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Small genes (40,000 small-gene families in ~2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enriches for small genes that are translated in microbiomes, suggesting the small genes identified are coding. More than 9,000 families encode potentially secreted or transmembrane proteins, more than 5,000 families encode predicted anti-CRISPR proteins, and more than 500 families encode predicted antimicrobial proteins. By combining homology and genomic-neighborhood analyses, we reveal substantial novelty and diversity within phage biology, including small phage genes found in multiple host phyla, small genes encoding proteins that play essential roles in host infection, and small genes that share genomic neighborhoods and whose encoded proteins may share related functions.

We recently reported the discovery of phenylacetate decarboxylase (PhdB), representing one of only ten glycyl‐radical‐enzyme reaction types known, and a promising biotechnological tool for first‐time biochemical synthesis of toluene from renewable resources. Here, we used experimental and computational data to evaluate the plausibility of three candidate PhdB mechanisms, involving either attack at the phenylacetate methylene carbon or carboxyl group [via H‐atom abstraction from COOH or single‐electron oxidation of COO− (Kolbe‐type decarboxylation)]. In vitro experimental data included assays with F‐labeled phenylacetate, kinetic studies, and tests with site‐directed PhdB mutants; computational data involved estimation of reaction energetics using density functional theory (DFT). The DFT results indicated that all three mechanisms are thermodynamically challenging (beyond the range of many known enzymes in terms of endergonicity or activation energy barrier), reflecting the formidable demands on PhdB for catalysis of this reaction. Evidence that PhdB was able to bind α,α‐difluorophenylacetate but was unable to catalyze its decarboxylation supported the enzyme's abstraction of a methylene H atom. Diminished activity of H327A and Y691F mutants was consistent with proposed proton donor roles for His327 and Tyr691. Collectively, these and other data most strongly support PhdB attack at the methylene carbon., Three candidate mechanisms for the novel glycyl radical enzyme (GRE) phenylacetate decarboxylase (PhdB) were evaluated using a combination of in vitro experimental data and computational data involving estimation of reaction energetics. The data best support a methylene carbon attack (green arrows) that differs substantially from the reported mechanism for p‐hydroxyphenylacetate decarboxylase.

Determining the carbon sources for active microbial populations in the subsurface is a challenging but highly informative component of subsurface microbial ecology. This work developed a method to provide ecological insights into groundwater microbial communities by characterizing community RNA through its radiocarbon and ribosomal RNA (rRNA) signatures. RNA was chosen as the biomolecule of interest because rRNA constitutes the majority of RNA in prokaryotes, represents recently active organisms, and yields detailed taxonomic information. The method was applied to a groundwater filter collected from a shallow alluvial aquifer in Colorado. RNA was extracted, radiometrically dated, and the 16S rRNA was analyzed by RNA-Seq. The RNA had a radiocarbon signature (Δ14C) of −193.4 ± 5.6‰. Comparison of the RNA radiocarbon signature to those of potential carbon pools in the aquifer indicated that at least 51% of the RNA was derived from autotrophy, in close agreement with the RNA-Seq data, which documented the prevalence of autotrophic taxa, such as Thiobacillus and Gallionellaceae. Overall, this hybrid method for RNA analysis provided cultivation-independent information on the in-situ carbon sources of active subsurface microbes and reinforced the importance of autotrophy and the preferential utilization of dissolved over sedimentary organic matter in alluvial aquifers.

Plants are an attractive sourceof renewable carbon for conversion to biofuels and bio-based chemicals. Conversion strategies often use a fraction of the biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components, such as aromatics and amino acids, may improve the efficiency of biomass conversion. Pseudomonas putida is a promising host for its ability to metabolize a wide variety of organic compounds. P. putida was engineered to produce methyl ketones, which are promising diesel blendstocks and potential platform chemicals, from glucose and lignin-related aromatics. Unexpectedly, P. putida methyl ketone production using Arabidopsis thaliana hydrolysates was enhanced 2-5-fold compared with sugar controls derived from engineered plants that overproduce lignin-related aromatics. This enhancement was more pronounced (~seven-fold increase) with hydrolysates from nonengineered switchgrass. Proteomic analysis of the methyl ketone-producing P. putida suggested that plant-derived amino acids may be the source of this enhancement. Mass spectrometry-based measurements of plant-derived amino acids demonstrated a high correlation between methyl ketone production and amino acid concentration in plant hydrolysates. Amendment of glucose-containing minimal media with a defined mixture of amino acids similar to those found in the hydrolysates studied led to a nine-fold increase in methyl ketone titer (1.1 g/L).

The long-term stability of U(IV) solid phases in anaerobic aquifers depends upon their reactivity in the presence of oxidizing chemical species and microbial catalysts. We performed flow-through column experiments under anaerobic conditions to investigate the mechanisms and dissolution rates of biogenic, noncrystalline UO2(s) by chemical oxidants (nitrate and/or nitrite) or by Thiobacillus denitrificans, a widespread, denitrifying, chemolithoautotrophic model bacterium. Dissolution rates of UO2(s) with dissolved nitrite were approximately 5 to 10 times greater than with nitrate alone. In the presence of wild-type T. denitrificans and nitrate, UO2(s) dissolution rates were similar to those of abiotic experiments with nitrite (from 1.15 × 10-14 to 4.94 × 10-13 mol m-2 s-1). Experiments with a T. dentrificans mutant strain defective in U(IV) oxidation supported microbially mediated U(IV) oxidation. X-ray absorption spectroscopy (XAS) analysis of post-reaction solids showed the presence of mononuclear U(VI) species rather than a solid U(VI) phase. At steady-state U release, kinetic and spectroscopic results suggest detachment of oxidized U(VI) from the UO2(s) surface as the rate-determining step rather than electron transfer or ion diffusion. Under anaerobic conditions, production of nitrite by nitrate-reducing microorganisms and enzymatically catalyzed, nitrate-dependent U(IV) oxidation are likely dual processes by which reduced U solids may be oxidized and mobilized in the aqueous phase.

We previously engineered Escherichia coli to overproduce medium- to long-chain saturated and monounsaturated methyl ketones, which could potentially be applied as diesel fuel blending agents or in the flavor and fragrance industry. Recent efforts at strain optimization have focused on cofactor balance, as fatty acid-derived pathways face the systematic metabolic challenge of net NADPH consumption (in large part, resulting from the key fatty acid biosynthetic enzyme FabG [β-ketoacyl-ACP reductase]) and net NADH production. In this study, we attempted to mitigate cofactor imbalance by heterologously expressing NADH-dependent, rather than NADPH-dependent, versions of FabG identified in previous studies. Of the four NADH-dependent versions of FabG tested in our previously best-reported methyl ketone-producing strain (EGS1895), the version from Acholeplasma laidlawii (Al_FabG) showed the greatest increase in methyl ketone yield in shake flasks (35-75% higher than for an RFP negative-control strain, depending on sugar loading). An improved strain (EGS2920) attained methyl ketone titers during fed-batch fermentation of 5.4 ± 0.5 g/L, which were, on average, ca. 40% greater than those for the base strain (EGS1895) under fermentation conditions optimized in this study. Shotgun proteomic data for strains EGS2920 and EGS1895 during fed-batch fermentation were consistent with the goal of alleviating NADPH limitation through expression of Al_FabG. For example, relative to strain EGS1895, strain EGS2920 significantly upregulated glucose-6-phosphate isomerase (directing flux into glycolysis rather than the NADPH-producing pentose phosphate pathway) and downregulated MaeB (a NADP+ -dependent malate dehydrogenase). Overall, the results suggest that heterologous expression of NADH-dependent FabG in E. coli may improve sustained production of fatty acid-derived renewable fuels and chemicals.

Author(s): Hubbard, SS; Williams, KH; Agarwal, D; Banfield, J; Beller, H; Bouskill, N; Brodie, E; Carroll, R; Dafflon, B; Dwivedi, D; Falco, N; Faybishenko, B; Maxwell, R; Nico, P; Steefel, C; Steltzer, H; Tokunaga, T; Tran, PA; Wainwright, H; Varadharajan, C | Abstract: Extreme weather, fires, and land use and climate change are significantly reshaping interactions within watersheds throughout the world. Although hydrological–biogeochemical interactions within watersheds can impact many services valued by society, uncertainty associated with predicting hydrologydriven biogeochemical watershed dynamics remains high. With an aim to reduce this uncertainty, an approximately 300-km2 mountainous headwater observatory has been developed at the East River, CO, watershed of the Upper Colorado River Basin. The site is being used as a testbed for the Department of Energy supported Watershed Function Project and collaborative efforts. Building on insights gained from research at the “sister” Rifle, CO, site, coordinated studies are underway at the East River site to gain a predictive understanding of how the mountainous watershed retains and releases water, nutrients, carbon, and metals. In particular, the project is exploring how early snowmelt, drought, and other disturbances influence hydrological–biogeochemical watershed dynamics at seasonal to decadal timescales. A system-of-systems perspective and a scale-adaptive simulation approach, involving the combined use of archetypal watershed subsystem “intensive sites” are being tested at the site to inform aggregated watershed predictions of downgradient exports. Complementing intensive site hydrological, geochemical, geophysical, microbiological, geological, and vegetation datasets are long-term, distributed measurement stations and specialized experimental and observational campaigns. Several recent research advances provide insights about the intensive sites as well as aggregated watershed behavior. The East River “community testbed” is currently hosting scientists from more than 30 institutions to advance mountainous watershed methods and understanding.

© 2017 American Chemical Society. Three-dimensional variably saturated flow and multicomponent biogeochemical reactive transport modeling, based on published and newly generated data, is used to better understand the interplay of hydrology, geochemistry, and biology controlling the cycling of carbon, nitrogen, oxygen, iron, sulfur, and uranium in a shallow floodplain. In this system, aerobic respiration generally maintains anoxic groundwater below an oxic vadose zone until seasonal snowmelt-driven water table peaking transports dissolved oxygen (DO) and nitrate from the vadose zone into the alluvial aquifer. The response to this perturbation is localized due to distinct physico-biogeochemical environments and relatively long time scales for transport through the floodplain aquifer and vadose zone. Naturally reduced zones (NRZs) containing sediments higher in organic matter, iron sulfides, and non-crystalline U(IV) rapidly consume DO and nitrate to maintain anoxic conditions, yielding Fe(II) from FeS oxidative dissolution, nitrite from denitrification, and U(VI) from nitrite-promoted U(IV) oxidation. Redox cycling is a key factor for sustaining the observed aquifer behaviors despite continuous oxygen influx and the annual hydrologically induced oxidation event. Depth-dependent activity of fermenters, aerobes, nitrate reducers, sulfate reducers, and chemolithoautotrophs (e.g., oxidizing Fe(II), S compounds, and ammonium) is linked to the presence of DO, which has higher concentrations near the water table. (Figure Presented).

Tropical forests absorb large amounts of atmospheric CO2 through photosynthesis but elevated temperatures suppress this absorption and promote monoterpene emissions. Using 13 CO2 labeling, here we show that monoterpene emissions from tropical leaves derive from recent photosynthesis and demonstrate distinct temperature optima for five groups (Groups 1-5), potentially corresponding to different enzymatic temperature-dependent reaction mechanisms within β-ocimene synthases. As diurnal and seasonal leaf temperatures increased during the Amazonian 2015 El Nino event, leaf and landscape monoterpene emissions showed strong linear enrichments of β-ocimenes (+4.4% °C-1 ) at the expense of other monoterpene isomers. The observed inverse temperature response of α-pinene (-0.8% °C-1 ), typically assumed to be the dominant monoterpene with moderate reactivity, was not accurately simulated by current global emission models. Given that β-ocimenes are highly reactive with respect to both atmospheric and biological oxidants, the results suggest that highly reactive β-ocimenes may play important roles in the thermotolerance of photosynthesis by functioning as effective antioxidants within plants and as efficient atmospheric precursors of secondary organic aerosols. Thus, monoterpene composition may represent a new sensitive 'thermometer' of leaf oxidative stress and atmospheric reactivity, and therefore a new tool in future studies of warming impacts on tropical biosphere-atmosphere carbon-cycle feedbacks.

The Design-Build-Test-Learn (DBTL) cycle, facilitated by exponentially improving capabilities in synthetic biology, is an increasingly adopted metabolic engineering framework that represents a more systematic and efficient approach to strain development than historical efforts in biofuels and biobased products. Here, we report on implementation of two DBTL cycles to optimize 1-dodecanol production from glucose using 60 engineered Escherichia coli MG1655 strains. The first DBTL cycle employed a simple strategy to learn efficiently from a relatively small number of strains (36), wherein only the choice of ribosome-binding sites and an acyl-ACP/acyl-CoA reductase were modulated in a single pathway operon including genes encoding a thioesterase (UcFatB1), an acyl-ACP/acyl-CoA reductase (Maqu_2507, Maqu_2220, or Acr1), and an acyl-CoA synthetase (FadD). Measured variables included concentrations of dodecanol and all proteins in the engineered pathway. We used the data produced in the first DBTL cycle to train several machine-learning algorithms and to suggest protein profiles for the second DBTL cycle that would increase production. These strategies resulted in a 21% increase in dodecanol titer in Cycle 2 (up to 0.83 g/L, which is more than 6-fold greater than previously reported batch values for minimal medium). Beyond specific lessons learned about optimizing dodecanol titer in E. coli, this study had findings of broader relevance across synthetic biology applications, such as the importance of sequencing checks on plasmids in production strains as well as in cloning strains, and the critical need for more accurate protein expression predictive tools.

Isoprenol (3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals. Biological production of isoprenol via the mevalonate pathway has been developed and optimized extensively in Escherichia coli, but high ATP requirements and isopentenyl diphosphate (IPP) toxicity have made it difficult to achieve high titer, yield, and large-scale production. To overcome these limitations, an IPP-bypass pathway was previously developed using the promiscuous activity of diphosphomevalonate decarboxylase, and enabled the production of isoprenol at a comparable yield and titer to the original pathway. In this study, we optimized this pathway, substantially improving isoprenol production. A titer of 3.7 g/L (0.14 g isoprenol per g glucose) was achieved in batch conditions using minimal medium by pathway optimization, and a further optimization of the fed-batch fermentation process enabled an isoprenol titer of 10.8 g/L (yield of 0.105 g/g and maximum productivity of 0.157 g L-1 h-1), which is the highest reported titer for this compound. The substantial increase in isoprenol titer via the IPP-bypass pathway in this study will facilitate progress toward commercialization.

Plant biomass is an attractive source of renewable carbon for conversion to biofuels and bio-based chemicals. Conversion strategies often use a fraction of the total biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components such as plant-derived aromatics and amino acids have the potential to improve the efficiency of overall biomass conversion.Pseudomonas putidais a promising host for biomass conversion for its ability to metabolize a wide variety of organic compounds, including aromatics derived from lignin.P. putidawas engineered to produce medium chain methyl ketones, which are promising diesel blendstocks and potential platform chemicals, from glucose and lignin-related aromatics, 4-hydroxybenzoate (4-HB) and protocatechuate (PCA). Unexpectedly,P. putidamethyl ketone production was enhanced 2-to 5-fold compared to sugar controls whenArabidopsis thalianahydrolysates derived from engineered plants that overproduce 4-HB and PCA, whileE. coliproduction was lowered in these hydrolysates. This enhancement was more pronounced (~7-fold increase) with hydrolysates derived from non-engineered switchgrass (Panicum virgatumL.) suggesting it did not arise from overproduction of 4-HB and PCA. Global proteomic analysis of the methyl ketone-producingP. putidasuggested that plant-derived amino acids may be the source of this enhancement. Mass spectrometry-based measurements of plant-derived amino acids demonstrated a high correlation between methyl ketone production and amino acid concentration in plant hydrolysates. Amendment of glucose-containing minimal media with a defined mixture of amino acids similar to those found in the hydrolysates studied led to a 9-fold increase in methyl ketone titer (1.1 g/L).

Hexavalent chromium Cr(VI) is a common inorganic contaminant in industrial areas and represents a serious threat to human health due its toxicity. Here we report experimental results from a field-scale investigation of Cr(VI) bio-immobilization at Hanford 100H reservation, a U.S Department of Energy facility (Washington State, USA). Microbial Cr(VI) reduction was stimulated via injection of a13C-labeled sodium lactate solution into the high-permeability aquifer consisting of gravel and coarse sand sediments. Concentrations and carbon isotope ratios of metabolites, including dissolved inorganic carbon and total organic carbon, and compound-specific analysis of acetate and propionate, together with phospholipid fatty acids (biomass) have been analyzed to help provide an understanding of the predominant redox processes accompanying Cr(VI) reduction. Results of our study indicate that the injection of an electron donor caused a sharp decrease of Cr(VI) concentration from ∼32 to ∼10 nM. Cr(VI) reduction was associated with a decrease in the concentration of carboxylic acids, such as lactate (∼6 mM to undetectable), propionate (∼9 mM to undetectable), and acetate (∼6 mM to undetectable), as well as dissolved inorganic carbon (30-10 mM C). Carbon isotope data indicate carbon transfers from the original substrate to organic byproducts and mineralized carbon. Concentrations of metabolites and stable isotope data as well as carbon isotope mass balance calculations were used to monitor biologically mediated reduction of Cr(VI).

© 2018 The Author(s). Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.

Covering: 2005 to 2015 Although natural products are best known for their use in medicine and agriculture, a number of fatty acid-derived and isoprenoid natural products are being developed for use as renewable biofuels and bio-based chemicals. This review summarizes recent work on fatty acid-derived compounds (fatty acid alkyl esters, fatty alcohols, medium- and short-chain methyl ketones, alkanes, α-olefins, and long-chain internal alkenes) and isoprenoids, including hemiterpenes (e.g., isoprene and isopentanol), monoterpenes (e.g., limonene), and sesquiterpenes (e.g., farnesene and bisabolene).

© 2017 American Chemical Society. Hexavalent chromium, Cr(VI), is a widespread and toxic groundwater contaminant. Reductive immobilization to Cr(III) is a treatment option, but its success depends on the long-term potential for reduced chromium precipitates to remain immobilized under oxidizing conditions. In this unique long-term study, aquifer sediments subjected to reductive Cr(VI) immobilization under different biogeochemical regimes were tested for their susceptibility to reoxidation. After reductive treatment for 1 year, sediments were exposed to oxygenated conditions for another 2 years in flow-through, laboratory columns. Under oxidizing conditions, immobilized chromium reduced under predominantly denitrifying conditions was mobilized at low concentrations (≪1 μM Cr(VI); ∼ 3% of Cr(III) deposited) that declined over time. A conceptual model of a limited pool of more soluble Cr(III), and a larger pool of relatively insoluble Cr(III), is proposed. In contrast, almost no chromium was mobilized from columns reduced under predominantly fermentative conditions, and where reducing conditions persisted for several months after introduction of oxidizing conditions, presumably due to the presence of a reservoir of reduced species generated during reductive treatment. The results from this 3-year study demonstrate that biogeochemical conditions present during reductive treatment, and the potential for buildup of reducing species, will impact the long-term sustainability of the remediation effort.

In this study of reductive chromium immobilization, we found that flow-through columns constructed with homogenized aquifer sediment and continuously infused with lactate, chromate, and various native electron acceptors diverged to have very different Cr(VI)-reducing biogeochemical regimes characterized by either denitrifying or fermentative conditions (as indicated by effluent chemical data, 16S rRNA pyrotag data, and metatranscriptome data). Despite the two dramatically different biogeochemical environments that evolved in the columns, these regimes created similar Cr(III)-Fe(III) hydroxide precipitates as the predominant Cr(VI) reduction product, as characterized by micro-X-ray fluorescence and micro-X-ray absorption near-edge structure analysis. We discuss two conflicting scenarios of microbially mediated formation of Cr(III)-Fe(III) precipitates, each of which is both supported and contradicted by different lines of evidence: (1) enzymatic reduction of Cr(VI) to Cr(III) followed by coprecipitation of Cr(III) and Fe(III) and (2) both regimes generated at least small amounts of Fe(II), which abiotically reduced Cr(VI) to form a Cr-Fe precipitate. Evidence of zones with different levels of Cr(VI) reduction suggest that local heterogeneity may have confounded interpretation of processes based on bulk measurements. This study indicates that the bulk redox status and biogeochemical regime, as categorized by the dominant electron-accepting process, do not necessarily control the final product of Cr(VI) reduction.

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H 2 and CO 2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[( R )-3-hydroxybutyrate] (PHB). Its ability to utilize CO 2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli ) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO 2 and H 2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.

© 2017 Jewell, Karaoz, Bill, Chakraborty, Brodie, Williams and Beller. Organic matter deposits in alluvial aquifers have been shown to result in the formation of naturally reduced zones (NRZs), which can modulate aquifer redox status and influence the speciation and mobility of metals, affecting groundwater geochemistry. In this study, we sought to better understand how natural organic matter fuels microbial communities within anoxic biogeochemical hot spots (NRZs) in a shallow alluvial aquifer at the Rifle (CO) site. We conducted a 20-day microcosm experiment in which NRZ sediments, which were enriched in buried woody plant material, served as the sole source of electron donors and microorganisms. The microcosms were constructed and incubated under anaerobic conditions in serum bottles with an initial N2headspace and were sampled every 5 days for metagenome and metatranscriptome profiles in combination with biogeochemical measurements. Biogeochemical data indicated that the decomposition of native organic matter occurred in different phases, beginning with mineralization of dissolved organic matter (DOM) to CO2during the first week of incubation, followed by a pulse of acetogenesis that dominated carbon flux after 2 weeks. A pulse of methanogenesis co-occurred with acetogenesis, but only accounted for a small fraction of carbon flux. The depletion of DOM over time was strongly correlated with increases in expression of many genes associated with heterotrophy (e.g., amino acid, fatty acid, and carbohydrate metabolism) belonging to a Hydrogenophaga strain that accounted for a relatively large percentage (~8%) of the metatranscriptome. This Hydrogenophaga strain also expressed genes indicative of chemolithoautotrophy, including CO2fixation, H2oxidation, S-compound oxidation, and denitrification. The pulse of acetogenesis appears to have been collectively catalyzed by a number of different organisms and metabolisms, most prominently pyruvate:ferredoxin oxidoreductase. Unexpected genes were identified among the most highly expressed (> 98th percentile) transcripts, including acetone carboxylase and cell-wall-associated hydrolases with unknown substrates (numerous lesser expressed cell-wall-associated hydrolases targeted peptidoglycan). Many of the most highly expressed hydrolases belonged to a Cα. Bathyarchaeota strain and may have been associated with recycling of bacterial biomass. Overall, these results highlight the complex nature of organic matter transformation in NRZs and the microbial metabolic pathways that interact to mediate redox status and elemental cycling.

As advances in biotechnology have continued at a rapid pace, interest in the biochemical production of so-called “drop-in” fuels has increased as a way to avoid the well-known shortcomings of ethanol as a fuel molecule and to potentially exploit the processing advantages of a water-immiscible fuel to reduce product recovery costs and energy requirements. In the current study, processes to produce either ethanol or a representative fatty acid ethyl ester (FAEE) via the fermentation of sugars liberated from lignocellulosic materials pretreated in acid or alkaline environments are analyzed in terms of economic and environmental metrics. Simplified process models are introduced and employed to estimate fuel production, greenhouse gas emissions, net energy consumption, minimum fuel selling price, and water consumption for both processes. Monte Carlo analyses were carried out to identify key sources of uncertainty and variability, and an analysis of the impact of potential improvements to the FAEE process was performed. We find that the near-term performance of processes to produce FAEE is significantly worse than that of ethanol production processes for all metrics considered, primarily due to poor fermentation yields and higher electricity demands for aerobic fermentation. Even if these issues are addressed in the longer term, the reduced cost and energy requirements of FAEE separation processes will be at least partially offset by inherent limitations in the relevant metabolic pathways that constrain the maximum yield potential of FAEE from biomass-derived sugars.

Tropical forests absorb large amounts of atmospheric CO

© 2016 International Society for Microbial Ecology All rights reserved. Groundwater ecosystems are conventionally thought to be fueled by surface-derived allochthonous organic matter and dominated by heterotrophic microbes living under often-oligotrophic conditions. However, in a 2-month study of nitrate amendment to a perennially suboxic aquifer in Rifle (CO), strain-resolved metatranscriptomic analysis revealed pervasive and diverse chemolithoautotrophic bacterial activity relevant to C, S, N and Fe cycling. Before nitrate injection, anaerobic ammonia-oxidizing (anammox) bacteria accounted for 16% of overall microbial community gene expression, whereas during the nitrate injection, two other groups of chemolithoautotrophic bacteria collectively accounted for 80% of the metatranscriptome: (1) members of the Fe(II)-oxidizing Gallionellaceae family and (2) strains of the S-oxidizing species, Sulfurimonas denitrificans. Notably, the proportion of the metatranscriptome accounted for by these three groups was considerably greater than the proportion of the metagenome coverage that they represented. Transcriptional analysis revealed some unexpected metabolic couplings, in particular, putative nitrate-dependent Fe(II) and S oxidation among nominally microaerophilic Gallionellaceae strains, including expression of periplasmic (NapAB) and membrane-bound (NarGHI) nitrate reductases. The three most active groups of chemolithoautotrophic bacteria in this study had overlapping metabolisms that allowed them to occupy different yet related metabolic niches throughout the study. Overall, these results highlight the important role that chemolithoautotrophy can have in aquifer biogeochemical cycling, a finding that has broad implications for understanding terrestrial carbon cycling and is supported by recent studies of geochemically diverse aquifers.

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

Environmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.

Cr(VI) is a widespread groundwater contaminant that is a potent toxin, mutagen, and carcinogen. In situ reductive immobilization is a favored approach for Cr(VI) bioremediation, and Cr(VI) reduction has been reported in a variety of aerobic, facultative, and anaerobic bacteria, including a number of pseudomonads. However, studies comparing Cr(VI) reduction under aerobic and denitrifying conditions in the same organism are not available. We have conducted studies with strain RCH2, a bacterium similar to Pseudomonas stutzeri that we isolated from a Cr-contaminated aquifer. Cell suspension studies with lactate demonstrated that Cr(VI) reduction could occur under either denitrifying or aerobic conditions (at comparable specific rates) and that reduction was at least 20-fold more rapid when the terminal electron acceptor (i.e., nitrate or O(2)) was present. Our results suggest that Cr(VI) reduction by strain RCH2 under either aerobic or denitrifying conditions is primarily cometabolic in the sense that the physiological electron acceptor (oxygen or nitrate) appears to be required. Under both aerobic and denitrifying conditions, the gene(s) associated with chromate reduction are not inducible by Cr. Continuous culture (chemostat) studies showed strong correlations (r(2) values >0.93) between nitrate reduction rate and the transcript copy number of either nirS (cytochrome cd(1) nitrite reductase) or narG (nitrate reductase α subunit). As our studies indicate that anaerobic Cr(VI) reduction by this pseudomonad requires active denitrification and that denitrification and chromate reduction rates are highly correlated (r(2) > 0.99), monitoring expression of such denitrification genes in biostimulated aquifers could provide valuable proxy information for in situ chromate reduction by similar bacteria even if the specific genes involved in chromate reduction have not been identified. We also report incomplete removal of reduced Cr from solution and on artifacts in the widely used diphenylcarbazide assay for Cr(VI), most notably, its complete inactivation in the presence of millimolar nitrite.

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila ), which 4 decades ago was reported to biosynthesize iso - and anteiso -branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M . luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C＝C bonds). Heterologous expression of Mlut_13230 ( oleA ) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C 27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M . luteus genes were responsible for production of these metabolites. Studies with wild-type M . luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (β-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during fatty acid biosynthesis.

© 2016 Javidpour et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and experimental results developed in this study to elucidate the biosynthetic pathway that produces unique and intriguing ladderane lipids.

Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2 and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.

Thiobacillus denitrificans is a widespread, chemolithoautotrophic bacterium with an unusual and environmentally relevant metabolic repertoire, which includes its ability to couple denitrification to sulfur compound oxidation; to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV); and to oxidize mineral electron donors. Recent analysis of its genome sequence also revealed the presence of genes encoding two [NiFe]hydrogenases, whose role in metabolism is unclear, as the sequenced strain does not appear to be able to grow on hydrogen as a sole electron donor under denitrifying conditions. In this study, we report the development of a genetic system for T. denitrificans , with which insertion mutations can be introduced by homologous recombination and complemented in trans . The antibiotic sensitivity of T. denitrificans was characterized, and a procedure for transformation with foreign DNA by electroporation was established. Insertion mutations were generated by in vitro transposition, the mutated genes were amplified by the PCR, and the amplicons were introduced into T. denitrificans by electroporation. The IncP plasmid pRR10 was found to be a useful vector for complementation. The effectiveness of the genetic system was demonstrated with the hynL gene, which encodes the large subunit of a [NiFe]hydrogenase. Interruption of hynL in a hynL ::kan mutant resulted in a 75% decrease in specific hydrogenase activity relative to the wild type, whereas complementation of the hynL mutation resulted in activity that was 50% greater than that of the wild type. The availability of a genetic system in T. denitrificans will facilitate our understanding of the genetics and biochemistry underlying its unusual metabolism.

Microbially mediated reductive immobilization of chromium is a possible remediation technique for sites contaminated with Cr(VI). This study is part of a broader effort investigating the biogeochemical mechanisms for Cr(VI) reduction in Hanford 100H aquifer sediments using flow-through laboratory columns. It had previously been shown that reduced chromium in the solid phase was in the form of freshly precipitated mixed-phase Cr(III)-Fe(III) (hydr)oxides, irrespective of the biogeochemical conditions in the columns. In this study, the reduced Cr phases in the columns were investigated further using spectroscopy to understand the structure and mechanisms involved in the formation of the end products. Several samples representing potential processes that could be occurring in the columns were synthesized in the laboratory and characterized using X-ray absorption near edge structure (XANES) and X-ray scattering. The XANES of Cr(III) particles in the columns most closely resembled those from synthetic samples produced by the abiotic reaction of Cr(VI) with microbially reduced Fe(II). Microbially mediated Cr-Fe reduction products were distinct from abiotic Cr-Fe (hydr)oxides [CrFe(OH)] and organically complexed Cr(III) sorbed onto the surface of a mixed ferrihydrite-goethite mineral phase. Furthermore, analyses of the abiotically synthesized samples revealed that even the end products of purely abiotic, iron-mediated reduction of Cr(VI) are affected by factors such as the presence of excess aqueous Fe(II) and cellular matter. These results suggest that CrFe(OH) phases made under realistic subsurface conditions or in biotic cultures are structurally different from pure Cr(OH) or laboratory-synthesized CrFe(OH). The observed structural differences imply that the reactivity and stability of biogenic CrFe(OH) could potentially be different from that of abiotic CrFe(OH).

The mechanism for anaerobic metabolism of benzene remains unknown. To date, there have been only a few studies reporting metabolites of anaerobic benzene biodegradation, in part because anaerobic benzene-degrading enrichment cultures are not very common and only two isolates have been characterized to date. Phenol and benzoate, metabolites consistent with benzene hydroxylation or benzene carboxylation, have been identified previously in mixed cultures, and more recently benzene methylation to toluene has been proposed as another possible mechanism for anaerobic benzene degradation. In this study, 13C6-benzene was added to nitrate-reducing and methanogenic enrichment cultures and specific 13C-labeled metabolites were monitored over time. The putative metabolites were detected by gas chromatography/mass spectrometry in ether extracts of 100-mL samples of culture taken at each time point. This method of analysis provided the sensitivity required to accurately quantify low concentrations of these compounds. In addition, benzoate trapping was used in an attemptto increase concentrations of upstream metabolites. In both cultures, in the presence and absence of unlabeled benzoate (trap), [ring-13C]-toluene and [ring-13C]benzoate were detected transiently during degradation. The data strongly support initial methylation of benzene to toluene, followed bytransformation to benzoate. Although benzene methylation has been proposed previously, this is the first direct evidence to supportthis pathway. In the methanogenic culture only, 13C6-phenol was also detected. The transient appearance of phenol, which appeared to be further transformed to benzoate, suggests that a pathway involving hydroxylation to phenol, as proposed in other studies, was also operative.

An interdisciplinary study was conducted to characterize the distribution and fate of NO3− in groundwater at Lawrence Livermore National Laboratory (LLNL) Site 300, a high-explosives test facility in the semi-arid Altamont Hills of California. Site 300 groundwater contains NO3− concentrations ranging from 200 mg NO3−/L. Several lines of evidence strongly suggest that denitrification is naturally attenuating NO3− in the confined, O2-depleted region of the bedrock aquifer under study (Tnbs2): (a) both NO3− and dissolved O2(DO) concentrations in groundwater decrease dramatically as groundwater flows from unconfined to confined aquifer conditions, (b) stable isotope signatures (i.e., δ15N and δ18O) of groundwater NO3− indicate a trend of isotopic enrichment that is characteristic of denitrification, and (c) dissolved N2 gas, the product of denitrification, was highly elevated in NO3−-depleted groundwater in the confined region of the Tnbs2 aquifer. Long-term NO3− concentrations were relatively high and constant in recharge-area monitoring wells (typically 70–100 mg NO3−/L) and relatively low and constant in the downgradient confined region (typically

Functionalization of bacterial cell surfaces has the potential to introduce new activities by chemical modification. Here we show that a bacteriophage-receptor complex can be used to functionalize the surface of two Gram-negative proteobacteria, Escherichia coli and Ralstonia eutropha with CdSe/ZnS nanoparticles. This work highlights the potential for using microbe-phage interactions to generate new functions on living cells.

We studied Cr isotopic fractionation during Cr(VI) reduction by Pseudomonas stutzeri strain RCH2. Despite the fact that strain RCH2 reduces Cr(VI) cometabolically under both aerobic and denitrifying conditions and at similar specific rates, fractionation was markedly different under these two conditions (ε was ∼2‰ aerobically and ∼0.4‰ under denitrifying conditions).

In this project, we combined molecular genetic, spectroscopic, and microscopic techniques with kinetic and reactive transport studies to describe and quantify biotic and abiotic mechanisms underlying anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation, which influences the long-term efficacy of in situ reductive immobilization of uranium at DOE sites. In these studies, Thiobacillus denitrificans, an autotrophic bacterium that catalyzes anaerobic U(IV) and Fe(II) oxidation, was used to examine coupled oxidation-reduction processes under either biotic (enzymatic) or abiotic conditions in batch and column experiments with biogenically produced UIVO2(s). Synthesis and quantitative analysis of coupled chemical and transport processes were done with the reactive transport modeling code Crunchflow. Research focused on identifying the primary redox proteins that catalyze metal oxidation, environmental factors that influence protein expression, and molecular-scale geochemical factors that control the rates of biotic and abiotic oxidation.

We have developed a real-time polymerase chain reaction (PCR) method that can quantify hydrocarbon-degrading bacteria in sediment samples based on a catabolic gene associated with the first step of anaerobic toluene and xylene degradation. The target gene, bssA, codes for the alpha-subunit of benzylsuccinate synthase. The primer-probe set for real-time PCR was based on consensus regions of bssA from four denitrifying bacterial strains; bssA sequences for two of these strains were determined during this study. The method proved to be sensitive (detection limit ca. 5 gene copies) and had a linear range of7 orders of magnitude. We used the method to investigate how gasohol releases from leaking underground storage tanks could affect indigenous toluene-degrading bacteria. Microcosms inoculated with aquifer sediments from four different sites were incubated anaerobically with BTEX (benzene, toluene, ethylbenzene, and xylenes) and nitrate in the presence and absence of ethanol. Overall, population trends were consistent with observed toluene degradation activity: the microcosms with the most rapid toluene degradation also had the largest numbers of bssA copies. In the microcosms with the most rapid toluene degradation, numbers of bssA copies increased 100-to 1000-fold over the first 4 days of incubation, during which time most of the toluene had been consumed. These results were supported by slot blot analyses with unamplified DNA and by cloning and sequencing of putative bssA amplicons, which confirmed the real-time PCR method's specificity for bssA. Use of a companion real-time PCR method for estimating total eubacterial populations (based on 16S rDNA) indicated that, in some cases, ethanol disproportionately supported the growth of bacteria that did not contain bssA. The real-time PCR method for bssA could be a powerful tool for monitored natural attenuation of BTEX in fuel-contaminated groundwater. To our knowledge, this is the first reported molecular method that targets anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene.

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) is a nitramine explosive that has contaminated soil and groundwater at military installations throughout the US. Although anaerobic RDX metabolism has been reported, the process is not well understood, as past studies have typically involved complex, undefined media with multiple potential electron donors and acceptors. In this study, bacteria enriched from RDX-contaminated aquifer sediments consumed RDX in a defined, bicarbonate-buffered, anaerobic medium containing hydrogen as the sole electron donor and RDX as a potential electron acceptor and sole nitrogen source. RDX was not consumed in live controls that did not contain hydrogen. Transient formation of mononitroso- and dinitroso-RDX metabolites (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine, respectively) was documented by liquid chromatography-mass spectrometry. However, studies with 14C-labeled RDX suggested that mineralization to carbon dioxide was negligible (

An important element of monitored natural attenuation is the detection in groundwater of distinctive products of pollutant degradation or transformation. In this study, three distinctive products of the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) were detected in contaminated groundwater from the Iowa Army Ammunition Plant; the products were MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine), and TNX (hexahydro-1,3,5-trinitroso-1,3,5-triazine). These compounds are powerful indicators of RDX transformation for several reasons: (a) they have unique chemical features that reveal their origin as RDX daughter products, (b) they have no known commercial, industrial, or natural sources, and (c) they are well documented as anaerobic RDX metabolites in laboratory studies. The products were analyzed by LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) with selected reaction monitoring and internal standard quantification using [ring-U-15N]RDX. Validation tests showed the novel LC/MS/MS method to be of favorable sensitivity (detection limits ca. 0.1 microg/L), accuracy, and precision. The products, which were detected in all groundwater samples with RDX concentrations ofca. 1 microg/L (25 out of 55 samples analyzed), were present at concentrations ranging from near the detection limit to 430 microg/L. MNX was the typically the most abundant of the three nitroso-substituted products; concentrations of the products seldom exceeded 4 mol % of the RDX concentration, although they ranged as high as 26 mol % (TNX). Geographic and temporal distributions of RDX, MNX, DNX, and TNX were assessed. A degradation product resulting from RDX ring cleavage, methylenedinitramine, was not detected by LC/MS/MS in any sample (detection limit ca. 0.6-4 microg/L). This extensive field characterization of MNX, DNX, and TNX distributions in groundwater by a highly selective analytical method (LC/MS/MS) is significant because very little is known about the occurrence of intrinsic RDX transformation in contaminated aquifers.

We previously reported development of a metabolic pathway in Escherichia coli for overproduction of medium-chain methyl ketones (MK), which are relevant to the biofuel and flavor-and-fragrance industries. This MK pathway was a re-engineered version of β-oxidation designed to overproduce β-ketoacyl-CoAs and involved overexpression of the fadM thioesterase gene. Here, we document metabolic engineering modifications that have led to a MK titer of 3.4 g/L after ~45 h of fed-batch glucose fermentation and attainment of 40% of the maximum theoretical yield (the best values reported to date for MK). Modifications included balancing overexpression of fadR and fadD to increase fatty acid flux into the pathway, consolidation of the pathway from two plasmids into one, codon optimization, and knocking out key acetate production pathways. In vitro studies confirmed that a decarboxylase is not required to convert β-keto acids into MK and that FadM is promiscuous and can hydrolyze several CoA-thioester pathway intermediates.

The potential for aerobic methyl tert -butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U- 14 C]MTBE was mineralized to 14 CO 2 . Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert -butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.

Monitoring programs for intrinsic bioremediation of fuel hydrocarbonsrequire indicators that can convincingly demonstrate in situ metabolism. In this evaluation of potential indicators of in situ anaerobic alkylbenzene metabolism, laboratory and field data are reviewed for two classes of aromatic acids: (i) benzylsuccinate, E-phenylitaconate, and their methyl homologs, and (ii) benzoate, and methyl-, dimethyl-, and trimethylbenzoates. The review includes previously unpublished field data from a hydrocarbon-contaminated site in Fallon (Nevada), at which both classes of metabolites were detected in groundwater. The two classes of compounds were evaluated with respect to specificity (i.e., unique biochemical relationship to a specific alkylbenzene), stability, and generation as degradation intermediates versus dead-end products; recent developments in the biochemistry of anaerobic toluene and xylene degradation were incorporated in this evaluation. In general, benzylsuccinates/E-phenylitaconates are superior to benzoates in terms of their very high specificity to their parent hydrocarbons and their lack of commercial and industrial sources. They are also uniquely indicative of anaerobic conditions. All of the benzoates, benzylsuccinates, and E-phenylitaconates are relatively stable chemically and (with the exceptionof benzoate) biologically under anaerobic conditions, based on the limited data available. Although benzoate, benzylsuccinate, and E-phenylitaconate are intermediates of anaerobic toluene mineralization to carbon dioxide, their methyl homologs can be either mineralization intermediates or cometabolic dead-end products of alkylbenzenes, depending on the bacteria involved. Benzoates are far more commonly reported in field studies of hydrocarbon-contaminated aquifers than are benzylsuccinates and E-phenylitaconates, although it is not clear whether this is an accurate representation of the relative occurrenceof these metabolites at contaminated sites, or whether it instead reflects the limited range of target analytes used in most field studies to date.

Benzylsuccinate synthase, which catalyzes the anaerobic addition of the methyl carbon of toluene to fumarate, has recently been reported in several denitrifying and sulfate-reducing, toluene-degrading bacteria. In substrate range studies with partially purified benzylsuccinate synthase from denitrifying Azoarcus sp. strain T, benzylsuccinate analogs were observed as a result of fumarate addition to the following toluene surrogates: xylenes, monofluorotoluenes, benzaldehyde, and 1-methyl-1-cyclohexene (but not 4-methyl-l-cyclohexene or methylcyclohexane). Benzylsuccinate was also observed as a result of toluene addition to maleate, but no products were observed from assays with toluene and either crotonate or trans-glutaconate. Toluene-maleate addition, like toluene-fumarate addition, resulted in highly stereospecific formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid]. The previously reported finding that the methyl H atom abstracted from toluene is retained in the succinyl moiety of benzylsuccinate was found to apply to several toluene surrogates. The implications of these observations for the mechanism of benzylsuccinate synthase will be discussed.

Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [( R )-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.

Major efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the proportion of reducing equivalents directed to biofuel biosynthesis rather than ATP production. A major concern with fermentative growth conditions is elevated NADH levels, which can adversely affect cell physiology. The purpose of this study was to identify homologs of Escherichia coli FabG, an essential reductase involved in fatty acid biosynthesis, that display a higher preference for NADH than for NADPH as a cofactor. Four potential NADH-dependent FabG variants were identified through bioinformatic analyses supported by crystallographic structure determination (1.3- to 2.0-Å resolution). In vitro assays of cofactor (NADH/NADPH) preference in the four variants showed up to ∼35-fold preference for NADH, which was observed with the Cupriavidus taiwanensis FabG variant. In addition, FabG homologs were overexpressed in fatty acid- and methyl ketone-overproducing E. coli host strains under anaerobic conditions, and the C. taiwanensis variant led to a 60% higher free fatty acid titer and 75% higher methyl ketone titer relative to the titers of the control strains. With further engineering, this work could serve as a starting point for establishing a microbial host strain for production of fatty acid-derived biofuels (e.g., methyl ketones) under anaerobic conditions.

Anaerobic assays conducted with strain T, a denitrifying bacterium capable of mineralizing toluene to carbon dioxide, demonstrated that toluene-grown, permeabilized cells catalyzed the addition of toluene to fumarate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP. In the presence of CoA, formation of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate formation from toluene and fumarate in assays with permeabilized cells was >30% of the specific rate of toluene consumption in whole-cell suspensions with nitrate; this observation suggests that benzylsuccinate formation may be the first reaction in anaerobic toluene degradation by strain T. Use of deuterium-labeled toluene and gas chromatography-mass spectrometry indicated that the H atom abstracted from the toluene methyl group during addition to fumarate was retained in the succinyl moiety of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-CoA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl)succinate. o-Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rates, the observed characteristics of the toluene-fumarate addition reaction (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction. Thus, addition to fumarate may be a biochemical strategy to anaerobically activate a range of methylbenzenes.

Background: The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. Results: We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5' mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts.Conclusion: We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well. © 2013 Bi et al.; licensee BioMed Central Ltd.