Arsenite Oxidase Research Articles

Characterization and exploration of an As(III)-oxidizing bacterium TMKU1 for plant growth promotion under arsenic stress

Arsenic (As) pollution has been recognized as a serious global environmental problem. Though various remediation strategies are being explored to cope up with the As-toxicity, currently microbe-assisted detoxification of As is found to be the most promising technique for restoring As-contaminated rhizosphere soil for its eco-friendly nature. As(III)-oxidizing bacteria were reported to most suitable in this regard. In this study, an As(III)-oxidizing bacterium, TMKU1 was isolated from As-contaminated rhizospheric soil that could withstand up to 20 mM As(III) and 95 mM As(V). The strain was closely related to the genus Acinetobacter, according to 16 S rDNA analysis. The strain could transform ∼70% of As(III) to As(V) under aerobic culture condition. The transformation of As(III) was found to be catalyzed by arsenite oxidase, which is constitutive nature in this strain. The As(III)-oxidase was found to be encoded by the functional gene aioA harbored on the genomic DNA. The enzyme was localized mostly in the periplasm, and the partially purified enzyme showed the Km = 95.76667 μM and Vmax = 0.356341 M min−1g−1 protein. The novelty of this strain is that it could express plant growth promoting features like phosphate solubilization, siderophore production, IAA production and N2-fixation, both under stress-free and As-stress conditions. Seed bacterization with TMKU1 not only significantly increased the germination of chickpea seeds, but also shielded the seedlings from the As toxicity and accumulation. Overall, the results suggest that this bacterial candidate would be of potential use in agricultural practices for detoxification of As in crop field.

Gamma irradiation in modulating arsenic bioremediation potential of Pseudomonas sp. AK1 and AK9

Purpose Present study deals with the role of gamma irradiation in modulating arsenic bioremediation of Pseudomonas sp. AK1 and AK9 strains Materials and methods The bacterial strains AK1 and AK9 of Pseudomonas sp. were irradiated at different doses (5 Gy, 10 Gy, 15 Gy and 20 Gy) of gamma irradiation. The effect of γ-irradiation on the growth and arsenic modulating ability of AK1 and AK9 strains was determined in the presence and absence of arsenic along with non-irradiated strains. Further, a comparative study of non-irradiated and irradiated strains by protein profiling in absence and presence of arsenic was carried out to confirm of the increased expression ofarsenite oxidase. Results Both strains were able to transform AsIII to AsV. Both strains AK1 and AK9 decrease the arsenic concentration by 626.68 ppb (13.36%) and 686.40 ppb (14.71%) after an incubation period of 96 h in presence of arsenic. Gamma irradiated AK9 strains showed doubled growth in presence of arsenic as compared to non-irradiated strains at 10 Gy treatment whereas no changes in growth was observed in irradiated AK1 strains. Gamma irradiated AK9 strain decrease 378.65 ppb (7.27%) more arsenic concentration from natural water sample supplemented with AsIII than non-irradiated AK9 strain. Further, in the protein profile, increased expression of arsenite oxidase (∼85 kDa) was observed in irradiated AK9 strains in presence of arsenic. Conclusions Overall, the results suggested that the gamma irradiated AK9 strain having potential for arsenic accumulation and increased arsenite tolerance may play a great role in the bioremediation of the arsenite at arsenic contaminated sites.

Novel insights into the co-selection of metal-driven antibiotic resistance in bacteria: a study of arsenic and antibiotic co-exposure.

The simultaneous development of antibiotic resistance in bacteria due to metal exposure poses a significant threat to the environment and human health. This study explored how exposure to both arsenic and antibiotics affects the ability of an arsenite oxidizer, Achromobacter xylosoxidans CAW4, to transform arsenite and its antibiotic resistance patterns. The bacterium was isolated from arsenic-contaminated groundwater in the Chandpur district of Bangladesh. We determined the minimum inhibitory concentration (MIC) of arsenite, cefotaxime, and tetracycline for A. xylosoxidans CAW4, demonstrating a multidrug resistance (MDR) trait. Following this determination, we aimed to mimic an environment where A. xylosoxidans CAW4 was exposed to both arsenite and antibiotics. We enabled the strain to grow in sub-MIC concentrations of 1mM arsenite, 40µg/mL cefotaxime, and 20µg/mL tetracycline. The expression dynamics of the arsenite oxidase (aioA) gene in the presence or absence of antibiotics were analyzed. The findings indicated that simultaneous exposure to arsenite and antibiotics adversely affected the bacteria's capacity to metabolize arsenic. However, when arsenite was present in antibiotics-containing media, it promoted bacterial growth. The study observed a global downregulation of the aioA gene in arsenic-antibiotic conditions, indicating the possibility of increased susceptibility through co-resistance across the entire bacterial population of the environment. This study interprets that bacterial arsenic-metabolizing ability can rescue the bacteria from antibiotic stress, further disseminating environmental cross-resistance. Therefore, the co-selection of metal-driven antibiotic resistance in bacteria highlights the need for effective measures to address this emerging threat to human health and the environment.

Purifying selection drives distinctive arsenic metabolism pathways in prokaryotic and eukaryotic microbes.

Microbes play a crucial role in the arsenic biogeochemical cycle through specific metabolic pathways to adapt to arsenic toxicity. However, the different arsenic-detoxification strategies between prokaryotic and eukaryotic microbes are poorly understood. This hampers our comprehension of how microbe-arsenic interactions drive the arsenic cycle and the development of microbial methods for remediation. In this study, we utilized conserved protein domains from 16 arsenic biotransformation genes (ABGs) to search for homologous proteins in 670 microbial genomes. Prokaryotes exhibited a wider species distribution of arsenic reduction- and arsenic efflux-related genes than fungi, whereas arsenic oxidation-related genes were more prevalent in fungi than in prokaryotes. This was supported by significantly higher acr3 (arsenite efflux permease) expression in bacteria (upregulated 3.72-fold) than in fungi (upregulated 1.54-fold) and higher aoxA (arsenite oxidase) expression in fungi (upregulated 5.11-fold) than in bacteria (upregulated 2.05-fold) under arsenite stress. The average values of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site (dN/dS) of homologous ABGs were higher in archaea (0.098) and bacteria (0.124) than in fungi (0.051). Significant negative correlations between the dN/dS of ABGs and species distribution breadth and gene expression levels in archaea, bacteria, and fungi indicated that microbes establish the distinct strength of purifying selection for homologous ABGs. These differences contribute to the distinct arsenic metabolism pathways in prokaryotic and eukaryotic microbes. These observations facilitate a significant shift from studying individual or several ABGs to characterizing the comprehensive microbial strategies of arsenic detoxification.

Isolation and characterization of rhizobacteria from lentil for arsenic resistance and plant growth promotion.

Low-cost microbial remediation strategies serve as a viable and potent weapon for curbing the arsenic menace. In the present study, two arsenic-resistant bacteria were isolated from the contaminated lentil rhizosphere in Gangetic plain of eastern India. LAR-21 (Burkholderia cepacia, MW356875) and LAR-25 (Burkholderia cenocepacia, MW356894) could remove 87.6% and 85.9% of arsenite (10mM) from the liquid culture medium in laboratory condition. They were highly resistant to arsenate and arsenite and also had a high arsenite oxidase activity. LAR-21 showed the highest level of minimum inhibitory concentration value of 390mM for arsenate and 31mM for arsenite. The same strain was found to show highest arsenite oxidase activity, i.e., 5.2nMmin-1mg-1of protein. These two strains further possess potential plant growth-promoting characteristics like indole acetic acid production (5-15mMIAAmL-1), 1-aminocyclopropane-1-carboxylate deaminase (8-21nMα-ketobutyratemgprotein-1h-1), nitrogenase activity (3-8.99nMethylenemgcellprotein-1h-1), siderophore production (17-22.1µMdeferoxaminemesylatemL-1), phosphate solubilization (261-453µgmL-1) under arsenic stress condition. The plant growth promotion of the strains was further validated by pot study of lentil by assessing their agronomic and growth-related traits, and potential to recover from arsenic stress (17.2-21.2% arsenic reduction in root and shoot, 16-19.2% in leaf and pod, and 15-23% reduction in seeds). The LAR-21 strain, thus, emerged as the most suited candidate for bioremediation and plant (lentil) growth promotion in arsenic polluted environment.

Characterization of As(III)-oxidizing bacteria Acinetobacter sp. TMKU7 having plant growth promoting features for possible application in arsenic-contaminated crop field

Arsenic pollution is considered as one of the global environmental threats. Arsenic is found in nature mostly in two different valence states, namely arsenite [As(III)] and arsenate [As(V)], the former is more toxic due to its solubility and reactivity. Certain microbial communities inhabiting in the As-rich environment contribute a lot in the geochemical cycling of As. This study focuses on the isolation and identification of As(III) oxidizing bacteria from soil sample, and furthermore assessment of its As(III) transforming potential. The study was also aimed to hunt its plant growth promoting features for possible application to promote crop yield As-contaminated soil. Among the 28 bacterial isolates, a strain designated as TMKU7 could transform ∼80% As(III) to As(V) under culture condition. Based on morphological, biochemical, and molecular characterization, the strain was identified as Acinetobacter sp. Arsenite oxidase, the key player for As(III) to As(V) conversion was found to be constitutive in this strain, and the enzymatic activity was mostly found in the periplasmic fraction. The K m and V max of the partially purified As(III) oxidase were determined to be 41.43 μM and 0.19 μM min−1 μg−1 protein, respectively. The presence of As(III) oxidase gene (aioA) in the genomic DNA was further confirmed by PCR amplification. Agarose entrapped partially purified enzyme showed a potential for As(III) removal from water as well. The expression of various plant growth promoting traits have added additional importance to this As-resistant strain, which could be utilized as a prospective As-detoxification candidate in the As-contaminated crop field for sustainable agriculture.

Genome sequence and characterisation of a freshwater photoarsenotroph, Cereibacter azotoformans strain ORIO, isolated from sediments capable of cyclic light-dark arsenic oxidation and reduction.

A freshwater photosynthetic arsenite-oxidizing bacterium, Cereibacter azotoformans strain ORIO, was isolated from Owens River, CA, USA. The waters from Owens River are elevated in arsenic and serve as the headwaters to the Los Angeles Aqueduct. The complete genome sequence of strain ORIO is 4.8 Mb genome (68% G + C content) and comprises two chromosomes and six plasmids. Taxonomic analysis placed ORIO within the Cereibacter genus (formerly Rhodobacter). The ORIO genome contains arxB2 AB1 CD (encoding an arsenite oxidase), arxXSR (regulators) and several ars arsenic resistance genes all co-localised on a 136 kb plasmid, named pORIO3. Phylogenetic analysis of ArxA, the molybdenum-containing arsenite oxidase catalytic subunit, demonstrated photoarsenotrophy is likely to occur within members of the Alphaproteobacteria. ORIO is a mixotroph, oxidises arsenite to arsenate (As(V)) photoheterotrophically, and expresses arxA in cultures grown with arsenite. Further ecophysiology studies with Owens River sediment demonstrated the interconversion of arsenite and As(V) was dependent on light-dark cycling. arxA and arrA (As(V) respiratory reductase) genes were detected in the light-dark cycled sediment metagenomes suggesting syntrophic interactions among arsenotrophs. This work establishes C. azotoformans str. ORIO as a new model organism for studying photoarsenotrophy and light-dark arsenic biogeochemical cycling.

Rapid arsenite oxidation by Paenarthrobacter nicotinovorans strain SSBW5: unravelling the role of GlpF, aioAB and aioE genes.

A novel arsenite resistant bacterial strain SSBW5 was isolated from the battery waste site of Corlim, Goa, India. This strain interestingly exhibited rapid arsenite oxidation with an accumulation of 5mM arsenate within 24h and a minimum inhibitory concentration (MIC) of 18mM. The strain SSBW5 was identified as Paenarthrobacter nicotinovorans using 16S rDNA sequence analysis. Fourier-transformed infrared (FTIR) spectroscopy of arsenite-exposed cells revealed the interaction of arsenite with several important functional groups present on the cell surface, possibly involved in the resistance mechanism. Interestingly, the whole genome sequence analysis also clearly elucidated the presence of genes, such as GlpF, aioAB and aioE encoding transporter, arsenite oxidase and oxidoreductase enzyme, respectively, conferring their role in arsenite resistance. Furthermore, this strain also revealed the presence of several other genes conferring resistance to various metals, drugs, antibiotics and disinfectants. Further suggesting the probable direct or indirect involvement of these genes in the detoxification of arsenite thereby increasing its tolerance limit. In addition, clumping of bacterial cells was observed through microscopic analysis which could also be a strategy to reduce arsenite toxicity thus indicating the existence of multiple resistance mechanisms in strain SSBW5. In the present communication, we are reporting for the first time the potential of P. nicotinovorans strain SSBW5 to be used in the bioremediation of arsenite via arsenite oxidation along with other toxic metals and metalloids.

Metagenomic and culture-dependent approaches unveil active microbial community and novel functional genes involved in arsenic mobilization and detoxification in groundwater

BackgroundArsenic (As) and its species are major pollutants in ecological bodied including groundwater in Bangladesh rendering serious public health concern. Bacteria with arsenotrophic genes have been found in the aquifer, converting toxic arsenite [As (III)] to less toxic arsenate [As (V)] that is easily removed using chemical and biological trappers. In this study, genomic and metagenomic approaches parallel to culture-based assay (Graphical abstract) have made it possible to decipher phylogenetic diversity of groundwater arsenotrophic microbiomes along with elucidation of their genetic determinants.ResultsSeventy-two isolates were retrieved from six As-contaminated (average As concentration of 0.23 mg/L) groundwater samples from Munshiganj and Chandpur districts of Bangladesh. Twenty-three isolates harbored arsenite efflux pump (arsB) gene with high abundance, and ten isolates possessing arsenite oxidase (aioA) gene, with a wide range of minimum inhibitory concentration, MICAs (2 to 32 mM), confirming their role in arsenite metabolism. There was considerable heterogeneity in species richness and microbial community structure. Microbial taxa from Proteobacteria, Firmicutes and Acidobacteria dominated these diversities. Through these combinatorial approaches, we have identified potential candidates such as, Pseudomonas, Acinetobacter, Stenotrophomonas, Achromobacter, Paraburkholderia, Comamonas and Klebsiella and associated functional genes (arsB, acr3, arsD, arsH, arsR) that could significantly contribute to arsenite detoxification, accumulation, and immobilization.ConclusionsCulture-dependent and -independent shotgun metagenomic investigation elucidated arsenotrophic microbiomes and their functions in As biogeochemical transformation. These findings laid a foundation for further large-scale researches on the arsenotrophic microbiomes and their concurrent functions in As biogeochemical transformation in As-contaminated areas of Bangladesh and beyond.

Bayesian network highlights the contributing factors for efficient arsenic phytoextraction by Pteris vittata in a contaminated field

Phytoextraction is a low-cost and eco-friendly method for removing pollutants, such as arsenic (As), from contaminated soil. One of the most studied As hyperaccumulators for soil remediation include Pteris vittata. Although phytoextraction using plant-assisted microbes has been considered a promising soil remediation method, microbial harnessing has not been achieved due to the complex and difficult to understand interactions between microbes and plants. This problem can possibly be addressed with a multi-omics approach using a Bayesian network. However, limited studies have used Bayesian networks to analyze plant–microbe interactions. Therefore, to understand this complex interaction and to facilitate efficient As phytoextraction using microbial inoculants, we conducted field cultivation experiments at two sites with different total As contents (62 and 8.9 mg/kg). Metabolome and microbiome data were obtained from rhizosphere soil samples using nuclear magnetic resonance and high-throughput sequencing, respectively, and a Bayesian network was applied to the obtained multi-omics data. In a highly As-contaminated site, inoculation with Pseudomonas sp. strain m307, which is an arsenite-oxidizing microbe having multiple copies of the arsenite oxidase gene, increased As concentration in the shoots of P. vittata to 157.5 mg/kg under this treatment; this was 1.5-fold higher than that of the other treatments. Bayesian network demonstrated that strain m307 contributed to As accumulation in P. vittata. Furthermore, the network showed that microbes belonging to the MND1 order positively contributed to As accumulation in P. vittata. Based on the ecological characteristics of MND1, it was suggested that the rhizosphere of P. vittata inoculated with strain m307 was under low-nitrogen conditions. Strain m307 may have induced low-nitrogen conditions via arsenite oxidation accompanied by nitrate reduction, potentially resulting in microbial iron reduction or the prevention of microbial iron oxidation. These conditions may have enhanced the bioavailability of arsenate, leading to increased As accumulation in P. vittata.

Arsenite oxidase in complex with antimonite and arsenite oxyanions: Insights into the catalytic mechanism

Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.

Arsenic removal in flue gas through anaerobic denitrification and sulfate reduction cocoupled arsenic oxidation

Arsenic in flue gas from municipal solid waste incineration can damage to human health and ecological environment. A sulfate-nitrate-reducing bioreactor (SNRBR) for flue gas arsenic removal was investigated. Arsenic removal efficiency attained 89.4%. An integrated metagenomic and metaproteomic investigation showed that three nitrate reductases (NapA, NapB and NarG), three sulfate reductases (Sat, AprAB and DsrAB), and arsenite oxidase (ArxA) regulated nitrate reduction, sulfate reduction and bacterial As(III)-oxidation, respectively. Citrobacter and Desulfobulbus could synthetically regulate the expression of arsenite-oxidizing gene, nitrate reductases and sulfate reducatases, which involved in As(III) oxidation, nitrate and sulfate reduction. A bacterial consortium containing Citrobacter, UG_Enterobacteriaceas, Desulfobulbus and Desulfovibrio could capable of simultaneously arsenic oxidation, sulfate reduction and denitrification. Anaerobic denitrification and sulfate reduction were cocoupled to arsenic oxidation. The biofilm was characterized by FTIR, XPS, XRD, EEM, and SEM. XRD and XPS spectra verified the formation of aarsenic species (As(V)) from flue gas As(III) conversion. Arsenic speciation in biofilms of SNRBR consisted of 77% residual arsenic, 15.9% organic matter-bound arsenic, and 4.3% strongly absorbed arsenic. Flue gas arsenic was bio-stabilized in the form of Fe–As–S and As-EPS through biodeposition, biosorption and biocomplexation. This provides a new way of flue gas arsenic removal using the sulfate-nitrate-reducing bioreactor.

Arsenic pollution and arsenic-resistant bacteria of drying Urmia Salt Lake

Extensive arsenic contamination of groundwater, rivers, and agricultural farms has led to widespread arsenic poisoning and consequent problems associated with health and economy. In recent years, many studies have been performed to understand bacterial arsenic detoxification and metabolism which has paved the way for bioremediation applications. This study attempted to isolate and characterize arsenic-resistant bacteria from the drying Urmia lake. As a result of the 16S rDNA analysis, six arsenic-resistant strains were identified as the members of Shouchella, Salipaludibacillus, and Evansella genera. For some of the strains, the maximum tolerance concentration for either arsenate or arsenite was considerably high, 320 and 16 mM, respectively. All the strains harbored the arsenate reductase gene (arsC). The arsenate permease (arsB) gene was identified in all strains except in strains S1, S12, and E15. The metabolic genes of respiratory arsenate reductase (arrB) and arsenite oxidase (arxA) were identified in none of these strains. This feature of the strains with the arsC gene region can be exploited to bioremediate arsenic from contaminated areas by using a two-step process. The second step can be carried out by the adsorption of arsenite to iron oxide or by precipitation with sulfide. Today, more and more ecosystems are being destroyed due to anthropogenic pollution, and it is important to reveal the negative effects and solutions of this situation on the ecosystems. So, the current study provides a potential source of bacteria for such studies that implement bioremediation practices to prevent arsenic catastrophe in vicinal territories, and reveals arsenic pollution in Urmia lake.

Enzyme-based electrochemical biosensor for antimonite detection in water.

Antimonite (SbIII) is a naturally occurring contaminant demanding on-site ultrasensitive detection. The enzyme-based electrochemical (EC) biosensors are promising, but the lack of specific SbIII oxidizing enzymes hindered the past efforts. Herein, we modulated the specificity of arsenite oxidase AioAB toward SbIII by regulating its spatial conformation from tight to loose using the metal-organic framework ZIF-8. The constructed EC biosensor, AioAB@ZIF-8, exhibited the substrate specificity toward SbIII at 12.8s-1μM-1, an order of magnitude higher than that of AsIII (1.1s-1μM-1). Relaxing AioAB structure in ZIF-8 was evidenced by the break of the S-S bond and the conversion of α helix to the random coil as suggested by Raman spectroscopy. Our AioAB@ZIF-8 EC sensor exhibited a dynamic linear range in 0.041-4.1μMat a response time of 5s, and the detection limit at 0.041μMat a high sensitivity of 1894nAμM-1. The insights into tuning the specificity of an enzyme shed new light on biosensing metal(loid)s without specific proteins.

Flue gas arsenic trioxide removal from sludge incineration by sulfate-reducing membrane biofilm reactor

Arsenic in flue gas from sludge incineration may damage to human health and ecological environment seriously. The sulfate-reducing membrane biofilm reactor (SRBR) was investigated for arsenic trioxide(As2O3) removal in flue gas from sludge incineration. Arsenic removal efficiency attained 94.4%. Desulfovibrio, UC_Clostridiales, and Pseudodesulfovibrio were the dominant genera. Desulfovibrio was the core arsenite-oxidizing-sulfate-reducing genus. Arsenite oxidase (ArxA) and sulfate-reducing enzymes (Sat, AprAB and DsrAB) responsible for bacterial As(III)-oxidation and sulfate reduction. Desulfovibrio could synthetically regulate the expression of functional proteins that involved in As oxidation, sulfate reduction, cell wall and membrane synthesis, antioxidant, DNA repair, glycolysis and amino acid synthesis under As stress, possessed resistant and removal ability to As. The biofilm in SRBR was characterized by FTIR, XPS, XRD, EEM, and SEM-EDS. XPS and XRD spectra indicate the formation of an arsenic species (As(V)) from arsenic oxidation. Humic acid complex arsenic (HA-As) and arsenopyrite-like phase (Fe-As-S) precipitation were formed in SRBR. Microbial arsenic oxidation was coupled sulfate reduction and biostabilization of arsenic from flue gas. Arsenic in flue gas was bio-stabilized in the form of Fe-As-S and HA-As via biocomplexation of humic acids in extracellular polymeric substances (EPS), biosorption and biodeposition. These results show that the sulfate-reducing membrane biofilm reactor is achievable and open new possibilities for applying the SRBR to removal of arsenic in flue gas from sludge incineration.

The structure of the complex between the arsenite oxidase from Pseudorhizobium banfieldiae sp. strain NT-26 and its native electron acceptor cytochrome c552.

The arsenite oxidase (AioAB) from Pseudorhizobium banfieldiae sp. strain NT-26 catalyzes the oxidation of arsenite to arsenate and transfers electrons to its cognate electron acceptor cytochrome c552 (cytc552). This activity underpins the ability of this organism to respire using arsenite present in contaminated environments. The crystal structure of the AioAB/cytc552 electron transfer complex reveals two A2B2/(cytc552)2 assemblies per asymmetric unit. Three of the four cytc552 molecules in the asymmetric unit dock to AioAB in a cleft at the interface between the AioA and AioB subunits, with an edge-to-edge distance of 7.5 Å between the heme of cytc552 and the [2Fe-2S] Rieske cluster in the AioB subunit. The interface between the AioAB and cytc552 proteins features electrostatic and nonpolar interactions and is stabilized by two salt bridges. A modest number of hydrogen bonds, salt bridges and relatively small, buried surface areas between protein partners are typical features of transient electron transfer complexes. Interestingly, the fourth cytc552 molecule is positioned differently between two AioAB heterodimers, with distances between its heme and the AioAB redox active cofactors that are outside the acceptable range for fast electron transfer. This unique cytc552 molecule appears to be positioned to facilitate crystal packing rather than reflecting a functional complex.

Cloning and functional characterization of arsenite oxidase (aoxB) gene associated with arsenic transformation in Pseudomonas sp. strain AK9

Arsenic transforming bacterial strains belong to genus Pseudomonas sp.AK9 (KY569424), were isolated from the middle Gangetic plains of Bihar, India. The Pseudomonas sp. AK9 strains were able to transform toxic arsenite to a less toxic arsenate. In the present work, the presence of different arsenic resistance genes (aoxB, arsB, acr3 and aoxAB) were observed in isolated strain. Furthermore, the aoxB gene was amplified from genomic DNA of AK9, cloned in E.coli/DH5αcells, and sequenced. The BLASTn results and phylogenetic study of the aoxB gene showed 95.32 % and 90.07 % identity with the large subunit of aoxB gene of previous reported Thiomonas arsenivorans strain DSM16361 and Thiomonas arsenivorans strain b6, respectively. Further overhang primers were designed for amplifications of full length aoxB gene (∼1200 bp), and cloned in to the expression vector and host E.coli/BL21 cells. The GST-aoxB gene was expressed in BL21 cells, and a profound expression product of ∼ 72 kDa was observed in SDS PAGE. The detection of a large subunit (aoxB) of arsenate oxidase protein in western blotting assay affirmed the expression of aoxB gene in recombinant E.coli/BL21 clone. Further, the recombinant E.coli/BL21cells showed increased growth than the normal E.coli/BL21 cells against As (III). Thus, this study showed the presence of aoxB gene in Pseudomonas sp. AK9 genome which regulates the resistant ability to arsenic toxicity.

Potential of methyltransferase containing Pseudomonas oleovorans for abatement of arsenic toxicity in rice

Arsenic (As) has become natural health hazard for millions of people across the world due to its distribution in the food chain. Naturally, it is present in different oxidative states of inorganic [As(V) and As(III)] and organic (DMA, MMA and TMA) forms. Among different mitigation approaches, microbe mediated mitigation of As toxicity is an effective and eco-friendly approach. The present study involves the characterization of bacterial strains containing arsenite methyltransferase (Pseudomonas oleovorans, B4.10); arsenate reductase (Sphingobacterium puteale, B4.22) and arsenite oxidase (Citrobacter sp., B5.12) activity with plant growth promoting (PGP) traits. Efficient reduction of grain As content by 61 % was observed due to inoculation of methyltransferase containing B4.10 as compared to B4.22 (47 %) and B5.12 (49 %). Reduced bioaccumulation of As in root (0.339) and shoot (0.166) in presence of B4.10 was found to be inversely related with translocation factor for Mn (3.28), Fe (0.073), and Se (1.82). Bioaccumulation of these micro elements was found to be associated with the modulated expression of different mineral transporters (OsIRT2, OsFRO2, OsTOM1, OsSultr4;1, and OsZIP2) in rice shoot. Improved dehydrogenase (407 %), and β-glucosidase (97 %) activity in presence of P. oleovorans (B4.10) as compared to arsenate reductase (198 and 50 %), and arsenite oxidase (134 and 69 %) containing bacteria was also observed. Our finding confers the potential of methyltransferase positive P. oleovorans (B4.10) for As stress amelioration. Reduced grain As uptake was found to be mediated by improved plant growth and nutrient uptake associated with enhanced soil microbial activity.

Different Regulatory Strategies of Arsenite Oxidation by Two Isolated Thermus tengchongensis Strains From Hot Springs.

Arsenic is a ubiquitous constituent in geothermal fluids. Thermophiles represented by Thermus play vital roles in its transformation in geothermal fluids. In this study, two Thermus tengchongensis strains, named as 15Y and 15W, were isolated from arsenic-rich geothermal springs and found different arsenite oxidation behaviors with different oxidation strategies. Arsenite oxidation of both strains occurred at different growth stages, and two enzyme-catalyzed reaction kinetic models were observed. The arsenite oxidase of Thermus strain 15W performed better oxidation activity, exhibiting typical Michaelis–Menten kinetics. The kinetic parameter of arsenite oxidation in whole cell showed a Vmax of 18.48 μM min–1 and KM of 343 μM. Both of them possessed the arsenite oxidase-coding genes aioB and aioA. However, the expression of gene aioBA was constitutive in strain 15W, whereas it was induced by arsenite in strain 15Y. Furthermore, strain 15Y harbored an intact aio operon including the regulatory gene of the ArsR family, whereas a genetic inversion of an around 128-kbp fragment produced the inactivation of this regulator in strain 15W, leading to the constitutive expression of aioBA genes. This study provides a valuable insight into the adaption of thermophiles to extreme environments.

Empirical Evidence of Arsenite Oxidase Gene as an Indicator Accounting for Arsenic Phytoextraction by Pteris vittata.

Arsenic (As) is a toxic semi-metallic element that is ubiquitous in the environment and poses serious human health risks. Phytoextraction by Pteris vittata is considered a low-cost and environmentally friendly approach to treat As-contaminated soil. P. vittata mainly absorbs arsenate thus the bioavailability of As to P. vittata depends on the chemical form of As. Microbial redox of As contributes to the biogeochemical cycling of As, and rhizobacterium-assisted phytoextraction by P. vittata was proposed. In this study, this microbe-assisted phytoextraction was applied to two fields, and the effectiveness of phytoextraction was evaluated. The results revealed that P. vittata was able to grow in temperate and subarctic climate zones. The biomass was influenced by the weather, and the As concentration in plants was dependent on the As content in the soil. The ratio of arsenite oxidase genes (aioA-like genes) to 16S rRNA genes was employed to evaluate the effect of As phytoextraction, and the results exhibited that the ratio was related to the As concentration in P. vittata. Our results showed that arsenite oxidation in the rhizosphere might not be achieved by single-strain inoculation, while this study provided empirical evidence that the rhizospheric aioA-like genes could be an indicator for evaluating the effectiveness of As phytoextraction.