Evaluating various composite sampling modes for detecting pathogenic SARS-CoV-2 virus in raw sewage.

Inadequate sampling approaches to wastewater analyses can introduce biases, leading to inaccurate results such as false negatives and significant over- or underestimation of average daily viral concentrations, due to the sporadic nature of viral input. To address this challenge, we conducted a field trial within the University of Tennessee residence halls, employing different composite sampling modes that encompassed different time intervals (1 h, 2 h, 4 h, 6 h, and 24 h) across various time windows (morning, afternoon, evening, and late-night). Our primary objective was to identify the optimal approach for generating representative composite samples of SARS-CoV-2 from raw wastewater. Utilizing reverse transcription-quantitative polymerase chain reaction, we quantified the levels of SARS-CoV-2 RNA and pepper mild mottle virus (PMMoV) RNA in raw sewage. Our findings consistently demonstrated that PMMoV RNA, an indicator virus of human fecal contamination in water environment, exhibited higher abundance and lower variability compared to pathogenic SARS-CoV-2 RNA. Significantly, both SARS-CoV-2 and PMMoV RNA exhibited greater variability in 1 h individual composite samples throughout the entire sampling period, contrasting with the stability observed in other time-based composite samples. Through a comprehensive analysis of various composite sampling modes using the Quade Nonparametric ANCOVA test with date, PMMoV concentration and site as covariates, we concluded that employing a composite sampler during a focused 6 h morning window for pathogenic SARS-CoV-2 RNA is a pragmatic and cost-effective strategy for achieving representative composite samples within a single day in wastewater-based epidemiology applications. This method has the potential to significantly enhance the accuracy and reliability of data collected at the community level, thereby contributing to more informed public health decision-making during a pandemic.

Distinct Depth-Discrete Profiles of Microbial Communities and Geochemical Insights in the Subsurface Critical Zone.

Microbial assembly and metabolic potential in the subsurface critical zone (SCZ) are substantially impacted by subsurface geochemistry and hydrogeology, selecting for microbes distinct from those in surficial soils. In this study, we integrated metagenomics and geochemistry to elucidate how microbial composition and metabolic potential are shaped and impacted by vertical variations in geochemistry and hydrogeology in terrestrial subsurface sediment. A sediment core from an uncontaminated, pristine well at Oak Ridge Field Research Center in Oak Ridge, Tennessee, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone, was used in this study. Our results showed that subsurface microbes were highly localized and that communities were rarely interconnected. Microbial community composition as well as metabolic potential in carbon and nitrogen cycling varied even over short vertical distances. Further analyses indicated a strong depth-related covariation of community composition with a subset of 12 environmental variables. An analysis of dissolved organic carbon (DOC) quality via ultrahigh resolution mass spectrometry suggested that the SCZ was generally a low-carbon environment, with the relative portion of labile DOC decreasing and that of recalcitrant DOC increasing along the depth, selecting microbes from copiotrophs to oligotrophs and also impacting the microbial metabolic potential in the carbon cycle. Our study demonstrates that sediment geochemistry and hydrogeology are vital in the selection of distinct microbial populations and metabolism in the SCZ. IMPORTANCE In this study, we explored the links between geochemical parameters, microbial community structure and metabolic potential across the depth of sediment, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone. Our results revealed that microbes in the terrestrial subsurface can be highly localized, with communities rarely being interconnected along the depth. Overall, our research demonstrates that sediment geochemistry and hydrogeology are vital in the selection of distinct microbial populations and metabolic potential in different depths of subsurface terrestrial sediment. Such studies correlating microbial community analyses and geochemistry analyses, including high resolution mass spectrometry analyses of natural organic carbon, will further the fundamental understanding of microbial ecology and biogeochemistry in subsurface terrestrial ecosystems and will benefit the future development of predictive models on nutrient turnover in these environments.

Decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA in raw sewage from university dormitories.

Introduction: Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA has been frequently detected in sewage from many university dormitories to inform public health decisions during the COVID-19 pandemic, a clear understanding of SARS-CoV-2 RNA persistence in site-specific raw sewage is still lacking. To investigate the SARS-CoV-2 RNA persistence, a field trial was conducted in the University of Tennessee dormitories raw sewage, similar to municipal wastewater. Methods: The decay of enveloped SARS-CoV-2 RNA and non-enveloped Pepper mild mottle virus (PMMoV) RNA was investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in raw sewage at 4°C and 20°C. Results: Temperature, followed by the concentration level of SARS-CoV-2 RNA, was the most significant factors that influenced the first-order decay rate constants (k) of SARS-CoV-2 RNA. The mean k values of SARS-CoV-2 RNA were 0.094 day-1 at 4°C and 0.261 day-1 at 20°C. At high-, medium-, and low-concentration levels of SARS-CoV-2 RNA, the mean k values were 0.367, 0.169, and 0.091 day-1, respectively. Furthermore, there was a statistical difference between the decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA at different temperature conditions. Discussion: The first decay rates for both temperatures were statistically comparable for SARS-CoV-2 RNA, which showed sensitivity to elevated temperatures but not for PMMoV RNA. This study provides evidence for the persistence of viral RNA in site-specific raw sewage at different temperature conditions and concentration levels.

Characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer.

The processing of sediment to accurately characterize the spatially-resolved depth profiles of geophysical and geochemical properties along with signatures of microbial density and activity remains a challenge especially in complex contaminated areas. This study processed cores from two sediment boreholes from background and contaminated core sediments and surrounding groundwater. Fresh core sediments were compared by depth to capture the changes in sediment structure, sediment minerals, biomass, and pore water geochemistry in terms of major and trace elements including pollutants, cations, anions, and organic acids. Soil porewater samples were matched to groundwater level, flow rate, and preferential flows and compared to homogenized groundwater-only samples from neighboring monitoring wells. Groundwater analysis of nearby wells only revealed high sulfate and nitrate concentrations while the same analysis using sediment pore water samples with depth was able to suggest areas high in sulfate- and nitrate-reducing bacteria based on their decreased concentration and production of reduced by-products that could not be seen in the groundwater samples. Positive correlations among porewater content, total organic carbon, trace metals and clay minerals revealed a more complicated relationship among contaminant, sediment texture, groundwater table, and biomass. The fluctuating capillary interface had high concentrations of Fe and Mn-oxides combined with trace elements including U, Th, Sr, Ba, Cu, and Co. This suggests the mobility of potentially hazardous elements, sediment structure, and biogeochemical factors are all linked together to impact microbial communities, emphasizing that solid interfaces play an important role in determining the abundance of bacteria in the sediments.

Small and mighty: adaptation of superphylum Patescibacteria to groundwater environment drives their genome simplicity.

BACKGROUND: The newly defined superphylum Patescibacteria such as Parcubacteria (OD1) and Microgenomates (OP11) has been found to be prevalent in groundwater, sediment, lake, and other aquifer environments. Recently increasing attention has been paid to this diverse superphylum including > 20 candidate phyla (a large part of the candidate phylum radiation, CPR) because it refreshed our view of the tree of life. However, adaptive traits contributing to its prevalence are still not well known. RESULTS: Here, we investigated the genomic features and metabolic pathways of Patescibacteria in groundwater through genome-resolved metagenomics analysis of > 600 Gbp sequence data. We observed that, while the members of Patescibacteria have reduced genomes (~ 1 Mbp) exclusively, functions essential to growth and reproduction such as genetic information processing were retained. Surprisingly, they have sharply reduced redundant and nonessential functions, including specific metabolic activities and stress response systems. The Patescibacteria have ultra-small cells and simplified membrane structures, including flagellar assembly, transporters, and two-component systems. Despite the lack of CRISPR viral defense, the bacteria may evade predation through deletion of common membrane phage receptors and other alternative strategies, which may explain the low representation of prophage proteins in their genomes and lack of CRISPR. By establishing the linkages between bacterial features and the groundwater environmental conditions, our results provide important insights into the functions and evolution of this CPR group. CONCLUSIONS: We found that Patescibacteria has streamlined many functions while acquiring advantages such as avoiding phage invasion, to adapt to the groundwater environment. The unique features of small genome size, ultra-small cell size, and lacking CRISPR of this large lineage are bringing new understandings on life of Bacteria. Our results provide important insights into the mechanisms for adaptation of the superphylum in the groundwater environments, and demonstrate a case where less is more, and small is mighty.

Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes.

Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses.IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.

Comparison of Thaumarchaeotal populations from four deep sea basins.

The nitrogen cycle in the marine environment is strongly affected by ammonia-oxidizing Thaumarchaeota. In some marine settings, Thaumarchaeotes can comprise a large percentage of the prokaryotic population. To better understand the biogeographic patterns of Thaumarchaeotes, we sought to investigate differences in their abundance and phylogenetic diversity between geographically distinct basins. Samples were collected from four marine basins (The Caspian Sea, the Great Australian Bight, and the Central and Eastern Mediterranean). The concentration of bacterial and archaeal 16S rRNA genes and archaeal amoA genes were assessed using qPCR. Minimum entropy decomposition was used to elucidate the fine-scale diversity of Thaumarchaeotes. We demonstrated that there were significant differences in the abundance and diversity of Thaumarchaeotes between these four basins. The diversity of Thaumarchaeotal oligotypes differed between basins with many oligotypes only present in one of the four basins, which suggests that their distribution showed biogeographic patterning. There were also significant differences in Thaumarchaeotal community structure between these basins. This would suggest that geographically distant, yet geochemically similar basins may house distinct Thaumarchaeaotal populations. These findings suggest that Thaumarchaeota are very diverse and that biogeography in part contributes in determining the diversity and distribution of Thaumarchaeotes.

Natural bacterial communities serve as quantitative geochemical biosensors.

UNLABELLED: Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. IMPORTANCE: Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.

Microbial community composition and diversity in Caspian Sea sediments.

The Caspian Sea is heavily polluted due to industrial and agricultural effluents as well as extraction of oil and gas reserves. Microbial communities can influence the fate of contaminants and nutrients. However, insight into the microbial ecology of the Caspian Sea significantly lags behind other marine systems. Here we describe microbial biomass, diversity and composition in sediments collected from three sampling stations in the Caspian Sea. Illumina sequencing of 16S rRNA genes revealed the presence of a number of known bacterial and archaeal heterotrophs suggesting that organic carbon is a primary factor shaping microbial communities. Surface sediments collected from bottom waters with low oxygen levels were dominated by Gammaproteobacteria while surface sediments collected from bottom waters under hypoxic conditions were dominated by Deltaproteobacteria, specifically sulfate-reducing bacteria. Thaumarchaeota was dominant across all surface sediments indicating that nitrogen cycling in this system is strongly influenced by ammonia-oxidizing archaea. This study provides a baseline assessment that may serve as a point of reference as this system changes or as the efficacy of new remediation efforts are implemented.

The unique chemistry of Eastern Mediterranean water masses selects for distinct microbial communities by depth.

The waters of the Eastern Mediterranean are characterized by unique physical and chemical properties within separate water masses occupying different depths. Distinct water masses are present throughout the oceans, which drive thermohaline circulation. These water masses may contain specific microbial assemblages. The goal of this study was to examine the effect of physical and geological phenomena on the microbial community of the Eastern Mediterranean water column. Chemical measurements were combined with phospholipid fatty acid (PLFA) analysis and high-throughput 16S rRNA sequencing to characterize the microbial community in the water column at five sites. We demonstrate that the chemistry and microbial community of the water column were stratified into three distinct water masses. The salinity and nutrient concentrations vary between these water masses. Nutrient concentrations increased with depth, and salinity was highest in the intermediate water mass. Our PLFA analysis indicated different lipid classes were abundant in each water mass, suggesting that distinct groups of microbes inhabit these water masses. 16S rRNA gene sequencing confirmed the presence of distinct microbial communities in each water mass. Taxa involved in autotrophic nitrogen cycling were enriched in the intermediate water mass suggesting that microbes in this water mass may be important to the nitrogen cycle of the Eastern Mediterranean. The Eastern Mediterranean also contains numerous active hydrocarbon seeps. We sampled above the North Alex Mud Volcano, in order to test the effect of these geological features on the microbial community in the adjacent water column. The community in the waters overlaying the mud volcano was distinct from other communities collected at similar depths and was enriched in known hydrocarbon degrading taxa. Our results demonstrate that physical phenomena such stratification as well as geological phenomena such as mud volcanoes strongly affect microbial community structure in the Eastern Mediterranean water column.

Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill.

The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of ∼4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using (14)C-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, both of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of (14)C-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)'s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem.

Microbial community response to addition of polylactate compounds to stimulate hexavalent chromium reduction in groundwater.

To evaluate the efficacy of bioimmobilization of Cr(VI) in groundwater at the Department of Energy Hanford site, we conducted a series of microcosm experiments using a range of commercial electron donors with varying degrees of lactate polymerization (polylactate). These experiments were conducted using Hanford Formation sediments (coarse sand and gravel) immersed in Hanford groundwater, which were amended with Cr(VI) and several types of lactate-based electron donors (Hydrogen Release Compound, HRC; primer-HRC, pHRC; extended release HRC) and the polylactate-cysteine form (Metal Remediation Compound, MRC). The results showed that polylactate compounds stimulated an increase in bacterial biomass and activity to a greater extent than sodium lactate when applied at equivalent carbon concentrations. At the same time, concentrations of headspace hydrogen and methane increased and correlated with changes in the microbial community structure. Enrichment of Pseudomonas spp. occurred with all lactate additions, and enrichment of sulfate-reducing Desulfosporosinus spp. occurred with almost complete sulfate reduction. The results of these experiments demonstrate that amendment with the pHRC and MRC forms result in effective removal of Cr(VI) from solution most likely by both direct (enzymatic) and indirect (microbially generated reductant) mechanisms.

Use of immunomagnetic separation for the detection of Desulfovibrio vulgaris from environmental samples.

Immunomagnetic separation (IMS) has proved highly efficient for recovering microorganisms from heterogeneous samples. Current investigation targeted the separation of viable cells of the sulfate-reducing bacterium, Desulfovibrio vulgaris. Streptavidin-coupled paramagnetic beads and biotin labeled antibodies raised against surface antigens of this microorganism were used to capture D. vulgaris cells in both bioreactor grown laboratory samples and from extremely low-biomass environmental soil and subsurface drilling samples. Initial studies on detection, recovery efficiency and viability for IMS were performed with laboratory grown D. vulgaris cells using various cell densities. Efficiency of cell isolation and recovery (i.e., release of the microbial cells from the beads following separation) was followed by microscopic imaging and acridine orange direct counts (AODC). Excellent recovery efficiency encouraged the use of IMS to capture Desulfovibrio spp. cells from low-biomass environmental samples. The environmental samples were obtained from a radionuclide-contaminated site in Germany and the chromium (VI)-contaminated Hanford site, an ongoing bioremediation project of the U.S. Department of Energy. Field deployable IMS technology may greatly facilitate environmental sampling and bioremediation process monitoring and enable transcriptomics and proteomics/metabolomics-based studies directly on cells collected from the field.

Deep-sea oil plume enriches indigenous oil-degrading bacteria.

The biological effects and expected fate of the vast amount of oil in the Gulf of Mexico from the Deepwater Horizon blowout are unknown owing to the depth and magnitude of this event. Here, we report that the dispersed hydrocarbon plume stimulated deep-sea indigenous γ-Proteobacteria that are closely related to known petroleum degraders. Hydrocarbon-degrading genes coincided with the concentration of various oil contaminants. Changes in hydrocarbon composition with distance from the source and incubation experiments with environmental isolates demonstrated faster-than-expected hydrocarbon biodegradation rates at 5°C. Based on these results, the potential exists for intrinsic bioremediation of the oil plume in the deep-water column without substantial oxygen drawdown.

Impact of elevated nitrate on sulfate-reducing bacteria: a comparative study of Desulfovibrio vulgaris.

Sulfate-reducing bacteria have been extensively studied for their potential in heavy-metal bioremediation. However, the occurrence of elevated nitrate in contaminated environments has been shown to inhibit sulfate reduction activity. Although the inhibition has been suggested to result from the competition with nitrate-reducing bacteria, the possibility of direct inhibition of sulfate reducers by elevated nitrate needs to be explored. Using Desulfovibrio vulgaris as a model sulfate-reducing bacterium, functional genomics analysis reveals that osmotic stress contributed to growth inhibition by nitrate as shown by the upregulation of the glycine/betaine transporter genes and the relief of nitrate inhibition by osmoprotectants. The observation that significant growth inhibition was effected by 70 mM NaNO(3) but not by 70 mM NaCl suggests the presence of inhibitory mechanisms in addition to osmotic stress. The differential expression of genes characteristic of nitrite stress responses, such as the hybrid cluster protein gene, under nitrate stress condition further indicates that nitrate stress response by D. vulgaris was linked to components of both osmotic and nitrite stress responses. The involvement of the oxidative stress response pathway, however, might be the result of a more general stress response. Given the low similarities between the response profiles to nitrate and other stresses, less-defined stress response pathways could also be important in nitrate stress, which might involve the shift in energy metabolism. The involvement of nitrite stress response upon exposure to nitrate may provide detoxification mechanisms for nitrite, which is inhibitory to sulfate-reducing bacteria, produced by microbial nitrate reduction as a metabolic intermediate and may enhance the survival of sulfate-reducing bacteria in environments with elevated nitrate level.

Response of Desulfovibrio vulgaris to alkaline stress.

The 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).

Cell-wide responses to low-oxygen exposure in Desulfovibrio vulgaris Hildenborough.

The responses of the anaerobic, sulfate-reducing organism Desulfovibrio vulgaris Hildenborough to low-oxygen exposure (0.1% O(2)) were monitored via transcriptomics and proteomics. Exposure to 0.1% O(2) caused a decrease in the growth rate without affecting viability. Concerted upregulation of the predicted peroxide stress response regulon (PerR) genes was observed in response to the 0.1% O(2) exposure. Several of the candidates also showed increases in protein abundance. Among the remaining small number of transcript changes was the upregulation of the predicted transmembrane tetraheme cytochrome c(3) complex. Other known oxidative stress response candidates remained unchanged during the low-O(2) exposure. To fully understand the results of the 0.1% O(2) exposure, transcriptomics and proteomics data were collected for exposure to air using a similar experimental protocol. In contrast to the 0.1% O(2) exposure, air exposure was detrimental to both the growth rate and viability and caused dramatic changes at both the transcriptome and proteome levels. Interestingly, the transcripts of the predicted PerR regulon genes were downregulated during air exposure. Our results highlight the differences in the cell-wide responses to low and high O(2) levels in D. vulgaris and suggest that while exposure to air is highly detrimental to D. vulgaris, this bacterium can successfully cope with periodic exposure to low O(2) levels in its environment.

Application of a high-density oligonucleotide microarray approach to study bacterial population dynamics during uranium reduction and reoxidation.

Reduction of soluble uranium U(VI) to less-soluble uranium U(IV) is a promising approach to minimize migration from contaminated aquifers. It is generally assumed that, under constant reducing conditions, U(IV) is stable and immobile; however, in a previous study, we documented reoxidation of U(IV) under continuous reducing conditions (Wan et al., Environ. Sci. Technol. 2005, 39:6162-6169). To determine if changes in microbial community composition were a factor in U(IV) reoxidation, we employed a high-density phylogenetic DNA microarray (16S microarray) containing 500,000 probes to monitor changes in bacterial populations during this remediation process. Comparison of the 16S microarray with clone libraries demonstrated successful detection and classification of most clone groups. Analysis of the most dynamic groups of 16S rRNA gene amplicons detected by the 16S microarray identified five clusters of bacterial subfamilies responding in a similar manner. This approach demonstrated that amplicons of known metal-reducing bacteria such as Geothrix fermentans (confirmed by quantitative PCR) and those within the Geobacteraceae were abundant during U(VI) reduction and did not decline during the U(IV) reoxidation phase. Significantly, it appears that the observed reoxidation of uranium under reducing conditions occurred despite elevated microbial activity and the consistent presence of metal-reducing bacteria. High-density phylogenetic microarrays constitute a powerful tool, enabling the detection and monitoring of a substantial portion of the microbial population in a routine, accurate, and reproducible manner.

Study of nitrate stress in Desulfovibrio vulgaris Hildenborough using iTRAQ proteomics.

The response of Desulfovibrio vulgaris Hildenborough (DvH), a sulphate-reducing bacterium, to nitrate stress was examined using quantitative proteomic analysis. DvH was stressed with 105 mM sodium nitrate (NaNO(3)), a level that caused a 50% inhibition in growth. The protein profile of stressed cells was compared with that of cells grown in the absence of nitrate using the iTRAQ peptide labelling strategy and tandem liquid chromatography separation coupled with mass spectrometry (quadrupole time-of-flight) detection. A total of 737 unique proteins were identified by two or more peptides, representing 22% of the total DvH proteome and spanning every functional category. The results indicate that this was a mild stress, as proteins involved in central metabolism and the sulphate reduction pathway were unperturbed. Proteins involved in the nitrate reduction pathway increased. Increases seen in transport systems for proline, glycine-betaine and glutamate indicate that the NaNO(3) exposure led to both salt stress and nitrate stress. Up-regulation observed in oxidative stress response proteins (Rbr, RbO, etc.) and a large number of ABC transport systems as well as in iron-sulphur-cluster-containing proteins, however, appear to be specific to nitrate exposure. Finally, a number of hypothetical proteins were among the most significant changers, indicating that there may be unknown mechanisms initiated upon nitrate stress in DvH.

Salt stress in Desulfovibrio vulgaris Hildenborough: an integrated genomics approach.

The ability of Desulfovibrio vulgaris Hildenborough to reduce, and therefore contain, toxic and radioactive metal waste has made all factors that affect the physiology of this organism of great interest. Increased salinity is an important and frequent fluctuation faced by D. vulgaris in its natural habitat. In liquid culture, exposure to excess salt resulted in striking elongation of D. vulgaris cells. Using data from transcriptomics, proteomics, metabolite assays, phospholipid fatty acid profiling, and electron microscopy, we used a systems approach to explore the effects of excess NaCl on D. vulgaris. In this study we demonstrated that import of osmoprotectants, such as glycine betaine and ectoine, is the primary mechanism used by D. vulgaris to counter hyperionic stress. Several efflux systems were also highly up-regulated, as was the ATP synthesis pathway. Increases in the levels of both RNA and DNA helicases suggested that salt stress affected the stability of nucleic acid base pairing. An overall increase in the level of branched fatty acids indicated that there were changes in cell wall fluidity. The immediate response to salt stress included up-regulation of chemotaxis genes, although flagellar biosynthesis was down-regulated. Other down-regulated systems included lactate uptake permeases and ABC transport systems. The results of an extensive NaCl stress analysis were compared with microarray data from a KCl stress analysis, and unlike many other bacteria, D. vulgaris responded similarly to the two stresses. Integration of data from multiple methods allowed us to develop a conceptual model for the salt stress response in D. vulgaris that can be compared to those in other microorganisms.