Search

Additional file 1:Figure S1. The experiment presented in Fig. 2 was repeated, and reproducible titers were obtained at a maximum of a C:N ratio of 40:1 for both urea and ammonium sulfate media, with a dip in TAL titers at a C:N of 8:1 in urea media. Error bars represent standard deviation of biological triplicates.Figure S2. Addition of buffer slightly improves TAL production. All buffers were added at a concentration of 100mM. Error bars represent standard deviation of biological triplicates.Figure S3. Antibiotic addition slows early growth on day 1 in YPD, but does not reduce maximum titers later on. TAL was measured after four days of growth. Antibiotic concentrations are listed in working concentration (µg/mL) under each condition in the top graph, which reflects the identical order in the bottom graph. Error bars represent standard deviation of biological triplicates.Figure S4. Comparison of hydrolysates derived from field-grown sorghum biomass that was stored via ensilage, or was dried prior to storage. Fermentation was performed in bench-scale flask cultures. (A) Titers obtained during fermentation over time. (B) Glucose during fermentation. (C) Xylose during fermentation. (D) Total viable colony forming units (CFUs) obtained at the end of the experiment. All error bars represent standard deviations of biological triplicates.Figure S5. The effect of addition of 1.25% DMSO to enhance TAL titers from ensiled hydrolysates produced from the same batch as in Supplementary Figure S4. Error bars represent standard deviation of biological triplicates.Figure S6. The same experiment performed in parallel to Fig. 4, but using only one day of pre-adaption in the final step of the seed culturing. In this case, a slight delay in growth in both dry and ensiled hydrolysates occurred during the first day. (A) Titers obtained during fermentation on filtered hydrolysates over time. (B) Growth in filtered hydrolysates, as measured by optical densities. (C) Primary carbon sources during fermentation on filtered hydrolysates, at the experiment start and end. (D) Titers obtained during fermentation on unfiltered, pasteurized hydrolysates over time. (E) Growth in unfiltered hydrolysates, as measured by viable colony forming units (CFUs) of R. toruloides on YPD plates. The limit of detectable colony units is indicated (1000 units/mL). (F) Primary carbon sources during fermentation on unfiltered hydrolysates, at the experiment start and end. All error bars represent standard deviations of biological triplicates.Figure S7. Analysis of partition coefficient of TAL between water and various common solvents used as overlays in bioreactor fermentations. Water was mixed at a 1:1 ratio with each solvent, and 6 g/L TAL was added. Subsequently, the aqueous phase was analyzed for TAL titers in five technical replicates. Error bars represent standard deviation. The partition coefficient between dodecane, durasyn164, and oleyl alcohol and water were calculated as 1.11, 0.59, and 2.68 respectively.

Additional file 1: Figure S1. Nitrogen physisorption isotherms of Pd/SiAl. Figure S2. Nitrogen physisorption isotherms of Pd/SiO2. Figure S3. Nitrogen physisorption isotherms of Pd/Al2O3. Figure S4. Nitrogen physisorption isotherms of Al2O3. Figure S5. Nitrogen physisorption isotherms of Ni/Al2O3. Figure S6. Nitrogen physisorption isotherms of Cu/Al2O3. Figure S7. Nitrogen physisorption isotherms of Pt/Al2O3. Figure S8. Mass spectrum of limonene. Figure S9. Mass spectrum of 1,8-cineole. Figure S10. Mass spectrum of p-cymene. Figure S11. GC–MS spectrum of products of Pd/SiO2 catalytic reaction with limonene for 3 h. Figure S12. GC–MS spectrum of products of Pd/SiO2 catalytic reaction with 1,8-cineole for 3 h. Figure S13. GC–MS spectrum of products of γ-Al2O3 catalytic reaction with limonene for 3 h. Figure S14. GC–MS spectrum of products of γ-Al2O3 catalytic reaction with 1,8-cineole for 3 h. Figure S15. Deactivation of 100 mg of 5 wt% Pd/Al2O3 with (A) limonene (B)1,8-cineole as the reactants under 250 °C. Figure S16. Production of 1,8-cineole. (A) production at different C/N ratios; (B) production at different induction conditions. Figure S17. Production of 1,8-cineole with HMGR_Da expressed from different plasmid configurations. (A) Plasmid configurations (HMGR_Da in the plasmid is highlighted); (B) 1, 8-cineole titers; (C) Pyruvate accumulation. Production was done in test tubes using fermentation media supplemented with 1.5% glucose, grown at 30 °C. Figure S18. Fed-batch fermentation for Ferm 3_Da. Figure S19. Glucose consumption comparison for Ferm 2 and Ferm 3.

The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual-inducible duet-expression vectors for P. putida KT2440. A number of inducible promoters (Plac , Ptac , PtetR/tetA and Pbad ) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.

Although the use of ionic liquids (ILs) for the pretreatment of lignocellulosic biomass has been limited due to high costs, recent efforts to develop low-cost protic ILs show promise for achieving cost-effectiveness for biorefineries. However, an additional challenge remains in that ILs present in biomass hydrolysates are toxic to most microbial hosts, resulting in poor growth phenotypes. To address this issue, we applied an adaptive laboratory evolution (ALE) approach for tolerizingPseudomonas putidaKT2440, an industrially relevant bacterial host, to two low-cost ILs (triethanolammonium acetate [TEOH][OAc] and triethylammonium hydrogen sulfate [TEA][HS]). After continuous cultivations with gradually increased IL levels, we obtained evolved strains showing significant improvements in their growth performance under high concentrations of the ILs (maximum 4% [TEOH][OAc] and 8% [TEA][HS], in w/v) at which the wild-type strain cannot grow. Sequencing of evolved strains revealed multiple regions where mutations were associated with improved performance in minimal media conditions (relA,gacS,oprB/PP_1446,fleQ,tktA, anduvrY/PP_4100) and in IL-specific conditions (PP_5350, PP_4929/emrE,oprD, and PP_5324). We further validated the causality of the PP_5350 andemrEgenes for improved IL toleranceviareverse engineering and transcriptomic analysis. A common mutation in the PP_5350 gene, encoding a RpiR family transcriptional regulator, was shown to significantly upregulate the glyoxylate cycle for efficient acetate catabolism. In addition, it was suggested that theemrEgene encodes an efflux pump which can export [TEA][HS]. Finally, the cultivation of two of the best performing evolved strains with IL-treated biomass hydrolysates demonstrated their considerable potential to be used as platform strains. Taken as a whole, this work provides strains for utilization of IL-treated biomass and a mechanistic understanding that could be further leveraged to develop efficient microbial bioprocesses.

Pseudomonas putida is a widely used host for metabolic engineering and synthetic biology. However, the use of P. putida has been hampered by the availability of a limited set of expression vectors for producing heterologous proteins. To widen the scope of expression vectors for gene co-expression studies, a previously established dual-inducible expression vector pRG_Duet2 developed for Corynebacterium glutamicum has been modified for use in P. putida. This expression vector, named pRGPDuo2, harbors two origins of replication, colE1 for replication in E. coli and pRO1600 for replication in P. putida. Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are individually controlled by inducible promoters Ptac or PtetR/tetA. Functional validation of pRGPDuo2 was confirmed by the co-expression of genes for the fluorescent proteins namely, superfolder green fluorescent protein (sfGFP), and red fluorescent protein (RFP). Moreover, the strength of the fluorescence signal was dependent on the inducer concentrations present in the culture medium. The expression vector pRGPDuo2 is an attractive addition to the existing repertoire of expression plasmids for expression profiling and adds to the tools available for P. putida metabolic engineering.

Studies on bacterial physiology are incomplete without knowledge of the signalling and regulatory systems that a bacterium uses to sense and respond to its environment. Two-component systems (TCSs) are among the most prevalent bacterial signalling systems, and they control essential and secondary physiological processes; however, even in model organisms, we lack a complete understanding of the signals sensed, the phosphotransfer partners and the functions regulated by these systems. In this review, we discuss several tools to map the genes targeted by transcriptionally acting TCSs. Many of these tools have been used for studying individual TCSs across diverse species, but systematic approaches to delineate entire signalling networks have been very few. Since genome sequences and high-throughput technologies are now readily available, the methods presented here can be applied to characterize the entire DNA-binding TCS signalling network in any bacterial species and are especially useful for non-model environmental bacteria.

Additional file 1. Supplementary figures and tables.

The rhizosphere microbiome (rhizobiome) plays a critical role in plant health and development. However the processes by which the constituent microbes interact to form and maintain a community are not well understood. To investigate these molecular processes, we examined pairwise interactions between 11 different microbial isolates under selected nutrient-rich and nutrient-limited conditions. We observed that when grown with media supplemented with 56 mM glucose, 2 microbial isolates were able to inhibit the growth of 6 out of 11 other microbes tested. The interaction between microbes persisted even after the antagonistic microbe was removed, upon exposure to spent media. To probe the genetic basis for these antagonistic interactions, we used a barcoded transposon library in a proxy bacterium, Pseudomonas putida , to identify genes which showed enhanced sensitivity to the antagonistic factor(s) secreted by Acinetobacter sp. 02. Iron metabolism-related gene clusters in P. putida were implicated by this systems-level analysis. The supplementation of iron prevented the antagonistic interaction in the original microbial pair supporting the hypothesis that iron limitation drives antagonistic microbial interactions between rhizobionts. We conclude that rhizobiome community composition is influenced by competition for limiting nutrients with implications for growth and development of the plant.

Advances in engineering biology have expanded the list of renewable compounds that can be produced at scale via biological routes from plant biomass. In most cases, these chemical products have not been evaluated for effects on biological systems, defined in the present study as bioactivity, that may be relevant to their manufacture. For sustainable chemical and fuel production, the industry needs to transition from fossil to renewable carbon sources, resulting in unprecedented expansion in the production and environmental distribution of chemicals used in biomanufacturing. Further, although some chemicals have been assessed for mammalian toxicity, environmental and agricultural hazards are largely unknown. We assessed 6 compounds that are representative of the emerging biofuel and bioproduct manufacturing process for their effect on model plants (Arabidopsis thaliana, Sorghum bicolor) and show that several alter plant seedling physiology at submillimolar concentrations. However, these responses change in the presence of individual bacterial species from the A. thaliana root microbiome. We identified 2 individual microbes that change the effect of chemical treatment on root architecture and a pooled microbial community with different effects relative to its constituents individually. The present study indicates that screening industrial chemicals for bioactivity on model organisms in the presence of their microbiomes is important for biologically and ecologically relevant risk analyses. Environ Toxicol Chem 2019;38:1911-1922. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Systems biology and synthetic biology are increasingly used to examine and modulate complex biological systems. As such, many issues arising during scaling-up microbial production processes can be addressed using these approaches. We review differences between laboratory-scale cultures and larger-scale processes to provide a perspective on those strain characteristics that are especially important during scaling. Systems biology has been used to examine a range of microbial systems for their response in bioreactors to fluctuations in nutrients, dissolved gases, and other stresses. Synthetic biology has been used both to assess and modulate strain response, and to engineer strains to improve production. We discuss these approaches and tools in the context of their use in engineering robust microbes for applications in large-scale production.

© 2018 John Wiley & Sons Ltd Bacterial response to metals can require complex regulation. We report an overlapping regulation for copper and zinc resistance genes in the denitrifying bacterium, Pseudomonas stutzeri RCH2, by three two-component regulatory proteins CopR1, CopR2 and CzcR. We conducted genome-wide evaluations to identify gene targets of two paralogous regulators, CopR1 and CopR2, annotated for copper signaling, and compared the results with the gene targets for CzcR, implicated in zinc signaling. We discovered that the CopRs and CzcR have largely common targets, and crossregulate a core set of P. stutzeri copper and zinc responsive genes. We established that this crossregulation is enabled by a conserved binding motif in the upstream regulatory regions of the target genes. The crossregulation is physiologically relevant as these regulators synergistically and antagonistically target multicopper oxidases, metal efflux and sequestration systems. CopR1 and CopR2 upregulate two cop operons encoding copper tolerance genes, while all three regulators downregulate a putative copper chaperone, Psest_1595. CzcR also upregulated the oprD gene and the CzcIABC Zn2+efflux system, while CopR1 and CopR2 downregulated these genes. Our study suggests that crossregulation of copper and zinc homeostasis can be advantageous, and in P. stutzeri this is enabled by shared binding motifs for multiple response regulators.

© 2016 American Chemical Society We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. In the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.

The scope of the project is to investigate efflux pump systems in engineered host microorganisms, such as E. coli, and develop a pump engineered to export a target compound. To initiate the project in coordination with other TOTAL driven projects, the first target compound to be studied was 1-hexene. However, we were investigating other chemicals as Styrene. The main goal of the project was to generate a set of optimized efflux pump systems for microorganisms (E. coli and Streptomyces or other host) engineered to contain biosynthetic pathways to export large titers of target compounds that are toxic (or accumulate and push back biosynthesis) to the host cell. An optimized microbial host will utilize specific and efficient cell wall located pumps to extrude harmful target compounds and enable greater production of these compounds.

© 2016 The Royal Society of Chemistry. Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multi-step process. Typically, each segment of the workflow is optimized separately, often generating conditions that may not be suitable for integration or consolidation with the upstream or downstream steps. While significant effort has gone into developing solutions to incompatibilities at discrete steps, very few studies report the consolidation of the multi-step workflow into a single pot reactor system. Here we demonstrate a one-pot biofuel production process that uses the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) for pretreatment of switchgrass biomass. [C2C1Im][OAc] is highly effective in deconstructing lignocellulose, but nonetheless leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. We report the discovery of an [C2C1Im]-tolerant E. coli strain, where [C2C1Im] tolerance is bestowed by a P7Q mutation in the transcriptional regulator encoded by rcdA. We establish that the causal impact of this mutation is the derepression of a hitherto uncharacterized major facilitator family transporter, YbjJ. To develop the strain for a one-pot process we engineered this [C2C1Im]-tolerant strain to express a recently reported d-limonene production pathway. We also screened previously reported [C2C1Im]-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the [C2C1Im]-tolerant E. coli strain so as to secrete this [C2C1Im]-tolerant cellulase. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel candidate precursor d-limonene in a one-pot process.

The trace elements molybdenum and tungsten are essential components of cofactors of many metalloenzymes. However, in sulfate-reducing bacteria, high concentrations of molybdate and tungstate oxyanions inhibit growth, thus requiring the tight regulation of their homeostasis. By a combination of bioinformatic and experimental techniques, we identified a novel regulator family, tungstate-responsive regulator (TunR), controlling the homeostasis of tungstate and molybdate in sulfate-reducing deltaproteobacteria. The effector-sensing domains of these regulators are similar to those of the known molybdate-responsive regulator ModE, while their DNA-binding domains are homologous to XerC/XerD site-specific recombinases. Using a comparative genomics approach, we identified DNA motifs and reconstructed regulons for 40 TunR family members. Positional analysis of TunR sites and putative promoters allowed us to classify most TunR proteins into two groups: (i) activators of modABC genes encoding a high-affinity molybdenum and tungsten transporting system and (ii) repressors of genes for toluene sulfonate uptake (TSUP) family transporters. The activation of modA and modBC genes by TunR in Desulfovibrio vulgaris Hildenborough was confirmed in vivo, and we discovered that the activation was diminished in the presence of tungstate. A predicted 30-bp TunR-binding motif was confirmed by in vitro binding assays. A novel TunR family of bacterial transcriptional factors controls tungstate and molybdate homeostasis in sulfate-reducing deltaproteobacteria. We proposed that TunR proteins participate in protection of the cells from the inhibition by these oxyanions. To our knowledge, this is a unique case of a family of bacterial transcriptional factors evolved from site-specific recombinases.

Lignocellulosic biomass is utilized as a renewable feedstock in various agro-industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these 'DP reducers' is achieved by expressing a bacterial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side-chain of hydroxycinnamoyl-CoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild-type plants. However, cell wall characterization of extract-free stems by thioacidolysis and by 2D-NMR revealed an increased amount of unusual C₆C₁ lignin monomers most likely linked with lignin as end-groups. Moreover the analysis of lignin isolated from these plants using size-exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C₆C₁ monomers ('DP reducers') into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis.

The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C(16) and C(18) unsubstituted, ω-hydroxy, and α,ω-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of ω-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm.

Microorganisms have been rich sources for natural products, some of which have found use as fuels, commodity chemicals, specialty chemicals, polymers, and drugs, to name a few. The recent interest in production of transportation fuels from renewable resources has catalyzed numerous research endeavors that focus on developing microbial systems for production of such natural products. Eliminating bottlenecks in microbial metabolic pathways and alleviating the stresses due to production of these chemicals are crucial in the generation of robust and efficient production hosts. The use of systems-level studies makes it possible to comprehensively understand the impact of pathway engineering within the context of the entire host metabolism, to diagnose stresses due to product synthesis, and provides the rationale to cost-effectively engineer optimal industrial microorganisms.

In an effort to investigate effects of environmental stressors on bacteria that are found to co-exist in several DOE-contaminated sites, we studied the effect of salt stress on Geobacter metallireducens strain GS15. A comparison of these results with salt stress in Desulfovibrio vulgaris and Shewanella oneidensis will provide a better understanding of bioremediation by these bacteria under hypersaline conditions. Strain GS15 was grown in the defined LS4D medium at 30oC containing 10mM acetate and 10mM Fe-NTA. In these growth conditions, the minimum inhibitory concentration of NaCl on this organism was determined to be 100mM. To examine salt stress response of GS15 in detail, 100mM NaCl was added to a midlog phase culture and cells were harvested after 1, 2 and 4 hours for different analyses including Fe(III) reduction, transcriptomics and phospholipids fatty acid analysis (PLFA). In stressed cultures, after 4 hours of exposure to salt, only 7mM of Fe(II) was reduced compared to more than 9mM in non-stressed controls. Transcriptomic analysis revealed that after 4 hrs, the most up-regulated genes observed were those that encode heat shock proteins, such as Hsp90, Hsp 20, GrpE, DnaK, and DnaJ. These heat shock proteins perform protein repair functions which can be in demand during stress. Also up-regulated were genes encoding proteins in the large and small subunits of the ribosome. The most down-regulated genes included those encoding the, sodium ion symporter, some genes of cytochrome C family and flagellar proteins (flagellin, FlaG, and FlgJ). It was also observed that several proteins involved in the process of chemotaxis like CheA, CheR, CheY, CheX along with several histidine kinases and response regulators were downregulated. PLFA analysis of stressed cells was performed and compared to non-stressed cells, showing small shifts in relative levels of saturated and unsaturated fatty acids in response to salt stress. The results of this study suggests that presence of 100mM NaCl causes some perturbation in the cell machinery of strain GS15 and that cells are able to overcome this stress without fatal consequences in response to stress.

The overview of changes in protein levels or states in response to a growth condition, stress, mutation or metabolic engineering is invaluable in understanding the physiology of a microbial system. The lipid profile of the cell is similarly a valuable diagnostic of the cellular response and health, especially in context of survival in a fluctuating environment. To obtain comprehensive cellular models, post-transcriptional cell wide surveys at the levels of proteins and lipids are required. Both these fields have been greatly bolstered by the development of high throughput methods using mass spectrometry. Multiple strategies now exist for the identification of proteins, and numerous workflows to quantify protein abundance have also been developed. Cellular profiling such as these allows us to assess the potential of a microbial system for environmental applications such as bioremediation and bio-energy.

We used isotopic labeling and metabolite analyses to test the predicted amino acid synthesis pathways in the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough. The data suggest several pathways that were not apparent in the initial genome annotation. First, D. vulgaris synthesizes isoleucine via citramalate synthase (DVU1914). Second, D. vulgaris synthesizes methionine via O-succinyl-homoserine, cystathionine, and homocysteine. DVU0171 is a candidate for cystathionine beta-lyase, but genes for the other steps are not apparent. Third, in vitro assays confirm the presence of Re-citrate-synthase (DVU0398), which is closely related to the recently identified Re-citrate-synthase found in some Clostridia. We also propose that D. vulgaris synthesizes 3-dehydroquinate, which is required for the synthesis of aromatic amino acids, via archaeal-like transaldolase and 3-dehydroquinate cyclase/deaminase (DVU0460,DVU0461).

The mechanisms of Desulfovibrio vulgaris Hildenborough responses to long-term NaCl exposure were studied by global transcriptional and metabolite analyses. The growth of D. vulgaris was inhibited by high salinity, and salt inhibition could be relieved by an addition of amino acids (e.g., glutamate, alanine) or yeast extract. Salt shock (sudden increase in salt concentration) and salt adaptation (inoculating cells in the medium containing high concentrations of salt) showed a significant difference in respective transcriptomes. Salt adaptation induced expression of genes involved in amino acid biosynthesis and transportation, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). Genes involved in energy metabolism, cell motility, and phage structures were repressed. Genes involved in Na+/H+ transport, K+ uptake and transportation, and proline biosynthesis and transportation were not significantly affected. Metabolite assays and external addition of amino acids into the growth medium of D. vulgaris suggest that amino acids, such as glutamate and alanine may accumulate as osmoprotectants in D. vulgaris. A conceptual model is proposed to link our observed results to currently available knowledge for further understanding the mechanisms of adaptation of D. vulgaris to sodium chloride.

A fundamental goal of the Environmental Stress Pathway Project (ESPP) of the Virtual Institute of Stress and survival (VIMSS) is a rigorous understanding of Desulfovibrio vulgaris Hildenborough physiology and its ability to survive in its environment. Such knowledge will be critical in discerning the biogeochemistry at metal contaminated sites, for bioremediation and natural attenuation for toxic metals. The Functional Genomics and Imaging Core (FGIC) focuses on the measurement of these responses at a cell wide level using systems biology approaches. Progress in the last one year built upon our optimized pipeline for generating biomass for various functional genomics studies and utilized improved genetic methods. Numerous additional transcriptomics data sets were added to our compendium of stress response studies. These included peroxide stress, low oxygen stress, high and low pH stress and alteration of growth conditions (e.g presence of methionine, alternate electron donors etc). To understand how genotype and environment interact to determine the phenotype and fitness of an organism, a long-term evolution experiment was also conducted to examine the dynamics and adaptation of D. vulgaris under extended salt exposure. For many of these stresses iTRAQ based quantitative proteomics and CE-MS based metabolite studies were also conducted. Improved genetic methods were employed to create several critical knock out mutants (e.g. echA, qmoABC and tatA) and several were characterized via growth and transcriptomics studies. Progress was also made in extending transcriptomics analysis to examine alternate D. vulgaris physiological states such as in biofilms and growth in syntrophic co-culture with Methanococcus maripaludis. Methods to conduct iTRAQ proteomics and stable isotopomer (13C) based metabolic flux analysis were also developed for studying co-cultures. A novel FTICR-MS based method for a comparative 12C/13C based metabolite analysis is being developed and will enable a direct comparison of control cultures to experimental samples. Additionally we continued to collect cell wide data in Shewanella oneidensis and Geobacter metallireducens for comparative studies. Great progress was made in improving extraction and high throughput of metabolite studies. Metabolite extraction and CE-MS detection for several hundred metabolites can now be conducted for these non-model organisms using high resolution separation and high resolution mass spectrometric methods. Continued studies to map cell wide responses have also emphasized the importance of changes that require orthologous measurements. With this in mind a novel protocol to monitor protein-protein interactions and redox state of the proteins has been developed. In an effort to supplement model development and elucidate intricacies of stress response cascades, comprehensive methods for identifying alternative regulatory mechanisms such as small non-coding RNAs are also underway. To optimize the use of the large amounts of data being collected, several data mining efforts were initiated. For example, iTRAQ data sets from the multiple stress response studies were mined for potential post translational modifications and confirmation of hypothetical proteins while 13C flux data were used to confirm gene annotation and assess missing steps in metabolic pathways. Work in underway in collaboration with the computational core to set up searchable databases of our proteomics and metabolite data also.

Desulfovibrio vulgaris Hildenborough (DvH) is an obligate anaerobe and has been used as a model organism for studying the energy metabolism of sulfate-reducing bacterium (SRB). However, experimental data about the transcriptional regulatory networks which are essential for understanding the cellular processes are very limited. In this study, several predicted global regulators are investigated via mutant characterization, transcriptomic assay and their in vivo gene regulations using ChIP-chip assay.

The Protein Complex Analysis Project (PCAP) has two major goals: 1. to develop an integrated set of high throughput pipelines to identify and characterize multi-protein complexes in a microbe more swiftly and comprehensively than currently possible and 2. to use these pipelines to elucidate and model the protein interaction networks regulating stress responses in Desulfovibrio vulgaris with the aim of understanding how this and similar microbes can be used in bioremediation of metal and radionuclides found in U.S. Department of Energy (DOE) contaminated sites. PCAP builds on the established research and infrastructure of another Genomics: GTL initiative conducted by the Environmental Stress Pathways Project (ESPP). ESPP has developed D. vulgaris as a model for stress responses and has used gene expression profiling to define specific sets of proteins whose expression changes after application of a stressor. Proteins, however, do not act in isolation. They participate in intricate networks of protein/protein interactions that regulate cellular metabolism. To understand and model how these identified genes affect the organism, therefore, it is essential to establish not only the other proteins that they directly contact, but the full repertoire of protein/protein interactions within the cell. In addition, there may well be genes whose activity is changed in response to stress not by regulating their expression level but by altering the protein partners that they bind, by modifying their structures, or by changing their subcellular locations. There may also be differences in the way proteins within individual cells respond to stress that are not apparent in assays that examine the average change in a population of cells. Therefore, we are extending ESPP's analysis to characterize the polypeptide composition of as many multi-protein complexes in the cell as possible and determine their stoichiometries, their quaternary structures, and their locations in planktonic cells and in individual cells within biofilms. PCAP will characterize complexes in wild type cells grown under normal conditions and also examine how these complexes are affected in cells perturbed by stress or by mutation of key stress regulatory genes. These data will all be combined with those of the ongoing work of the ESPP to understand, from a physical-chemical, control-theoretical, and evolutionary point of view, the role of multi-protein complexes in stress pathways involved in the biogeochemistry of soil microbes under a wide variety of conditions. Essential to this endeavor is the development of automated high throughput methods that are robust and allow for the comprehensive analysis of many protein complexes. Biochemical purification of endogenous complexes and identification by mass spectrometry is being coupled with in vitro and in vivo EM molecular imaging methods. Because no single method can isolate all complexes, we are developing two protein purification pipelines, one the current standard Tandem Affinity Purification approach, the other a novel tagless strategy. Specific variants ofeach of these are being developed for water soluble and membrane proteins. Our Bioinstrumentation group is developing highly parallel micro-scale protein purification and protein sample preparation platforms, and mass spectrometry data analysis is being automated to allow the throughput required. The stoichiometries of the purified complexes are being determined and the quaternary structures of complexes larger than 250 kDa are being solved by single particle EM. We are developing EM tomography approaches to examine whole cells and sectioned, stained material to detect complexes in cells and determine their localization and structures. New image analysis methods will be applied to speed determination of quaternary structures from EM data. Once key components in the interaction network are defined, to test and validate our pathway models, mutant strains not expressing these

Sulfate reducing bacteria (SRB), found widely in nature, use sulfate as the terminal electron acceptor in their respiratory cycle, leading to the production of hydrogen sulfide. These bacteria have both ecological and economic importance. SRB play a role in various biogeochemical cycles including the sulfur and carbon cycles. They have a negative economic impact on the oil industry, where their metabolism causes corrosion and clogging of machinery, and fouling of oil wells. However, they have also been shown to reduce and/or immobilize toxic water-soluble metals such as copper (II), chromium (IV) and uranium (VI), and thus are candidates for bioremediation applications. Desulfovibrio vulgaris Hildenborough (DvH) is a member of the most well studied genus of SRBs. A goal of the Environmental Stress Pathway Project (ESPP) in the Virtual Institute for Microbial Stress and Survival (VIMSS) is to understand the regulatory networks in DvH for applications to bioremediation. One aspect of this is the elucidation of protein post-translational modifications (PTMs) in DvH.PTMs play various roles in the cell. Some modifications play a role in protein structure, such as lipid anchors or some disulfide bonds. Others are directly involved in regulation of protein function such as phosphorylation and glycosylation. Still others arise through cellular damage such as irreversible oxidation events. Whatever the role these PTMs play, they must be characterized at the protein level because they are not directly coded for in the genome. Furthermore, DvH may be particularly likely to use PTMs as a regulatory mechanism: Evidence for this includes the observation that the DvH genome encodes an abnormal number of histidine kinases. Our goal is to determine the types of protein modifications that arise in DvH and how these modifications affect the ability of DvH to survive or adapt to its environment. This work leverages the unique resources of the Virtual Institute for Microbial Stress and Survival: Quality controlled biomass produced at LBL (Hazen lab) is used for all proteomic LC/MS/MS measurements at LBL (Keasling lab). Our initial survey of PTMs in DvH was obtained by mining these numerous proteomic LC/MS/MS data sets acquired over the course of ESPP for evidence of modified peptides. Data mining for PTMs is performed at Sandia National Labs. The searched-for modifications were determined based on literature precedence and a genome search for the existence of relevant transferases. To date we have found preliminary evidence for cysteine oxidation, lysine acetylation, and methylation of lysine and arginine. Data mining for additional PTMs is ongoing. Future work will focus on validation of these findings and determining which, if any, of these modifications play a regulatory role in DvH. Validation will require selective isolation of the proteins of interest for further characterization. Here, protein isolation is made possible through the work being performed at LBL and the University of Missouri to generate DvH mutants containing tagged versions of DvH proteins.

nifgenes, found primarily in bacteria, encode the nitrogenase complex and other enzymes involved in nitrogen fixation. Though found in aerobic bacteria, nitrogen fixation occurs optimally in anaerobic conditions and nif genes are present in many obligate and facultative anaerobic bacteria as well as some archaea. The consensus sequences in these genes are highly conserved across nitrogen fixing bacteria, but variability exists in their regulation. In the anaerobic sulfate reducing soil bacterium Desulfovibrio vulgaris Hildenborough the nif genes are encoded on a 200kb megaplasmid which harbors approximately 5% of all of the genes in D. vulgaris. The function of the megaplasmid in the growth and survival of D. vulgaris is not well understood and the megaplasmid is documented to be lost in growth conditions that do not require nitrogen fixation (such as excess ammonium salts). In order determine the growth conditions that playa role in nitrogen utilization a wild type D. vulgaris strain containing the megaplasmid was compared with a D. vulgaris strain lacking the megaplasmid (MP(-)). Nitrogen source utilization is evaluated by direct growth response using Omnilog Phenotype Microarray assays. The selected nitrogen utilization phenotype microarray consists of a single 96 wellplate loaded with 95 different nitrogen containing compounds. These assays, were used to track and compare the nitrogen utilization profiles and assess specific metabolic pathways for nitrogen assimilation. Activated megaplasmid nif gene function is seen with the growth of D. vulgaris on Nz in the absence of added inorganic nitrogen compounds. For the megaplasmid containing wild type strain, growth on N2 alone yields three-fold longer generation times which is not observed with the MP(-) strain. Genes on the megaplasmid may also confer resistance to toxic ions as D. vulgaris growth with NOz and N03 reaches higher final yields than the MP(-) strain. Here we demonstrate how phenotypic mapping of the two strains reveal some of the functions of the D. vulgaris megaplasmid.

Though considered obligate anaerobes for many years after their discovery, sulfate reducing bacteria like Desulfovibrio vulgaris Hildenborough (DvH) are found in environments with very low sulfate and in many environments that are regularly exposed to oxygen or are normally aerobic. The best growth condition for DvH, measured as increase in biomass, remains a completely anaerobic environment. However, DvH is clearly able to tolerate sub-aerobic environments and can survive exposure to air for up to 20 days. Controlled experiments were conducted to expose DvH to aerobic and microaerobic conditions (0.1% O2). Cell-wide responses were monitored via transcriptomics and proteomics measurements. Microaerobic conditions caused an overall decrease in growth without affecting the viability of the bacterium. Cellular responses to microaerobic conditions were mild and primarily included up-regulation of the putative PerR regulon, but other known oxidative stress response candidates remained unchanged. Other transcripts that show an expression profile similar to the PerR regulon genes included the cydA/B operon, encoding putative oxidative phosphorylation proteins. However, comparison with data from prior DvH functional genomics studies suggested that many of these changes could be part of a general stress response in DvH. In contrast, exposure to air produced drastic changes at both the transcriptome and proteome levels and had a detrimental effect on both growth and viability of DvH. During aerobic stress, increases in proteases and chaperones signified air exposure to be a very harsh stress in DvH. However, quantitative proteomics also indicated an accumulation of superoxide-dismutase, catalase as well as ferritins and thioredoxins, and these candidates may be critical for the survival of the small fraction of cells which survive air exposure. Our results indicated that DvH has very different responses towards microaerobic vs. aerobic exposure. Growth of DvH strains under these different O2 exposures and the data from our integrated genomics experiment are presented and have been used to improve the model for O2 stress response in DvH.

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

Fatty acid-derived biofuels can be a better solution than bioethanol to replace petroleum fuel, since they have similar energy content and combustion properties as current transportation fuels. The environmentally friendly microbial fermentation process has been used to synthesize advanced biofuels from renewable feedstock. Due to their robustness as well as the high tolerance to fermentation inhibitors and phage contamination, yeast strains such as Saccharomyces cerevisiae and Yarrowia lipolytica have attracted tremendous attention in recent studies regarding the production of fatty acid-derived biofuels, including fatty acids, fatty acid ethyl esters, fatty alcohols, and fatty alkanes. However, the native yeast strains cannot produce fatty acids and fatty acid-derived biofuels in large quantities. To this end, we have summarized recent publications in this review on metabolic engineering of yeast strains to improve the production of fatty acid-derived biofuels, identified the bottlenecks that limit the productivity of biofuels, and categorized the appropriate approaches to overcome these obstacles. Virginia Tech. Start-up fund - #175323 Virginia Tech. Open Access Subvention Fund