ABSTRACT
OBJECTIVES: The objective of this study was to investigate the effects of medium composition on CO fermentation by Clostridium carboxidivorans. The focus was to reduce the medium cost preserving acceptable levels of solvent production. METHODS: Yeast extract (YE) concentration was set in the range of 0-3 g/L. Different reducing agents were investigated, including cysteine-HCl 0.6 g/L, pure cysteine 0.6 g/L, sodium sulphide (Na2S) 0.6 g/L, cysteine-sodium sulphide 0.6 g/L and cysteine-sodium sulphide 0.72 g/L. The concentration of the metal solution was decreased down to 25 % of the standard value. Fermentation tests were also carried out with and without tungsten or selenium. RESULTS: The results demonstrated that under optimized conditions, namely yeast extract (YE) concentration set at 1 g/L, pure cysteine as the reducing agent and trace metal concentration reduced to 75 % of the standard value, reasonable solvent production was achieved in less than 150 h. Under these operating conditions, the production levels were found to be 1.39 g/L of ethanol and 0.27 g/L of butanol. Furthermore, the study revealed that selenium was not necessary for C. carboxidivorans fermentation, whereas the presence of tungsten played a crucial role in both cell growth and solvent production. CONCLUSIONS: The optimization of the medium composition in CO fermentation by Clostridium carboxidivorans is crucial for cost-effective solvent production. Tuning the yeast extract (YE) concentration, using pure cysteine as the reducing agent and reducing trace metal concentration contribute to reasonable solvent production within a relatively short fermentation period. Tungsten is essential for cell growth and solvent production, while selenium is not required.
Subject(s)
Bioreactors , Clostridium , Culture Media , Fermentation , Clostridium/metabolism , Clostridium/growth & development , Culture Media/chemistry , Bioreactors/microbiology , Carbon Monoxide/metabolism , Ethanol/metabolism , Selenium/metabolism , Butanols/metabolism , Tungsten/metabolismABSTRACT
Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium Aromatoleum aromaticum (AORAa) consists of three different subunits (AorABC) and uses nicotinamide adenine dinucleotide (NAD) as an electron acceptor. Here, we reveal that the enzyme forms filaments of repeating AorAB protomers that are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with quantum mechanics:molecular mechanics (QM:MM)-based modeling for the coordination of the W-co, enables formulation of a hypothetical catalytic mechanism that paves the way to further engineering for applications in synthetic biology and biotechnology.
Subject(s)
Aldehyde Oxidoreductases , Nanowires , Aldehyde Oxidoreductases/chemistry , Aldehyde Oxidoreductases/metabolism , Tungsten/metabolism , NAD , Cryoelectron Microscopy , Aldehyde DehydrogenaseABSTRACT
Tungsten is used in several applications and human exposure may occur. To assess its pulmonary toxicity, we exposed male mice to nose-only inhalation of tungsten particles at 9, 23 or 132 mg/m3 (Low, Mid and High exposure) (45 min/day, 5 days/week for 2 weeks). Increased genotoxicity (assessed by comet assay) was seen in bronchoalveolar (BAL) fluid cells at Low and High exposure. We measured acellular ROS production, and cannot exclude that ROS contributed to the observed genotoxicity. We saw no effects on body weight gain, pulmonary inflammation, lactate dehydrogenase or protein in BAL fluid, pathology of liver or kidney, or on sperm counts. In conclusion, tungsten showed non-dose dependent genotoxicity in the absence of inflammation and therefore interpreted to be primary genotoxicity. Based on genotoxicity, a Lowest Observed Adverse Effect Concentration (LOAEC) could be set at 9 mg/m3. It was not possible to establish a No Adverse Effect Concentration (NOAEC).
Subject(s)
Semen , Tungsten , Humans , Mice , Male , Animals , Tungsten/metabolism , Tungsten/pharmacology , Reactive Oxygen Species/metabolism , Semen/metabolism , DNA Damage , Inflammation/pathology , Inhalation Exposure/adverse effects , Bronchoalveolar Lavage Fluid , LungABSTRACT
The effect of tungsten and selenium on cell growth and production of metabolites such as acetic acid and ethanol when fermenting syngas using "Clostridium autoethanogenum" was investigated to improve the process efficiency. General concentrations of selenium and tungsten in the medium are 0.01 µM during acetogenic syngas fermentation. We conducted culture experiments at concentrations of 0, 0.001, 0.01 and 0.1 µM for each heavy metal. The effect of selenium on cell growth and total metabolite production was greater than that of tungsten as the effect of selenium on formate dehydrogenase, an important enzyme of the Wood-Ljungdahl pathway, is greater than that of tungsten. Although an increase in tungsten had a marginal effect on total metabolite production, the ethanol/acetic acid production ratio increased significantly due to a decrease in acetic acid and an increase in ethanol production. Thus, tungsten plays a key role in activating aldehyde:ferredoxin oxidoreductase, a key enzyme in the reduction of acetate to ethanol. A specific ethanol productivity of 0.462 g ethanol/g DCWâd was obtained in a culture using 0.01 µM selenium and 0.1 µM tungsten, which was 2.18 times higher than when using 0.01 µM of both selenium and tungsten.
Subject(s)
Selenium , Tungsten , Acetic Acid/metabolism , Clostridium/metabolism , Ethanol/metabolism , Fermentation , Selenium/metabolism , Tungsten/metabolismABSTRACT
The present work describes an easy way to prepare a Chloroplast/PDA@WO3 biohybrid platform based on the deposition of chloroplasts on WO3 substrate previously modified with polydopamine (PDA) film as anchoring agent. The use of PDA as an immobilization matrix for chloroplasts, and also as an electron mediator under LED irradiation, resulted in enhanced photocurrents. The use of the chloroplasts amplified the photocurrent, when compared to the bare substrate (WO3). The best electrode performance was obtained using high intensity LED irradiation at 395 nm, for the electrode exposed for 10 min to 150 µg mL-1 of intact chloroplasts. Amperometric curves obtained by on/off cycles using an applied potential of +0.50 V, in PBS electrolyte (pH 7.0), showed that the presence of 0.2 × 10-3 mol L-1 of simazine caused an approximately 50% decrease of the photobiocurrent. Preliminary studies indicated that the synthesized platform based on intact chloroplasts is a good strategy for studying the behavior of photosynthetic entities, using an LED light-responsive WO3 semiconductor substrate. This work contributes to the understanding of photobiocatalysts that emerge as a new class of materials with sophisticated and intricate structures. These are promising materials with remarkably improved quantum efficiency with potential applications in photobioelectrocatalysis.
Subject(s)
Oxides , Tungsten , Chloroplasts/metabolism , Electrodes , Oxides/chemistry , Photosynthesis , Tungsten/chemistry , Tungsten/metabolismABSTRACT
Pseudomonas strains are a promising host cell in metabolic engineering for bioconversion, environmental remediation, and most recently for bioelectrochemical applications. This study isolated an electrochemically active Pseudomonas sp. from an anaerobic sludge using a colorimetric and electrochromic WO3 nanorod (WO3-NR) probe. A strategy was developed to determine the presence of electroactive species from enriched cultures. A mixed consortium was enriched using Pseudomonas isolation media containing betaine and triclosan as the carbon source and antibacterial reagent, respectively. A single blue colony was isolated using WO3-NR sandwiched agar plates. The isolate was sequenced by 16 s rRNA and designated Pseudomonas aeruginosa PBH03, producing phenazines and pyocyanin aerobically. The isolate exhibited clear electrochemical characteristics from cyclic voltammetry and linear sweep voltammetry and produced a current density of 9.01 µA cm-2 in a microbial fuel cell.
Subject(s)
Nanotubes , Tungsten , Colorimetry , Pseudomonas , Pseudomonas aeruginosa/metabolism , Pyocyanine/metabolism , Tungsten/metabolismABSTRACT
Molybdenum- and tungsten-dependent proteins catalyze essential processes in living organisms and biogeochemical cycles. Among these enzymes, members of the dimethyl sulfoxide (DMSO) reductase superfamily are considered the most diverse, facilitating a wide range of chemical transformations that can be categorized as oxygen atom installation, removal, and transfer. Importantly, DMSO reductase enzymes provide high efficiency and excellent selectivity while operating under mild conditions without conventional oxidants such as oxygen or peroxides. Despite the potential utility of these enzymes as biocatalysts, such applications have not been fully explored. In addition, the vast majority of DMSO reductase enzymes still remain uncharacterized. In this review, we describe the reactivities, proposed mechanisms, and potential synthetic applications of selected enzymes in the DMSO reductase superfamily. We also highlight emerging opportunities to discover new chemical activity and current challenges in studying and engineering proteins in the DMSO reductase superfamily.
Subject(s)
Iron-Sulfur Proteins , Oxidoreductases , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Oxidoreductases/metabolism , Oxygen/metabolism , Tungsten/metabolismABSTRACT
This paper reports the synthesis of a new nitrogen-doped porous bio-graphene (NPBG) with a specific biomorphic structure, using Pistacia lentiscus as a natural carbon source containing nitrogen that also acts as a bio-template. The obtained NPBG demonstrated the unique feature of doped nitrogen with a 3D nanoporous structure. Next, a WO3/N-doped porous bio-graphene nanocomposite (WO3/NPBG-NC) was synthesized, and the products were characterized using XPS, SEM, TEM, FT-IR, EDX, XRD, and Raman analyses. The presence of nitrogen doped in the structure of the bio-graphene (BG) was confirmed to be pyridinic-N and pyrrolic-N with N1 peaks at 398.3 eV and 400.5 eV, respectively. The photocatalytic degradation of the anionic azo dyes and drugs was investigated, and the results indicated that the obtained NPBG with a high surface area (151.98 m2/g), unique electronic properties, and modified surface improved the adsorption and photocatalytic properties in combination with WO3 nanoparticles (WO3-NPs) as an effective visible-light-driven photocatalyst. The synthesized WO3/NPBG-NC with a surface area of 226.92 m2/g displayed lower bandgap and higher electron transfer compared with blank WO3-NPs, leading to an increase in the photocatalytic performance through the enhancement of the separation of charge and a reduction in the recombination rate. At the optimum conditions of 0.015 g of the nanocomposite, a contact time of 15 min, and 100 mg/L of dyes, the removal percentages were 100%, 99.8%, and 98% for methyl red (MR), Congo red (CR), and methyl orange (MO), respectively. In the case of the drugs, 99% and 87% of tetracycline and acetaminophen, respectively, at a concentration of 10 mg/L, were removed after 20 min.
Subject(s)
Graphite/metabolism , Light , Nanoparticles/chemistry , Oxides/chemistry , Pistacia/chemistry , Tungsten/chemistry , Catalysis , Graphite/chemistry , Nanoparticles/metabolism , Oxides/metabolism , Particle Size , Photochemical Processes , Pistacia/metabolism , Porosity , Surface Properties , Tungsten/metabolismABSTRACT
Tungsten (W) is a metal that is generally thought to be seldom used in biology. We show here that a W-containing oxidoreductase (WOR) family is diverse and widespread in the microbial world. Surprisingly, WORs, along with the tungstate-specific transporter Tup, are abundant in the human gut microbiome, which contains 24 phylogenetically distinct WOR types. Two model gut microbes containing six types of WOR and Tup were shown to assimilate W. Two of the WORs were natively purified and found to contain W. The enzymes catalyzed the conversion of toxic aldehydes to the corresponding acid, with one WOR carrying out an electron bifurcation reaction coupling aldehyde oxidation to the simultaneous reduction of NAD+ and of the redox protein ferredoxin. Such aldehydes are present in cooked foods and are produced as antimicrobials by gut microbiome metabolism. This aldehyde detoxification strategy is dependent on the availability of W to the microbe. The functions of other WORs in the gut microbiome that do not oxidize aldehydes remain unknown. W is generally beyond detection (<6 parts per billion) in common foods and at picomolar concentrations in drinking water, suggesting that W availability could limit some gut microbial functions and might be an overlooked micronutrient.
Subject(s)
Aldehydes/metabolism , Food , Gastrointestinal Microbiome , Tungsten/metabolism , Aldehyde Oxidoreductases/metabolism , Humans , Oxidation-ReductionABSTRACT
Inhalation of tungsten particulates is a relevant route of exposure in occupational and military settings. Exposure to tungsten alloys is associated with increased incidence of lung pathologies, including interstitial lung disease and cancer. We have demonstrated, oral exposure to soluble tungsten enhances breast cancer metastasis to the lungs through changes in the surrounding microenvironment. However, more research is required to investigate if changes in the lung microenvironment, following tungsten particulate exposure, can drive tumorigenesis or metastasis to the lung niche. This study examined if inhalation to environmentally relevant concentrations of tungsten particulates caused acute damage to the microenvironment in the lungs and/or systemically using a whole-body inhalation system. Twenty-four female BALB/c mice were exposed to Filtered Air, 0.60 mg/m3, or 1.7 mg/m3 tungsten particulates (<1 µm) for 4 h. Tissue samples were collected at days 1 and 7 post-exposure. Tungsten accumulation in the lungs persisted up to 7 days post-exposure and produced acute changes to the lung microenvironment including increased macrophage and neutrophil infiltration, increased levels of proinflammatory cytokines interleukin 1 beta and C-X-C motif chemokine ligand 1, and an increased percentage of activated fibroblasts (alpha-smooth muscle actin+). Exposure to tungsten also resulted in systemic effects on the bone, including tungsten deposition and transient increases in gene expression of proinflammatory cytokines. Taken together, acute whole-body inhalation of tungsten particulates, at levels commonly observed in occupational and military settings, resulted in changes to the lung and bone microenvironments that may promote tumorigenesis or metastasis and be important molecular drivers of other tungsten-associated lung pathologies such as interstitial lung disease.
Subject(s)
Lung , Tungsten , Administration, Inhalation , Animals , Dust , Female , Inhalation Exposure/adverse effects , Lung/pathology , Mice , Neutrophil Infiltration , Tungsten/metabolism , Tungsten/toxicityABSTRACT
Constructing synthetic models of the Mo/Cu active site of aerobic carbon monoxide dehydrogenase (CODH) has been a long-standing synthetic challenge thought to be crucial for understanding how atmospheric concentrations of CO and CO2 are regulated in the global carbon cycle by chemolithoautotrophic bacteria and archaea. Here we report a W/Cu complex that is among the closest synthetic mimics constructed to date, enabled by a silyl protection/deprotection strategy that provided access to a kinetically stabilized complex with mixed O2-/S2- ligation between (bdt)(O)WVI and CuI(NHC) (bdt = benzene dithiolate, NHC = N-heterocyclic carbene) sites. Differences between the inorganic core's structural and electronic features outside the protein environment relative to the native CODH cofactor point to a biochemical CO oxidation mechanism that requires a strained active site geometry, with Lewis acid/base frustration enforced by the protein secondary structure. This new mechanistic insight has the potential to inform synthetic design strategies for multimetallic energy storage catalysts.
Subject(s)
Aldehyde Oxidoreductases/metabolism , Carbon Monoxide/metabolism , Copper/metabolism , Lewis Acids/metabolism , Molybdenum/metabolism , Multienzyme Complexes/metabolism , Tungsten/metabolism , Aldehyde Oxidoreductases/chemistry , Base Pairing , Carbon Monoxide/chemistry , Copper/chemistry , Density Functional Theory , Lewis Acids/chemistry , Models, Molecular , Molecular Structure , Molybdenum/chemistry , Multienzyme Complexes/chemistry , Oxidation-Reduction , Tungsten/chemistryABSTRACT
Genetic circuits that encode extracellular electron transfer (EET) pathways allow the intracellular state of Escherichia coli to be electronically monitored and controlled. However, relatively low electron flux flows through these pathways, limiting the degree of control by these circuits. Since the EET pathway is composed of multiple multiheme cytochromes c (cyts c) from Shewanella oneidensis MR-1, we hypothesized that lower expression levels of cyt c may explain this low EET flux and may be caused by the differences in the cyt c maturation (ccm) machinery between these two species. Here, we constructed random mutations within ccmH by error-prone PCR and screened for increased cyt c production. We identified two ccmH mutants, ccmH-132 and ccmH-195, that exhibited increased heterologous cyt c expression, but had different effects on EET. The ccmH-132 strain reduced WO3 nanoparticles faster than the parental control, whereas the ccmH-195 strain reduced more slowly. The same trend is reflected in electrical current generation: ccmH-132, which has only a single mutation from WT, drastically increased current production by 77%. The percentage of different cyt c proteins in these two mutants suggests that the stoichiometry of the S. oneidensis cyts c is a key determinant of current production by Mtr-expressing E. coli. Thus, we conclude that modulating cyt c maturation effectively improves genetic circuits governing EET in engineered biological systems, enabling better bioelectronic control of E. coli.
Subject(s)
Bioelectric Energy Sources , Cytochromes c/genetics , Escherichia coli/genetics , Genetic Engineering/methods , Shewanella/genetics , Amino Acid Transport Systems/metabolism , Bacterial Proteins/genetics , Cytochromes c/metabolism , Electrochemistry , Electron Transport/genetics , Electrons , Mutation , Nanoparticles/chemistry , Operon , Oxides/metabolism , Tungsten/metabolismABSTRACT
The tungsten containing Aldehyde:ferredoxin oxidoreductases (AOR) offer interesting opportunities for biocatalytic approaches towards aldehyde oxidation and carboxylic acid reduction. The hyperthermophilic archaeon Pyrococcus furiosus encodes five different AOR family members: glyceraldehyde-3-phosphate oxidoreductase (GAPOR), aldehyde oxidoreductase (AOR), and formaldehyde oxidoreductase (FOR), WOR4 and WOR5. GAPOR functions as a glycolytic enzyme and is highly specific for the substrate glyceraldehyde-3-phosphate (GAP). AOR, FOR and WOR5 have a broad substrate spectrum, and for WOR4 no substrate has been identified to date. As ambiguous kinetic parameters have been reported for different AOR family enzymes the steady state kinetics under different physiologically relevant conditions was explored. The GAPOR substrate GAP was found to degrade at 60 °C by non-enzymatic elimination of the phosphate group to methylglyoxal with a half-life t1/2 = 6.5 min. Methylglyoxal is not a substrate or inhibitor of GAPOR. D-GAP was identified as the only substrate oxidized by GAPOR, and the kinetics of the enzyme was unaffected by the presence of L-GAP, which makes GAPOR the first enantioselective enzyme of the AOR family. The steady-state kinetics of GAPOR showed partial substrate inhibition, which assumes the GAP inhibited form of the enzyme retains some activity. This inhibition was found to be alleviated completely by a 1 M NaCl resulting in increased enzyme activity at high substrate concentrations. GAPOR activity was strongly pH dependent, with the optimum at pH 9. At pH 9, the substrate is a divalent anion and, therefore, positively charged amino acid residues are likely to be involved in the binding of the substrate. FOR exhibited a significant primary kinetic isotope effect of the apparent Vmax for the deuterated substrate, formaldehyde-d2, which shows that the rate-determining step involves a CH bond break from the aldehyde. The implications of these results for the reaction mechanism of tungsten-containing AORs, are discussed.
Subject(s)
Aldehyde Oxidoreductases/metabolism , Archaeal Proteins/metabolism , Pyrococcus furiosus/enzymology , Tungsten/metabolism , Aldehyde Oxidoreductases/antagonists & inhibitors , Aldehydes/metabolism , Archaeal Proteins/antagonists & inhibitors , Enzyme Inhibitors , Glyceraldehyde 3-Phosphate/chemistry , Glyceraldehyde 3-Phosphate/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/antagonists & inhibitors , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Hydrogen-Ion Concentration , Kinetics , Oxidation-Reduction , Sodium Chloride , Substrate Specificity , TemperatureABSTRACT
Nanoconstructions composed of lipid vesicles and inorganic units (nanoparticles, metal complexes) arouse much interest across materials science and nanotechnology as hybrid materials combining useful functionalities from both parts. Ideally, these units are to be embedded into the bilayer to keep the biophysical performance of lipid vesicles having inorganic moieties screened from the environment. This can be achieved by doping a lipid bilayer with cluster complexes of transition metals. In this work, we report the preparation of nanoparticles from trinuclear W3S4 cluster complexes and egg phosphatidylcholine. A systematic study of their properties was performed by the differential scanning calorimetry, NMR spectroscopy, dynamic light scattering, and transmission electron microscopy. Phospholipids and clusters have been found to spontaneously self-assemble into novel cluster-lipid hybrid materials. The behavior of clusters in the hydrophobic lipid environment is determined by the structure of the ligands and cluster-to-lipid ratio. Intact cluster complexes bearing compact hydrophobic ligands are embedded into the hydrophobic midplane of a lipid bilayer, whereas cluster complexes bearing larger ligands drive the aggregation of lipids and cluster complexes. Considering these differences, it could be possible to obtain different self-assembled associates such as cluster-doped liposomes or lipid-covered crystals. These cluster-lipid hybrids can be a platform for the design of new materials for nanotechnology.
Subject(s)
Lipid Bilayers/metabolism , Liposomes/metabolism , Phospholipids/metabolism , Tungsten/metabolism , Dynamic Light Scattering , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Liposomes/chemistry , Nanotechnology , Phospholipids/chemistry , Tungsten/chemistryABSTRACT
Dimethyl sulfoxide reductases (DMSO) are molybdoenzymes widespread in all domains of life. They catalyse not only redox reactions, but also hydroxylation/hydration and oxygen transfer processes. Although literature on DMSO is abundant, the biological significance of these enzymes in anaerobic respiration and the molecular mechanisms beyond the expression of genes coding for them are still scarce. In this review, a deep revision of the literature reported on DMSO as well as the use of bioinformatics tools and free software has been developed in order to highlight the relevance of DMSO reductases on anaerobic processes connected to different biogeochemical cycles. Special emphasis has been addressed to DMSO from extremophilic organisms and their role in nitrogen cycle. Besides, an updated overview of phylogeny of DMSOs as well as potential applications of some DMSO reductases on bioremediation approaches are also described.
Subject(s)
Extremophiles , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Isoenzymes , Multigene Family , Oxidoreductases/genetics , Oxidoreductases/metabolism , Phylogeny , Coenzymes/chemistry , Coenzymes/metabolism , Extremophiles/genetics , Extremophiles/metabolism , Iron-Sulfur Proteins/chemistry , Metabolic Networks and Pathways , Metalloproteins/chemistry , Metalloproteins/metabolism , Molybdenum/chemistry , Molybdenum/metabolism , Molybdenum Cofactors , Nitrogen Cycle , Oxidation-Reduction , Oxidoreductases/chemistry , Pteridines/chemistry , Pteridines/metabolism , Structure-Activity Relationship , Tungsten/chemistry , Tungsten/metabolismABSTRACT
Tungsten is an emerging contaminant because of its potential toxicity to humans. However, tungsten-plant-microbe interactions remains unknown. The objective of the study was to evaluate the effect of tungsten-resistant bacteria on tungsten species in plants and microbial community structure in soil. Although bacterial inoculation did not affect lettuce (Lactuca sativa L.) growth or tungsten uptake via root, tungsten-resistant bacteria increased translocation of tungsten from root to shoot. Bacterial inoculation slightly oxidized tungsten in lettuce based on tungsten L3 x-ray absorption near-edge structure (XANES). Tungsten in lettuce roots and shoots grown in tungsten(VI)-spiked soil existed as a mixture of tungsten(IV) and tungsten(VI). Tungsten accumulated as polytungstate in the root and monotungstate in the shoot. Inoculation with tungsten-resistant bacteria and plant growth increased microbial diversity in tungsten-contaminated soil. In tungsten-spiked soils without plants, metal-resistant or reducing bacteria were found while bacteria growing in rhizosphere were detected in soils supporting plant growth. These results indicate a role of the bacteria and plants in phytoremediation of tungsten-contaminated soil.
Subject(s)
Enterobacteriaceae/drug effects , Lactuca/metabolism , Proteobacteria/drug effects , Soil Microbiology , Soil Pollutants/toxicity , Tungsten/toxicity , Bioaccumulation , Biodegradation, Environmental , Drug Resistance, Bacterial , Enterobacteriaceae/metabolism , Lactuca/growth & development , Lactuca/microbiology , Plant Roots/metabolism , Plant Roots/microbiology , Plant Shoots/metabolism , Plant Shoots/microbiology , Proteobacteria/metabolism , Soil Pollutants/metabolism , Tungsten/metabolismABSTRACT
Tungsten (W) is a valuable element with considerable industrial and economic importance that belongs to the European Union list of critical metals with a high supply risk. Therefore, the development of effective and new methods for W recovery is essential to ensure a sustainable supply. In the present study, the Sulfitobacter dubius W transport system TupABC was explored in order to demonstrate both its functionality in Escherichia coli cells and to construct a bioaccumulator (EcotupW). The complete gene cluster tupBCA or partial gene cluster tupBC were cloned in an expression vector and transformed into E. coli. Metal accumulation was evaluated when each construct strain was grown with three separate metal oxyanions (tungstate, molybdate or chromate). The specificity of the bioaccumulator was determined by competition assays using cells grown with mixed solutions of metal oxyanions (W/Mo and W/Cr). The results showed the relevance of the TupA protein in the TupABC transporter system to W-uptake and also allowed Mo and Cr accumulations, although with amounts 1.7 and 2.9-fold lower than W, respectively. To identify the importance of the valine residue in the accumulation efficiency of the VTTS motif, site-directed mutagenesis of tupA was performed. A mutant with a threonine residue, instead of the respective valine, confirmed that W was internalized by nearly double the amount compared to the native form. The findings indicated that cells carrying the native S. dubius TupABC system were great W-bioaccumulators and could be promising tools for W recovery.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Bacterial Proteins/genetics , Escherichia coli/genetics , Rhodobacteraceae/genetics , Tungsten/metabolism , ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/metabolism , Chromates/metabolism , Conservation of Natural Resources , Gene Expression , Molybdenum/metabolism , Multigene Family , Mutation , Protein Binding , Tungsten Compounds/metabolismABSTRACT
Serovars of Salmonella enterica are common food-borne bacterial pathogens. Salmonella typhi, which causes typhoid, is the most dangerous of them. Though detailed molecular pathogenesis studies reveal many virulence factors, inability to identify their biochemical functions hampers the development of diagnostic methods and therapeutic leads. Lack of quicker diagnosis is an impediment in starting early antibiotic treatment to reduce the severe morbidity and mortality in typhoid. In this study, employing bioinformatic prediction, biochemical analysis, and recombinantly cloning the active region, we show that extracellularly secreted virulence-associated protein, small intestinal invasion factor E (SiiE), possesses a sulfite oxidase (SO) domain that catalyzes the conversion of sodium sulfite to sodium sulfate using tungsten as the cofactor. This activity common to Salmonella enterica serovars seems to be specific to them from bioinformatic analysis of available bacterial genomes. Along with the ability of this large non-fimbrial adhesin of 600 kDa binding to sialic acid on the host cells, this activity could aid in subverting the host defense mechanism by destroying sulfites released by the immune cells and colonize the host gastrointestinal epithelium. Being an extracellular enzyme, it could be an ideal candidate for developing diagnostics of S. enterica, particularly S. typhi.
Subject(s)
Adhesins, Bacterial/metabolism , Salmonella enterica/enzymology , Salmonella enterica/pathogenicity , Sulfite Oxidase/metabolism , Virulence Factors/metabolism , Bacterial Adhesion , Computational Biology , Salmonella enterica/genetics , Salmonella typhimurium , Sulfates/metabolism , Sulfite Oxidase/genetics , Sulfites/metabolism , Tungsten/metabolism , VirulenceABSTRACT
An overview is provided of the molybdenum- and tungsten-containing enzymes that catalyze the interconversion of formate and CO2 , focusing on common structural and mechanistic themes, as well as a consideration of the manner in which the mature Mo- or W-containing cofactor is inserted into apoprotein.
Subject(s)
Aldehyde Oxidoreductases/chemistry , Coenzymes/chemistry , Formate Dehydrogenases/chemistry , Molybdenum/chemistry , Tungsten/chemistry , Aldehyde Oxidoreductases/metabolism , Catalysis , Coenzymes/metabolism , Formate Dehydrogenases/metabolism , Molybdenum/metabolism , Structure-Activity Relationship , Tungsten/metabolismABSTRACT
Two factors, climate change brought on by rising atmospheric CO2 levels and the accelerating shift toward renewable energy sources, have together worked to heighten interest in understanding how biological catalysts so effectively bring about the reduction of CO2 to formate, with potential applications for both bioremediation and energy storage. Most metal-dependent formate dehydrogenases, containing either molybdenum or tungsten in their active sites, function physiologically in the direction of formate oxidation to CO2, but it has become clear that many, if not all, are also effective in catalyzing the reverse reaction. In this chapter, we describe methods for isolating and characterizing these enzymes.