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Methylmercury (MeHg) is a bioaccumulative toxic contaminant in many ecosystems, but factors governing its production are poorly understood. Recent work has shown that the anaerobic microbial conversion of mercury (Hg) to MeHg requires the Hg-methylation genes hgcAB and that these genes can be used as biomarkers in PCR-based estimators of Hg-methylator abundance. In an effort to determine reliable methods for assessing hgcA abundance and diversity and linking them to MeHg concentrations, multiple approaches were compared including metagenomic shotgun sequencing, 16S rRNA gene pyrosequencing and cloning/sequencing hgcAB gene products. Hg-methylator abundance was also determined by quantitative hgcA qPCR amplification and metaproteomics for comparison to the above measurements. Samples from eight sites were examined covering a range of total Hg (HgT; 0.03-14 mg kg -1 dry wt. soil) and MeHg (0.05-27 μg kg -1 dry wt. soil) concentrations. In the metagenome and amplicon sequencing of hgcAB diversity, the Deltaproteobacteria were the dominant Hg-methylators while Firmicutes and methanogenic Archaea were typically ∼50% less abundant. This was consistent with metaproteomics estimates where the Deltaproteobacteria were steadily higher. The 16S rRNA gene pyrosequencing did not have sufficient resolution to identify hgcAB + species. Metagenomic and hgcAB results were similar for Hg-methylator diversity and clade-specific qPCR-based approaches for hgcA are only appropriate when comparing the abundance of a particular clade across various samples. Weak correlations between Hg-methylating bacteria and soil Hg concentrations were observed for similar environmental samples, but overall total Hg and MeHg concentrations poorly correlated with Hg-cycling genes.

Desulfovibrio alaskensis G20 biofilms were cultivated on 316 steel, 1018 steel, or borosilicate glass under steady-state conditions in electron-acceptor limiting (EAL) and electron-donor limiting (EDL) conditions with lactate and sulfate in a defined medium. Increased corrosion was observed on 1018 steel under EDL conditions compared to 316 steel, and biofilms on 1018 carbon steel under the EDL condition had at least twofold higher corrosion rates compared to the EAL condition. Protecting the 1018 metal coupon from biofilm colonization significantly reduced corrosion, suggesting that the corrosion mechanism was enhanced through attachment between the material and the biofilm. Metabolomic mass spectrometry analyses demonstrated an increase in a flavin-like molecule under the 1018 EDL condition and sulfonates under the 1018 EAL condition. These data indicate the importance of S-cycling under the EAL condition, and that the EDL is associated with increased biocorrosion via indirect extracellular electron transfer mediated by endogenously produced flavin-like molecules.

Phylogenetic and geological evidence supports the hypothesis that life on Earth originated in thermal environments and conserved energy through methanogenesis or sulfur reduction. Here we describe two populations of the deeply rooted archaeal phylum Korarchaeota, which were retrieved from the metagenome of a circumneutral, suboxic hot spring that contains high levels of sulfate, sulfide, methane, hydrogen and carbon dioxide. One population is closely related to 'Candidatus Korarchaeum cryptofilum OPF8', while the more abundant korarchaeote, 'Candidatus Methanodesulfokores washburnensis', contains genes that are necessary for anaerobic methane and dissimilatory sulfur metabolisms. Phylogenetic and ancestral reconstruction analyses suggest that methane metabolism originated in the Korarchaeota, whereas genes for dissimilatory sulfite reduction were horizontally transferred to the Korarchaeota from the Firmicutes. Interactions among enzymes involved in both metabolisms could facilitate exergonic, sulfite-dependent, anaerobic oxidation of methane to methanol; alternatively, 'Ca. M. washburnensis' could conduct methanogenesis and sulfur reduction independently. Metabolic reconstruction suggests that 'Ca. M. washburnensis' is a mixotroph, capable of amino acid uptake, assimilation of methane-derived carbon and/or CO 2 fixation by archaeal type III-b RuBisCO for scavenging ribose carbon. Our findings link anaerobic methane metabolism and dissimilatory sulfur reduction within a single deeply rooted archaeal population and have implications for the evolution of these traits throughout the Archaea.

The Carpathian Basin is a lowland plain located mainly in Hungary. Due to the nature of the bedrock, alluvial deposits, and a bowl shape, many lakes and ponds of the area are characterized by high alkalinity. In this study, we characterized temporal changes in eukaryal and bacterial community dynamics with high throughput sequencing and relate the changes to environmental conditions in Lake Velence located in Fejér county, Hungary. The sampled Lake Velence microbial populations (algal and bacterial) were analyzed to identify potential correlations with other community members and environmental parameters at six timepoints over 6 weeks in the Spring of 2012. Correlations between community members suggest a positive relationship between certain algal and bacterial populations (e.g. Chlamydomondaceae with Actinobacteria and Acidobacteria), while other correlations allude to changes in these relationships over time. During the study, high nitrogen availability may have favored non-nitrogen fixing cyanobacteria, such as the toxin-producing Microcystis aeruginosa, and the eutrophic effect may have been exacerbated by high phosphorus availability as well as the high calcium and magnesium content of the Carpathian Basin bedrock, potentially fostering exopolymer production and cell aggregation. Cyanobacterial bloom formation could have a negative environmental impact on other community members and potentially affect overall water quality as well as recreational activities. To our knowledge, this is the first prediction for relationships between photoautotrophic eukaryotes and bacteria from an alkaline, Hungarian lake.

Resource scarcity is a common stress in nature and has a major impact on microbial physiology. This review highlights microbial acclimations to resource scarcity, focusing on resource investment strategies for chemoheterotrophs from the molecular level to the pathway level. Competitive resource allocation strategies often lead to a phenotype known as overflow metabolism; the resulting overflow byproducts can stabilize cooperative interactions in microbial communities and can lead to cross-feeding consortia. These consortia can exhibit emergent properties such as enhanced resource usage and biomass productivity. The literature distilled here draws parallels between in silico and laboratory studies and ties them together with ecological theories to better understand microbial stress responses and mutualistic consortia functioning., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Desulfovibrio spp. are capable of heavy metal reduction and are well-studied systems for understanding metal fate and transport in anaerobic environments. Desulfovibrio vulgaris Hildenborough was grown under environmentally relevant conditions (i.e., temperature, nutrient limitation) to elucidate the impacts on Cr(VI) reduction on cellular physiology. Growth at 20 °C was slower than 30 °C and the presence of 50 μM Cr(VI) caused extended lag times for all conditions, but once growth resumed the growth rate was similar to that without Cr(VI). Cr(VI) reduction rates were greatly diminished at 20 °C for both 50 and 100 μM Cr(VI), particularly for the electron acceptor limited (EAL) condition in which Cr(VI) reduction was much slower, the growth lag much longer (200 h), and viability decreased compared to balanced (BAL) and electron donor limited (EDL) conditions. When sulfate levels were increased in the presence of Cr(VI), cellular responses improved via a shorter lag time to growth. Similar results were observed between the different resource (donor/acceptor) ratio conditions when the sulfate levels were normalized (10 mM), and these results indicated that resource ratio (donor/acceptor) impacted D. vulgaris response to Cr(VI) and not merely sulfate limitation. The results suggest that temperature and resource ratios greatly impacted the extent of Cr(VI) toxicity, Cr(VI) reduction, and the subsequent cellular health via Cr(VI) influx and overall metabolic rate. The results also emphasized the need to perform experiments at lower temperatures with nutrient limitation to make accurate predictions of heavy metal reduction rates as well as physiological states in the environment.

Contamination from anthropogenic activities has significantly impacted Earth's biosphere. However, knowledge about how environmental contamination affects the biodiversity of groundwater microbiomes and ecosystem functioning remains very limited. Here, we used a comprehensive functional gene array to analyze groundwater microbiomes from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN), representing a wide pH range and uranium, nitrate, and other contaminants. We hypothesized that the functional diversity of groundwater microbiomes would decrease as environmental contamination (e.g., uranium or nitrate) increased or at low or high pH, while some specific populations capable of utilizing or resistant to those contaminants would increase, and thus, such key microbial functional genes and/or populations could be used to predict groundwater contamination and ecosystem functioning. Our results indicated that functional richness/diversity decreased as uranium (but not nitrate) increased in groundwater. In addition, about 5.9% of specific key functional populations targeted by a comprehensive functional gene array (GeoChip 5) increased significantly ( P < 0.05) as uranium or nitrate increased, and their changes could be used to successfully predict uranium and nitrate contamination and ecosystem functioning. This study indicates great potential for using microbial functional genes to predict environmental contamination and ecosystem functioning. IMPORTANCE Disentangling the relationships between biodiversity and ecosystem functioning is an important but poorly understood topic in ecology. Predicting ecosystem functioning on the basis of biodiversity is even more difficult, particularly with microbial biomarkers. As an exploratory effort, this study used key microbial functional genes as biomarkers to provide predictive understanding of environmental contamination and ecosystem functioning. The results indicated that the overall functional gene richness/diversity decreased as uranium increased in groundwater, while specific key microbial guilds increased significantly as uranium or nitrate increased. These key microbial functional genes could be used to successfully predict environmental contamination and ecosystem functioning. This study represents a significant advance in using functional gene markers to predict the spatial distribution of environmental contaminants and ecosystem functioning toward predictive microbial ecology, which is an ultimate goal of microbial ecology., (Copyright © 2018 He et al.)

Biofilms of sulfate-reducing bacteria (SRB) are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here we show that two supposedly identical wild-type cultures of the SRB Desulfovibrio vulgaris Hildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS), was sufficient to eliminate biofilm formation in D. vulgaris Hildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient) was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered. IMPORTANCE The growth of bacteria attached to a surface (i.e., biofilm), specifically biofilms of sulfate-reducing bacteria, has a profound impact on the economy of developed nations due to steel and concrete corrosion in industrial pipelines and processing facilities. Furthermore, the presence of sulfate-reducing bacteria in oil wells causes oil souring from sulfide production, resulting in product loss, a health hazard to workers, and ultimately abandonment of wells. Identification of the required genes is a critical step for determining the mechanism of biofilm formation by sulfate reducers. Here, the transporter by which putative biofilm structural proteins are exported from sulfate-reducing Desulfovibrio vulgaris Hildenborough cells was discovered, and a single nucleotide change within the gene coding for this transporter was found to be sufficient to completely stop formation of biofilm., (Copyright © 2017 De León et al.)

Recent discoveries have shown that the marker gene for anaerobic methane cycling (mcrA) is more widespread in the Archaea than previously thought. However, it remains unclear whether novel mcrA genes associated with the Bathyarchaeota and Verstraetearchaeota are distributed across diverse environments. We examined two geochemically divergent but putatively methanogenic regions of Yellowstone National Park to investigate whether deeply-rooted archaea possess and express novel mcrA genes in situ. Small-subunit (SSU) rRNA gene analyses indicated that Bathyarchaeota were predominant in seven of ten sediment layers, while the Verstraetearchaeota and Euryarchaeota occurred in lower relative abundance. Targeted amplification of novel mcrA genes suggested that diverse taxa contribute to alkane cycling in geothermal environments. Two deeply-branching mcrA clades related to Bathyarchaeota were identified, while highly abundant verstraetearchaeotal mcrA sequences were also recovered. In addition, detection of SSU rRNA and mcrA transcripts from one hot spring suggested that predominant Bathyarchaeota were also active, and that methane cycling genes are expressed by the Euryarchaeota, Verstraetearchaeota, and an unknown lineage basal to the Bathyarchaeota. These findings greatly expand the diversity of the key marker gene for anaerobic alkane cycling and outline the need for greater understanding of the functional capacity and phylogenetic affiliation of novel mcrA variants.

Alkaliphilus metalliredigens strain QYMF is an anaerobic, alkaliphilic, and metal-reducing bacterium associated with phylum Firmicutes QYMF was isolated from alkaline borax leachate ponds. The genome sequence will help elucidate the role of metal-reducing microorganisms under alkaline environments, a capability that is not commonly observed in metal respiring-microorganisms., (Copyright © 2016 Hwang et al.)

Active data screening is an integral part of many scientific activities, and mobile technologies have greatly facilitated this process by minimizing the reliance on large hardware instrumentation. In order to meet with the increasingly growing field of metabolomics and heavy workload of data processing, we designed the first remote metabolomic data screening platform for mobile devices. Two mobile applications (apps), XCMS Mobile and METLIN Mobile, facilitate access to XCMS and METLIN, which are the most important components in the computer-based XCMS Online platforms. These mobile apps allow for the visualization and analysis of metabolic data throughout the entire analytical process. Specifically, XCMS Mobile and METLIN Mobile provide the capabilities for remote monitoring of data processing, real time notifications for the data processing, visualization and interactive analysis of processed data (e.g., cloud plots, principle component analysis, box-plots, extracted ion chromatograms, and hierarchical cluster analysis), and database searching for metabolite identification. These apps, available on Apple iOS and Google Android operating systems, allow for the migration of metabolomic research onto mobile devices for better accessibility beyond direct instrument operation. The utility of XCMS Mobile and METLIN Mobile functionalities was developed and is demonstrated here through the metabolomic LC-MS analyses of stem cells, colon cancer, aging, and bacterial metabolism.

Much research has been invested into engineering microorganisms to perform desired biotransformations; nonetheless, these efforts frequently fall short of expected results due to the unforeseen effects of biofeedback regulation and functional incompatibility. In nature, metabolic function is compartmentalized into diverse organisms assembled into robust consortia, in which the division of labor is thought to lead to increased community efficiency and productivity. Here we consider whether and how consortia can be designed to perform bioprocesses of interest beyond the metabolic flexibility limitations of a single organism. Advances in post-genomic analysis of microbial consortia and application of high-resolution global measurements now offer the promise of systems-level understanding of how microbial consortia adapt to changes in environmental variables and inputs of carbon and energy. We argue that, when combined with appropriate modeling frameworks, systems-level knowledge can markedly improve our ability to predict the fate and functioning of consortia. Here we articulate our collective perspective on the current and future state of microbial community engineering and control while placing specific emphasis on ecological principles that promote control over community function and emergent properties.

Algal biofuels and valuable co-products are being produced in both open and closed cultivation systems. Growing algae in open pond systems may be a more economical alternative, but this approach allows environmental microorganisms to colonize the pond and potentially infect or outcompete the algal "crop." In this study, we monitored the microbial community of an outdoor, open raceway pond inoculated with a high lipid-producing alkaliphilic alga, Chlorella vulgaris BA050. The strain C. vulgaris BA050 was previously isolated from Soap Lake, Washington, a system characterized by a high pH (∼9.8). An outdoor raceway pond (200 L) was inoculated with C. vulgaris and monitored for 10 days and then the culture was transferred to a 2,000 L raceway pond and cultivated for an additional 6 days. Community DNA samples were collected over the 16-day period in conjunction with water chemistry analyses and cell counts. Universal primers for the SSU rRNA gene sequences for Eukarya, Bacteria, and Archaea were used for barcoded pyrosequence determination. The environmental parameters that most closely correlated with C. vulgaris abundance were pH and phosphate. Community analyses indicated that the pond system remained dominated by the Chlorella population (93% of eukaryotic sequences), but was also colonized by other microorganisms. Bacterial sequence diversity increased over time while archaeal sequence diversity declined over the same time period. Using SparCC co-occurrence network analysis, a positive correlation was observed between C. vulgaris and Pseudomonas sp. throughout the experiment, which may suggest a symbiotic relationship between the two organisms. The putative relationship coupled with high pH may have contributed to the success of C. vulgaris. The characterization of the microbial community dynamics of an alkaliphilic open pond system provides significant insight into open pond systems that could be used to control photoautotrophic biomass productivity in an open, non-sterile environment.

Cr(VI) is a widespread environmental contaminant that is highly toxic and soluble. Previous work indicated that a one-time amendment of polylactate hydrogen-release compound (HRC) reduced groundwater Cr(VI) concentrations for >3.5 years at a contaminated aquifer; however, microbial communities responsible for Cr(VI) reduction are poorly understood. In this study, we hypothesized that HRC amendment would significantly change the composition and structure of groundwater microbial communities, and that the abundance of key functional genes involved in HRC degradation and electron acceptor reduction would increase long-term in response to this slowly degrading, complex substrate. To test these hypotheses, groundwater microbial communities were monitored after HRC amendment for >1 year using a comprehensive functional gene microarray. The results showed that the overall functional composition and structure of groundwater microbial communities underwent sequential shifts after HRC amendment. Particularly, the abundance of functional genes involved in acetate oxidation, denitrification, dissimilatory nitrate reduction, metal reduction, and sulfate reduction significantly increased. The overall community dynamics was significantly correlated with changes in groundwater concentrations of microbial biomass, acetate, NO3-, Cr(VI), Fe(II) and SO4(2-). Our results suggest that HRC amendment primarily stimulated key functional processes associated with HRC degradation and reduction of multiple electron acceptors in the aquifer toward long-term Cr(VI) reduction.

To investigate the genetic basis of microbial evolutionary adaptation to salt (NaCl) stress, populations of Desulfovibrio vulgaris Hildenborough (DvH), a sulfate-reducing bacterium important for the biogeochemical cycling of sulfur, carbon and nitrogen, and potentially the bioremediation of toxic heavy metals and radionuclides, were propagated under salt stress or non-stress conditions for 1200 generations. Whole-genome sequencing revealed 11 mutations in salt stress-evolved clone ES9-11 and 14 mutations in non-stress-evolved clone EC3-10. Whole-population sequencing data suggested the rapid selective sweep of the pre-existing polymorphisms under salt stress within the first 100 generations and the slow fixation of new mutations. Population genotyping data demonstrated that the rapid selective sweep of pre-existing polymorphisms was common in salt stress-evolved populations. In contrast, the selection of pre-existing polymorphisms was largely random in EC populations. Consistently, at 100 generations, stress-evolved population ES9 showed improved salt tolerance, namely increased growth rate (2.0-fold), higher biomass yield (1.8-fold) and shorter lag phase (0.7-fold) under higher salinity conditions. The beneficial nature of several mutations was confirmed by site-directed mutagenesis. All four tested mutations contributed to the shortened lag phases under higher salinity condition. In particular, compared with the salt tolerance improvement in ES9-11, a mutation in a histidine kinase protein gene lytS contributed 27% of the growth rate increase and 23% of the biomass yield increase while a mutation in hypothetical gene DVU2472 contributed 24% of the biomass yield increase. Our results suggested that a few beneficial mutations could lead to dramatic improvements in salt tolerance.

To understand patterns of geochemical cycling in pristine versus contaminated groundwater ecosystems, pristine shallow groundwater (FW301) and contaminated groundwater (FW106) samples from the Oak Ridge Integrated Field Research Center (OR-IFRC) were sequenced and compared to each other to determine phylogenetic and metabolic difference between the communities. Proteobacteria (e.g., Burkholderia, Pseudomonas) are the most abundant lineages in the pristine community, though a significant proportion ( >55%) of the community is composed of poorly characterized low abundance (individually <1%) lineages. The phylogenetic diversity of the pristine community contributed to a broader diversity of metabolic networks than the contaminated community. In addition, the pristine community encodes redundant and mostly complete geochemical cycles distributed over multiple lineages and appears capable of a wide range of metabolic activities. In contrast, many geochemical cycles in the contaminated community appear truncated or minimized due to decreased biodiversity and dominance by Rhodanobacter populations capable of surviving the combination of stresses at the site. These results indicate that the pristine site contains more robust and encodes more functional redundancy than the stressed community, which contributes to more efficient nutrient cycling and adaptability than the stressed community.

The genome of Pelosinus fermentans JBW45, isolated from a chromium-contaminated site in Hanford, Washington, USA, has been completed with PacBio sequencing. Nine copies of the rRNA gene operon and multiple transposase genes with identical sequences resulted in breaks in the original draft genome and may suggest genomic instability of JBW45., (Copyright © 2015 De León et al.)

Although many Archaea have AMP-Acs (acetyl-coenzyme A synthetase) and ADP-Acs, the extant methanogenic genus Methanosarcina is the only identified Archaeal genus that can utilize acetate via acetate kinase (Ack) and phosphotransacetylase (Pta). Despite the importance of ack as the potential urkinase in the ASKHA phosphotransferase superfamily, an origin hypothesis does not exist for the acetate kinase in Bacteria, Archaea, or Eukarya. Here we demonstrate that Archaeal AMP-Acs and ADP-Acs contain paralogous ATPase motifs previously identified in Ack, which demonstrate a novel relation between these proteins in Archaea. The identification of ATPase motif conservation and resulting structural features in AMP- and ADP-acetyl-CoA synthetase proteins in this study expand the ASKHA superfamily to include acetyl-CoA synthetase. Additional phylogenetic analysis showed that Pta and MaeB sequences had a common ancestor, and that the Pta lineage within the halophilc archaea was an ancestral lineage. These results suggested that divergence of a duplicated maeB within an ancient halophilic, archaeal lineage formed a putative pta ancestor. These results provide a potential scenario for the establishment of the Ack/Pta pathway and provide novel insight into the evolution of acetate metabolism for all three domains of life.

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., (Copyright © 2015 Smith et al.)

Desulfovibrio carbinoliphilus subsp. oakridgensis FW-101-2B is an anaerobic, organic acid/alcohol-oxidizing, sulfate-reducing δ-proteobacterium. FW-101-2B was isolated from contaminated groundwater at The Field Research Center at Oak Ridge National Lab after in situ stimulation for heavy metal-reducing conditions. The genome will help elucidate the metabolic potential of sulfate-reducing bacteria during uranium reduction., (Copyright © 2015 Ramsay et al.)

We report the genome sequence of Anaeromyxobacter sp. Fw109-5, isolated from nitrate- and uranium-contaminated subsurface sediment of the Oak Ridge Integrated Field-Scale Subsurface Research Challenge (IFC) site, Oak Ridge Reservation, TN. The bacterium's genome sequence will elucidate its physiological potential in subsurface sediments undergoing in situ uranium bioremediation and natural attenuation., (Copyright © 2015 Hwang et al.)

An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. As a result of this unique integration, we can analyze large profiling datasets and simultaneously obtain structural identifications. Validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometry data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level.

Fluorocarbons are lipophobic and non-polar molecules that exhibit remarkable biocompatibility, with applications in liquid ventilation and synthetic blood. The unique properties of these compounds have also enabled mass spectrometry imaging of tissues where the fluorocarbons act as a Teflon-like coating for nanostructured surfaces to assist in desorption/ionization. Here we report fluorinated gold nanoparticles (f-AuNPs) designed to facilitate nanostructure imaging mass spectrometry. Irradiation of f-AuNPs results in the release of the fluorocarbon ligands providing a driving force for analyte desorption. The f-AuNPs allow for the mass spectrometry analysis of both lipophilic and polar (central carbon) metabolites. An important property of AuNPs is that they also act as contrast agents for X-ray microtomography and electron microscopy, a feature we have exploited by infusing f-AuNPs into tissue via fluorocarbon liquids to facilitate multimodal (molecular and anatomical) imaging.

Sulfate-reducing bacteria (SRB) can interact syntrophically with other community members in the absence of sulfate, and interactions with hydrogen-consuming methanogens are beneficial when these archaea consume potentially inhibitory H2 produced by the SRB. A dual continuous culture approach was used to characterize population structure within a syntrophic biofilm formed by the SRB Desulfovibrio vulgaris Hildenborough and the methanogenic archaeum Methanococcus maripaludis. Under the tested conditions, monocultures of D. vulgaris formed thin, stable biofilms, but monoculture M. maripaludis did not. Microscopy of intact syntrophic biofilm confirmed that D. vulgaris formed a scaffold for the biofilm, while intermediate and steady-state images revealed that M. maripaludis joined the biofilm later, likely in response to H2 produced by the SRB. Close interactions in structured biofilm allowed efficient transfer of H2 to M. maripaludis, and H2 was only detected in cocultures with a mutant SRB that was deficient in biofilm formation (ΔpilA). M. maripaludis produced more carbohydrate (uronic acid, hexose, and pentose) as a monoculture compared to total coculture biofilm, and this suggested an altered carbon flux during syntrophy. The syntrophic biofilm was structured into ridges (∼300 × 50 μm) and models predicted lactate limitation at ∼50 μm biofilm depth. The biofilm had structure that likely facilitated mass transfer of H2 and lactate, yet maximized biomass with a more even population composition (number of each organism) when compared to the bulk-phase community. Total biomass protein was equivalent in lactate-limited and lactate-excess conditions when a biofilm was present, but in the absence of biofilm, total biomass protein was significantly reduced. The results suggest that multispecies biofilms create an environment conducive to resource sharing, resulting in increased biomass retention, or carrying capacity, for cooperative populations.

We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibrio vulgaris Hildenborough that are predicted to function via two-component signaling. Using purified proteins, we examined cyclic-di-GMP (c-di-GMP) production or turnover in vitro of all eight proteins. The two RRs containing only GGDEF domains (DVU2067, DVU0636) demonstrated c-di-GMP production activity in vitro. Of the remaining proteins, three RRs with HD-GYP domains (DVU0722, DVUA0086, and DVU2933) were confirmed to be Mn(2+)-dependent phosphodiesterases (PDEs) in vitro and converted c-di-GMP to its linear form, pGpG. DVU0408, containing both c-di-GMP production (GGDEF) and degradation domains (EAL), showed c-di-GMP turnover activity in vitro also with production of pGpG. No c-di-GMP related activity could be assigned to the RR DVU0330, containing a metal-dependent phosphohydrolase HD-OD domain, or to the HD-GYP domain RR, DVU1181. Studies included examining the impact of overexpressed cyclic-di-GMP-modulating RRs in the heterologous host E. coli and led to the identification of one RR, DVU0636, with increased cellulose production. Evaluation of a transposon mutant in DVU0636 indicated that the strain was impaired in biofilm formation and demonstrated an altered carbohydrate:protein ratio relative to the D. vulgaris wild type biofilms. However, grown in liquid lactate/sulfate medium, the DVU0636 transposon mutant showed no growth impairment relative to the wild-type strain. Among the eight candidates, only the transposon disruption mutant in the DVU2067 RR presented a growth defect in liquid culture. Our results indicate that, of the two diguanylate cyclases (DGCs) that function as part of two-component signaling, DVU0636 plays an important role in biofilm formation while the function of DVU2067 has pertinence in planktonic growth.

Regardless of current market conditions and availability of conventional petroleum sources, alternatives are needed to circumvent future economic and environmental impacts from continued exploration and harvesting of conventional hydrocarbons. Diatoms and green algae (microalgae) are eukaryotic photoautotrophs that can utilize inorganic carbon (e.g., CO2) as a carbon source and sunlight as an energy source, and many microalgae can store carbon and energy in the form of neutral lipids. In addition to accumulating useful precursors for biofuels and chemical feed stocks, the use of autotrophic microorganisms can further contribute to reduced CO2 emissions through utilization of atmospheric CO2. Because of the inherent connection between carbon, nitrogen, and phosphorus in biological systems, macronutrient deprivation has been proven to significantly enhance lipid accumulation in different diatom and algae species. However, much work is needed to understand the link between carbon, nitrogen, and phosphorus in controlling resource allocation at different levels of biological resolution (cellular versus ecological). An improved understanding of the relationship between the effects of N, P, and micronutrient availability on carbon resource allocation (cell growth versus lipid storage) in microalgae is needed in conjunction with life cycle analysis. This mini-review will briefly discuss the current literature on the use of nutrient deprivation and other conditions to control and optimize microalgal growth in the context of cell and lipid accumulation for scale-up processes.

FISH (fluorescence in situ hybridization) is a valuable technique to visualize and quantify localization of different microbial species within biofilms. Biofilm conformation can be altered during typical sample preparation for FISH, which can impact observations in multispecies biofilms, including the relative positions of cells. Here, we describe methods to preserve 3-D structure during FISH for visualization of an anaerobic coculture biofilm of Desulfovibrio vulgaris Hildenborough and Methanococcus maripaludis.

The Heart Lake Geyser Basin (HLGB) is remotely located at the base of Mount Sheridan in southern Yellowstone National Park (YNP), Wyoming, USA and is situated along Witch Creek and the northwestern shore of Heart Lake. Likely because of its location, little is known about the microbial community structure of springs in the HLGB. Bacterial and archaeal populations were monitored via small subunit (SSU) rRNA gene pyrosequencing over 3 years in 3 alkaline (pH 8.5) hot springs with varying temperatures (44°C, 63°C, 75°C). The bacterial populations were generally stable over time, but varied by temperature. The dominant bacterial community changed from moderately thermophilic and photosynthetic members (Cyanobacteria and Chloroflexi) at 44°C to a mixed photosynthetic and thermophilic community (Deinococcus-Thermus) at 63°C and a non-photosynthetic thermophilic community at 75°C. The archaeal community was more variable across time and was predominantly a methanogenic community in the 44 and 63°C springs and a thermophilic community in the 75°C spring. The 75°C spring demonstrated large shifts in the archaeal populations and was predominantly Candidatus Nitrosocaldus, an ammonia-oxidizing crenarchaeote, in the 2007 sample, and almost exclusively Thermofilum or Candidatus Caldiarchaeum in the 2009 sample, depending on SSU rRNA gene region examined. The majority of sequences were dissimilar (≥10% different) to any known organisms suggesting that HLGB possesses numerous new phylogenetic groups that warrant cultivation efforts.

Knowledge of taxis (directed swimming) in the Archaea is currently expanding through identification of novel receptors, effectors, and proteins involved in signal transduction to the flagellar motor. Although the ability for biological cells to sense and swim toward hydrogen gas has been hypothesized for many years, this capacity has yet to be observed and demonstrated. Here we show that the average swimming velocity increases in the direction of a source of hydrogen gas for the methanogen, Methanococcus maripaludis using a capillary assay with anoxic gas-phase control and time-lapse microscopy. The results indicate that a methanogen couples motility to hydrogen concentration sensing and is the first direct observation of hydrogenotaxis in any domain of life. Hydrogenotaxis represents a strategy that would impart a competitive advantage to motile microorganisms that compete for hydrogen gas and would impact the C, S and N cycles.

Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post-resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.

A detailed physiological and molecular analysis of lipid accumulation under a suite of conditions including nitrogen limitation, alkaline pH stress, bicarbonate supplementation, and organic acid supplementation was performed on the marine diatom Phaeodactylum tricornutum. For all tested conditions, nitrogen limitation was a prerequisite for lipid accumulation and the other culturing strategies only enhanced accumulation highlighting the importance of compounded stresses on lipid metabolism. Volumetric lipid levels varied depending on condition; the observed rankings from highest to lowest were for inorganic carbon addition (15 mM bicarbonate), organic acid addition (15 carbon mM acetate), and alkaline pH stress (pH 9.0). For all lipid-accumulating cultures except acetate supplementation, a common series of physiological steps were observed. Upon extracellular nitrogen exhaustion, culture growth continued for approximately 1.5 cell doublings with decreases in specific protein and photosynthetic pigment content. As nitrogen limitation arrested cell growth, carbohydrate content decreased with a corresponding increase in lipid content. Addition of the organic carbon source acetate appeared to activate alternative metabolic pathways for lipid accumulation. Molecular level data on more than 50 central metabolism transcripts were measured using real-time PCR. Analysis of transcripts suggested the central metabolism pathways associated with bicarbonate transport, carbonic anhydrases, and C4 carbon fixations were important for lipid accumulation. Transcriptomic data also suggested that repurposing of phospholipids may play a role in lipid accumulation. This study provides a detailed physiological and molecular-level foundation for improved understanding of diatom nutrient cycling and contributes to a metabolic blueprint for controlling lipid accumulation in diatoms.

Patterns of spatial positioning of individuals within microbial communities are often critical to community function. However, understanding patterning in natural communities is hampered by the multitude of cell-cell and cell-environment interactions as well as environmental variability. Here, through simulations and experiments on communities in defined environments, we examined how ecological interactions between two distinct partners impacted community patterning. We found that in strong cooperation with spatially localized large fitness benefits to both partners, a unique pattern is generated: partners spatially intermixed by appearing successively on top of each other, insensitive to initial conditions and interaction dynamics. Intermixing was experimentally observed in two obligatory cooperative systems: an engineered yeast community cooperating through metabolite-exchanges and a methane-producing community cooperating through redox-coupling. Even in simulated communities consisting of several species, most of the strongly-cooperating pairs appeared intermixed. Thus, when ecological interactions are the major patterning force, strong cooperation leads to partner intermixing.DOI:http://dx.doi.org/10.7554/eLife.00230.001.

Pelosinus fermentans JBW45 is an anaerobic, lactate-fermenting bacterium isolated from Cr(VI)-contaminated groundwater at the Hanford Nuclear Reservation 100-H site (Washington) that was collected after stimulation with a polylactate compound. The genome sequence of this organism will provide insight into the metabolic potential of a predominant population during stimulation for metal-reducing conditions.

Due to potential sequencing errors in pyrosequencing data, species richness and diversity indices of microbial systems can be miscalculated. The "traditional" sequence refinement method is not sufficient to account for overestimations (e.g., length, primer errors, ambiguous nucleotides). Recent in silico and single-organism studies have revealed the importance of sequence quality scores in the estimation of ecological indices; however, this is the first study to compare quality-score stringencies across four regions of the SSU rRNA gene sequence (V1V2, V3, V4, and V6) with actual environmental samples compared directly to corresponding clone libraries produced from the same primer sets. The nucleic acid sequences determined via pyrosequencing were subjected to varying quality-score cutoffs that ranged from 25 to 32, and at each quality-score cutoff, either 10 or 15 % of the nucleotides were allowed to be below the cutoff. When species richness estimates were compared for the tested samples, the cutoff values of Q27(15%), Q30(10%), and Q32(15%) for V1V2, V4, and V6, respectively, estimated similar values as obtained with clone libraries and Sanger sequencing. The most stringent Q tested (Q32(10%)) was not enough to account for species richness inflation of the V3 region pyrosequence data. Results indicated that quality-score assessment greatly improved estimates of ecological indices for environmental samples (species richness and α-diversity) and that the effect of quality-score filtering was region-dependent.

Background: Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination., Results: The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases)., Conclusions: The results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.

Background: Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations., Results: The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells., Conclusions: Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion.

Crp/Fnr-type global transcriptional regulators regulate various metabolic pathways in bacteria and typically function in response to environmental changes. However, little is known about the function of four annotated Crp/Fnr homologs (DVU0379, DVU2097, DVU2547, and DVU3111) in Desulfovibrio vulgaris Hildenborough. A systematic study using bioinformatic, transcriptomic, genetic, and physiological approaches was conducted to characterize their roles in stress responses. Similar growth phenotypes were observed for the crp/fnr deletion mutants under multiple stress conditions. Nevertheless, the idea of distinct functions of Crp/Fnr-type regulators in stress responses was supported by phylogeny, gene transcription changes, fitness changes, and physiological differences. The four D. vulgaris Crp/Fnr homologs are localized in three subfamilies (HcpR, CooA, and cc). The crp/fnr knockout mutants were well separated by transcriptional profiling using detrended correspondence analysis (DCA), and more genes significantly changed in expression in a ΔDVU3111 mutant (JW9013) than in the other three paralogs. In fitness studies, strain JW9013 showed the lowest fitness under standard growth conditions (i.e., sulfate reduction) and the highest fitness under NaCl or chromate stress conditions; better fitness was observed for a ΔDVU2547 mutant (JW9011) under nitrite stress conditions and a ΔDVU2097 mutant (JW9009) under air stress conditions. A higher Cr(VI) reduction rate was observed for strain JW9013 in experiments with washed cells. These results suggested that the four Crp/Fnr-type global regulators play distinct roles in stress responses of D. vulgaris. DVU3111 is implicated in responses to NaCl and chromate stresses, DVU2547 in nitrite stress responses, and DVU2097 in air stress responses.

Recent studies evaluating bulk soap in public restroom soap dispensers have demonstrated up to 25% of open refillable bulk-soap dispensers were contaminated with ~ 6 log(10)(CFU ml(-1)) heterotrophic bacteria. In this study, plastic counter-mounted, plastic wall-mounted and stainless steel wall-mounted dispensers were analyzed for suspended and biofilm bacteria using total cell and viable plate counts. Independent of dispenser type or construction material, the bulk soap was contaminated with 4-7 log(10)(CFU ml(-1)) bacteria, while 4-6 log(10)(CFU cm(-2)) biofilm bacteria were isolated from the inside surfaces of the dispensers (n = 6). Dispenser remediation studies, including a 10 min soak with 5000 mg l(-1) sodium hypochlorite, were then conducted to determine the efficacy of cleaning and disinfectant procedures against established biofilms. The testing showed that contamination of the bulk soap returned to pre-test levels within 7-14 days. These results demonstrate biofilm is present in contaminated bulk-soap dispensers and remediation studies to clean and sanitize the dispensers are temporary.

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B₁₂ biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.

Although little is known of potential function for conserved signaling proteins, it is hypothesized that such proteins play important roles to coordinate cellular responses to environmental stimuli. In order to elucidate the function of a putative sensory box protein (PAS domains) in Shewanella oneidensis MR-1, the physiological role of SO3389 was characterized. The predicted open reading frame (ORF) encodes a putative sensory box protein that has PAS, GGDEF, and EAL domains, and an in-frame deletion mutant was constructed (ΔSO3389) with approximately 95% of the ORF deleted. Under aerated conditions, wild-type and mutant cultures had similar growth rates, but the mutant culture had a lower growth rate under static, aerobic conditions. Oxygen consumption rates were lower for mutant cultures (1.5-fold), and wild-type cultures also maintained lower dissolved oxygen concentrations under aerated growth conditions. When transferred to anoxic conditions, the mutant did not grow with fumarate, iron(III), or dimethyl sulfoxide (DMSO) as electron acceptors. Biochemical assays demonstrated the expression of different c-type cytochromes as well as decreased fumarate reductase activity in the mutant transferred to anoxic growth conditions. Transcriptomic studies showed the inability of the mutant to up-express and down-express genes, including c-type cytochromes (e.g., SO4047/SO4048, SO3285/SO3286), reductases (e.g., SO0768, SO1427), and potential regulators (e.g., SO1329). The complemented strain was able to grow when transferred from aerobic to anoxic growth conditions with the tested electron acceptors. The modeled structure for the SO3389 PAS domains was highly similar to the crystal structures of FAD-binding PAS domains that are known O2/redox sensors. Based on physiological, genomic, and bioinformatic results, we suggest that the sensory box protein, SO3389, is an O2/redox sensor that is involved in optimization of aerobic growth and transitions to anoxia in S. oneidensis MR-1.

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.

To understand how sulphate-reducing bacteria respond to oxidative stresses, the responses of Desulfovibrio vulgaris Hildenborough to H(2)O(2)-induced stresses were investigated with transcriptomic, proteomic and genetic approaches. H(2)O(2) and induced chemical species (e.g. polysulfide, ROS) and redox potential shift increased the expressions of the genes involved in detoxification, thioredoxin-dependent reduction system, protein and DNA repair, and decreased those involved in sulfate reduction, lactate oxidation and protein synthesis. A gene coexpression network analysis revealed complicated network interactions among differentially expressed genes, and suggested possible importance of several hypothetical genes in H(2)O(2) stress. Also, most of the genes in PerR and Fur regulons were highly induced, and the abundance of a Fur regulon protein increased. Mutant analysis suggested that PerR and Fur are functionally overlapped in response to stresses induced by H(2)O(2) and reaction products, and the upregulation of thioredoxin-dependent reduction genes was independent of PerR or Fur. It appears that induction of those stress response genes could contribute to the increased resistance of deletion mutants to H(2)O(2)-induced stresses. In addition, a conceptual cellular model of D. vulgaris responses to H(2)O(2) stress was constructed to illustrate that this bacterium may employ a complicated molecular mechanism to defend against the H(2)O(2)-induced stresses., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Understanding adaptation of biological communities to environmental change is a central issue in ecology and evolution. Metagenomic analysis of a stressed groundwater microbial community reveals that prolonged exposure to high concentrations of heavy metals, nitric acid and organic solvents ( approximately 50 years) has resulted in a massive decrease in species and allelic diversity as well as a significant loss of metabolic diversity. Although the surviving microbial community possesses all metabolic pathways necessary for survival and growth in such an extreme environment, its structure is very simple, primarily composed of clonal denitrifying gamma- and beta-proteobacterial populations. The resulting community is overabundant in key genes conferring resistance to specific stresses including nitrate, heavy metals and acetone. Evolutionary analysis indicates that lateral gene transfer could have a key function in rapid response and adaptation to environmental contamination. The results presented in this study have important implications in understanding, assessing and predicting the impacts of human-induced activities on microbial communities ranging from human health to agriculture to environmental management, and their responses to environmental changes.

The response of Desulfovibrio vulgaris Hildenborough to salt adaptation (long-term NaCl exposure) was examined by performing physiological, global transcriptional, and metabolite analyses. Salt adaptation was reflected by increased expression of genes involved in amino acid biosynthesis and transport, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). The expression of genes involved in carbon metabolism, cell growth, and phage structures was decreased. Transcriptome profiles of D. vulgaris responses to salt adaptation were compared with transcriptome profiles of D. vulgaris responses to salt shock (short-term NaCl exposure). Metabolite assays showed that glutamate and alanine accumulated under salt adaptation conditions, suggesting that these amino acids may be used as osmoprotectants in D. vulgaris. Addition of amino acids (glutamate, alanine, and tryptophan) or yeast extract to the growth medium relieved salt-related growth inhibition. A conceptual model that links the observed results to currently available knowledge is proposed to increase our understanding of the mechanisms of D. vulgaris adaptation to elevated NaCl levels.

Determining the transient chemical properties of the intracellular environment can elucidate the paths through which a biological system adapts to changes in its environment, for example, the mechanisms that enable some obligate anaerobic bacteria to survive a sudden exposure to oxygen. Here we used high-resolution Fourier transform infrared (FTIR) spectromicroscopy to continuously follow cellular chemistry within living obligate anaerobes by monitoring hydrogen bond structures in their cellular water. We observed a sequence of well orchestrated molecular events that correspond to changes in cellular processes in those cells that survive, but only accumulation of radicals in those that do not. We thereby can interpret the adaptive response in terms of transient intracellular chemistry and link it to oxygen stress and survival. This ability to monitor chemical changes at the molecular level can yield important insights into a wide range of adaptive responses.

Hypothetical (HyP) and conserved HyP genes account for >30% of sequenced bacterial genomes. For the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, 347 of the 3634 genes were annotated as conserved HyP (9.5%) along with 887 HyP genes (24.4%). Given the large fraction of the genome, it is plausible that some of these genes serve critical cellular roles. The study goals were to determine which genes were expressed and provide a more functionally based annotation. To accomplish this, expression profiles of 1234 HyP and conserved genes were used from transcriptomic datasets of 11 environmental stresses, complemented with shotgun LC-MS/MS and AMT tag proteomic data. Genes were divided into putatively polycistronic operons and those predicted to be monocistronic, then classified by basal expression levels and grouped according to changes in expression for one or multiple stresses. One thousand two hundred and twelve of these genes were transcribed with 786 producing detectable proteins. There was no evidence for expression of 17 predicted genes. Except for the latter, monocistronic gene annotation was expanded using the above criteria along with matching Clusters of Orthologous Groups. Polycistronic genes were annotated in the same manner with inferences from their proximity to more confidently annotated genes. Two targeted deletion mutants were used as test cases to determine the relevance of the inferred functional annotations.

Bacterial 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-year 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 were 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 with the population variation through 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 bioreduction; however, the two biostimulated 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.

The definition and delineation of microbial species are of great importance and challenge due to the extent of evolution and diversity. Whole-genome DNA-DNA hybridization is the cornerstone for defining procaryotic species relatedness, but obtaining pairwise DNA-DNA reassociation values for a comprehensive phylogenetic analysis of procaryotes is tedious and time consuming. A previously described microarray format containing whole-genomic DNA (the community genome array or CGA) was rigorously evaluated as a high-throughput alternative to the traditional DNA-DNA reassociation approach for delineating procaryotic species relationships. DNA similarities for multiple bacterial strains obtained with the CGA-based hybridization were comparable to those obtained with various traditional whole-genome hybridization methods (r=0.87, P<0.01). Significant linear relationships were also observed between the CGA-based genome similarities and those derived from small subunit (SSU) rRNA gene sequences (r=0.79, P<0.0001), gyrB sequences (r=0.95, P<0.0001) or REP- and BOX-PCR fingerprinting profiles (r=0.82, P<0.0001). The CGA hybridization-revealed species relationships in several representative genera, including Pseudomonas, Azoarcus and Shewanella, were largely congruent with previous classifications based on various conventional whole-genome DNA-DNA reassociation, SSU rRNA and/or gyrB analyses. These results suggest that CGA-based DNA-DNA hybridization could serve as a powerful, high-throughput format for determining species relatedness among microorganisms.