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1.  “Artifactual” arsenate DNA 
Artificial DNA, PNA & XNA  2012;3(1):1-2.
The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate-containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA1 arose much skepticism, primarily due to the very limited chemical stability of arsenate esters (see ref. 2 and references therein). A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present evidence that the identification of arsenate DNA was artifactual.
PMCID: PMC3368811  PMID: 22679526
arsenate; bacteria; DNA; genetic material; life
2.  Arsenate replacing phosphate - alternative life chemistries and ion promiscuity 
Biochemistry  2011;50(7):1128-1134.
A newly identified bacterial strain that can grow in the presence of arsenate, and possibly in the absence of phosphate, has raised much interest, but also fueled an active debate. Can arsenate substitute for phosphate in some, or possibly in most, of the absolutely essential phosphate-based biomolecules, including DNA? If so, then the possibility of alternative, arsenic-based life forms must be considered. The physicochemical similarity of these two oxyanions speaks in favor of this idea. However, arsenate-esters, and arsenate-diesters in particular, are extremely unstable in aqueous media. Here we explore the potential of arsenate to be used as substrate by phosphate-utilizing enzymes. We review the existing literature on arsenate enzymology, that intriguingly, dates back to the 1930s, and Otto Warburg. We address the issue of how and to what degree proteins can distinguish between arsenate and phosphate, and what is known in general about oxyanion specificity. And, we also discuss how phosphate-arsenate promiscuity may affect evolutionary transitions between phosphate and arsenate based biochemistry. Finally, we highlight potential applications of arsenate as a structural and mechanistic probe of enzymes whose catalyzed reactions involve the making or breaking of phosphoester bonds.
PMCID: PMC3070116  PMID: 21214261
3.  Life and death with arsenic 
Arsenic and phosphorus are group 15 elements with similar chemical properties. Is it possible that arsenate could replace phosphate in some of the chemicals that are required for life? Phosphate esters are ubiquitous in biomolecules and are essential for life, from the sugar phosphates of intermediary metabolism to ATP to phospholipids to the phosphate backbone of DNA and RNA. Some enzymes that form phosphate esters catalyze the formation of arsenate esters. Arsenate esters hydrolyze very rapidly in aqueous solution, which makes it improbable that phosphorous could be completely replaced with arsenic to support life. Studies of bacterial growth at high arsenic:phosphorus ratios demonstrate that relatively high arsenic concentrations can be tolerated, and that arsenic can become involved in vital functions in the cell, though likely much less efficiently than phosphorus. Recently Wolfe-Simon et al. [1] reported the isolation of a microorganism that they maintain uses arsenic in place of phosphorus for growth. Here, we examine and evaluate their data and conclusions.
PMCID: PMC3801090  PMID: 21387349
arsenate; arsenic life; ester hydrolysis; phosphate
4.  Arsenate Resistance in the Unicellular Marine Diazotroph Crocosphaera watsonii 
The toxic arsenate ion can behave as a phosphate analog, and this can result in arsenate toxicity especially in areas with elevated arsenate to phosphate ratios like the surface waters of the ocean gyres. In these systems, cellular arsenate resistance strategies would allow phytoplankton to ameliorate the effects of arsenate transport into the cell. Despite the potential coupling between arsenate and phosphate cycling in oligotrophic marine waters, relatively little is known about arsenate resistance in the nitrogen-fixing marine cyanobacteria that are key components of the microbial community in low nutrient systems. The unicellular diazotroph, Crocosphaera watsonii WH8501, was able to grow at reduced rates with arsenate additions up to 30 nM, and estimated arsenate to phosphate ratios of 6:1. The genome of strain WH8501 contains homologs for arsA, arsH, arsB, and arsC, allowing for the reduction of arsenate to arsenite and the pumping of arsenite out of the cell. The short-term addition of arsenate to the growth medium had no effect on nitrogen fixation. However, arsenate addition did result in the up-regulation of the arsB gene with increasing arsenate concentrations, indicating the induction of the arsenate detoxification response. The arsB gene was also up-regulated by phosphorus stress in concert with a gene encoding the high-affinity phosphate binding protein pstS. Both genes were down-regulated when phosphate was re-fed to phosphorus-stressed cells. A field survey of surface water from the low phosphate western North Atlantic detected expression of C. watsonii arsB, suggestive of the potential importance of arsenate resistance strategies in this and perhaps other systems.
PMCID: PMC3201022  PMID: 22046174
cyanobacteria; phosphorus; marine; arsenate; diazotroph; Crocosphaera
5.  Structural and Functional Consequences of Phosphate–Arsenate Substitutions in Selected Nucleotides: DNA, RNA, and ATP 
The Journal of Physical Chemistry. B  2012;116(16):4801-4811.
A recent finding of a bacterial strain (GFAJ-1) that can rely on arsenic instead of phosphorus raised the questions of if and how arsenate can replace phosphate in biomolecules that are essential to sustain cell life. Apart from questions related to chemical stability, there are those of the structural and functional consequences of phosphate-arsenate substitutions in vital nucleotides in GFAJ1-like cells. In this study we selected three types of molecules (ATP/ADP as energy source and replication regulation; DNA–protein complexes for DNA replication and transcription initiation; and a tRNA–protein complex and ribosome for protein synthesis) to computationally probe if arsenate nucleotides can retain the structural and functional features of phosphate nucleotides. Hydrolysis of adenosine triarsenate provides 2–3 kcal/mol less energy than ATP hydrolysis. Arsenate DNA/RNA interacts with proteins slightly less strongly than phosphate DNA/RNA, mainly due to the weaker electrostatic interactions of arsenate. We observed that the weaker arsenate RNA–protein interactions may hamper rRNA assembly into a functional ribosome. We further compared the experimental EXAFS spectra of the arsenic bacteria with theoretical EXAFS spectra for arsenate DNA and rRNA. Our results demonstrate that while it is possible that dried GFAJ-1 cells contain linear arsenate DNA, the arsenate 70S ribosome does not contribute to the main arsenate depository in the GFAJ-1 cell. Our study indicates that evolution has optimized the inter-relationship between proteins and DNA/RNA, which requires overall changes at the molecular and systems biology levels when replacing phosphate by arsenate.
PMCID: PMC3337691  PMID: 22480264
6.  Sequencing and expression of two arsenic resistance operons with different functions in the highly arsenic-resistant strain Ochrobactrum tritici SCII24T 
BMC Microbiology  2008;8:95.
Arsenic (As) is a natural metalloid, widely used in anthropogenic activities, that can exist in different oxidation states. Throughout the world, there are several environments contaminated with high amounts of arsenic where many organisms can survive. The most stable arsenical species are arsenate and arsenite that can be subject to chemically and microbiologically oxidation, reduction and methylation reactions. Organisms surviving in arsenic contaminated environments can have a diversity of mechanisms to resist to the harmful effects of arsenical compounds.
The highly metal resistant Ochrobactrum tritici SCII24 was able to grow in media with arsenite (50 mM), arsenate (up to 200 mM) and antimonite (10 mM). This strain contains two arsenic and antimony resistance operons (ars1 and ars2), which were cloned and sequenced. Sequence analysis indicated that ars1 operon contains five genes encoding the following proteins: ArsR, ArsD, ArsA, CBS-domain-containing protein and ArsB. The ars2 operon is composed of six genes that encode two other ArsR, two ArsC (belonging to different families of arsenate reductases), one ACR3 and one ArsH-like protein. The involvement of ars operons in arsenic resistance was confirmed by cloning both of them in an Escherichia coli ars-mutant. The ars1 operon conferred resistance to arsenite and antimonite on E. coli cells, whereas the ars2 operon was also responsible for resistance to arsenite and arsenate. Although arsH was not required for arsenate resistance, this gene seems to be important to confer high levels of arsenite resistance. None of ars1 genes were detected in the other type strains of genus Ochrobactrum, but sequences homologous with ars2 operon were identified in some strains.
A new strategy for bacterial arsenic resistance is described in this work. Two operons involved in arsenic resistance, one giving resistance to arsenite and antimonite and the other giving resistance to arsenate were found in the same bacterial strain.
PMCID: PMC2440759  PMID: 18554386
7.  Analysis of Genes Involved in Arsenic Resistance in Corynebacterium glutamicum ATCC 13032†  
Applied and Environmental Microbiology  2005;71(10):6206-6215.
Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1′) located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1′, arsR2, arsB2, and arsC2 being inducible by arsenite.
PMCID: PMC1266000  PMID: 16204540
8.  Effect of arsenate on inorganic phosphate transport in Escherichia coli. 
Journal of Bacteriology  1980;144(1):366-374.
The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium. The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined. The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio. Growth in arsenate-containing medium was not due to detoxification of the arsenate. Kinetic studies revealed that cells grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vmax) for Pi, but maintained the same affinity (Km). Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 microM arsenate was determined. The Pst-dependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium. The Pi transport-dependent strain ceased growth in arsenate-containing media. This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium. Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide. Previously accumulated arsenate could exchange with arsenate or Pi in the medium.
PMCID: PMC294657  PMID: 6998959
9.  Arsenic speciation in saliva of acute promyelocytic leukemia patients undergoing arsenic trioxide treatment 
Analytical and Bioanalytical Chemistry  2013;405(6):1903-1911.
Arsenic trioxide has been successfully used as a therapeutic in the treatment of acute promyelocytic leukemia (APL). Detailed monitoring of the therapeutic arsenic and its metabolites in various accessible specimens of APL patients can contribute to improving treatment efficacy and minimizing arsenic-induced side effects. This article focuses on the determination of arsenic species in saliva samples from APL patients undergoing arsenic treatment. Saliva samples were collected from nine APL patients over three consecutive days. The patients received 10 mg arsenic trioxide each day via intravenous infusion. The saliva samples were analyzed using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry. Monomethylarsonous acid and monomethylmonothioarsonic acid were identified along with arsenite, dimethylarsinic acid, monomethylarsonic acid, and arsenate. Arsenite was the predominant arsenic species, accounting for 71.8 % of total arsenic in the saliva. Following the arsenic infusion each day, the percentage of methylated arsenicals significantly decreased, possibly suggesting that the arsenic methylation process was saturated by the high doses immediately after the arsenic infusion. The temporal profiles of arsenic species in saliva following each arsenic infusion over 3 days have provided information on arsenic exposure, metabolism, and excretion. These results suggest that saliva can be used as an appropriate clinical biomarker for monitoring arsenic species in APL patients.
FigureArsenic species and temporal profiles over three days from nine patients
PMCID: PMC3565090  PMID: 23318765
Acute promyelocytic leukemia; Arsenic speciation; Saliva; Metabolism; Arsenic trioxide treatment
10.  Occurrence and distribution of arsenic in soils and plants 
Inorganic arsenicals have been used in agriculture as pesticides or defoliants for many years and, in localized areas, oxides of arsenic have contaminated soils as a result of fallout from ore-smelting operations and coal-fired power plants. Use of inorganic arsenicals is no longer permitted in most agricultural operations, and recent air pollution controls have markedly reduced contamination from smelters. Thus, this paper will concentrate on the effect of past applications on arsenic accumulation in soil, phytotoxicity to and uptake by plants as influenced by soil properties, and alleviation of the deleterious effects of arsenic.
Once incorporated into the soil, inorganic arsenical pesticides and arsenic oxides revert to arsenates, except where the soil is under reducing conditions. The arsenate ion has properties similar to that of orthophosphate, and is readily sorbed by iron and aluminum components. This reaction greatly restricts the downward movement (leaching) of arsenic in soils and the availability of arsenic to plants.
Several methods of estimating plant available arsenic in soils have been developed. They involve extraction of the soil with reagents used to estimate phosphorus availability. This extractable arsenic is reasonably well correlated with reduced plant growth by, and plant uptake of arsenic. For most plants, levels of arsenic in the edible portion of the plant are well below the critical concentration for animal or human consumption, even when severe phytotoxicity occurs.
Alleviation of arsenic phytotoxicity has been attempted by increasing the soil pH, by use of iron or aluminum sulfate, by desorbing arsenate with phosphate and subsequent leaching, and by cultural practices such as deep plowing. Only limited benefits have accrued from these procedures the cost of which is often prohibitively high. Since attempts to reduce arsenic toxicity have not been very successful, its excessive accumulation in soils should be avoided.
PMCID: PMC1637429  PMID: 908315
11.  Genome-wide Association Mapping Identifies a New Arsenate Reductase Enzyme Critical for Limiting Arsenic Accumulation in Plants 
PLoS Biology  2014;12(12):e1002009.
A genome-wide association study identifies the enzyme in plants that transforms arsenate into arsenite, allowing its extrusion into the soil and thereby controlling arsenic accumulation.
Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional arsenate reductase confirmed the arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We also confirmed the previous observation that the ACR2 arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice.
Author Summary
Arsenic is a human carcinogen that accumulates from soil into many different food crops, where it presents a significantly increased cancer risk when foods derived from these crops are consumed. Plants naturally control the amount of arsenic they accumulate by first chemically converting arsenate into arsenite, which is then extruded from the roots back into the soil. Because arsenate is a chemical analogue of phosphate, conversion of arsenate in the root to arsenite may also prevent arsenic being efficiently transported to the shoots via the phosphate transport system. The chemical reduction of arsenate to generate arsenite is therefore clearly a key component of a plant's detoxification strategy. Here, we use genetic methods to identify the enzyme responsible for this crucial reaction—HAC1. We show that HAC1 is responsible for arsenate reductase activity in both the outer layer of the root (epidermis) and the inner layer adjacent to the xylem (pericycle). In its absence, the roots return less arsenic to the soil and the shoots accumulate up to 300 times more arsenic. This knowledge creates new opportunities to limit arsenic accumulation in food crops, thereby helping to reduce the cancer risk from this food-chain contaminant.
PMCID: PMC4251824  PMID: 25464340
12.  Testing for bacterial resistance to arsenic in monitoring well water by the direct viable counting method. 
Applied and Environmental Microbiology  1987;53(12):2929-2934.
Direct viable counting of metal-resistant bacteria (DVCMR) has been found to be useful in both enumerating and differentiating metal-resistant and metal-sensitive strains of bacteria. The DVCMR bioassay was used to detect effects of low and high concentrations of arsenic and arsenicals on bacterial populations in groundwater. The level of resistance of the bacterial populations to arsenate was determined by the DVCMR bioassay, and the results showed a linear correlation with the total arsenic concentrations in the monitoring well water samples; no correlation was observed by culture methods with the methods employed. Bacteria resistant to 2,000 micrograms of arsenate per ml were isolated from all monitoring well water samples studied. Strains showed similar antibiotic and heavy-metal profiles, suggesting that the arsenic was not a highly selective pressure for arsenic alone. The monitoring well water samples were amended with arsenate and nutrients to determine the biotransformation mechanisms involved. Preliminary results suggest that bacteria indigenous to the monitoring well water samples did not directly transform, i.e., precipitate or volatilize, dissolved arsenic. It was concluded that arsenic contamination of the groundwater can be monitored by the DVCMR bioassay.
PMCID: PMC204224  PMID: 3324971
13.  Isolation and characterization of arsenic resistant bacteria from wastewater 
Brazilian Journal of Microbiology  2015;45(4):1309-1315.
The present study proposed the isolation of arsenic resistant bacteria from wastewater. Only three bacterial isolates (MNZ1, MNZ4 and MNZ6) were able to grow in high concentrations of arsenic. The minimum inhibitory concentrations of arsenic against MNZ1, MNZ4 and MNZ6 were 300 mg/L, 300 mg/L and 370 mg/L respectively. The isolated strains showed maximum growth at 37 °C and at 7.0 pH in control but in arsenite stress Luria Bertani broth the bacterial growth is lower than control. All strains were arsenite oxidizing. All strains were biochemically characterized and ribotyping (16S rRNA) was done for the purpose of identification which confirmed that MNZ1 was homologous to Enterobacter sp. while MNZ4 and MNZ6 showed their maximum homology with Klebsiella pneumoniae. The protein profiling of these strains showed in arsenic stressed and non stressed conditions, so no bands of induced proteins appeared in stressed conditions. The bacterial isolates can be exploited for bioremediation of arsenic containing wastes, since they seem to have the potential to oxidize the arsenite (more toxic) into arsenate (less toxic) form.
PMCID: PMC4323304  PMID: 25763035
Arsenite oxidizing bacteria; Bioremediation. Enterobacter sp.; Klebsiella pneumoniae
14.  Interaction of Arsenate with Phosphate-Transport Systems in Wild-Type and Mutant Streptococcus faecalis 
Journal of Bacteriology  1966;91(6):2257-2262.
Harold, F. M. (National Jewish Hospital, Denver, Colo.), and J. R. Baarda. Interaction of arsenate with phosphate-transport systems in wild-type and mutant Streptococcus faecalis. J. Bacteriol. 91:2257–2262. 1966.—Arsenate competitively inhibits the growth of Streptococcus faecalis, primarily by competition with phosphate for a common transport system. Arsenate is itself accumulated by the cells; the uptake requires metabolic energy, and the intracellular arsenate level may reach 0.01 m. Cells loaded with arsenate have lost the capacity to take up radioactive glutamate, rubidium, phosphate, or arsenate itself, apparently by the uncoupling of adenosine triphosphate generation. The pH dependence of arsenate uptake is complex. At low concentrations of extracellular arsenate, uptake by the wild-type strain 9790 exhibits a single maximum about pH 8; mutant PT-1, previously shown to be defective in phosphate uptake, takes up essentially no arsenate. At high concentrations of arsenate, uptake by the wild type is bimodal with maxima at pH 5.5 and 9; the uptake curve for mutant PT-1 corresponds to the shoulder in the curve for the wild type. The apparent dissociation constant for arsenate uptake by the wild type is approximately 10−5m from pH 5 to 9, whereas that for mutant PT-1 is about 5 × 10−5 M at pH 5 and rises rapidly with increasing pH. The results confirm the earlier conclusion that the lesion in mutant PT-1 resides in the transport of phosphate and arsenate. It is proposed that the wild type has two distinct transport systems, whereas the mutant has lost the one with alkaline pH optimum.
PMCID: PMC316203  PMID: 4957614
15.  Shewanella sp. O23S as a Driving Agent of a System Utilizing Dissimilatory Arsenate-Reducing Bacteria Responsible for Self-Cleaning of Water Contaminated with Arsenic 
The purpose of this study was a detailed characterization of Shewanella sp. O23S, a strain involved in arsenic transformation in ancient gold mine waters contaminated with arsenic and other heavy metals. Physiological analysis of Shewanella sp. O23S showed that it is a facultative anaerobe, capable of growth using arsenate, thiosulfate, nitrate, iron or manganite as a terminal electron acceptor, and lactate or citrate as an electron donor. The strain can grow under anaerobic conditions and utilize arsenate in the respiratory process in a broad range of temperatures (10–37 °C), pH (4–8), salinity (0%–2%), and the presence of heavy metals (Cd, Co, Cr, Cu, Mn, Mo, Se, V and Zn). Under reductive conditions this strain can simultaneously use arsenate and thiosulfate as electron acceptors and produce yellow arsenic (III) sulfide (As2S3) precipitate. Simulation of As-removal from water containing arsenate (2.5 mM) and thiosulfate (5 mM) showed 82.5% efficiency after 21 days of incubation at room temperature. Based on the obtained results, we have proposed a model of a microbially mediated system for self-cleaning of mine waters contaminated with arsenic, in which Shewanella sp. O23S is the main driving agent.
PMCID: PMC4519849  PMID: 26121297
Shewanella spp.; dissimilatory arsenate reduction; arsenic removal
16.  Temporal transcriptomic response during arsenic stress in Herminiimonas arsenicoxydans 
BMC Genomics  2010;11:709.
Arsenic is present in numerous ecosystems and microorganisms have developed various mechanisms to live in such hostile environments. Herminiimonas arsenicoxydans, a bacterium isolated from arsenic contaminated sludge, has acquired remarkable capabilities to cope with arsenic. In particular our previous studies have suggested the existence of a temporal induction of arsenite oxidase, a key enzyme in arsenic metabolism, in the presence of As(III).
Microarrays were designed to compare gene transcription profiles under a temporal As(III) exposure. Transcriptome kinetic analysis demonstrated the existence of two phases in arsenic response. The expression of approximatively 14% of the whole genome was significantly affected by an As(III) early stress and 4% by an As(III) late exposure. The early response was characterized by arsenic resistance, oxidative stress, chaperone synthesis and sulfur metabolism. The late response was characterized by arsenic metabolism and associated mechanisms such as phosphate transport and motility. The major metabolic changes were confirmed by chemical, transcriptional, physiological and biochemical experiments. These early and late responses were defined as general stress response and specific response to As(III), respectively.
Gene expression patterns suggest that the exposure to As(III) induces an acute response to rapidly minimize the immediate effects of As(III). Upon a longer arsenic exposure, a broad metabolic response was induced. These data allowed to propose for the first time a kinetic model of the As(III) response in bacteria.
PMCID: PMC3022917  PMID: 21167028
17.  Construction of a Modular Arsenic-Resistance Operon in E. coli and the Production of Arsenic Nanoparticles 
Arsenic is a widespread contaminant of both land and water around the world. Current methods of decontamination such as phytoremediation and chemical adsorbents can be resource and time intensive, and may not be suitable for some areas such as remote communities where cost and transportation are major issues. Bacterial decontamination, with strict controls preventing environmental release, may offer a cost-effective alternative or provide a financial incentive when used in combination with other remediation techniques. In this study, we have produced Escherichia coli strains containing arsenic-resistance genes from a number of sources, overexpressing them and testing their effects on arsenic resistance. While the lab E. coli strain JM109 (the “wild-type”) is resistant up to 20 mM sodium arsenate, the strain containing our plasmid pEC20 is resistant up to 80 mM. When combined with our construct pArsRBCC arsenic-­containing nanoparticles were observed at the cell surface; the elements of pEC20 and pArsRBCC were therefore combined in a modular construct, pArs, in order to evaluate the roles and synergistic effects of the components of the original plasmids in arsenic resistance and nanoparticle formation. We have also investigated introducing the lac operator in order to more tightly control expression from pArs. We demonstrate that our strains are able to reduce toxic forms of arsenic into stable, insoluble metallic As(0), providing one way to remove arsenate contamination, and which may also be of benefit for other heavy metals.
PMCID: PMC4611968  PMID: 26539432
biogenic nanoparticles; arsenic nanoparticles; arsenic decontamination; arsenic reduction; modular arsenic-resistance construct
18.  In silico analysis of bacterial arsenic islands reveals remarkable synteny and functional relatedness between arsenate and phosphate 
In order to construct a more universal model for understanding the genetic requirements for bacterial AsIII oxidation, an in silico examination of the available sequences in the GenBank was assessed and revealed 21 conserved 5–71 kb arsenic islands within phylogenetically diverse bacterial genomes. The arsenic islands included the AsIII oxidase structural genes aioBA, ars operons (e.g., arsRCB) which code for arsenic resistance, and pho, pst, and phn genes known to be part of the classical phosphate stress response and that encode functions associated with regulating and acquiring organic and inorganic phosphorus. The regulatory genes aioXSR were also an island component, but only in Proteobacteria and orientated differently depending on whether they were in α-Proteobacteria or β-/γ-Proteobacteria. Curiously though, while these regulatory genes have been shown to be essential to AsIII oxidation in the Proteobacteria, they are absent in most other organisms examined, inferring different regulatory mechanism(s) yet to be discovered. Phylogenetic analysis of the aio, ars, pst, and phn genes revealed evidence of both vertical inheritance and horizontal gene transfer (HGT). It is therefore likely the arsenic islands did not evolve as a whole unit but formed independently by acquisition of functionally related genes and operons in respective strains. Considering gene synteny and structural analogies between arsenate and phosphate, we presumed that these genes function together in helping these microbes to be able to use even low concentrations of phosphorus needed for vital functions under high concentrations of arsenic, and defined these sequences as the arsenic islands.
PMCID: PMC3834237  PMID: 24312089
arsenic islands; arsenite oxidase; AioBA; phosphorus; synteny
19.  Phosphate fertilizer is a main source of arsenic in areas affected with chronic kidney disease of unknown etiology in Sri Lanka 
SpringerPlus  2015;4:90.
Chronic Kidney Disease of unknown etiology (CKDu) has escalated into an epidemic in North Central Province (NCP) and adjacent farming areas in the dry zone of Sri Lanka. Studies have shown that this special type of CKD is a toxic nephropathy and arsenic may play a causative role along with a number of other heavy metals. We investigated the hypothesis that chemical fertilizers and pesticide could be a source of arsenic. 226 samples of Fertilizers and 273 samples of pesticides were collected and analyzed using atomic absorption spectrometry and inductively coupled plasma mass spectrometry for arsenic and other heavy metals in two university laboratories. Almost all the agrochemicals available to the farmers in the study area are contaminated with arsenic. The highest amount was in triple super phosphate (TSP) with a mean value of 31 mg/kg. Also TSP is a rich source of other nephrotoxic metals including Cr, Co, Ni, Pb and V. Annually more than 0.1 million tons of TSP is imported to Sri Lanka containing approximately 2100 kg of arsenic. The next highest concentration was seen in the rock phosphate obtained from an open pit mine in NCP (8.56 mg/kg). Organic fertilizer contained very low amounts of arsenic. Arsenic contamination in pesticides varied from 0.18 mg/kg to 2.53 mg/kg although arsenic containing pesticides are banned in Sri Lanka. Glyphosate the most widely used pesticide in Sri Lanka contains average of 1.9 mg/kg arsenic. Findings suggest that agrochemicals especially phosphate fertilizers are a major source of inorganic arsenic in CKDu endemic areas. Organic fertilizer available in Sri Lanka is comparatively very low in arsenic and hence the farmers in CKDu endemic areas in Sri Lanka should be encouraged to minimize the use of imported chemical fertilizer and use organic fertilizers instead.
PMCID: PMC4348354  PMID: 25763302
Arsenic; Pesticides; Fertilizer; Chronic kidney disease of unknown etiology; Sri Lanka
20.  Phosphate transport in arsenate-resistant mutants of Micrococcus lysodeikticus. 
Journal of Bacteriology  1979;137(1):69-72.
Two types of arsenate-resistant mutants of Micrococcus lysodeikticus were found: (i) mutants that grow in the presence of 10 mM but not 1 mM phosphate (Pi) with low uptake rate for Pi and arsenate, and (ii) mutants able to grow in the presence of 10 mM and 1 mM Pi, with a near-normal uptake rate for Pi but a low one for arsenate. The Km values for Pi transport and the Ki values for its competitive inhibition by arsenate were similar for the mutants and the wild type. Similar to the wild type, the mutants also accumulated Pi to high concentrations. In all strains, the transport of Pi was subject to repression by Pi. Mutant types showed lower Vmax but unaltered Km values for arsenate as compared to the wild type, and they accumulated arsenate to markedly lower levels. The results suggest a two-component transport system common to Pi and arsenate.
PMCID: PMC218419  PMID: 762027
21.  Arsenic Contamination in Food-chain: Transfer of Arsenic into Food Materials through Groundwater Irrigation 
Arsenic contamination in groundwater in Bangladesh has become an additional concern vis-à-vis its use for irrigation purposes. Even if arsenic-safe drinking-water is assured, the question of irrigating soils with arsenic-laden groundwater will continue for years to come. Immediate attention should be given to assess the possibility of accumulating arsenic in soils through irrigation-water and its subsequent entry into the food-chain through various food crops and fodders. With this possibility in mind, arsenic content of 2,500 water, soil and vegetable samples from arsenic-affected and arsenic-unaffected areas were analyzed during 1999–2004. Other sources of foods and fodders were also analyzed. Irrigating a rice field with groundwater containing 0.55 mg/L of arsenic with a water requirement of 1,000 mm results in an estimated addition of 5.5 kg of arsenic per ha per annum. Concentration of arsenic as high as 80 mg per kg of soil was found in an area receiving arsenic-contaminated irrigation. A comparison of results from affected and unaffected areas revealed that some commonly-grown vegetables, which would usually be suitable as good sources of nourishment, accumulate substantially-elevated amounts of arsenic. For example, more than 150 mg/kg of arsenic has been found to be accumulated in arum (kochu) vegetable. Implications of arsenic ingested in vegetables and other food materials are discussed in the paper.
PMCID: PMC3013251  PMID: 17366772
Arsenic; Arsenic contamination; Food; Plants; Colocassia antiquorum; Bioavailability; Bangladesh
22.  Genomic Evidence Reveals the Extreme Diversity and Wide Distribution of the Arsenic-Related Genes in Burkholderiales 
PLoS ONE  2014;9(3):e92236.
So far, numerous genes have been found to associate with various strategies to resist and transform the toxic metalloid arsenic (here, we denote these genes as “arsenic-related genes”). However, our knowledge of the distribution, redundancies and organization of these genes in bacteria is still limited. In this study, we analyzed the 188 Burkholderiales genomes and found that 95% genomes harbored arsenic-related genes, with an average of 6.6 genes per genome. The results indicated: a) compared to a low frequency of distribution for aio (arsenite oxidase) (12 strains), arr (arsenate respiratory reductase) (1 strain) and arsM (arsenite methytransferase)-like genes (4 strains), the ars (arsenic resistance system)-like genes were identified in 174 strains including 1,051 genes; b) 2/3 ars-like genes were clustered as ars operon and displayed a high diversity of gene organizations (68 forms) which may suggest the rapid movement and evolution for ars-like genes in bacterial genomes; c) the arsenite efflux system was dominant with ACR3 form rather than ArsB in Burkholderiales; d) only a few numbers of arsM and arrAB are found indicating neither As III biomethylation nor AsV respiration is the primary mechanism in Burkholderiales members; (e) the aio-like gene is mostly flanked with ars-like genes and phosphate transport system, implying the close functional relatedness between arsenic and phosphorus metabolisms. On average, the number of arsenic-related genes per genome of strains isolated from arsenic-rich environments is more than four times higher than the strains from other environments. Compared with human, plant and animal pathogens, the environmental strains possess a larger average number of arsenic-related genes, which indicates that habitat is likely a key driver for bacterial arsenic resistance.
PMCID: PMC3954881  PMID: 24632831
23.  Arsenic-Lipid Complex Formation During the Active Transport of Arsenate in Yeast 
Journal of Bacteriology  1969;97(2):658-662.
In studying formation of an arsenic-lipid complex during the active transport of 74As-arsenate in yeast, it was found that adaptation of yeast to arsenate resulted in cell populations which showed a deficient inflow of arsenate as compared to the nonadapted yeast. Experiments with both types of cells showed a direct correlation between the arsenate taken up and the amount of As-lipid complex formed. 74As-arsenate was bound exclusively to the phosphoinositide fraction of the cellular lipids. When arsenate transport was inhibited by dinitrophenol and sodium azide, the formation of the As-lipid complex was also inhibited. Phosphate did not interfere with the arsenate transport at a non-inhibitory concentration of external arsenate (10−9m). The As-adapted cells but not the unadapted cells were able to take up phosphate when growing in the presence of 10−2m arsenate.
PMCID: PMC249742  PMID: 5773018
24.  The ArsR Repressor Mediates Arsenite-Dependent Regulation of Arsenate Respiration and Detoxification Operons of Shewanella sp. Strain ANA-3▿ † 
Journal of Bacteriology  2009;191(21):6722-6731.
Microbial arsenate reduction affects the fate and transport of arsenic in the environment. Arsenate respiratory (arr) and detoxifying (ars) reduction pathways in Shewanella sp. strain ANA-3 are induced by arsenite and under anaerobic conditions. Here it is shown that an ArsR family protein, called ArsR2, regulates the arsenate respiratory reduction pathway in response to elevated arsenite under anaerobic conditions. Strains lacking arsR2 grew faster in the presence of high levels of arsenite (3 mM). Moreover, expression of arrA and arsC (arsenate reductase-encoding genes) in the ΔarsR2 mutant of ANA-3 were increased in cells grown under anaerobic conditions and in the absence of arsenic. Mutations in putative arsenic binding amino acid residues in ArsR2 (substitutions of Cys-30 and Cys-32 with Ser) resulted in ANA-3 strains that exhibited anaerobic growth deficiencies with high levels of arsenite and arsenate. DNA binding studies with purified ArsR2 showed that ArsR2 binding to the arr promoter region was impaired by trivalent arsenicals such as arsenite and phenylarsine oxide. However, ArsR2 binding occurred in the presence of arsenate. A second known regulator of the arr operon, cyclic AMP (cAMP)-cAMP receptor protein (CRP), could bind simultaneously with ArsR2 within the arr promoter region. It is concluded that ArsR2 is most likely the major arsenite-dependent regulator of arr and ars operons in Shewanella sp. strain ANA-3. However, anaerobic growth on arsenate will require coregulation with global regulators such as cAMP-CRP.
PMCID: PMC2795299  PMID: 19717602
25.  Arsenic Resistance and Prevalence of Arsenic Resistance Genes in Campylobacter jejuni and Campylobacter coli Isolated from Retail Meats  
Studies that investigate arsenic resistance in the foodborne bacterium Campylobacter are limited. A total of 552 Campylobacter isolates (281 Campylobacter jejuni and 271 Campylobacter coli) isolated from retail meat samples were subjected to arsenic resistance profiling using the following arsenic compounds: arsanilic acid (4–2,048 μg/mL), roxarsone (4–2048 μg/mL), arsenate (16–8,192 μg/mL) and arsenite (4–2,048 μg/mL). A total of 223 of these isolates (114 Campylobacter jejuni and 109 Campylobacter coli) were further analyzed for the presence of five arsenic resistance genes (arsP, arsR, arsC, acr3, and arsB) by PCR. Most of the 552 Campylobacter isolates were able to survive at higher concentrations of arsanilic acid (512–2,048 μg/mL), roxarsone (512–2,048 μg/mL), and arsenate (128–1,024 μg/mL), but at lower concentrations for arsenite (4–16 μg/mL). Ninety seven percent of the isolates tested by PCR showed the presence of arsP and arsR genes. While 95% of the Campylobacter coli isolates contained a larger arsenic resistance operon that has all of the four genes (arsP, arsR, arsC and acr3), 85% of the Campylobacter jejuni isolates carried the short operon (arsP, and arsR). The presence of arsC and acr3 did not significantly increase arsenic resistance with the exception of conferring resistance to higher concentrations of arsenate to some Campylobacter isolates. arsB was prevalent in 98% of the tested Campylobacter jejuni isolates, regardless of the presence or absence of arsC and acr3, but was completely absent in Campylobacter coli. To our knowledge, this is the first study to determine arsenic resistance and the prevalence of arsenic resistance genes in such a large number of Campylobacter isolates.
PMCID: PMC3774448  PMID: 23965921
Campylobacter; arsenic resistance; arsP; arsR; arsC; acr3; arsB; arsenic operon; retail meats

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