z., He, Z., He, Q., Zhou, J., Huang, K.H., Alm, E.J., Hazen, T.C. and Wall, J.D. (2007) Analysis of a ferric uptake regulator (Fur) mutant of Desulfovibrio vulgaris Hildenborough. Appl Environ. Mirobiol., 73(17):5389–5400 [View the Publication]closeAbstractPrevious experiments examining the transcriptional profile of the anaerobe
Desulfovibrio vulgaris have demonstrated up-regulation of the Fur regulon in response to various environmental stressors. To test the involvement of Fur in the growth response and transcriptional regulation of D. vulgaris, a targeted mutagenesis procedure was used for deleting the fur gene. Growth of the resulting Dfur mutant (JW707) was not affected by iron availability, but did exhibit increased sensitivity to nitrite and osmotic stresses when compared to the wild type. Transcriptional profiling of JW707 indicated that iron bound Fur acts as a traditional repressor for ferrous iron uptake genes (feoAB) and other genes containing a predicted Fur binding site within their promoter. Despite the apparent lack of siderophore biosynthesis genes within the D. vulgaris genome, a large 12 gene operon encoding orthologs to TonB and TolQR also appeared to be repressed by iron bound Fur. While other genes predicted to be involved in iron homeostasis were unaffected by the presence or absence of Fur, alternative expression patterns that could be interpreted as repression or activation by iron-free Fur were observed. Both the physiological and transcriptional data implicate a global 1 regulatory role for Fur in the sulfate-reducing bacterium D. vulgaris. Funding SourceESPP, Metabolomics KeywordsEnvironmental Genomics, Functional Genomics, Metabolomics, Stress Response, Sulfate Reducers, Transcriptomics |
| 12 |
Berube, Paul M., Samudrala, Ram and Stahl, David A. (2007) Transcription of All amoC Copies Is Associated with Recovery of Nitrosomonas europaea from Ammonia Starvation. J. Bacteriol. , 189(11):3935-3944 [View the Publication]closeAbstractThe chemolithotrophic ammonia-oxidizing bacterium Nitrosomonas europaea is known to be highly resistant to starvation conditions. The transcriptional response of N. europaea to ammonia addition following short- and long-term starvation was examined by primer extension and S1 nuclease protection analyses of genes encoding enzymes for ammonia oxidation (amoCAB operons) and CO2 fixation (cbbLS), a third, lone copy of amoC (amoC3), and two representative housekeeping genes (glyA and rpsJ). Primer extension analysis of RNA isolated from growing, starved, and recovering cells revealed two differentially regulated promoters upstream of the two amoCAB operons. The distal {sigma}70 type amoCAB promoter was constitutively active in the presence of ammonia, but the proximal promoter was only active when cells were recovering from ammonia starvation. The lone, divergent copy of amoC (amoC3) was expressed only during recovery. Both the proximal amoC1,2 promoter and the amoC3 promoter are similar to gram-negative {sigma}E promoters, thus implicating {sigma}E in the regulation of the recovery response. Although modeling of subunit interactions suggested that a nonconservative proline substitution in AmoC3 may modify the activity of the holoenzyme, characterization of a {Delta}amoC3 strain showed no significant difference in starvation recovery under conditions evaluated. In contrast to the amo transcripts, a delayed appearance of transcripts for a gene required for CO2 fixation (cbbL) suggested that its transcription is retarded until sufficient energy is available. Overall, these data revealed a programmed exit from starvation likely involving regulation by {sigma}E and the coordinated regulation of catabolic and anabolic genes. Funding SourceESPP KeywordsEnvironmental Genomics, Evolutionary Biology, Extremophiles, Functional Genomics, Stress Response, Transcriptomics |
| 13 |
Brodie, Eoin L.
DeSantis, Todd Z.
Parker, Jordan P. Moberg
Zubietta, Ingrid X.
Piceno, Yvette M.
Andersen, Gary L. (2007) Urban aerosols harbor diverse and dynamic bacterial populations. PNAS, 104(1):299-304 [View the Publication]closeAbstractConsidering the importance of its potential implications for human health, agricultural productivity, and ecosystem stability, surprisingly little is known regarding the composition or dynamics of the atmosphere's microbial inhabitants. Using a custom high-density DNA microarray, we detected and monitored bacterial populations in two U.S. cities over 17 weeks. These urban aerosols contained at least 1,800 diverse bacterial types, a richness approaching that of some soil bacterial communities. We also reveal the consistent presence of bacterial families with pathogenic members including environmental relatives of select agents of bioterrorism significance. Finally, using multivariate regression techniques, we demonstrate that temporal and meteorological influences can be stronger factors than location in shaping the biological composition of the air we breathe. Funding SourceESPP, MAGGIE, ERSP KeywordsBioremediation, Environmental Genomics, Field Studies, Microarrays |
| 14 |
Caffrey, Sean M., Park, Hyung-Soo, Voordouw, Johanna K., He, Zhili, Zhou, Jizhong and Voordouw, Gerrit (2007) Function of Periplasmic Hydrogenases in the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough. J. Bacteriol., 189(17):6159-6167 [View the Publication]closeAbstractThe sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen > 50% hydrogen > lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase. Funding SourceESPP KeywordsFunctional Genomics, Metabolism, Microarrays, Sulfate Reducers |
| 15 |
DeSantis, Todd Z., Brodie, Eoin L., Moberg, Jordan P., Zubieta, Ingrid X., Piceno, Yvette M. and Andersen, Gary L. (2007) High-Density Universal 16S rRNA Microarray Analysis Reveals Broader Diversity than Typical Clone Library When Sampling the Environment. Microb. Ecol., 3: [View the Publication]closeAbstractMolecular approaches aimed at detection of a broadrange of prokaryotes in the environment routinely rely
on classifying heterogeneous 16S rRNA genes amplified by polymerase chain reaction (PCR) using primers with broad specificity. The general method of sampling and categorizing DNA has been to clone then sequence the PCR products. However, the number of clones required to adequately catalog the majority of taxa in a sample is unwieldy. Alternatively, hybridizing target sequences to a universal 16S rRNA gene microarray may provide a more rapid and comprehensive view of prokaryotic community composition. This study investigated the breadth and accuracy of a microarray in detecting diverse 16S rRNA gene sequence types compared to clone-and-sequencing using three environmental samples: urban aerosol, subsurface soil, and subsurface water. PCR products generated from universal 16S rRNA gene-targeted primers were classified by using either the clone-and-sequence method or by hybridization to a novel high-density microarray of 297,851 probes complementary to 842 prokaryotic subfamilies. The three clone libraries comprised 1391 highquality
sequences. Approximately 8% of the clones could not be placed into a known subfamily and were
considered novel. The microarray results confirmed the majority of clone-detected subfamilies and additionally
demonstrated greater amplicon diversity extending into phyla not observed by the cloning method. Sequences
matching operational taxonomic units within the phyla Nitrospira, Planctomycetes, and TM7, which were
uniquely detected by the array, were verified with specific primers and subsequent amplicon sequencing. Subfamily richness detected by the array corresponded well with nonparametric richness predictions extrapolated from clone libraries except in the water community where clone-based richness predictions were greatly exceeded. It was concluded that although the microarray is unreliable in identifying novel prokaryotic taxa, it reveals greater diversity in environmental samples than sequencing a typically sized clone library. Furthermore, the microarray allowed samples to be rapidly evaluated with replication, a significant advantage in studies of microbial ecology. Funding SourceESPP, ERSP KeywordsBioremediation, Environmental Genomics, Extremophiles, Microarrays |
| 16 |
Gao, H., Yang, Zamin K., Gentry, Terry J., Wu, Liyou, Schadt, Christopher W. and Zhou, Jizhong (2007) Microarray-based Analysis of Microbial Community RNAs by Whole Community RNA Amplification (WCRA). Appl. Environ. Microbiol., 73(2):563-571 [View the Publication]closeAbstractA new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis fur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion. Funding SourceESPP KeywordsFunctional Genomics, Microarrays, Sequencing |
| 17 |
Garczarek, Florian, Dong, Ming, Typke, Dieter, Witkowska, H. Ewa, Hazen, Terry C., Nogales, Eva, Biggin, Mark D., Glaeser, Robert M. (2007) Octomeric pyruvate-ferredoxin oxidoreductase from Desulfovibrio vulgaris. Journal of Structural Biology, 159(1):9-18 [View the Publication]closeAbstractPyruvate-ferredoxin oxidoreductatse (PFOR) carries out the central step in oxidative decarboxylation of pyruvate to acetyl-CoA. We have purified this enzyme from Desulfovibrio vulgaris Hildenborough (DvH) as part of a systematic characterization of as many multiprotein complexes as possible for this organism, and the three-dimensional structure of this enzyme has been determined by a combination of electron microscopy (EM), single particle image analysis, homology modeling and computational molecular docking. Our results show that the 1 MDa DvH PFOR complex is a homo-octomer, or more precisely, a tetramer of the dimeric form of the related enzyme found in Desulfovibrio africanus (Da), with which it shares a sequence identity of 69%. Our homology model of the DvH PFOR dimer is based on the Da PFOR X-ray structure. Docking of this model into our 17 A resolution EM-reconstruction of negatively stained DvH PFOR octomers strongly suggests that the difference in oligomerization state for the two species is due to the insertion of a single valine residue (Val383) within a surface loop of the DvH enzyme. This study demonstrates that the strategy of intermediate resolution EM reconstruction coupled to homology modeling and docking can be powerful enough to infer the functionality of single amino acid residues. Funding SourcePCAP KeywordsProteomics, Sulfate Reducers |
| 18 |
He, Zhili, Terry J. Gentry, Christopher W. Schadt, Liyou Wu, Jost Liebich, Song C. Chong, Weimin Wu, Baohua Gu, Phil Jardine, Craig Criddle, and Jizhong Zhou (2007) GeoChip: A comprehensive microarray for investigating biogeochemical, ecological, and environmental processes. The ISME Journal, 1(1):67-77 [View the Publication]closeAbstractDue to their vast diversity and as-yet uncultivated status, detection, characterization and quantification of microorganisms in natural settings are very challenging, and linking microbial diversity to ecosystem processes and functions is even more difficult. Microarray-based genomic technology for detecting functional genes and processes has a great promise of overcoming such obstacles. Here, a novel comprehensive microarray, termed GeoChip, has been developed, containing 24,243 oligonucleotide (50mer) probes and covering > 10,000 genes in >150 functional groups involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance, and organic contaminant degradation. The developed GeoChip was successfully used for tracking the dynamics of metal-reducing bacteria and associated communities for an in situ bioremediation study, which is the first time to demonstrate that uranium can be bioremediated to the concentrations below the USA EPA maximum contaminant level (MCL) for drinking water. This is the first comprehensive microarray available for studying biogeochemical processes and functional activities of microbial communities important to human health, agriculture, energy, global climate change, ecosystem management, and environmental cleanup and restoration. It is particularly useful for providing direct linkages of microbial genes/populations to ecosystem processes and functions.
Funding SourceESPP KeywordsBioremediation, Functional Genomics, Microarrays |
| 19 |
Jacobsen, Janet,
Joyner, Dominique C.
Borglin, Sharon E.
Hazen, Terry C.
Arkin, Adam P.
Bethel, E. Wes (2007) Visualization of Growth Curve Data from Phenotype Microarray Experiments. 11th International Conference on Information Visualization IV 2007, Zurich, Switzerland(IEEE Computer Society Press, Los Alamitos, CA):July 4-6, 2007closeAbstractPhenotype microarrays provide a technology to simultaneously survey the response of an organism to nearly 2,000 substrates, including carbon, nitrogen and potassium sources; varying pH; varying salt concentrations; and antibiotics. In order to more quickly and easily view and compare the large number of growth
curves produced by phenotype microarray experiments, we have developed software to produce and display color images, each of which corresponds to a set of 96 growth curves. Using color images to represent growth curves data has proven to be a valuable way to assess experiment quality, compare replicates, facilitate comparison of the responses of different organisms, and identify significant phenotypes. The color images are linked to traditional plots of growth versus time, as well as to information about the experiment, organism, and
substrate. In order to share and view information and data project-wide, all information, plots, and data are accessible using only a Web browser. Funding SourceESPP KeywordsImaging, Microarrays, Phenomics |
| 20 |
Kazakov, Alexei E., Cipriano, Michael J., Novichkov, Pavel S., Minovitsky, Simon, Vinogradov, Dmitry V., Arkin, Adam P., Mironov, Andrey A., Gelfand, Mikhail S., and Dubchak, Inna (2007) RegTransBase - a database of regulatory sequences and interactions in a wide range of prokaryotic genomes. Nucleic Acids Research, 35(Database Issue): D407-D412 [View the Publication]closeAbstractRegTransBase, a manually curated database of regulatory interactions in prokaryotes, captures the knowledge in published scientific literature using a controlled vocabulary. Although a number of databases describing interactions between regulatory proteins and their binding sites are currently being maintained, they focus mostly on the model organisms Escherichia coli and Bacillus subtilis, or are entirely computationally derived. RegTransBase describes a large number of regulatory interactions reported in many organisms and contains various types of experimental data, in particular: the activation or repression of transcription by an identified direct regulator; determining the transcriptional regulatory function of a protein (or RNA) directly binding to DNA (RNA); mapping or prediction of binding site for a regulatory protein; characterization of regulatory mutations.
Currently, the RegTransBase content is derived from about 3000 relevant articles describing over 7000 experiments in relation to 128 microbes. It contains data on the regulation of about 7500 genes and evidence for 6500 interactions with 650 regulators. RegTransBase also contains manually created position weight matrices (PWM) that can be used to identify candidate regulatory sites in over 60 species. RegTransBase is available at http://regtransbase.lbl.gov
Funding SourceESPP KeywordsBioinformatics, Comparative Genomics |
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