Alexa fluor-labeled fluorescent cellulose nanocrystals for bioimaging solid cellulose in spatially structured microenvironments.

Methods to covalently conjugate Alexa Fluor dyes to cellulose nanocrystals, at limiting amounts that retain the overall structure of the nanocrystals as model cellulose materials, were developed using two approaches. In the first, aldehyde groups are created on the cellulose surfaces by reaction with limiting amounts of sodium periodate, a reaction well-known for oxidizing vicinal diols to create dialdehyde structures. Reductive amination reactions were then applied to bind Alexa Fluor dyes with terminal amino-groups on the linker section. In the absence of the reductive step, dye washes out of the nanocrystal suspension, whereas with the reductive step, a colored product is obtained with the characteristic spectral bands of the conjugated dye. In the second approach, Alexa Fluor dyes were modified to contain chloro-substituted triazine ring at the end of the linker section. These modified dyes then were reacted with cellulose nanocrystals in acetonitrile at elevated temperature, again isolating material with the characteristic spectral bands of the Alexa Fluor dye. Reactions with Alexa Fluor 546 are given as detailed examples, labeling on the order of 1% of the total glucopyranose rings of the cellulose nanocrystals at dye loadings of ca. 5 µg/mg cellulose. Fluorescent cellulose nanocrystals were deposited in pore network microfluidic structures (PDMS) and proof-of-principle bioimaging experiments showed that the spatial localization of the solid cellulose deposits could be determined, and their disappearance under the action of Celluclast enzymes or microbes could be observed over time. In addition, single molecule fluorescence microscopy was demonstrated as a method to follow the disappearance of solid cellulose deposits over time, following the decrease in the number of single blinking dye molecules with time instead of fluorescent intensity.

Global transcriptomics analysis of the Desulfovibrio vulgaris change from syntrophic growth with Methanosarcina barkeri to sulfidogenic metabolism.

Desulfovibrio vulgaris is a metabolically flexible micro-organism. It can use sulfate as an electron acceptor to catabolize a variety of substrates, or in the absence of sulfate can utilize organic acids and alcohols by forming a syntrophic association with a hydrogen-scavenging partner to relieve inhibition by hydrogen. These alternative metabolic types increase the chance of survival for D. vulgaris in environments where one of the potential external electron acceptors becomes depleted. In this work, whole-genome D. vulgaris microarrays were used to determine relative transcript levels as D. vulgaris shifted its metabolism from syntrophic in a lactate-oxidizing dual-culture with Methanosarcina barkeri to a sulfidogenic metabolism. Syntrophic dual-cultures were grown in two independent chemostats and perturbation was introduced after six volume changes with the addition of sulfate. The results showed that 132 genes were differentially expressed in D. vulgaris 2 h after addition of sulfate. Functional analyses suggested that genes involved in cell envelope and energy metabolism were the most regulated when comparing syntrophic and sulfidogenic metabolism. Upregulation was observed for genes encoding ATPase and the membrane-integrated energy-conserving hydrogenase (Ech) when cells shifted to a sulfidogenic metabolism. A five-gene cluster encoding several lipoproteins and membrane-bound proteins was downregulated when cells were shifted to a sulfidogenic metabolism. Interestingly, this gene cluster has orthologues found only in another syntrophic bacterium, Syntrophobacter fumaroxidans, and four recently sequenced Desulfovibrio strains. This study also identified several novel c-type cytochrome-encoding genes, which may be involved in the sulfidogenic metabolism.

Microbial dynamics in upflow anaerobic sludge blanket (UASB) bioreactor granules in response to short-term changes in substrate feed.

The upflow anaerobic sludge blanket (UASB) reactor is a microcosm for the methanogenic degradation of organic matter in anaerobic environments, and depends on the auto-formation of dense 3D biofilms of 1-3 mm in diameter, referred to as granular sludge (biogranules). Past research has shown that UASB and other methanogenic reactors are extremely stable functionally, but the underlying basis of the functional stability is not well understood. In this study, microbial dynamics in the communities residing in UASB biogranules were analysed to determine responses to short-term perturbations (change in reactor feed). The reactor was fed with simulated brewery wastewater (SBWW) for 1.5 months (phase 1), acetate/sulfate for 2 months (phase 2), acetate alone for 3 months (phase 3) and then a return to SBWW for 2 months (phase 4). Analysis of 16S rRNA, methanogen-associated mcrA and sulfate reducer-associated dsrAB gene-based-clone libraries showed a relatively simple community composed mainly of the methanogenic archaea (Methanobacterium and Methanosaeta), members of the green non-sulfur (Chloroflexi) group of bacteria and Syntrophobacter, Spirochaeta, Acidobacteria and Cytophaga-related bacterial sequences. The mcrA clone libraries were dominated throughout by Methanobacterium- and Methanospirillum-related sequences. Although the reactor performance remained relatively stable throughout the experiment, community diversity levels generally decreased for all libraries in response to a change from SBWW to acetate alone feed. There was a large transitory increase noted in 16S diversity at the 2 month sampling on acetate alone, entirely related to an increase in bacterial diversity. Upon return to SBWW conditions in phase 4, all diversity measures returned to near phase 1 levels. Our results demonstrated that microbial communities, even highly structured ones such as in UASB biogranules, are very capable of responding to rapid and major changes in their environment.

Use of gene probes to assess the impact and effectiveness of aerobic in situ bioremediation of TCE.

Gene probe hybridization was used to determine distribution and expression of co-metabolic genes at a contaminated site as it underwent in situ methanotrophic bioremediation of trichloroethylene (TCE). The bioremediation strategies tested included a series of air, air:methane, and air:methane:nutrient pulses of the test plot using horizontal injection wells. During the test period, the levels of TCE reduced drastically in almost all test samples. Sediment core samples (n=367) taken from 0 m (surface)-43 m depth were probed for gene coding for methanotrophic soluble methane monooxygenase (sMMO) and heterotrophic toluene dioxygenase (TOD), which are known to co-metabolize TCE. The same sediment samples were also probed for genes coding for methanol dehydrogenase (MDH) (catalyzing the oxidation of methanol to formaldehyde) to assess specifically changes in methylotrophic bacterial populations in the site. Gene hybridization results showed that the frequency of detection of sMMO genes were stimulated approximately 250% following 1% methane:air (v/v) injection. Subsequent injection of 4% methane:air (v/v) resulted in an 85% decline probably due to nutrient limitations, since addition of nutrients (gaseous nitrogen and phosphorus) thereafter caused an increase in the frequency of detection of sMMO genes. Detection of TOD genes declined during the process, and eventually they were non-detectable by the final treatment, suggesting that methanotrophs displaced the TOD gene containing heterotrophs. Active transcription of sMMO and TOD was evidenced by hybridization to mRNA. These analyses combined with results showing the concomitant decline in TCE concentrations, increases in chloride concentration and increases in methanotroph viable counts, provide multiple lines of evidence that TCE remediation was caused specifically by methanotrophs. Our results suggest that sMMO genes are responsible for most, if not all, of the observed biodegradation of TCE. This study demonstrates that the use of nucleic acid analytical methods provided a gene specific assessment of the effects of in situ treatment technologies.

Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri.

We present a genome-scale metabolic model for the archaeal methanogen Methanosarcina barkeri. We characterize the metabolic network and compare it to reconstructions from the prokaryotic, eukaryotic and archaeal domains. Using the model in conjunction with constraint-based methods, we simulate the metabolic fluxes and resulting phenotypes induced by different environmental and genetic conditions. This represents the first large-scale simulation of either a methanogen or an archaeal species. Model predictions are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for wild type and mutants of the methanogenic pathway have a high level of agreement with experimental findings. We further examine the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction, and determine a stoichiometry for the nitrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation and further uncover the metabolic characteristics of methanogenesis.

mRNA-targeted fluorescent in situ hybridization (FISH) of Gram-negative bacteria without template amplification or tyramide signal amplification.

Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multi-copy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact that there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate that the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.

Optimization of RNA isolation from the archaebacterium Methanosarcina barkeri and validation for oligonucleotide microarray analysis.

The recent completion of a draft genome sequence for Methanosarcina barkeri has allowed the application of various high throughput post-genomics technologies, such as nucleic acid microarrays and mass spectrometry of proteins to detect global changes in transcription and translation that occur in response to experimental treatments. However, due to the production of a thick heteropolysaccharide outer layer, M. barkeri usually grows in large aggregates of cells rather than as individual, planktonic cells. Complete disruption of these aggregates and lysis of the released cells presents technical difficulties in ensuring the isolation of intact RNA from the entire population of cells. Initial attempts at isolating RNA from M. barkeri using several standard extraction protocols gave incomplete lysis of cells and resulted in low yields of poor quality RNA. In this study, we tested several chemical and mechanical disruption modifications of standard RNA extraction methods to optimize the extraction efficiency and minimize the number of unlysed cells remaining after extraction. As a further test of the quality of the resulting RNAs, their performance in replicate microarray analyses were determined. The results showed that inclusion of a liquid nitrogen grinding step prior to Trizol extraction, combined with moderate bead beating, yielded the most complete cell lysis, the highest yield of RNA and the most reproducible microarray results for M. barkeri. From these results it is clear that the methods used to isolate RNA can have a significant impact on the variability, trend and, presumably, the accuracy of microarray data. In addition, functional analysis of the microarray results obtained with RNA from the optimized protocol showed that, as expected, the genes involved in methanogenesis were among the most highly expressed genes in M. barkeri.

Microbial reduction of hexavalent chromium under vadose zone conditions.

Hexavalent chromium [Cr(VI)] is a common contaminant associated with nuclear reactors and fuel processing. Improper disposal at facilities in and and semiarid regions has contaminated underlying vadose zones and aquifers. The objectives of this study were to assess the potential for immobilizing Cr(VI) using a native microbial community to reduce soluble Cr(VI) to insoluble Cr(III) under conditions similar to those in the vadose zone, and to evaluate the potential for enhancing biological Cr(VI) reduction through nutrient addition. Batch microcosm and unsaturated flow column experiments were performed. Native microbial communities in subsurface sediments with no prior Cr(VI) exposure were shown to be capable of Cr(VI) reduction. In both the batch and column experiments, Cr(VI) reduction and loss from the aqueous phase were enhanced by adding high levels of both nitrate (NO3-) and organic C (molasses). Nutrient amendments resulted in up to 87% reduction of the initial 67 mg L(-1) Cr(VI) in an unsaturated batch experiment. Molasses and nitrate additions to 15 cm long unsaturated flow columns receiving 65 mg L(-1) Cr(VI) resulted in microbially mediated reduction and immobilization of 10% of the Cr during a 45-d experiment. All of the immobilized Cr was in the form of Cr(III), as shown by XANES analysis. This suggests that biostimulation of microbial Cr(VI) reduction in vadose zones by nutrient amendment is a promising strategy, and that immobilization of close to 100% of Cr contamination could be achieved in a thick vadose zone with longer flow paths and longer contact times than in this experiment.

Environmental genomics reveals a single-species ecosystem deep within Earth.

DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator, composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earth's crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.

Evolution of the syntrophic interaction between Desulfovibrio vulgaris and Methanosarcina barkeri: Involvement of an ancient horizontal gene transfer.

The sulfate reducing bacteria Desulfovibrio vulgaris and the methanogenic archaea Methanosarcina barkeri can grow syntrophically on lactate. In this study, a set of three closely located genes, DVU2103, DVU2104, and DVU2108 of D. vulgaris, was found to be up-regulated 2- to 4-fold following the lifestyle shift from syntroph to sulfate reducer; moreover, none of the genes in this gene set were differentially regulated when comparing gene expression from various D. vulgaris pure culture experiments. Although exact function of this gene set is unknown, the results suggest that it may play roles related to the lifestyle change of D. vulgaris from syntroph to sulfate reducer. This hypothesis is further supported by phylogenomic analyses showing that homologies of this gene set were only narrowly present in several groups of bacteria, most of which are restricted to a syntrophic lifestyle, such as Pelobacter carbinolicus, Syntrophobacter fumaroxidans, Syntrophomonas wolfei, and Syntrophus aciditrophicus. Phylogenetic analysis showed that all three individual genes in the gene set tended to be clustered with their homologies from archaeal genera, and they were rooted on archaeal species in the phylogenetic trees, suggesting that they were horizontally transferred from archaeal methanogens. In addition, no significant bias in codon and amino acid usages was detected between these genes and the rest of the D. vulgaris genome, suggesting the gene transfer may have occurred early in the evolutionary history so that sufficient time has elapsed to allow an adaptation to the codon and amino acid usages of D. vulgaris. This report provides novel insights into the origin and evolution of bacterial genes linked to the lifestyle change of D. vulgaris from a syntrophic to a sulfate-reducing lifestyle.

Development and assessment of whole-genome oligonucleotide microarrays to analyze an anaerobic microbial community and its responses to oxidative stress.

The application of DNA microarray technology to investigate multiple-species microbial communities presents great challenges. In this study, we reported the design and quality assessment of four whole genome oligonucleotide microarrays for two syntroph bacteria, Desulfovibrio vulgaris and Syntrophobacter fumaroxidans, and two archaeal methanogens, Methanosarcina barkeri, and Methanospirillum hungatei, and their application to analyze global gene expression in a four-species microbial community in response to oxidative stress. In order to minimize the possibility of cross-hybridization, cross-genome comparison was performed to assure all probes unique to each genome so that the microarrays could provide species-level resolution. Microarray quality was validated by the good reproducibility of experimental measurements of multiple biological and analytical replicates. This study showed that S. fumaroxidans and M. hungatei responded to the oxidative stress with up-regulation of several genes known to be involved in reactive oxygen species (ROS) detoxification, such as catalase and rubrerythrin in S. fumaroxidans and thioredoxin and heat shock protein Hsp20 in M. hungatei. However, D. vulgaris seemed to be less sensitive to the oxidative stress as a member of a four-species community, since no gene involved in ROS detoxification was up-regulated. Our work demonstrated the successful application of microarrays to a multiple-species microbial community, and our preliminary results indicated that this approach could provide novel insights on the metabolism within microbial communities.

DNA microarray analysis of anaerobic Methanosarcina barkeri reveals responses to heat shock and air exposure.

Methanosarcina barkeri is a methanogenic archaeon that can digest cellulose and other polysaccharides to produce methane. It can only grow under strictly anoxic conditions, but which can survive air exposure. To obtain further knowledge of cellular changes occurring in M. barkeri in response to air exposure and other environmental stresses, we constructed the first oligonucleotide microarray for M. barkeri and used it to investigate the global transcriptomic responses of M. barkeri to air exposure and heat shock at 45 degrees C for 1 h. The results showed that various house-keeping genes, such as genes involved in DNA replication recombination and repair, energy production and conversion, and protein turnover were regulated by environmental stimuli. In response to air exposure, up-regulation of a large number of transposase encoding genes was observed. However, no differential expression of genes encoding superoxide dismutase, catalase, nonspecific peroxidases or thioredoxin reductase was observed in response to air exposure, implying that no significant level of reactive oxygen species has been formed under air exposure. In response to heat shock, both Hsp70 (DnaK-DnaJ-GrpE chaperone system) the Hsp60 (GroEL) systems were up-regulated, suggesting that they may play an important role in protein biogenesis in M. barkeri during heat stress.

Integrated analysis of transcriptomic and proteomic data of Desulfovibrio vulgaris: zero-inflated Poisson regression models to predict abundance of undetected proteins.

MOTIVATION: Integrated analysis of global scale transcriptomic and proteomic data can provide important insights into the metabolic mechanisms underlying complex biological systems. However, because the relationship between protein abundance and mRNA expression level is complicated by many cellular and physical processes, sophisticated statistical models need to be developed to capture their relationship. RESULTS: In this study, we describe a novel data-driven statistical model to integrate whole-genome microarray and proteomic data collected from Desulfovibrio vulgaris grown under three different conditions. Based on the Poisson distribution pattern of proteomic data and the fact that a large number of proteins were undetected (excess zeros), zero-inflated Poisson (ZIP)-based models were proposed to define the correlation pattern between mRNA and protein abundance. In addition, by assuming that there is a probability mass at zero representing unexpressed genes and expressed proteins that were undetected owing to technical limitations, a Potential ZIP model was established. Two significant improvements introduced by this approach are (1) the predicted protein abundance level values for experimentally detected proteins are corrected by considering their mRNA levels and (2) protein abundance values can be predicted for undetected proteins (in the case of this study, approximately 83% of the proteins in the D.vulgaris genome) for better biological interpretation. We demonstrated the use of these statistical models by comparatively analyzing proteomic and microarray results from D.vulgaris grown on lactate-based versus formate-based media. These models correctly predicted increased expression of Ech hydrogenase and decreased expression of Coo hydrogenase for D.vulgaris grown on formate.

Two-component signal transduction systems of Desulfovibrio vulgaris: structural and phylogenetic analysis and deduction of putative cognate pairs.

A large number of two-component signal transduction systems (TCSTS) including 59 putative sensory histidine kinases (HK) and 55 response regulators (RR) were identified from the Desulfovibrio vulgaris genome. In this study, the structural and phylogenetic analyses of all putative TCSTSs in D. vulgaris were performed. The results showed that D. vulgaris contained 21 hybrid-type HKs, implying that multiple-step phosphorelay may be a common signal transduction mechanism in D. vulgaris. Despite the low sequence similarity that restricted the resolution of the phylogenetic analyses, most TCSTS components of D. vulgaris were found clustered into several subfamilies previously recognized in Escherichia coli and Bacillus subtilis. An attempt was made in this investigation to identify the possible cognate HK-RR pairs not linked on the chromosome in D. vulgaris based on similar expression patterns in response to various environmental and genetic changes. Expression levels of all HK and RR genes were measured using whole-genome microarrays. Five groups of HK-RR genes not linked on the chromosome were identified as possible cognate pairs in D. vulgaris. The results provided a preliminary list of possible cognate HK-RR pairs and constitute a basis for further exploration of interaction and physiological function of TCSTSs in D. vulgaris.

Oxidative stress and heat-shock responses in Desulfovibrio vulgaris by genome-wide transcriptomic analysis.

Sulfate-reducing bacteria such as Desulfovibrio vulgaris have developed a set of responses that allow them to survive in hostile environments. To obtain further knowledge of the protective mechanisms employed by D. vulgaris in response to oxidative stress and heat shock, we performed a genome-wide transcriptomic analysis to determine the cellular responses to both stimuli. The results showed that 130 genes were responsive to oxidative stress, while 427 genes were responsive to heat-shock. Functional analyses suggested that the genes regulated were involved in a variety of cellular functions. Amino acid biosynthetic pathways were induced by both oxidative stress and heat shock treatments, while fatty acid metabolism, purine and cofactor biosynthesis were induced by heat shock only. The rubrerythrin gene (rbr) was up-regulated in response to oxidative stress, suggesting an important role for this protein in the oxidative damage resistance response in D. vulgaris. In addition, thioredoxin reductase (trxB) was also responsive to oxidative stress, suggesting that the thiol-specific redox system might also be involved in oxidative protection in this organism. In contrast, the expression of rubredoxin oxidoreductase (rbo), superoxide dismutase (sodB) and catalase (katA) genes were not regulated in response to oxidative stress. Comparison of cellular responses to oxidative stress and heat-shock allowed the identification of 66 genes that showed a similar drastic response to both environmental perturbations, implying that these genes might be part of the general stress response (GSR) network in D. vulgaris. This hypothesis was further supported by the identification of a conserved motif upstream of these stress-responsive genes.

Global transcriptomic analysis of Desulfovibrio vulgaris on different electron donors.

Whole-genome microarrays of Desulfovibrio vulgaris were used to determine relative transcript levels in cells grown to exponential or stationary phase on a medium containing either lactate or formate as electron donor. The results showed that 158 and 477 genes were differentially expressed when comparing exponential to stationary phase in lactate- or formate-based media, respectively; and 505 and 355 genes were responsive to the electron donor used at exponential or stationary phase, respectively. Functional analyses suggested that the differentially regulated genes were involved in almost every aspect of cellular metabolism, with genes involved in protein synthesis, carbon, and energy metabolism being the most regulated. The results suggested that HynBA-1 might function as a primary periplasmic hydrogenase responsible for oxidation of H2 linked to the proton gradient in lactate-based medium, while several periplasmic hydrogenases including HynBA-1 and Hyd might carry out this role in formate-based medium. The results also indicated that the alcohol dehydrogenase and heterodisulfide reductase catalyzed pathway for proton gradient formation might be actively functioning for ATP synthesis in D. vulgaris. In addition, hierarchical clustering analysis using expression data across different electron donors and growth phases allowed the identification of the common electron donor independent changes in gene expression specifically associated with the exponential to stationary phase transition, and those specifically associated with the different electron donors independent of growth phase. The study provides the first global description and functional interpretation of transcriptomic response to growth phase and electron donor in D. vulgaris.

Molecular analysis of deep subsurface Cretaceous rock indicates abundant Fe(III)- and S(zero)-reducing bacteria in a sulfate-rich environment.

A multilevel sampler (MLS) was emplaced in a borehole straddling anaerobic, sulfate-rich Cretaceous-era shale and sandstone rock formations approximately 200 m below ground surface at Cerro Negro, New Mexico. Sterile quartzite sand contained in chambers in the sampler allowed in situ colonization and recovery of nucleic acids for molecular analyses. Denaturing gradient gel electrophoresis and 16S rRNA gene cloning results indicated a homogeneously distributed bacterial community across the shale-sandstone interface. delta-Proteobacteria sequences were common at all depths, and were dominated by members of the Geobacteraceae family (Pelobacter, Desulphuromonas and Geobacter). Other members of this group are capable of dissimilatory Fe(III) and/or S degrees reduction, but not sulfate reduction. RNA hybridization data also suggested that Fe(III)-/S degrees -reducing bacteria were predominant. These findings are striking considering the lack of significant concentrations of these electron acceptors in this environment. The next most abundant bacterial group indicated was the sulfate reducers, including Desulfobacterium, Desulfocapsa and Desulfobulbus. Sequences related to fermenters, denitrifiers and acetogens were also recovered. The presence of a phylogenetically and functionally diverse microbial community in this deep subsurface environment likely reflects the complex nature of the primary energy and carbon sources, kerogen associated with the shale.

A proteomic view of Desulfovibrio vulgaris metabolism as determined by liquid chromatography coupled with tandem mass spectrometry.

Direct LC-MS/MS was used to examine the proteins extracted from exponential or stationary phase Desulfovibrio vulgaris cells that had been grown on a minimal medium containing either lactate or formate as the primary carbon source. Across all four growth conditions, 976 gene products were identified with high confidence, which is equal to approximately 28% of all predicted proteins in the D. vulgaris genome. Bioinformatic analysis showed that the proteins identified were distributed among almost all functional classes, with the energy metabolism category containing the greatest number of identified proteins. At least 154 ORFs originally annotated as hypothetical proteins were found to encode the expressed proteins, which provided verification for the authenticity of these hypothetical proteins. Proteomic analysis showed that proteins potentially involved in ATP biosynthesis using the proton gradient across membrane, such as ATPase, alcohol dehydrogenases, heterodisulfide reductases, and [NiFe] hydrogenase (HynAB-1) of the hydrogen cycling were highly expressed in all four growth conditions, suggesting they may be the primary pathways for ATP synthesis in D. vulgaris. Most of the enzymes involved in substrate-level phosphorylation were also detected in all tested conditions. However, no enzyme involved in CO cycling or formate cycling was detected, suggesting that they are not the primary ATP-biosynthesis pathways under the tested conditions. This study provides the first proteomic overview of the cellular metabolism of D. vulgaris. The complete list of proteins identified in this study and their abundances (peptide hits) is provided in Supplementary Table 1.

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris.

High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.

Bioinformatic insights from metagenomics through visualization.

Cutting-edge biological and bioinformatics research seeks a systems perspective through the analysis of multiple types of high-throughput and other experimental data for the same sample. Systems-level analysis requires the integration and fusion of such data, typically through advanced statistics and mathematics. Visualization is a complementary computational approach that supports integration and analysis of complex data or its derivatives. We present a bioinformatics visualization prototype, Juxter, which depicts categorical information derived from or assigned to these diverse data for the purpose of comparing patterns across categorizations. The visualization allows users to easily discern correlated and anomalous patterns in the data. These patterns, which might not be detected automatically by algorithms, may reveal valuable information leading to insight and discovery. We describe the visualization and interaction capabilities and demonstrate its utility in a new field, metagenomics, which combines molecular biology and genetics to identify and characterize genetic material from multi-species microbial samples.