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Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that plays an important role in biogeochemical iron and sulfur cycling and is a member of the consortia used in industrial hydrometallurgical processing of copper. Metal sulfide bioleaching is catalyzed by the regeneration of ferric iron, however, bioleaching of chalcopyrite, the dominant unmined form of copper on Earth, is inhibited by surface passivation. Here, we report the implementation of CRISPR interference (CRISPRi) using the catalytically inactive Cas12a (dCas12a) in A. ferrooxidans to knockdown the expression of genes in the petI and petII operons. These operons encode bc1 complex proteins and knockdown of these genes enabled the manipulation (enhancement or repression) of iron oxidation. The petB2 gene knockdown strain enhanced iron oxidation, leading to enhanced pyrite and chalcopyrite oxidation, which correlated with reduced biofilm formation and decreased surface passivation of the minerals. These findings highlight the utility of CRISPRi/dCas12a technology for engineering A. ferrooxidans while unveiling a new strategy to manipulate and improve bioleaching efficiency.
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Self-assembled quantum dots (QDs) are potential candidates for photoelectric and photovoltaic devices, because of their discrete energy levels. The characterization of QDs at the atomic level using a multimodal approach is crucial to improving device performance because QDs are nanostructures with highly correlated structural parameters. In this study, scanning transmission electron microscopy, geometric phase analysis, and atom probe tomography were employed to characterize structural parameters such as the shape, strain, and composition of self-assembled InAs-QDs with InGaAlAs spacer layers. The measurements revealed characteristic AlAs-rich regions above the QDs and InAs-rich regions surrounding the QD columns, which can be explained by the relationship between the effect of strain and surface curvature around the QD. The methodology described in this study accelerates the development of future QD devices because its multiple perspectives reveal phenomena such as atomic-scale segregations and allow for more detailed discussions of the mechanisms of these phenomena.
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As global demands for rare-earth elements (REEs) continue to grow, the biological recovery of REEs has been explored as a promising strategy, driven by potential economic and environmental benefits. It is known that calcium-binding domains, including helix-loop-helix EF hands and repeats-in-toxin (RTX) domains, can bind lanthanide ions due to their similar ionic radii and coordination preference to calcium. Recently, the lanmodulin protein from Methylorubrum extorquens was reported, which has evolved a high affinity for lanthanide ions over calcium. Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile, which has been explored for use in bioleaching for metal recovery. In this report, A. ferrooxidans was engineered for the recombinant intracellular expression of lanmodulin. In addition, an RTX domain from the adenylate cyclase protein of Bordetella pertussis, which has previously been shown to bind Tb3+, was expressed periplasmically via fusion with the endogenous rusticyanin protein. The binding of lanthanides (Tb3+, Pr3+, Nd3+, and La3+) was improved by up to 4-fold for cells expressing lanmodulin and 13-fold for cells expressing the RTX domains in both pure and mixed metal solutions. Interestingly, the presence of lanthanides in the growth media enhanced protein expression, likely by influencing protein stability. Both engineered cell lines exhibited higher recoveries and selectivities for four tested lanthanides (Tb3+, Pr3+, Nd3+, and La3+) over non-REEs (Fe2+ and Co2+) in a synthetic magnet leachate, demonstrating the potential of these new strains for future REE reclamation and recycling applications.
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Acidithiobacillus , Elementos da Série dos Lantanídeos , Metais Terras Raras , Cálcio/metabolismo , Acidithiobacillus/genética , Acidithiobacillus/química , Acidithiobacillus/metabolismo , Elementos da Série dos Lantanídeos/metabolismo , Íons/metabolismoRESUMO
Cell adhesion is generally a prerequisite to the microbial bioleaching of sulfide minerals, and surface biofilm formation is modulated via quorum sensing (QS) communication. We explored the impact of the overexpression of endogenous QS machinery on the covellite bioleaching capabilities of Acidithiobacillus ferrooxidans, a representative acidophilic chemolithoautotrophic bacterium. Cells were engineered to overexpress the endogenous qs-I operon or just the afeI gene under control of the tac promoter. Both strains exhibited increased transcriptional gene expression of afeI and improved cell adhesion to covellite, including increased production of extracellular polymeric substances and increased biofilm formation. Under low iron conditions, the improved bioleaching of covellite was more evident when afeI was overexpressed alone as compared to the native operon. These observations demonstrate the potential for the genetic modulation of QS as a mechanism for increasing the bioleaching efficiency of covellite, and potentially other copper sulfide minerals.
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A hydrometallurgical process is developed to lower the costs of copper production and thereby sustain the use of copper throughout the global transition to renewable energy technologies. The unique feature of the hydrometallurgical process is the reductive treatment of chalcopyrite, which is in contrast to the oxidative treatment more commonly pursued in the literature. Chalcopyrite reduction by chromium(II) ion is described for the first time and superior kinetics are shown. At high concentrate loadings of 39, 78, and 117â g L-1 , chalcopyrite reacted completely within minutes at room temperature and pressure. The XRD, SEM-EDS, and XPS measurements indicate that chalcopyrite reacts to form copper(I) chloride (CuCl). After the reductive treatment, the mineral products are leached by iron(III) sulfate to demonstrate the complete extraction of copper. The chromium(II) ion may be regenerated by an electrolysis unit inspired by an iron chromium flow battery in a practical industrial process.
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Cloretos , Cobre , Compostos Férricos , Cromo , FerroRESUMO
We report electron-beam induced dynamics at PbS-Pb solid-liquid interfaces. We fabricated PbS-Pb solid-liquid nanointerfaces by heating PbS nanocrystals under vacuum to observe the entire process of structural evolution at the atomic scale. We investigated the dynamics using time-resolved high-angle annular dark-field imaging. Electron-beam irradiation caused layer-by-layer dissolution of PbS at the interface, resulting in the formation of the Pb nanodroplet. Ordered liquid layers were observed adjacent to the interface even under continuous electron-beam irradiation and followed the movement of the interface. Instantaneous epitaxial growth of PbS was observed as a reverse process of the dissolution. The resultant Pb nanodroplet provides indisputable evidence for selective sputtering of sulfur atoms via electron-beam irradiation. This paper demonstrates atomic resolution in situ observations of selective and complete sputtering. The observed dynamics can be explained by the intermittent phase transition via nonequilibrium states of the solid-liquid nanointerface triggered by selective sputtering.
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BACKGROUND: Epigenetics is crucial for connecting environmental stresses with physiological responses in humans. Mongolia, where nomadic livestock pastoralism has been the primal livelihood, has a higher prevalence of various chronic diseases than the surrounding East Asian regions, which are more suitable for crop farming. The genes related to dietary stress and pathogenesis of related disorders may have varying epigenetic statuses among the human populations with diverse dietary cultures. Hence, to understand such epigenetic differences, we conducted a comparative analysis of genome-wide DNA methylation of Mongolians and crop-farming East Asians. METHODS: Genome-wide DNA methylation status of peripheral blood cells (PBCs) from 23 Mongolian adults and 24 Thai adults was determined using the Infinium Human Methylation 450K arrays and analyzed in combination with previously published 450K data of 20 Japanese and 8 Chinese adults. CpG sites/regions differentially methylated between Mongolians and crop-farming East Asians were detected using a linear model adjusted for sex, age, ethnicity, and immune cell heterogeneity on RnBeads software. RESULTS: Of the quality-controlled 389,454 autosomal CpG sites, 223 CpG sites were significantly differentially methylated among Mongolians and the four crop farming East Asian populations (false discovery rate < 0.05). Analyses focused on gene promoter regions revealed that PM20D1 (peptidase M20 domain containing 1), which is involved in mitochondrial uncoupling and various processes, including cellular protection from reactive oxygen species (ROS) and thermogenesis, was the top differentially methylated gene. Moreover, gene ontology enrichment analysis revealed that biological processes related to ROS metabolism were overrepresented among the top 1% differentially methylated genes. The promoter regions of these genes were generally hypermethylated in Mongolians, suggesting that the metabolic pathway detoxifying ROS might be globally suppressed in Mongolians, resulting in the high susceptibility of this population to various chronic diseases. CONCLUSIONS: This study showed a significantly diverse DNA methylation status among Mongolians and crop-farming East Asians. Further, we found an association between the differentially methylated genes and various metabolic and neurodegenerative diseases. Knowledge of the epigenetic regulators might help in proper understanding, treatment, and control of such disorders, and physiological adaptation in the future.
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Metilação de DNA , Epigênese Genética , Adaptação Fisiológica , Metilação de DNA/genética , Estudo de Associação Genômica Ampla , Humanos , Estilo de Vida , Espécies Reativas de OxigênioRESUMO
Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies.Acidithiobacillus ferrooxidansis an iron- and sulfur-oxidizing chemolithoautotroph that is known to participate in metal bioleaching, and its metabolic capabilities have been exploited for industrial-scale copper and gold biomining. In addition to bioleaching, microorganisms could also be engineered for selective metal binding, enabling new opportunities for metal bioseparation and recovery. Here, we explored the ability of polyhistidine (polyHis) tags appended to two recombinantly expressed endogenous proteins to enhance the metal binding capacity of A. ferrooxidans. The genetically engineered cells achieved enhanced cobalt and copper binding capacities, and the Langmuir isotherm captures their interaction behavior with these divalent metals. Additionally, the cellular localization of the recombinant proteins correlated with kinetic modeling of the binding interactions, where the outer membrane-associated polyHis-tagged licanantase peptide bound the metals faster than the periplasmically expressed polyHis-tagged rusticyanin protein. The selectivity of the polyHis sequences for cobalt over copper from mixed metal solutions suggests potential utility in practical applications, and further engineering could be used to create metal-selective bioleaching microorganisms.
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Acidithiobacillus , Proteínas de Membrana , Acidithiobacillus/química , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Cátions Bivalentes , Cobre/metabolismo , Histidina , Proteínas de Membrana/metabolismoRESUMO
There are several natural and anthropomorphic environments where iron- and/or sulfur-oxidizing bacteria thrive in extremely acidic conditions. These acidophilic chemolithautotrophs play important roles in biogeochemical iron and sulfur cycles, are critical catalysts for industrial metal bioleaching operations, and have underexplored potential in future biotechnological applications. However, their unique growth conditions complicate the development of genetic techniques. Over the past few decades genetic tools have been successfully developed for Acidithiobacillus ferrooxidans, which serves as a model organism that exhibits both iron- and sulfur-oxidizing capabilities. Conjugal transfer of plasmids has enabled gene overexpression, gene knockouts, and some preliminary metabolic engineering. We highlight the development of genetic systems and recent genetic engineering of A. ferrooxidans, and discuss future perspectives.
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Acidithiobacillus , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Ferro/metabolismo , Engenharia Metabólica , Oxirredução , Enxofre/metabolismoRESUMO
Acidithiobacillus ferrooxidans is a well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is exploited for its ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate-cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild-type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild-type cells. Furthermore, we found that improved iron oxidation ability in the presence of chloride also resulted in higher levels of intracellular reactive oxygen species (ROS) in the strain. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria. IMPORTANCE The use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits, including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for the use of freshwater resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.
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Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Glutationa Sintase/genética , Ferro/metabolismo , Tolerância ao Sal/genética , Acidithiobacillus/efeitos dos fármacos , Escherichia coli/genética , Glutationa/biossíntese , Concentração de Íons de Hidrogênio , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Cloreto de Sódio/farmacologiaRESUMO
Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is commonly reported to exhibit diauxic population growth behavior where ferrous iron is oxidized before elemental sulfur when both are available, despite the higher energy content of sulfur. We have discovered sulfur dispersion formulations that enables sulfur oxidation before ferrous iron oxidation. The oxidation of dispersed sulfur can lower the culture pH within days below the range where aerobic ferrous iron oxidation can occur. Thus, ferric iron reduction can be observed quickly which had previously been reported over extended incubation periods with untreated sulfur. Therefore, we demonstrate that this substrate utilization pattern is strongly dependent on the cell loading in relation to sulfur concentration, sulfur surface hydrophobicity, and the pH of the culture. Our dispersed sulfur formulation, lig-sulfur, can be used to support the rapid antibiotic selection of plasmid-transformed cells, which is not possible in liquid cultures where ferrous iron is the main source of energy for these acidophiles. Furthermore, we find that media containing lig-sulfur supports higher production of green fluorescent protein compared to media containing ferrous iron. The use of dispersed sulfur is a valuable new tool for the development of engineered A. ferrooxidans strains and it provides a new method to control iron and sulfur oxidation behaviors.
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Acidithiobacillus/crescimento & desenvolvimento , Meios de Cultura/química , Ferro/metabolismo , Enxofre/metabolismo , OxirreduçãoRESUMO
Acidithiobacillus ferrooxidans cells can oxidize iron and sulfur and are key members of the microbial biomining communities that are exploited in the large-scale bioleaching of metal sulfide ores. Some minerals are recalcitrant to bioleaching due to the presence of other inhibitory materials in the ore bodies. Additives are intentionally included in processed metals to reduce environmental impacts and microbially influenced corrosion. We have previously reported a new aerobic corrosion mechanism where A. ferrooxidans cells combined with pyrite and chloride can oxidize low-grade stainless steel (SS304) with a thiosulfate-mediated mechanism. Here we explore process conditions and genetic engineering of the cells that enable corrosion of a higher grade steel (SS316). The addition of elemental sulfur and an increase in the cell loading resulted in a 74% increase in the corrosion of SS316 as compared to the initial sulfur- and cell-free control experiments containing only pyrite. The overexpression of the endogenous rus gene, which is involved in the cellular iron oxidation pathway, led to a further 85% increase in the corrosion of the steel in addition to the improvements made by changes to the process conditions. Thus, the modification of the culturing conditions and the use of rus-overexpressing cells led to a more than threefold increase in the corrosion of SS316 stainless steel, such that 15% of the metal coupons was dissolved in just 2 weeks. This study demonstrates how the engineering of cells and the optimization of their cultivation conditions can be used to discover conditions that lead to the corrosion of a complex metal target.
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Acidithiobacillus , Azurina , Aço Inoxidável , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Azurina/genética , Azurina/metabolismo , Corrosão , Ferro/metabolismo , Oxirredução , Sulfetos/metabolismoRESUMO
Microbially influenced corrosion (MIC) results in significant damage to metallic materials in many industries. Anaerobic sulfate-reducing bacteria (SRB) have been well studied for their involvement in these processes. Highly corrosive environments are also found in pulp and paper processing, where chloride and thiosulfate lead to the corrosion of stainless steels. Acidithiobacillus ferrooxidans is a critically important chemolithotrophic acidophile exploited in metal biomining operations, and there is interest in using A. ferrooxidans cells for emerging processes such as electronic waste recycling. We explored conditions under which A. ferrooxidans could enable the corrosion of stainless steel. Acidic medium with iron, chloride, low sulfate, and pyrite supplementation created an environment where unstable thiosulfate was continuously generated. When combined with the chloride, acid, and iron, the thiosulfate enabled substantial corrosion of stainless steel (SS304) coupons (mass loss, 5.4 ± 1.1 mg/cm2 over 13 days), which is an order of magnitude higher than what has been reported for SRB. There results were verified in an abiotic flow reactor, and the importance of mixing was also demonstrated. Overall, these results indicate that A. ferrooxidans and related pyrite-oxidizing bacteria could produce aggressive MIC conditions in certain environmental milieus.IMPORTANCE MIC of industrial equipment, gas pipelines, and military material leads to billions of dollars in damage annually. Thus, there is a clear need to better understand MIC processes and chemistries as efforts are made to ameliorate these effects. Additionally, A. ferrooxidans is a valuable acidophile with high metal tolerance which can continuously generate ferric iron, making it critical to copper and other biomining operations as well as a potential biocatalyst for electronic waste recycling. New MIC mechanisms may expand the utility of these cells in future metal resource recovery operations.
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Acidithiobacillus/metabolismo , Ferro/química , Aço Inoxidável/química , Sulfatos/química , Tiossulfatos/química , Ligas , Crescimento Quimioautotrófico , Cobre , Corrosão , Elétrons , Microbiologia Industrial , Mineração , Oxidantes , Oxirredução , Sulfetos , Propriedades de SuperfícieRESUMO
Context: Adipose tissue is the main organ that stores energy and participates in adaptive thermogenesis of the human body. The adipose tissue content in an individual is determined by a combination of genetic factors and lifestyle related factors. While Japanese people, along with the closely related East Asians, are generally thinner than individuals of European ancestry, they are prone to accumulating visceral adipose tissues. Genome-wide discovery of loci influencing obesity-related traits, and application of the genome sequence data to assess natural selection, provides evidence that the obesity-related traits in East Asians might be shaped by natural selection. Objective: This review aims to summarise health and evolutionary implications of genetic variants influencing obesity-related traits in Japanese. Methods: This study gathered recently published papers of medical, genetic and evolutionary studies regarding obesity-related traits in the Japanese and closely related East Asians. Results and conclusion: A high susceptibility to central obesity of Japanese and closely related East Asians might have been shaped by natural selection favouring thrifty genotypes. Moreover, natural selection favouring higher thermogenic activity of brown adipose tissues would contribute to increased non-thrifty alleles in ancestors of East Asians.
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Evolução Biológica , Predisposição Genética para Doença/genética , Variação Genética , Obesidade/genética , Tecido Adiposo Marrom/metabolismo , Humanos , Japão , Obesidade Abdominal/genética , Seleção GenéticaRESUMO
The development of Acidithiobacillus ferrooxidans as a non-model host organism for synthetic biology is hampered by a lack of genetic tools and techniques. New plating and liquid-based selection methods were developed to improve the identification of transformed cell lines. Enabled by these methods, a hyperactive transposase was used to generate mutants with integrated genes for the expression of the superfolder green fluorescent protein (sfGFP) gene or a 2-keto decarboxylase (KDC) gene, which enabled the production and secretion of isobutyric acid (IBA). An inverse PCR method was used to identify the insertion sites of the KDC gene in several mutants, leading to the identification of a region on the chromosome that may be suitable for future genetic insertions. These results demonstrate that functional exogenous metabolic genes have been chromosomally integrated into A. ferrooxidans, and this advance will facilitate the future development of these cells for new biotechnology applications.IMPORTANCEAcidithiobacillus ferrooxidans is an iron- and sulfur-oxidizing chemolithoautotroph and is a key member of the microbial consortia used in industrial biomining applications. There is interest in exploiting these cells for other metal recovery applications as well as in developing them as unique nonmodel microbial cell factories. Plasmid-driven expression of exogenous genes has been reported, and homologous recombination has been used to knock out some gene expression. Here, new selection protocols facilitated the development of a transposition method for chromosomal integration of exogenous genes into A. ferrooxidans This greatly expands the available genetic toolbox, which will open the door to greater metabolic engineering efforts for these cells.
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Acidithiobacillus/genética , Cromossomos Bacterianos/genética , Engenharia Genética/métodos , Transposases/metabolismo , Acidithiobacillus/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutagênese Insercional , Transposases/genéticaRESUMO
Complex I (NADH:quinone oxidoreductase) is central to cellular aerobic energy metabolism, and its deficiency is involved in many human mitochondrial diseases. Complex I translocates protons across the membrane using electron transfer energy. Semiquinone (SQ) intermediates appearing during catalysis are suggested to be key for the coupling mechanism in complex I. However, the existence of SQ has remained controversial due to the extreme difficulty in detecting unstable and low intensity SQ signals. Here, for the first time with Escherichia coli complex I reconstituted in proteoliposomes, we successfully resolved and characterized three distinct SQ species by EPR. These species include: fast-relaxing SQ (SQNf) with P1/2 (half-saturation power level)>50mW and a wider linewidth (12.8 G); slow-relaxing SQ (SQNs) with P1/2=2-3mW and a 10G linewidth; and very slow-relaxing SQ (SQNvs) with P1/2= ~0.1mW and a 7.5G linewidth. The SQNf signals completely disappeared in the presence of the uncoupler gramicidin D or squamotacin, a potent E. coli complex I inhibitor. The pH dependency of the SQNf signals correlated with the proton-pumping activities of complex I. The SQNs signals were insensitive to gramicidin D, but sensitive to squamotacin. The SQNvs signals were insensitive to both gramicidin D and squamotacin. Our deuterium exchange experiments suggested that SQNf is neutral, while SQNs and SQNvs are anion radicals. The SQNs signals were lost in the ΔNuoL mutant missing transporter module subunits NuoL and NuoM. The roles and relationships of the SQ intermediates in the coupling mechanism are discussed.