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Abstract

Interspecies hydrogen transfer between organisms producing and consuming hydrogen promotes the decomposition
of organic matter in most anoxic environments. Although syntrophic coupling between hydrogen
producers and consumers is a major feature of the carbon cycle, mechanisms for energy recovery at the
extremely low free energies of reactions typical of these anaerobic communities have not been established. In
this study, comparative transcriptional analysis of a model sulfate-reducing microbe, Desulfovibrio vulgaris
Hildenborough, suggested the use of alternative electron transfer systems dependent on growth modality.
During syntrophic growth on lactate with a hydrogenotrophic methanogen, numerous genes involved in
electron transfer and energy generation were upregulated in D. vulgaris compared with their expression in
sulfate-limited monocultures. In particular, genes coding for the putative membrane-bound Coo hydrogenase,
two periplasmic hydrogenases (Hyd and Hyn), and the well-characterized high-molecular-weight cytochrome
(Hmc) were among the most highly expressed and upregulated genes. Additionally, a predicted operon
containing genes involved in lactate transport and oxidation exhibited upregulation, further suggesting an
alternative pathway for electrons derived from lactate oxidation during syntrophic growth. Mutations in a
subset of genes coding for Coo, Hmc, Hyd, and Hyn impaired or severely limited syntrophic growth but had
little effect on growth via sulfate respiration. These results demonstrate that syntrophic growth and sulfate
respiration use largely independent energy generation pathways and imply that to understand microbial
processes that sustain nutrient cycling, lifestyles not captured in pure culture must be considered.

Funding Source

ESPP

Keywords

Environmental Genomics

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Abstract

Recent 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 Source

ESPP, Metabolomics

Keywords

Functional Genomics, Metabolomics, Proteomics, Sulfate Reducers

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Abstract

Signal-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 Source

ESPP, ERSP

Keywords

Comparative Genomics, Microarrays, Transcriptomics

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Abstract

Abstract 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 Source

ESPP, ERSP

Keywords

Bioremediation, Environmental Genomics, Metabolism, Stress Response, Sulfate Reducers

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Abstract

Comparing 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 Source

ESPP

Keywords

Environmental Genomics, Evolutionary Biology, Models

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Abstract

Bacteria 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 Source

ESPP

Keywords

Comparative Genomics, Microarrays, Sequencing, Sulfate Reducers, Transcriptomics

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Abstract

Previous 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 Source

ESPP, Metabolomics

Keywords

Environmental Genomics, Functional Genomics, Metabolomics, Stress Response, Sulfate Reducers, Transcriptomics

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Abstract

Considering 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 Source

ESPP, MAGGIE, ERSP

Keywords

Bioremediation, Environmental Genomics, Field Studies, Microarrays

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Abstract

Molecular 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 Source

ESPP, ERSP

Keywords

Bioremediation, Environmental Genomics, Extremophiles, Microarrays

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Abstract

Pyruvate-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 Source

PCAP

Keywords

Proteomics, Sulfate Reducers