Antimicrobial Activity of Metals and Metalloids.

Competition shapes evolution. Toxic metals and metalloids have exerted selective pressure on life since the rise of the first organisms on the Earth, which has led to the evolution and acquisition of resistance mechanisms against them, as well as mechanisms to weaponize them. Microorganisms exploit antimicrobial metals and metalloids to gain competitive advantage over other members of microbial communities. This exerts a strong selective pressure that drives evolution of resistance. This review describes, with a focus on arsenic and copper, how microorganisms exploit metals and metalloids for predation and how metal- and metalloid-dependent predation may have been a driving force for evolution of microbial resistance against metals and metalloids.

Arsenate reductase of Rufibacter tibetensis is a metallophosphoesterase evolved to catalyze redox reactions.

An arsenate reductase (Car1) from the Bacteroidetes species Rufibacter tibetensis 1351T was isolated from the Tibetan Plateau. The strain exhibits resistance to arsenite [As(III)] and arsenate [As(V)] and reduces As(V) to As(III). Here we shed light on the mechanism of enzymatic reduction by Car1. AlphaFold2 structure prediction, active site energy minimization, and steady-state kinetics of wild-type and mutant enzymes give insight into the catalytic mechanism. Car1 is structurally related to calcineurin-like metallophosphoesterases (MPPs). It functions as a binuclear metal hydrolase with limited phosphatase activity, particularly relying on the divalent metal Ni2+. As an As(V) reductase, it displays metal promiscuity and is coupled to the thioredoxin redox cycle, requiring the participation of two cysteine residues, Cys74 and Cys76. These findings suggest that Car1 evolved from a common ancestor of extant phosphatases by incorporating a redox function into an existing MPP catalytic site. Its proposed mechanism of arsenate reduction involves Cys74 initiating a nucleophilic attack on arsenate, leading to the formation of a covalent intermediate. Next, a nucleophilic attack of Cys76 leads to the release of As(III) and the formation of a surface-exposed Cys74-Cys76 disulfide, ready for reduction by thioredoxin.

The ArsQ permease and transport of the antibiotic arsinothricin.

The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacterium Burkholderia gladioli GSRB05. AST is a broad-spectrum antibiotic effective against human pathogens such as carbapenem-resistant Enterobacter cloacae. It is a non-proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three-gene cluster, arsQML. ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R-AST-OH), which is methylated by ArsM to the reduced trivalent form of AST (R-AST). In the culture medium of B. gladioli, both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study, B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and its derivatives. Cells of E. coli accumulate R-AST, and exponentially growing cells expressing arsQ take up less R-AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.

Fundamentals and application in phytoremediation of an efficient arsenate reducing bacterium Pseudomonas putida ARS1.

Arsenic is a ubiquitous environmental pollutant. Microbe-mediated arsenic bio-transformations significantly influence arsenic mobility and toxicity. Arsenic transformations by soil and aquatic organisms have been well documented, while little is known regarding effects due to endophytic bacteria. An endophyte Pseudomonas putida ARS1 was isolated from rice grown in arsenic contaminated soil. P. putida ARS1 shows high tolerance to arsenite (As(III)) and arsenate (As(V)), and exhibits efficient As(V) reduction and As(III) efflux activities. When exposed to 0.6 mg/L As(V), As(V) in the medium was completely converted to As(III) by P. putida ARS1 within 4 hr. Genome sequencing showed that P. putida ARS1 has two chromosomal arsenic resistance gene clusters (arsRCBH) that contribute to efficient As(V) reduction and As(III) efflux, and result in high resistance to arsenicals. Wolffia globosa is a strong arsenic accumulator with high potential for arsenic phytoremediation, which takes up As(III) more efficiently than As(V). Co-culture of P. putida ARS1 and W. globosa enhanced arsenic accumulation in W. globosa by 69%, and resulted in 91% removal of arsenic (at initial concentration of 0.6 mg/L As(V)) from water within 3 days. This study provides a promising strategy for in situ arsenic phytoremediation through the cooperation of plant and endophytic bacterium.

Anoxygenic phototrophic arsenite oxidation by a Rhodobacter strain.

Microbially mediated arsenic redox transformations are key for arsenic speciation and mobility in rice paddies. Whereas anaerobic anoxygenic photosynthesis coupled to arsenite (As(III)) oxidation has been widely examined in arsenic-replete ecosystems, it remains unknown whether this light-dependent process exists in paddy soils. Here, we isolated a phototrophic purple bacteria, Rhodobacter strain CZR27, from an arsenic-contaminated paddy soil and demonstrated its capacity to oxidize As(III) to arsenate (As(V)) using malate as a carbon source photosynthetically. Genome sequencing revealed an As(III)-oxidizing gene cluster (aioXSRBA) encoding an As(III) oxidase. Functional analyses showed that As(III) oxidation under anoxic phototrophic conditions correlated with transcription of the large subunit of the As(III) oxidase aioA gene. Furthermore, the non-As(III) oxidizer Rhodobacter capsulatus SB1003 heterologously expressing aioBA from strain CZR27 was able to oxidize As(III), indicating that aioBA was responsible for the observed As(III) oxidation in strain CZR27. Our study provides evidence for the presence of anaerobic photosynthesis-coupled As(III) oxidation in paddy soils, highlighting the importance of light-dependent, microbe-mediated arsenic redox changes in paddy arsenic biogeochemistry.

Arsenite Methyltransferase Diversity and Optimization of Methylation Efficiency.

Arsenic is methylated by arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferases (ArsMs). ArsM crystal structures show three domains (an N-terminal SAM binding domain (A domain), a central arsenic binding domain (B domain), and a C-terminal domain of unknown function (C domain)). In this study, we performed a comparative analysis of ArsMs and found a broad diversity in structural domains. The differences in the ArsM structure enable ArsMs to have a range of methylation efficiencies and substrate selectivities. Many small ArsMs with 240-300 amino acid residues have only A and B domains, represented by RpArsM from Rhodopseudomonas palustris. These small ArsMs have higher methylation activity than larger ArsMs with 320-400 residues such as Chlamydomonas reinhardtii CrArsM, which has A, B, and C domains. To examine the role of the C domain, the last 102 residues in CrArsM were deleted. This CrArsM truncation exhibited higher As(III) methylation activity than the wild-type enzyme, suggesting that the C-terminal domain has a role in modulating the rate of catalysis. In addition, the relationship of arsenite efflux systems and methylation was examined. Lower rates of efflux led to higher rates of methylation. Thus, the rate of methylation can be modulated in multiple ways.

Widespread Distribution of the arsO Gene Confers Bacterial Resistance to Environmental Antimony.

Microbial oxidation of environmental antimonite (Sb(III)) to antimonate (Sb(V)) is an antimony (Sb) detoxification mechanism. Ensifer adhaerens ST2, a bacterial isolate from a Sb-contaminated paddy soil, oxidizes Sb(III) to Sb(V) under oxic conditions by an unknown mechanism. Genomic analysis of ST2 reveals a gene of unknown function in an arsenic resistance (ars) operon that we term arsO. The transcription level of arsO was significantly upregulated by the addition of Sb(III). ArsO is predicted to be a flavoprotein monooxygenase but shows low sequence similarity to other flavoprotein monooxygenases. Expression of arsO in the arsenic-hypersensitive Escherichia coli strain AW3110Δars conferred increased resistance to Sb(III) but not arsenite (As(III)) or methylarsenite (MAs(III)). Purified ArsO catalyzes Sb(III) oxidation to Sb(V) with NADPH or NADH as the electron donor but does not oxidize As(III) or MAs(III). The purified enzyme contains flavin adenine dinucleotide (FAD) at a ratio of 0.62 mol of FAD/mol protein, and enzymatic activity was increased by addition of FAD. Bioinformatic analyses show that arsO genes are widely distributed in metagenomes from different environments and are particularly abundant in environments affected by human activities. This study demonstrates that ArsO is an environmental Sb(III) oxidase that plays a significant role in the detoxification of Sb(III).

Arsenic in medicine: past, present and future.

Arsenicals are one of the oldest treatments for a variety of human disorders. Although infamous for its toxicity, arsenic is paradoxically a therapeutic agent that has been used since ancient times for the treatment of multiple diseases. The use of most arsenic-based drugs was abandoned with the discovery of antibiotics in the 1940s, but a few remained in use such as those for the treatment of trypanosomiasis. In the 1970s, arsenic trioxide, the active ingredient in a traditional Chinese medicine, was shown to produce dramatic remission of acute promyelocytic leukemia similar to the effect of all-trans retinoic acid. Since then, there has been a renewed interest in the clinical use of arsenicals. Here the ancient and modern medicinal uses of inorganic and organic arsenicals are reviewed. Included are antimicrobial, antiviral, antiparasitic and anticancer applications. In the face of increasing antibiotic resistance and the emergence of deadly pathogens such as the severe acute respiratory syndrome coronavirus 2, we propose revisiting arsenicals with proven efficacy to combat emerging pathogens. Current advances in science and technology can be employed to design newer arsenical drugs with high therapeutic index. These novel arsenicals can be used in combination with existing drugs or serve as valuable alternatives in the fight against cancer and emerging pathogens. The discovery of the pentavalent arsenic-containing antibiotic arsinothricin, which is effective against multidrug-resistant pathogens, illustrates the future potential of this new class of organoarsenical antibiotics.

The Great Oxidation Event expanded the genetic repertoire of arsenic metabolism and cycling.

The rise of oxygen on the early Earth about 2.4 billion years ago reorganized the redox cycle of harmful metal(loids), including that of arsenic, which doubtlessly imposed substantial barriers to the physiology and diversification of life. Evaluating the adaptive biological responses to these environmental challenges is inherently difficult because of the paucity of fossil records. Here we applied molecular clock analyses to 13 gene families participating in principal pathways of arsenic resistance and cycling, to explore the nature of early arsenic biogeocycles and decipher feedbacks associated with planetary oxygenation. Our results reveal the advent of nascent arsenic resistance systems under the anoxic environment predating the Great Oxidation Event (GOE), with the primary function of detoxifying reduced arsenic compounds that were abundant in Archean environments. To cope with the increased toxicity of oxidized arsenic species that occurred as oxygen built up in Earth's atmosphere, we found that parts of preexisting detoxification systems for trivalent arsenicals were merged with newly emerged pathways that originated via convergent evolution. Further expansion of arsenic resistance systems was made feasible by incorporation of oxygen-dependent enzymatic pathways into the detoxification network. These genetic innovations, together with adaptive responses to other redox-sensitive metals, provided organisms with novel mechanisms for adaption to changes in global biogeocycles that emerged as a consequence of the GOE.

Functional and structural characterization of AntR, an Sb(III) responsive transcriptional repressor.

The ant operon of the antimony-mining bacterium Comamonas testosterone JL40 confers resistance to Sb(III). The operon is transcriptionally regulated by the product of the first gene in the operon, antR. AntR is a member of ArsR/SmtB family of metal/metalloid-responsive repressors resistance. We purified and characterized C. testosterone AntR and demonstrated that it responds to metalloids in the order Sb(III) = methylarsenite (MAs(III) >> As(III)). The protein was crystallized, and the structure was solved at 2.1 Å resolution. The homodimeric structure of AntR adopts a classical ArsR/SmtB topology architecture. The protein has five cysteine residues, of which Cys103a from one monomer and Cys113b from the other monomer, are proposed to form one Sb(III) binding site, and Cys113a and Cys103b forming a second binding site. This is the first report of the structure and binding properties of a transcriptional repressor with high selectivity for environmental antimony.

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-18T , 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.

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.

A PadR family transcriptional repressor controls transcription of a trivalent metalloid resistance operon of Azospirillum halopraeferens strain Au 4.

Methylarsenite [MAs(III)] is a highly toxic arsenical produced by some microbes as an antibiotic. In this study, we demonstrate that a PadR family transcriptional regulator, PadRars , from Azospirillum halopraeferens strain Au 4 directly binds to the promoter region of the arsenic resistance (ars) operon (consisting of padRars , arsV, and arsW) and represses transcription of arsV and arsW genes involved in MAs(III) resistance. Quantitative reverse transcriptase PCR and transcriptional reporter assays showed that transcription of the ars operon is induced strongly by MAs(III) and less strongly by arsenite and antimonite. Electrophoretic mobility shift assays with recombinant PadRars showed that it represses transcription of the ars operon by binding to two inverted-repeat sequences within the ars promoter. PadRars has two conserved cysteine pairs, Cys56/57 and Cys133/134; mutation of the first pair to serine abolished the transcriptional response of the ars operon to trivalent metalloids, suggesting that Cys56/57 form a binding site for trivalent metalloids. Either C133S or C134S derivative responses to MAs(III) but not As(III) or Sb(III), suggesting that it is a third ligand to trivalent metalloids. PadRars represents a new type of repressor proteins regulating transcription of an ars operon involved in the resistance to trivalent metalloids, especially MAs(III).

ArsZ from Ensifer adhaerens ST2 is a novel methylarsenite oxidase.

Trivalent methylarsenite [MAs(III)] produced by biomethylation is more toxic than inorganic arsenite [As(III)]. Hence, MAs(III) has been proposed to be a primordial antibiotic. Other bacteria evolved mechanisms to detoxify MAs(III). In this study, the molecular mechanisms of MAs(III) resistance of Ensifer adhaerens ST2 were investigated. In the chromosome of E. adhaerens ST2 is a gene encoding a protein of unknown function. Here, we show that this gene, designated arsZ, encodes a novel MAs(III) oxidase that confers resistance by oxidizing highly toxic MAs(III) to relatively nontoxic MAs(V). Two other genes, arsRK, are adjacent to arsZ but are divergently encoded in the opposite direction. Heterologous expression of arsZ in Escherichia coli confers resistance to MAs(III) but not to As(III). Purified ArsZ catalyses thioredoxin- and NAPD+ -dependent oxidation of MAs(III). Mutational analysis of ArsZ suggests that Cys59 and Cys123 are involved in the oxidation of MAs(III). Expression of arsZ, arsR and arsK genes is induced by MAs(III) and As(III) and is likely controlled by the ArsR transcriptional repressor. These results demonstrate that ArsZ is a novel MAs(III) oxidase that contributes to E. adhaerens tolerance to environmental organoarsenicals. The arsZRK operon is widely present in bacteria within the Rhizobiaceae family.

Functional characterization of the methylarsenite-inducible arsRM operon from Noviherbaspirillum denitrificans HC18.

Microbial arsenic methylation by arsenite (As(III)) S-adenosylmethionine methyltransferases (ArsMs) can produce the intermediate methylarsenite (MAs(III)), which is highly toxic and is used by some microbes as an antibiotic. Other microbes have evolved mechanisms to detoxify MAs(III). In this study, an arsRM operon was identified in the genome of an MAs(III)-methylation strain Noviherbaspirillum denitrificans HC18. The arsM gene (NdarsM) is located downstream of an open reading frame encoding an MAs(III)-responsive transcriptional regulator (NdArsR). The N. denitrificans arsRM genes are co-transcribed whose expression is significantly induced by MAs(III), likely by alleviating the repressive effect of ArsR on arsRM transcription. Both in vivo and in vitro assays showed that NdArsM methylates MAs(III) to dimethyl- and trimethyl-arsenicals but does not methylate As(III). Heterologous expression of NdarsM in arsenic-sensitive Escherichia coli AW3110 conferred resistance to MAs(III) but not As(III). NdArsM has the four conserved cysteine residues present in most ArsMs, but only two of them are essential for MAs(III) methylation. The ability to methylate MAs(III) by enzymes such as NdArsM may be an evolutionary step originated from enzymes capable of methylating As(III). This finding reveals a mechanism employed by microbes such as N. denitrificans HC18 to detoxify MAs(III) by further methylation.

Oxidation of organoarsenicals and antimonite by a novel flavin monooxygenase widely present in soil bacteria.

Arsenic can be biomethylated to form a variety of organic arsenicals differing in toxicity and environmental mobility. Trivalent methylarsenite (MAs(III)) produced in the methylation process is more toxic than inorganic arsenite (As(III)). MAs(III) also serves as a primitive antibiotic and, consequently, some environmental microorganisms have evolved mechanisms to detoxify MAs(III). However, the mechanisms of MAs(III) detoxification are not well understood. In this study, we identified an arsenic resistance (ars) operon consisting of three genes, arsRVK, that contribute to MAs(III) resistance in Ensifer adhaerens ST2. ArsV is annotated as an NADPH-dependent flavin monooxygenase with unknown function. Expression of arsV in the arsenic hypersensitive Escherichia coli strain AW3110Δars conferred resistance to MAs(III) and the ability to oxidize MAs(III) to MAs(V). In the presence of NADPH and either FAD or FMN, purified ArsV protein was able to oxidize both MAs(III) to MAs(V) and Sb(III) to Sb(V). Genes with arsV-like sequences are widely present in soils and environmental bacteria. Metagenomic analysis of five paddy soils showed the abundance of arsV-like sequences of 0.12-0.25 ppm. These results demonstrate that ArsV is a novel enzyme for the detoxification of MAs(III) and Sb(III) and the genes encoding ArsV are widely present in soil bacteria.

Regulation of arsenic methylation: identification of the transcriptional region of the human AS3MT gene.

The human enzyme As(III) S-adenosylmethionine methyltransferase (AS3MT) catalyzes arsenic biotransformations and is considered to contribute to arsenic-related diseases. AS3MT is expressed in various tissues and cell types including liver, brain, adrenal gland, and peripheral blood mononuclear cells but not in human keratinocytes, urothelial, or brain microvascular endothelial cells. This indicates that AS3MT expression is regulated in a tissue/cell type-specific manner, but the mechanism of transcriptional regulation of expression of the AS3MT gene is not known. In this study, we define the DNA sequence of the core promoter region of the human AS3MT gene. We identify a GC box in the promoter to which the stress-related transcription factor Sp1 binds, indicating involvement of regulatory elements in AS3MT gene expression.

Selective Methylation by an ArsM S-Adenosylmethionine Methyltransferase from Burkholderia gladioli GSRB05 Enhances Antibiotic Production.

Arsenic methylation contributes to the formation and diversity of environmental organoarsenicals, an important process in the arsenic biogeochemical cycle. The arsM gene encoding an arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferase is widely distributed in members of every kingdom. A number of ArsM enzymes have been shown to have different patterns of methylation. When incubated with inorganic As(III), Burkholderia gladioli GSRB05 has been shown to synthesize the organoarsenical antibiotic arsinothricin (AST) but does not produce either methylarsenate (MAs(V)) or dimethylarsenate (DMAs(V)). Here, we show that cells of B. gladioli GSRB05 synthesize DMAs(V) when cultured with either MAs(III) or MAs(V). Heterologous expression of the BgarsM gene in Escherichia coli conferred resistance to MAs(III) but not As(III). The cells methylate MAs(III) and the AST precursor, reduced trivalent hydroxyarsinothricin (R-AST-OH) but do not methylate inorganic As(III). Similar results were obtained with purified BgArsM. Compared with ArsM orthologs, BgArsM has an additional 37 amino acid residues in a linker region between domains. Deletion of the additional 37 residues restored As(III) methylation activity. Cells of E. coli co-expressing the BgarsL gene encoding the noncanonical radical SAM enzyme that catalyzes the synthesis of R-AST-OH together with the BgarsM gene produce much more of the antibiotic AST compared with E. coli cells co-expressing BgarsL together with the CrarsM gene from Chlamydomonas reinhardtii, which lacks the sequence for additional 37 residues. We propose that the presence of the insertion reduces the fitness of B. gladioli because it cannot detoxify inorganic arsenic but concomitantly confers an evolutionary advantage by increasing the ability to produce AST.

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.

Identification of a MarR Subfamily That Regulates Arsenic Resistance Genes.

In this study, comprehensive analyses were performed to determine the function of an atypical MarR homolog in Achromobacter sp. strain As-55. Genomic analyses of Achromobacter sp. As-55 showed that this marR is located adjacent to an arsV gene. ArsV is a flavin-dependent monooxygenase that confers resistance to the antibiotic methylarsenite [MAs(III)], the organoarsenic compound roxarsone(III) [Rox(III)], and the inorganic antimonite [Sb(III)]. Similar marR genes are widely distributed in arsenic-resistant bacteria. Phylogenetic analyses showed that these MarRs are found in operons predicted to be involved in resistance to inorganic and organic arsenic species, so the subfamily was named MarRars. MarRars orthologs have three conserved cysteine residues, which are Cys36, Cys37, and Cys157 in Achromobacter sp. As-55, mutation of which compromises the response to MAs(III)/Sb(III). GFP-fluorescent biosensor assays show that AdMarRars (MarR protein of Achromobacter deleyi As-55) responds to trivalent As(III) and Sb(III) but not to pentavalent As(V) or Sb(V). The results of RT-qPCR assays show that arsV is expressed constitutively in a marR deletion mutant, indicating that marR represses transcription of arsV. Moreover, electrophoretic mobility shift assays (EMSAs) demonstrate that AdMarRars binds to the promoters of both marR and arsV in the absence of ligands and that DNA binding is relieved upon binding of As(III) and Sb(III). Our results demonstrate that AdMarRars is a novel As(III)/Sb(III)-responsive transcriptional repressor that controls expression of arsV, which confers resistance to MAs(III), Rox(III), and Sb(III). AdMarRars and its orthologs form a subfamily of MarR proteins that regulate genes conferring resistance to arsenic-containing antibiotics. IMPORTANCE In this study, a MarR family member, AdMarRars was shown to regulate the arsV gene, which confers resistance to arsenic-containing antibiotics. It is a founding member of a distinct subfamily that we refer to as MarRars, regulating genes conferring resistance to arsenic and antimony antibiotic compounds. AdMarRars was shown to be a repressor containing conserved cysteine residues that are required to bind As(III) and Sb(III), leading to a conformational change and subsequent derepression. Here we show that members of the MarR family are involved in regulating arsenic-containing compounds.