ArsV and ArsW provide synergistic resistance to the antibiotic methylarsenite

Toxic organoarsenicals enter the environment from biogenic and anthropogenic activities such as microbial methylation of inorganic arsenic and pentavalent herbicides such as monosodium methylarsenate (MSMA or MAs(V)). Trivalent MAs(III) is considerably more toxic than arsenite or arsenate. Microbes have evolved mechanisms to detoxify organoarsenicals. We previously identified ArsV, a flavin-linked monooxygenase and demonstrated that it confers resistance to methylarsenite by oxidation to methylarsenate. The arsV gene is usually in an arsenic resistance (ars) operon controlled by an ArsR repressor and adjacent to a methylarsenite efflux gene, either arsK or a gene for a putative transporter. Here we show that Paracoccus sp. SY oxidizes methylarsenite. It has an ars operon with three genes, arsR, arsV and a transport gene termed arsW. Heterologous expression of arsV in Escherichia coli conferred resistance to MAs(III), while arsW did not. Co-expression of arsV and arsW increased resistance compared with either alone. The cells oxidized methylarsenite and accumulated less methylarsenate. Everted membrane vesicles from E. coli cells expressing arsW-accumulated methylarsenate. We propose that ArsV is a monooxygenase that oxidizes methylarsenite to methylarsenate, which is extruded by ArsW, one of only a few known pentavalent organoarsenical efflux permeases, a novel pathway of organoarsenical resistance.     

The antibiotic action of methylarsenite is an emergent property of microbial communities

Arsenic is the most ubiquitous environmental toxin. Here, we demonstrate that bacteria have evolved the ability to use arsenic to gain a competitive advantage over other bacteria at least twice. Microbes generate toxic methylarsenite (MAs(III)) by methylation of arsenite (As(III)) or reduction of methylarsenate (MAs(V)). MAs(III) is oxidized aerobically to MAs(V), making methylation a detoxification process. MAs(V) is continually re‐reduced to MAs(III) by other community members, giving them a competitive advantage over sensitive bacteria. Because generation of a sustained pool of MAs(III) requires microbial communities, these complex interactions are an emergent property. We show that reduction of MAs(V) by Burkholderia sp. MR1 produces toxic MAs(III) that inhibits growth of Escherichia coli in mixed culture. There are three microbial mechanisms for resistance to MAs(III). ArsH oxidizes MAs(III) to MAs(V). ArsI degrades MAs(III) to As(III). ArsP confers resistance by efflux. Cells of E. coli expressing arsI, arsH or arsP grow in mixed culture with Burkholderia sp. MR1 in the presence of MAs(V). Thus MAs(III) has antibiotic properties: a toxic organic compound produced by one microbe to kill off competitors. Our results demonstrate that life has adapted to use environmental arsenic as a weapon in the continuing battle for dominance.     

Novel Hyper Antimony-Oxidizing Bacteria Isolated from Contaminated Mine Soils in China

Antimony (Sb)-oxidizing bacteria play an important role in environmental Sb bioremediation because of their ability to convert the more toxic Sb(III) to the less toxic Sb(V). So far, the information about the Sb(III)-oxidizing bacteria species is still limited. In this study, three highly Sb(III)-resistant bacterial strains were isolated from contaminated mine soils after aerobic enrichment culturing with Sb(III) (1 mM). The morphological, biochemical, and 16S rRNA gene sequencing analysis suggested that the three novel bacterial isolates fell within Cupriavidus, Moraxella, and Bacillus, respectively. Among the strains, Moraxella sp. S2 isolated from soils with the highest Sb content exhibited the highest minimum inhibitory concentration for Sb(III) but the lowest Sb(III) oxidation efficiency, which could not completely oxidize 50 μM Sb(III) in 15 days. Cupriavidus sp. S1 was able to oxidize 50 μM Sb(III) completely in 12 days, but could not oxidize 100 μM Sb(III) even with extended time of incubation, while Bacillus sp. S3 with the lowest resistance to Sb(III) could aerobically oxidize 100 µM Sb(III) within 2 days, showing high Sb(III) oxidation efficiency. Our research demonstrated that indigenous microorganisms associated with Sb mine soils were capable of Sb oxidation, and the novel bacteria isolated could represent good candidates for Sb remediation in heavily polluted sites.     

ArsH is an organoarsenical oxidase that confers resistance to trivalent forms of the herbicide monosodium methylarsenate and the poultry growth promoter roxarsone

Environmental organoarsenicals are produced by microorganisms and are introduced anthropogenically as herbicides and antimicrobial growth promoters for poultry and swine. Nearly every prokaryote has an ars (arsenic resistance) operon, and some have an arsH gene encoding an atypical flavodoxin. The role of ArsH in arsenic resistance has been unclear. Here we demonstrate that ArsH is an organoarsenical oxidase that detoxifies trivalent methylated and aromatic arsenicals by oxidation to pentavalent species. Escherichia coli, which does not have an arsH gene, is very sensitive to the trivalent forms of the herbicide monosodium methylarsenate [MSMA or MAs(V)] and antimicrobial growth promoter roxarsone [Rox(V)], as well as to phenylarsenite [PhAs(III), also called phenylarsine oxide or PAO]. Pseudomonas putida has two chromosomally encoded arsH genes and is highly resistant to the trivalent forms of these organoarsenicals. A derivative of P. putida with both arsH genes deleted is sensitive to MAs(III), PhAs(III) or Rox(III). P. putida arsH expressed in E. coli conferred resistance to each trivalent organoarsenical. Cells expressing PpArsH oxidized the trivalent organoarsenicals. PpArsH was purified, and the enzyme in vitro similarly oxidized the trivalent organoarsenicals. These results suggest that ArsH catalyzes a novel biotransformation that confers resistance to environmental methylated and aromatic arsenicals.     

An ArsRC fusion protein enhances arsenate sensing and detoxification

Arsenical resistance (ars) operons encode genes for arsenic resistance and biotransformation. The majority are composed of individual genes, but fusion of ars genes is not uncommon, although it is not clear if the fused gene products are functional. Here we report identification of a four-gene ars operon from Paracoccus sp. SY that has two arsR-arsC gene fusions. ArsRC1 and ArsRC2 are related proteins that consist of an N-terminal ArsR arsenite (As(III))-responsive repressor with a C-terminal ArsC arsenate reductase. The other two genes in the operon are gapdh and arsJ. GAPDH, glyceraldehyde 3-phosphate dehydrogenase, forms 1-arseno-3-phosphoglycerate (1As3PGA) from 3-phosphoglyceraldehyde and arsenate (As(V)), ArsJ is an efflux permease for 1As3PGA that dissociates into extracellular As(V) and 3-phosphoglycerate. The net effect is As(V) extrusion and resistance. ArsRs are usually selective for As(III) and do not respond to As(V). However, the substrates and products of this operon are pentavalent, which would not be inducers of the operon. We propose that ArsRC fusions overcome this limitation by channelling the ArsC product into the ArsR binding site without diffusion through the cytosol, a de facto mechanism for As(V) induction. This novel mechanism for arsenate sensing can confer an evolutionary advantage for detoxification of inorganic arsenate.     

DNA sequence homology analysis of<Emphasis Type="Italic">ars</Emphasis> genes in arsenic-resistant bacteria

Homology ofars (arsenic-resistance system) genes was examined among the indigenous bacteria isolated from the soils and sediments of two abandened Au mines, which are highly contaminated with arsenic. The DNA and amino acid sequence homology of thears determinants were investigated using anars genotype. The isolated showed As(III)-oxidation ability containedarsAB genes encoding the efflux pump as well asarsR andarsD regulator genes. ThearsR andarsD leader gene are required for an arsenic resistance system when the high-homology genes (arsR; pl258 52.09% andarsD;Shewanell sp. 42.33%) are controlled by thears inducer-independent regulatory amino acid sequence. These leader gene were observed under weak acidic conditions in the Myoung-bong (pH; 5.0 to 6.0) and Duck-um (pH; 4.0 to 7.0) mines In addition, the strains with the ability of As (V)-reduction involved thearsC gene homologues, as in the strain CW-16 (Pseudomonas putida). The arsenic-resistance genes in the isolated indigenous bacteria showed varying degrees of amino acid similarity to the homologous genes found in the database (GenBank) such asP. putida KT2440: 39–53% forarsR, 22–42% forarsD, 16–84% forarsA, 26–45% forarsB, 17–44% forarsAB, 37–41% forarsC, and 14–47% forarsH. These findings suggested that the function of the variousars gene in indigenous bacteria existing in weakly oxidative conditions may be the key factor for redox mechanisms and biogeochemical systems in arsenic contaminated soils.     

Six New Families of Aerobic Arsenate Reducing Bacteria: Leclercia,Raoultella, Kosakonia,Lelliottia, Yokenella,and Kluyvera

Elevated levels of arsenate can occur in the environment due to processes such as mining activities, and microbes must utilize various detoxification mechanisms to adapt to the associated pressure. The aim of this study was to identify as many aerobic arsenate-reducing bacteria (aARB) as possible in order to investigate their phylogenetic diversity and molecular mechanisms of arsenic resistance. We isolated 24 strains of aARB from a long-standing arsenic contaminated environment and detected the ars genotype in them. All 24 strains could reduce approximately 90% of arsenate, and 23 of them exhibited (6–59%) arsenic removal ability. The 16S rRNA gene analyses revealed aARB representing 16 genera were abundant. The included six genera, namely Leclercia, Raoultella, Kosakonia, Lelliottia, Yokenella, and Kluyvera, that were not previously known to reduce or exhibit resistance to arsenic. Twenty-one of 24 aARB were positive for ars amplification and 17 of them harbored a putative arsC gene, which is well-known for its involvement in arsenate reduction. However, the arsenic resistance associated with aARB strains is not always determined by the ars operon system. These results have provided additional insight into aARB and their potential for arsenic transformation and bioremediation.     

Organoarsenical tolerance in Sphingobacterium wenxiniae,a bacterium isolated from activated sludge

Organoarsenicals enter the environment from biogenic and anthropogenic sources. Trivalent inorganic arsenite (As(III)) is microbially methylated to more toxic methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)) that oxidize in air to MAs(V) and DMAs(V). Sources include the herbicide monosodium methylarsenate (MSMA or MAs(V)), which is microbially reduced to MAs(III), and the aromatic arsenical roxarsone (3-nitro-4-hydroxybenzenearsonic acid or Rox), an antimicrobial growth promoter for poultry and swine. Here we show that Sphingobacterium wenxiniae LQY-18^T, isolated from activated sludge, is resistant to trivalent MAs(III) and Rox(III). Sphingobacterium wenxiniae detoxifies MAs(III) and Rox(III) by oxidation to MAs(V) and Rox(V). Sphingobacterium wenxiniae has a novel chromosomal gene, termed arsU1. Expressed in Escherichia coli arsU1 confers resistance to MAs(III) and Rox(III) but not As(III) or pentavalent organoarsenicals. Purified ArsU1 catalyses oxidation of trivalent methylarsenite and roxarsone. ArsU1 has six conserved cysteine residues. The DNA sequence for the three C-terminal cysteines was deleted, and the other three were mutated to serines. Only C45S and C122S lost activity, suggesting that Cys45 and Cys122 play a role in ArsU1 function. ArsU1 requires neither FMN nor FAD for activity. These results demonstrate that ArsU1 is a novel MAs(III) oxidase that contributes to S. wenxiniae tolerance to organoarsenicals.     

Identification of an arsenic resistance mechanism in rhizobial strains

Arsenic (As) is a very toxic metalloid to a great number of organisms. It is one of the most important global environmental pollutants. To resist the arsenate invasion, some microorganisms have developed or acquired genes that permit the cell to neutralize the toxic effects of arsenic through the exclusion of arsenic from the cells. In this work, two arsenic resistance genes, arsA and arsC, were identified in three strains of Rhizobium isolated from nodules of legumes that grew in contaminated soils with effluents from the chemical and fertilizer industry containing heavy-metals, in the industrial area of Estarreja, Portugal. The arsC gene was identified in strains of Sinorhizobium loti [DQ398936], Rhizobium leguminosarum [DQ398938] and Mesorhizobium loti [DQ398939]. This is the first time that arsenic resistance genes, namely arsC, have been identified in Rhizobium leguminosarum strains. The search for the arsA gene revealed that not all the strains with the arsenate reductase gene had a positive result for ArsA, the ATPase for the arsenite-translocating system. Only in Mesorhizobium loti was the arsA gene amplified [DQ398940]. The presence of an arsenate reductase in these strains and the identification of the arsA gene in Mesorhizobium loti, confirm the presence of an ars operon and consequently arsenate resistance.     

ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase

The toxic metalloid arsenic is widely disseminated in the environment and causes a variety of health and environment problems. As an adaptation to arsenic-contaminated environments, organisms have developed resistance systems. Many ars operons contain only three genes, arsRBC. Five gene ars operons have two additional genes, arsD and arsA, and these two genes are usually adjacent to each other. ArsA from Escherichia coli plasmid R773 is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump. ArsD was recently identified as an arsenic chaperone to the ArsAB pump, transferring the trivalent metalloids As(III) and Sb(III) to the ArsA subunit of the pump. This increases the affinity of ArsA for As(III), resulting in increased rates if extrusion and resistance to environmentally relevant concentrations of arsenite. ArsD is a homodimer with three vicinal cysteine pairs, Cys12–Cys13, Cys112–Cys113 and Cys119–Cys120, in each subunit. Each vicinal pair binds one As(III) or Sb(III). ArsD mutants with alanines substituting for Cys112, Cys113, Cys119 or Cys120, individually or in pairs or truncations lacking the vicinal pairs, retained ability to interact with ArsA, to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys12, Cys13 or Cys18, individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. It is proposed that ArsD residues Cys12, Cys13 and Cys18, but not Cys112, Cys113, Cys119 or Cys120, are required for delivery of As(III) to and activation of the ArsAB pump.     

Conserved cysteine residues determine substrate specificity in a novel As(III) S‐adenosylmethionine methyltransferase from Aspergillus fumigatus

Methylation of inorganic arsenic is a central process in the organoarsenical biogeochemical cycle. Members of every kingdom have ArsM As(III) S‐adenosylmethionine (SAM) methyltransferases that methylates inorganic As(III) into mono‐ (MAs(III)), di‐ (DMAs(III)) and tri‐ (TMAs(III)) methylarsenicals. Every characterized ArsM to date has four conserved cysteine residues. All four cysteines are required for methylation of As(III) to MAs(III), but methylation of MAs(III) to DMAs(III) requires only the two cysteines closest to the C‐terminus. Fungi produce volatile and toxic arsines, but the physiological roles of arsenic methylation and the biochemical basis is unknown. Here they demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues. The genome of Aspergillus fumigatus has four arsM genes encoding ArsMs with only the second, third and fourth conserved cysteine residues. AfArsM1 methylates MAs(III) but not As(III). Heterologous expression of AfarsM1 in an Escherichia coli conferred resistance to MAs(III) but not As(III). The existence of ArsMs with only three conserved cysteine residues suggest that the ability to methylate MAs(III) may be an evolutionary step toward enzymes capable of methylating As(III), the result of a loss of function mutation in organisms with infrequent exposure to inorganic As(III) or as a resistance mechanism for MAs(III).     

Antimony-Oxidizing Bacteria Isolated from Antimony-Contaminated Sediment – A Phylogenetic Study

Twelve antimony-resistant bacteria were isolated from sediment collected in the vicinity of an antimony oxide-producing factory in Korea. Eight of these strains were heterotrophic Sb(III)-oxidizing bacteria. Phylogenetic study showed that the Sb(III)-oxidizing bacteria fell within two subdivisions of Proteobacteria. Cupriavidus sp. NL4 and Comamonas sp. NL11 belong to the subdivision β-Proteobacteria. Acinetobacter sp. NL1, Acinetobacter sp. NL12, Pseudomonas sp. NL2, Pseudomonas sp. NL5, Pseudomonas sp. NL6, and Pseudomonas sp. NL10 are the members of the γ-subdivision of the Proteobacteria. Among them, Cupriavidus sp. NL4 completely oxidized 100 μmoles of Sb(III) per liter of medium in 500 h, while the other strains were not able to oxidize all of the Sb(III) in the medium, even with longer incubation. The results imply that diverse bacterial lineages are able to detoxify sites polluted with Sb(III) by oxidizing it to Sb(V), and to contribute to antimony cycling in natural environments.     

Caenorhabditis elegans expresses a functional ArsA

Because arsenic is the most prevalent environmental toxin, it is imperative that we understand the mechanisms of metalloid detoxification. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid-borne operon-encoded efflux systems. Bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite (As(III)) and antimonite (Sb(III)). Here, we describe the identification of a Caenorhabditis elegans homolog (asna-1) that encodes the ATPase component of the Escherichia coli As(III) and Sb(III) transporter. We evaluated the responses of wild-type and asna-1-mutant nematodes to various metal ions and found that asna-1-mutant nematodes are more sensitive to As(III) and Sb(III) toxicity than are wild-type animals. These results provide evidence that ASNA-1 is required for C. elegans' defense against As(III) and Sb(III) toxicity. A purified maltose-binding protein (MBP)-ASNA-1 fusion protein was biochemically characterized, and its properties compared with those of ArsAs. The ATPase activity of the ASNA-1 protein was dependent on the presence of As(III) or Sb(III). As(III) stimulated ATPase activity by 2 +/- 0.2-fold, whereas Sb(III) stimulated it by 4.6 +/- 0.15-fold. The results indicate that As(III)- and Sb(III)-stimulated ArsA ATPase activities are not restricted to bacteria, but extend to animals, by demonstrating that the asna-1 gene from the nematode, C. elegans, encodes a functional ArsA ATPase whose activity is stimulated by As(III) and Sb(III) and which is critical for As(III) and Sb(III) tolerance in the intact organism.     

Role of extracellular polymeric substance (EPS) in toxicity response of soil bacteria Bacillus sp. S3 to multiple heavy metals

Heavy metal resistant bacteria are of great interest because of their potential use in bioremediation. Understanding the survival and adaptive strategies of these bacteria under heavy metal stress is important for better utilization of these bacteria in remediation. The objective of this study was to investigate the role of bacterial extracellular polymeric substance (EPS) in detoxifying against different heavy metals in Bacillus sp. S3, a new hyper antimony-oxidizing bacterium previously isolated from contaminated mine soils. The results showed that Bacillus sp. S3 is a multi-metal resistant bacterial strain, especially to Sb(III), Cu(II) and Cr(VI). Toxic Cd(II), Cr(VI) and Cu(II) could stimulate the secretion of EPS in Bacillus sp. S3, significantly enhancing the adsorption and detoxification capacity of heavy metals. Both Fourier transform infrared spectroscopy (FTIR) and three-dimensional excitation–emission matrix (3D-EEM) analysis further confirmed that proteins were the main compounds of EPS for metal binding. In contrast, the EPS production was not induced under Sb(III) stress. Furthermore, the TEM–EDX micrograph showed that Bacillus sp. S3 strain preferentially transported the Sb(III) to the inside of the cell rather than adsorbed it on the extracellular surface, indicating intracellular detoxification rather than extracellular EPS precipitation played an important role in microbial resistance towards Sb(III). Together, our study suggests that the toxicity response of EPS to heavy metals is associated with difference in EPS properties, metal types and corresponding environmental conditions, which is likely to contribute to microbial-mediated remediation.