MetFish: a Metabolomics Pipeline for Studying Microbial Communities in Chemically Extreme Environments.

Metabolites have essential roles in microbial communities, including as mediators of nutrient and energy exchange, cell-to-cell communication, and antibiosis. However, detecting and quantifying metabolites and other chemicals in samples having extremes in salt or mineral content using liquid chromatography-mass spectrometry (LC-MS)-based methods remains a significant challenge. Here, we report a facile method based on in situ chemical derivatization followed by extraction for analysis of metabolites and other chemicals in hypersaline samples, enabling for the first time direct LC-MS-based exometabolomics analysis in sample matrices containing up to 2 M total dissolved salts. The method, MetFish, is applicable to molecules containing amine, carboxylic acid, carbonyl, or hydroxyl functional groups, and it can be integrated into either targeted or untargeted analysis pipelines. In targeted analyses, MetFish provided limits of quantification as low as 1 nM, broad linear dynamic ranges (up to 5 to 6 orders of magnitude) with excellent linearity, and low median interday reproducibility (e.g., 2.6%). MetFish was successfully applied in targeted and untargeted exometabolomics analyses of microbial consortia, quantifying amino acid dynamics in the exometabolome during community succession; in situ in a native prairie soil, whose exometabolome was isolated using a hypersaline extraction; and in input and produced fluids from a hydraulically fractured well, identifying dramatic changes in the exometabolome over time in the well. IMPORTANCE The identification and accurate quantification of metabolites using electrospray ionization-mass spectrometry (ESI-MS) in hypersaline samples is a challenge due to matrix effects. Clean-up and desalting strategies that typically work well for samples with lower salt concentrations are often ineffective in hypersaline samples. To address this gap, we developed and demonstrated a simple yet sensitive and accurate method-MetFish-using chemical derivatization to enable mass spectrometry-based metabolomics in a variety of hypersaline samples from varied ecosystems and containing up to 2 M dissolved salts.

Corrigendum: Saliniramus fredricksonii gen. nov., sp. nov., a heterotrophic halophile isolated from Hot Lake, Washington, a member of a novel lineage (Salinarimonadaceae fam. nov.) within the order Rhizobiales, and reclassification of the genus Salinarimonas Liu et al. 2010 into Salinarimonadaceae.

There was an error in the proposed genus name in the published article, in that the genus 'Salinivirga' was effectively published while this article was in review. Therefore, the genus 'Salinivirga' should be replaced with 'Saliniramus'. For the convenience of future readers, we have included the complete corrected article below, in which all occurrences of the incorrect genus name have been amended: A halophilic bacterial strain, HL-109T, was isolated from the unicyanobacterial consortium UCC-O, which was obtained from the photosynthetic mat of Hot Lake (Washington, USA). A polyphasic approach using phenotypic, genotypic and chemotaxonomic data was used to classify the strain within the order Rhizobiales. The organism stained Gram-negative and was a moderate thermophile with a growth optimum of 45 °C. It was obligately aerobic, heterotrophic and halophilic, growing in both NaCl and MgSO4 brines. The novel isolate had a polymorphic cellular morphology of short rods with occasional branching, and cells were monotrichous. The major fatty acids detected were C18 : 1, C18 : 0, C16 : 0 and C18 : cyc. Phylogenetic analysis of the 16S rRNA gene placed the strain in the order Rhizobiales and it shared 94 % identity with the type strain of its nearest relative, Salinarimonas ramus. Morphological, chemotaxonomic and phylogenetic results did not affiliate the novel organism with any of the families in the Rhizobiales; therefore, HL-109T is representative of a new lineage, for which the name Saliniramus fredricksonii gen. nov., sp. nov. is proposed, with the type strain HL-109T (=JCM 31876T=DSM 102886T). In addition, examination of the phylogenetics of strain HL-109T and its nearest relatives, Salinarimonas ramus and Salinarimonasrosea, demonstrates that these halophiles form a clade distinct from the described families of the Rhizobiales. We further propose the establishment of a new family, Salinarimonadaceae fam. nov., to accommodate the genera Saliniramus and Salinarimonas (the type genus of the family).

Salinivirga fredricksonii gen. nov., sp. nov., a heterotrophic halophile isolated from a photosynthetic mat, a member of a novel lineage (Salinarimonadaceae fam. nov.) within the order Rhizobiales, and reclassification of the genus Salinarimonas Liu et al. 2010 into Salinarimonadaceae.

A halophilic bacterial strain, HL-109T, was isolated from the unicyanobacterial consortium UCC-O, which was obtained from the photosynthetic mat of Hot Lake (Washington, USA). A polyphasic approach using phenotypic, genotypic and chemotaxonomic data was used to classify the strain within the order Rhizobiales. The organism stained Gram-negative and was a moderate thermophile with a growth optimum of 45 °C. It was obligately aerobic, heterotrophic and halophilic, growing in both NaCl and MgSO4 brines. The novel isolate had a polymorphic cellular morphology of short rods with occasional branching, and cells were monotrichous. The major fatty acids detected were C18 : 1, C18 : 0, C16 : 0 and C18 : cyc. Phylogenetic analysis of the 16S rRNA gene placed the strain in the order Rhizobiales and it shared 94 % identity with the type strain of its nearest relative, Salinarimonas ramus. Morphological, chemotaxonomic and phylogenetic results did not affiliate the novel organism with any of the families in the Rhizobiales; therefore, HL-109T is representative of a new lineage, for which the name Salinivirga fredricksonii gen. nov., sp. nov. is proposed, with the type strain HL-109T (=JCM 31876T=DSM 102886T). In addition, examination of the phylogenetics of strain HL-109T and its nearest relatives, Salinarimonas ramus and Salinarimonasrosea, demonstrates that these halophiles form a clade distinct from the described families of the Rhizobiales. We further propose the establishment of a new family, Salinarimonadaceae fam. nov., to accommodate the genera Salinivirga and Salinarimonas (the type genus of the family).

Genomic Reconstruction of Carbohydrate Utilization Capacities in Microbial-Mat Derived Consortia.

Two nearly identical unicyanobacterial consortia (UCC) were previously isolated from benthic microbial mats that occur in a heliothermal saline lake in northern Washington State. Carbohydrates are a primary source of carbon and energy for most heterotrophic bacteria. Since CO2 is the only carbon source provided, the cyanobacterium must provide a source of carbon to the heterotrophs. Available genomic sequences for all members of the UCC provide opportunity to investigate the metabolic routes of carbon transfer between autotroph and heterotrophs. Here, we applied a subsystem-based comparative genomics approach to reconstruct carbohydrate utilization pathways and identify glycohydrolytic enzymes, carbohydrate transporters and pathway-specific transcriptional regulators in 17 heterotrophic members of the UCC. The reconstructed metabolic pathways include 800 genes, near a one-fourth of which encode enzymes, transporters and regulators with newly assigned metabolic functions resulting in discovery of novel functional variants of carbohydrate utilization pathways. The in silico analysis revealed the utilization capabilities for 40 carbohydrates and their derivatives. Two Halomonas species demonstrated the largest number of sugar catabolic pathways. Trehalose, sucrose, maltose, glucose, and beta-glucosides are the most commonly utilized saccharides in this community. Reconstructed regulons for global regulators HexR and CceR include central carbohydrate metabolism genes in the members of Gammaproteobacteria and Alphaproteobacteria, respectively. Genomics analyses were supplemented by experimental characterization of metabolic phenotypes in four isolates derived from the consortia. Measurements of isolate growth on the defined medium supplied with individual carbohydrates confirmed most of the predicted catabolic phenotypes. Not all consortia members use carbohydrates and only a few use complex polysaccharides suggesting a hierarchical carbon flow from cyanobacteria to each heterotroph. In summary, the genomics-based identification of carbohydrate utilization capabilities provides a basis for future experimental studies of carbon flow in UCC.

Predicting Species-Resolved Macronutrient Acquisition during Succession in a Model Phototrophic Biofilm Using an Integrated 'Omics Approach.

The principles governing acquisition and interspecies exchange of nutrients in microbial communities and how those exchanges impact community productivity are poorly understood. Here, we examine energy and macronutrient acquisition in unicyanobacterial consortia for which species-resolved genome information exists for all members, allowing us to use multi-omic approaches to predict species' abilities to acquire resources and examine expression of resource-acquisition genes during succession. Metabolic reconstruction indicated that a majority of heterotrophic community members lacked the genes required to directly acquire the inorganic nutrients provided in culture medium, suggesting high metabolic interdependency. The sole primary producer in consortium UCC-O, cyanobacterium Phormidium sp. OSCR, displayed declining expression of energy harvest, carbon fixation, and nitrate and sulfate reduction proteins but sharply increasing phosphate transporter expression over 28 days. Most heterotrophic members likewise exhibited signs of phosphorus starvation during succession. Though similar in their responses to phosphorus limitation, heterotrophs displayed species-specific expression of nitrogen acquisition genes. These results suggest niche partitioning around nitrogen sources may structure the community when organisms directly compete for limited phosphate. Such niche complementarity around nitrogen sources may increase community diversity and productivity in phosphate-limited phototrophic communities.

Indirect Interspecies Regulation: Transcriptional and Physiological Responses of a Cyanobacterium to Heterotrophic Partnership.

The mechanisms by which microbes interact in communities remain poorly understood. Here, we interrogated specific interactions between photoautotrophic and heterotrophic members of a model consortium to infer mechanisms that mediate metabolic coupling and acclimation to partnership. This binary consortium was composed of a cyanobacterium, Thermosynechococcus elongatus BP-1, which supported growth of an obligate aerobic heterotroph, Meiothermus ruber strain A, by providing organic carbon, O2, and reduced nitrogen. Species-resolved transcriptomic analyses were used in combination with growth and photosynthesis kinetics to infer interactions and the environmental context under which they occur. We found that the efficiency of biomass production and resistance to stress induced by high levels of dissolved O2 increased, beyond axenic performance, as a result of heterotrophic partnership. Coordinated transcriptional responses transcending both species were observed and used to infer specific interactions resulting from the synthesis and exchange of resources. The cyanobacterium responded to heterotrophic partnership by altering expression of core genes involved with photosynthesis, carbon uptake/fixation, vitamin synthesis, and scavenging of reactive oxygen species (ROS). IMPORTANCE This study elucidates how a cyanobacterial primary producer acclimates to heterotrophic partnership by modulating the expression levels of key metabolic genes. Heterotrophic bacteria can indirectly regulate the physiology of the photoautotrophic primary producers, resulting in physiological changes identified here, such as increased intracellular ROS. Some of the interactions inferred from this model system represent putative principles of metabolic coupling in phototrophic-heterotrophic partnerships.

Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism.

Only a small fraction of vitamin B12-requiring organisms are able to synthesize B12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B12-binding enzymes and regulatory roles for B12 Here we report the development and use of a B12-based chemical probe to identify B12-binding proteins in a nonphototrophic B12-producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B12-binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B12 likely modulates growth, and by limiting its availability to auxotrophs, B12-producing organisms may facilitate coordination of community metabolism.

Underlying mechanisms for syntrophic metabolism of essential enzyme cofactors in microbial communities.

Many microorganisms are unable to synthesize essential B vitamin-related enzyme cofactors de novo. The underlying mechanisms by which such microbes survive in multi-species communities are largely unknown. We previously reported the near-complete genome sequence of two ~18-member unicyanobacterial microbial consortia that maintain stable membership on defined medium lacking vitamins. Here we have used genome analysis and growth studies on isolates derived from the consortia to reconstruct pathways for biogenesis of eight essential cofactors and predict cofactor usage and precursor exchange in these communities. Our analyses revealed that all but the two Halomonas and cyanobacterial community members were auxotrophic for at least one cofactor. We also observed a mosaic distribution of salvage routes for a variety of cofactor precursors, including those produced by photolysis. Potentially bidirectional transporters were observed to be preferentially in prototrophs, suggesting a mechanism for controlled precursor release. Furthermore, we found that Halomonas sp. do not require cobalamin nor control its synthesis, supporting the hypothesis that they overproduce and export vitamins. Collectively, these observations suggest that the consortia rely on syntrophic metabolism of cofactors as a survival strategy for optimization of metabolic exchange within a shared pool of micronutrients.

Functional roles of CymA and NapC in reduction of nitrate and nitrite by Shewanella putrefaciens W3-18-1.

Shewanella putrefaciens W3-18-1 harbours two periplasmic nitrate reductase (Nap) gene clusters, NapC-associated nap-alpha (napEDABC) and CymA-dependent nap-beta (napDAGHB), for dissimilatory nitrate respiration. CymA is a member of the NapC/NirT quinol dehydrogenase family and acts as a hub to support different respiratory pathways, including those on iron [Fe(III)] and manganese [Mn(III, IV)] (hydr)oxide, nitrate, nitrite, fumarate and arsenate in Shewanella strains. However, in our analysis it was shown that another NapC/NirT family protein, NapC, was only involved in nitrate reduction, although both CymA and NapC can transfer quinol-derived electrons to a periplasmic terminal reductase or an electron acceptor. Furthermore, our results showed that NapC could only interact specifically with the Nap-alpha nitrate reductase while CymA could interact promiscuously with Nap-alpha, Nap-beta and the NrfA nitrite reductase for nitrate and nitrite reduction. To further explore the difference in specificity, site-directed mutagenesis on both CymA and NapC was conducted and the phenotypic changes in nitrate and nitrite reduction were tested. Our analyses demonstrated that the Lys-91 residue played a key role in nitrate reduction for quinol oxidation and the Asp-166 residue might influence the maturation of CymA. The Asp-97 residue might be one of the key factors that influence the interaction of CymA with the cytochromes NapB and NrfA.

Formaldehyde as a carbon and electron shuttle between autotroph and heterotroph populations in acidic hydrothermal vents of Norris Geyser Basin, Yellowstone National Park.

The Norris Geyser Basin in Yellowstone National Park contains a large number of hydrothermal systems, which host microbial populations supported by primary productivity associated with a suite of chemolithotrophic metabolisms. We demonstrate that Metallosphaera yellowstonensis MK1, a facultative autotrophic archaeon isolated from a hyperthermal acidic hydrous ferric oxide (HFO) spring in Norris Geyser Basin, excretes formaldehyde during autotrophic growth. To determine the fate of formaldehyde in this low organic carbon environment, we incubated native microbial mat (containing M. yellowstonensis) from a HFO spring with (13)C-formaldehyde. Isotopic analysis of incubation-derived CO2 and biomass showed that formaldehyde was both oxidized and assimilated by members of the community. Autotrophy, formaldehyde oxidation, and formaldehyde assimilation displayed different sensitivities to chemical inhibitors, suggesting that distinct sub-populations in the mat selectively perform these functions. Our results demonstrate that electrons originally resulting from iron oxidation can energetically fuel autotrophic carbon fixation and associated formaldehyde excretion, and that formaldehyde is both oxidized and assimilated by different organisms within the native microbial community. Thus, formaldehyde can effectively act as a carbon and electron shuttle connecting the autotrophic, iron oxidizing members with associated heterotrophic members in the HFO community.

Ecophysiology of an uncultivated lineage of Aigarchaeota from an oxic, hot spring filamentous 'streamer' community.

The candidate archaeal phylum 'Aigarchaeota' contains microorganisms from terrestrial and subsurface geothermal ecosystems. The phylogeny and metabolic potential of Aigarchaeota has been deduced from several recent single-cell amplified genomes; however, a detailed description of their metabolic potential and in situ transcriptional activity is absent. Here, we report a comprehensive metatranscriptome-based reconstruction of the in situ metabolism of Aigarchaeota in an oxic, hot spring filamentous 'streamer' community. Fluorescence in situ hybridization showed that these newly discovered Aigarchaeota are filamentous, which is consistent with the presence and transcription of an actin-encoding gene. Aigarchaeota filaments are intricately associated with other community members, which include both bacteria (for example, filamentous Thermocrinis spp.) and archaea. Metabolic reconstruction of genomic and metatranscriptomic data suggests that this aigarchaeon is an aerobic, chemoorganoheterotroph with autotrophic potential. A heme copper oxidase complex was identified in the environmental genome assembly and highly transcribed in situ. Potential electron donors include acetate, fatty acids, amino acids, sugars and aromatic compounds, which may originate from extracellular polymeric substances produced by other microorganisms shown to exist in close proximity and/or autochthonous dissolved organic carbon (OC). Transcripts related to genes specific to each of these potential electron donors were identified, indicating that this aigarchaeon likely utilizes several OC substrates. Characterized members of this lineage cannot synthesize heme, and other cofactors and vitamins de novo, which suggests auxotrophy. We propose the name Candidatus 'Calditenuis aerorheumensis' for this aigarchaeon, which describes its filamentous morphology and its primary electron acceptor, oxygen.

Identification and Resolution of Microdiversity through Metagenomic Sequencing of Parallel Consortia.

To gain a predictive understanding of the interspecies interactions within microbial communities that govern community function, the genomic complement of every member population must be determined. Although metagenomic sequencing has enabled the de novo reconstruction of some microbial genomes from environmental communities, microdiversity confounds current genome reconstruction techniques. To overcome this issue, we performed short-read metagenomic sequencing on parallel consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition. The differences in species abundance between the two consortia allowed reconstruction of near-complete (at an estimated >85% of gene complement) genome sequences for 17 of the 20 detected member species. Two Halomonas spp. indistinguishable by amplicon analysis were found to be present within the community. In addition, comparison of metagenomic reads against the consensus scaffolds revealed within-species variation for one of the Halomonas populations, one of the Rhodobacteraceae populations, and the Rhizobiales population. Genomic comparison of these representative instances of inter- and intraspecies microdiversity suggests differences in functional potential that may result in the expression of distinct roles in the community. In addition, isolation and complete genome sequence determination of six member species allowed an investigation into the sensitivity and specificity of genome reconstruction processes, demonstrating robustness across a wide range of sequence coverage (9× to 2,700×) within the metagenomic data set.

Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions.

The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are needed to shorten the gap between characterizing a microbial genome and experimentally validating bioinformatically predicted functions. Of particular importance are transport mechanisms, which shuttle nutrients such as B vitamins and metabolites across cell membranes and are required for the survival of microbes ranging from members of environmental microbial communities to pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, along with characterization of intracellular enzyme-cofactor associations, are needed to enable a significantly improved understanding of microbial biochemistry and physiology, microbial interactions, and microbial responses to perturbations. Chemical probes derived from B vitamins B1, B2, and B7 have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular enzyme-cofactor associations through live cell labeling of the filamentous anoxygenic photoheterotroph, Chloroflexus aurantiacus J-10-fl, known to employ mechanisms for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by identifying B vitamin transport and cofactor-dependent interactions required for survival.

Multi-Omic Dynamics Associate Oxygenic Photosynthesis with Nitrogenase-Mediated H2 Production in Cyanothece sp. ATCC 51142.

To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process.

Genomic distribution of B-vitamin auxotrophy and uptake transporters in environmental bacteria from the Chloroflexi phylum.

Bacteria from the Chloroflexi phylum are dominant members of phototrophic microbial mat communities in terrestrial thermal environments. Vitamins of B group are key intermediates (precursors) in the biosynthesis of indispensable enzyme cofactors driving numerous metabolic processes in all forms of life. A genomics-based reconstruction and comparative analysis of respective biosynthetic and salvage pathways and riboswitch regulons in over 20 representative Chloroflexi genomes revealed a widespread auxotrophy for some of the vitamins. The most prominent predicted phenotypic signature, auxotrophy for vitamins B1 and B7 was experimentally confirmed for the best studied model organism Chloroflexus aurantiacus. These observations along with identified candidate genes for the respective uptake transporters pointed to B vitamin cross-feeding as an important aspect of syntrophic metabolism in microbial communities. Inferred specificities of homologous substrate-binding components of ABC transporters for vitamins B1 (ThiY) and B2 (RibY) were verified by thermofluorescent shift approach. A functional activity of the thiamine-specific transporter ThiXYZ from C. aurantiacus was experimentally verified by genetic complementation in E. coli. Expanding the integrative approach, which was applied here for a comprehensive analysis of B-vitamin metabolism in Chloroflexi would allow reconstruction of metabolic interdependencies in microbial communities.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Conservation of transcription start sites within genes across a bacterial genus.

Transcription start sites (TSSs) lying inside annotated genes, on the same or opposite strand, have been observed in diverse bacteria, but the function of these unexpected transcripts is unclear. Here, we use the metal-reducing bacterium Shewanella oneidensis MR-1 and its relatives to study the evolutionary conservation of unexpected TSSs. Using high-resolution tiling microarrays and 5'-end RNA sequencing, we identified 2,531 TSSs in S. oneidensis MR-1, of which 18% were located inside coding sequences (CDSs). Comparative transcriptome analysis with seven additional Shewanella species revealed that the majority (76%) of the TSSs within the upstream regions of annotated genes (gTSSs) were conserved. Thirty percent of the TSSs that were inside genes and on the sense strand (iTSSs) were also conserved. Sequence analysis around these iTSSs showed conserved promoter motifs, suggesting that many iTSS are under purifying selection. Furthermore, conserved iTSSs are enriched for regulatory motifs, suggesting that they are regulated, and they tend to eliminate polar effects, which confirms that they are functional. In contrast, the transcription of antisense TSSs located inside CDSs (aTSSs) was significantly less likely to be conserved (22%). However, aTSSs whose transcription was conserved often have conserved promoter motifs and drive the expression of nearby genes. Overall, our findings demonstrate that some internal TSSs are conserved and drive protein expression despite their unusual locations, but the majority are not conserved and may reflect noisy initiation of transcription rather than a biological function. Importance: The first step of gene expression is the initiation of transcription from promoters, which have been traditionally thought to be located upstream of genes. Recently, studies showed that in diverse bacteria, promoters are often located inside genes. It has not been clear if these unexpected promoters are important to the organism or if they result from transcriptional noise. Here, we identify and examine promoters in eight related bacterial species. Promoters that lie within genes on the sense strand are often conserved as locations and in their sequences. Furthermore, these promoters often affect the bacterium's growth. Thus, many of these unexpected promoters are likely functional. Fewer promoters that lie within genes on the antisense strand are conserved, but the conserved ones seem to drive the expression of nearby genes.

Comparisons of Shewanella strains based on genome annotations, modeling, and experiments.

BACKGROUND: Shewanella is a genus of facultatively anaerobic, Gram-negative bacteria that have highly adaptable metabolism which allows them to thrive in diverse environments. This quality makes them an attractive bacterial target for research in bioremediation and microbial fuel cell applications. Constraint-based modeling is a useful tool for helping researchers gain insights into the metabolic capabilities of these bacteria. However, Shewanella oneidensis MR-1 is the only strain with a genome-scale metabolic model constructed out of 21 sequenced Shewanella strains. RESULTS: In this work, we updated the model for Shewanella oneidensis MR-1 and constructed metabolic models for three other strains, namely Shewanella sp. MR-4, Shewanella sp. W3-18-1, and Shewanella denitrificans OS217 which span the genus based on the number of genes lost in comparison to MR-1. We also constructed a Shewanella core model that contains the genes shared by all 21 sequenced strains and a few non-conserved genes associated with essential reactions. Model comparisons between the five constructed models were done at two levels - for wildtype strains under different growth conditions and for knockout mutants under the same growth condition. In the first level, growth/no-growth phenotypes were predicted by the models on various carbon sources and electron acceptors. Cluster analysis of these results revealed that the MR-1 model is most similar to the W3-18-1 model, followed by the MR-4 and OS217 models when considering predicted growth phenotypes. However, a cluster analysis done based on metabolic gene content revealed that the MR-4 and W3-18-1 models are the most similar, with the MR-1 and OS217 models being more distinct from these latter two strains. As a second level of comparison, we identified differences in reaction and gene content which give rise to different functional predictions of single and double gene knockout mutants using Comparison of Networks by Gene Alignment (CONGA). Here, we showed how CONGA can be used to find biomass, metabolic, and genetic differences between models. CONCLUSIONS: We developed four strain-specific models and a general core model that can be used to do various in silico studies of Shewanella metabolism. The developed models provide a platform for a systematic investigation of Shewanella metabolism to aid researchers using Shewanella in various biotechnology applications.

Live cell chemical profiling of temporal redox dynamics in a photoautotrophic cyanobacterium.

Protein reduction-oxidation (redox) modification is an important mechanism that allows microorganisms to sense environmental changes and initiate cellular responses. We have developed a quantitative chemical probe approach for live cell labeling and imaging of proteins that are sensitive to redox modifications. We utilize this in vivo strategy to identify 176 proteins undergoing ∼5-10-fold dynamic redox change in response to nutrient limitation and subsequent replenishment in the photoautotrophic cyanobacterium Synechococcus sp. PCC 7002. We detect redox changes in as little as 30 s after nutrient perturbation and oscillations in reduction and oxidation for 60 min following the perturbation. Many of the proteins undergoing dynamic redox transformations participate in the major components for the production (photosystems and electron transport chains) or consumption (Calvin-Benson cycle and protein synthesis) of reductant and/or energy in photosynthetic organisms. Thus, our in vivo approach reveals new redox-susceptible proteins and validates those previously identified in vitro.

Changes in translational efficiency is a dominant regulatory mechanism in the environmental response of bacteria.

To understand how cell physiological state affects mRNA translation, we used Shewanella oneidensis MR-1 grown under steady state conditions at either 20% or 8.5% O2. Using a combination of quantitative proteomics and RNA-Seq, we generated high-confidence data on >1000 mRNA and protein pairs. By using a steady state model, we found that differences in protein-mRNA ratios were primarily due to differences in the translational efficiency of specific genes. When oxygen levels were lowered, 28% of the proteins showed at least a 2-fold change in expression. Transcription levels were sp. significantly altered for 26% of the protein changes; translational efficiency was significantly altered for 46% and a combination of both was responsible for the remaining 28%. Changes in translational efficiency were significantly correlated with the codon usage pattern of the genes and measurable tRNA pools changed in response to altered O2 levels. Our results suggest that changes in the translational efficiency of proteins, in part due to altered tRNA pools, is a major determinant of regulated alterations in protein expression levels in bacteria.