High-throughput protein characterization by complementation using DNA barcoded fragment libraries.

Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation.

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface.

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting.

Large-scale genetic characterization of the model sulfate-reducing bacterium, Hildenborough.

Sulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions.

A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria.

Exometabolomics is an approach to assess how microorganisms alter, or react to their environments through the depletion and production of metabolites. It allows the examination of how soil microbes transform the small molecule metabolites within their environment, which can be used to study resource competition and cross-feeding. This approach is most powerful when used with defined media that enable tracking of all metabolites.

A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis.

Bacterial communities in water, soil, and humans play an essential role in environmental ecology and human health. PCR-based amplicon analysis, such as 16S rRNA sequencing, is a fundamental tool for quantifying and studying microbial composition, dynamics, and interactions. However, given the complexity of microbial communities, a substantial number of samples becomes necessary for analyses that parse the factors that determine microbial composition.

Deletion Mutants, Archived Transposon Library, and Tagged Protein Constructs of the Model Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough.

The dissimilatory sulfate-reducing Hildenborough (ATCC 29579) was chosen by the research collaboration ENIGMA to explore tools and protocols for bringing this anaerobe to model status. Here, we describe a collection of genetic constructs generated by ENIGMA that are available to the research community.

Selective carbon sources influence the end products of microbial nitrate respiration.

Respiratory and catabolic genes are differentially distributed across microbial genomes. Thus, specific carbon sources may favor different respiratory processes. We profiled the influence of 94 carbon sources on the end products of nitrate respiration in microbial enrichment cultures from diverse terrestrial environments.

Correction: Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics.

[This corrects the article DOI: 10.1371/journal.pgen.

The selective pressures on the microbial community in a metal-contaminated aquifer.

In many environments, toxic compounds restrict which microorganisms persist. However, in complex mixtures of inhibitory compounds, it is challenging to determine which specific compounds cause changes in abundance and prevent some microorganisms from growing. We focused on a contaminated aquifer in Oak Ridge, Tennessee, USA that has large gradients of pH and widely varying concentrations of uranium, nitrate, and many other inorganic ions.

Dissimilatory Sulfate Reduction Under High Pressure by G20.

Biosouring results from production of HS by sulfate-reducing microorganisms (SRMs) in oil reservoirs. HS is toxic, corrosive, and explosive, and as such, represents a significant threat to personnel, production facilities, and transportation pipelines. Since typical oil reservoir pressures can range from 10 to 50 MPa, understanding the role that pressure plays in SRM metabolism is important to improving souring containment strategies.

Mutant phenotypes for thousands of bacterial genes of unknown function.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function.

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics.

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways.

System-Wide Adaptations of Desulfovibrio alaskensis G20 to Phosphate-Limited Conditions.

The prevalence of lipids devoid of phosphorus suggests that the availability of phosphorus limits microbial growth and activity in many anoxic, stratified environments. To better understand the response of anaerobic bacteria to phosphate limitation and starvation, this study combines microscopic and lipid analyses with the measurements of fitness of pooled barcoded transposon mutants of the model sulfate reducing bacterium Desulfovibrio alaskensis G20. Phosphate-limited G20 has lower growth rates and replaces more than 90% of its membrane phospholipids by a mixture of monoglycosyl diacylglycerol (MGDG), glycuronic acid diacylglycerol (GADG) and ornithine lipids, lacks polyphosphate granules, and synthesizes other cellular inclusions.

Complete Genome Sequence of Cupriavidus basilensis 4G11, Isolated from the Oak Ridge Field Research Center Site.

Cupriavidus basilensis 4G11 was isolated from groundwater at the Oak Ridge Field Research Center (FRC) site. Here, we report the complete genome sequence and annotation of Cupriavidus basilensis 4G11. The genome contains 8,421,483 bp, 7,661 predicted protein-coding genes, and a total GC content of 64.

Monofluorophosphate is a selective inhibitor of respiratory sulfate-reducing microorganisms.

Despite the environmental and economic cost of microbial sulfidogenesis in industrial operations, few compounds are known as selective inhibitors of respiratory sulfate reducing microorganisms (SRM), and no study has systematically and quantitatively evaluated the selectivity and potency of SRM inhibitors. Using general, high-throughput assays to quantitatively evaluate inhibitor potency and selectivity in a model sulfate-reducing microbial ecosystem as well as inhibitor specificity for the sulfate reduction pathway in a model SRM, we screened a panel of inorganic oxyanions. We identified several SRM selective inhibitors including selenate, selenite, tellurate, tellurite, nitrate, nitrite, perchlorate, chlorate, monofluorophosphate, vanadate, molydate, and tungstate.

Mechanisms of direct inhibition of the respiratory sulfate-reduction pathway by (per)chlorate and nitrate.

We investigated perchlorate (ClO(4)(-)) and chlorate (ClO(3)(-)) (collectively (per)chlorate) in comparison with nitrate as potential inhibitors of sulfide (H(2)S) production by mesophilic sulfate-reducing microorganisms (SRMs). We demonstrate the specificity and potency of (per)chlorate as direct SRM inhibitors in both pure cultures and undefined sulfidogenic communities. We demonstrate that (per)chlorate and nitrate are antagonistic inhibitors and resistance is cross-inducible implying that these compounds share at least one common mechanism of resistance.

The genetic basis of energy conservation in the sulfate-reducing bacterium Desulfovibrio alaskensis G20.

Sulfate-reducing bacteria play major roles in the global carbon and sulfur cycles, but it remains unclear how reducing sulfate yields energy. To determine the genetic basis of energy conservation, we measured the fitness of thousands of pooled mutants of Desulfovibrio alaskensis G20 during growth in 12 different combinations of electron donors and acceptors. We show that ion pumping by the ferredoxin:NADH oxidoreductase Rnf is required whenever substrate-level phosphorylation is not possible.

Functional genomics with a comprehensive library of transposon mutants for the sulfate-reducing bacterium Desulfovibrio alaskensis G20.

Unlabelled: The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5' RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase.

Genetic basis for nitrate resistance in Desulfovibrio strains.

Nitrate is an inhibitor of sulfate-reducing bacteria (SRB). In petroleum production sites, amendments of nitrate and nitrite are used to prevent SRB production of sulfide that causes souring of oil wells. A better understanding of nitrate stress responses in the model SRB, Desulfovibrio vulgaris Hildenborough and Desulfovibrio alaskensis G20, will strengthen predictions of environmental outcomes of nitrate application.

Control of methionine metabolism by the SahR transcriptional regulator in Proteobacteria.

Sulphur is an essential element in the metabolism. The sulphur-containing amino acid methionine is a metabolic precursor for S-adenosylmethionine (SAM), which serves as a coenzyme for ubiquitous methyltrtansferases. Recycling of organic sulphur compounds, e.

Flexibility of syntrophic enzyme systems in Desulfovibrio species ensures their adaptation capability to environmental changes.

The mineralization of organic matter in anoxic environments relies on the cooperative activities of hydrogen producers and consumers obligately linked by interspecies metabolite exchange in syntrophic consortia that may include sulfate reducing species such as Desulfovibrio. To evaluate the metabolic flexibility of syntrophic Desulfovibrio to adapt to naturally fluctuating methanogenic environments, we studied Desulfovibrio alaskensis strain G20 grown in chemostats under respiratory and syntrophic conditions with alternative methanogenic partners, Methanococcus maripaludis and Methanospirillum hungatei, at different growth rates. Comparative whole-genome transcriptional analyses, complemented by G20 mutant strain growth experiments and physiological data, revealed a significant influence of both energy source availability (as controlled by dilution rate) and methanogen on the electron transfer systems, ratios of interspecies electron carriers, energy generating systems, and interspecies physical associations.

Indirect and suboptimal control of gene expression is widespread in bacteria.

Gene regulation in bacteria is usually described as an adaptive response to an environmental change so that genes are expressed when they are required. We instead propose that most genes are under indirect control: their expression responds to signal(s) that are not directly related to the genes' function. Indirect control should perform poorly in artificial conditions, and we show that gene regulation is often maladaptive in the laboratory.

The Enhancer of split complex arose prior to the diversification of schizophoran flies and is strongly conserved between Drosophila and stalk-eyed flies (Diopsidae).

Background: In Drosophila, the Enhancer of split complex (E(spl)-C) comprises 11 bHLH and Bearded genes that function during Notch signaling to repress proneural identity in the developing peripheral nervous system. Comparison with other insects indicates that the basal state for Diptera is a single bHLH and Bearded homolog and that the expansion of the gene complex occurred in the lineage leading to Drosophila. However, comparative genomic data from other fly species that would elucidate the origin and sequence of gene duplication for the complex is lacking.

Evidence-based annotation of transcripts and proteins in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

We used high-resolution tiling microarrays and 5' RNA sequencing to identify transcripts in Desulfovibrio vulgaris Hildenborough, a model sulfate-reducing bacterium. We identified the first nucleotide position for 1,124 transcripts, including 54 proteins with leaderless transcripts and another 72 genes for which a major transcript initiates within the upstream protein-coding gene, which confounds measurements of the upstream gene's expression. Sequence analysis of these promoters showed that D.