Identification of genes conferring arsenic resistance to Escherichia coli from an effluent treatment plant sludge metagenomic library

The majority of bacteria elude culture in the laboratory. A metagenomic approach provides culture-independent access to the gene pool of the whole bacterial community. A metagenomic library was constructed from an industrial effluent treatment plant sludge containing about 1.25 Gb of microbial community DNA. Two arsenic-resistant clones were selected from the metagenomic library. Clones MT3 and MT6 had eight- and 18-fold higher resistance to sodium arsenate in comparison with the parent strain, respectively. The clones also showed increased resistance to arsenite but not to antimony. Sequence analysis of the clones revealed genes encoding for putative arsenate reductases and arsenite efflux pumps. A novel arsenate resistance gene ( arsN ) encoding a protein with similarity to acetyltransferases was identified from clone MT6. ArsN homologues were found to be closely associated with arsenic resistance genes in many bacterial genomes. ArsN homologues were found fused to putative arsenate reductases in Methylibium petroleiphilum PM1 and Anaeromyxobacter dehalogenans 2CP-C and with a putative arsenite chaperone in Burkholderia vietnamiensis G4. ArsN alone resulted in an approximately sixfold higher resistance to sodium arsenate in wild-type Escherichia coli W3110.     

Genomic Responses to Arsenic in the Cyanobacterium Synechocystis sp. PCC 6803

Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As^III] and arsenate [As^V]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.     

arsRBOCT arsenic resistance system encoded by linear plasmid pHZ227 in Streptomyces sp. strain FR-008

In the arsenic resistance gene cluster from the large linear plasmid pHZ227, two novel genes, arsO (for a putative flavin-binding monooxygenase) and arsT (for a putative thioredoxin reductase), were coactivated and cotranscribed with arsR1-arsB and arsC, respectively. Deletion of the ars gene cluster on pHZ227 in Streptomyces sp. strain FR-008 resulted in sensitivity to arsenic, and heterologous expression of the ars gene cluster in the arsenic-sensitive Streptomyces strains conferred resistance on the new hosts. The pHZ227 ArsB protein showed homology to the yeast arsenite transporter Acr3p. The pHZ227 ArsC appears to be a bacterial thioredoxin-dependent ArsC-type arsenate reductase with four conserved cysteine thioredoxin-requiring motifs.     

Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana tolerance to arsenic stress

? Arsenic contamination has a negative impact on crop cultivation and on human health. As yet, no proteins have been identified in plants that mediate the extrusion of arsenic. Here, we heterologously expressed the yeast (Saccharomyces cerevisiae) arsenite efflux transporter ACR3 into Arabidopsis to evaluate how this affects plant tolerance and tissue arsenic contents. ? ACR3 was cloned from yeast and transformed into wild-type and nip7;1 Arabidopsis. Arsenic tolerance was determined at the cellular level using vitality stains in protoplasts, in intact seedlings grown on agar plates and in mature plants grown hydroponically. Arsenic efflux was measured from protoplasts and from intact plants, and arsenic levels were measured in roots and shoots of plants exposed to arsenate. ? At the cellular level, all transgenic lines showed increased tolerance to arsenite and arsenate and a greater capacity for arsenate efflux. With intact plants, three of four stably transformed lines showed improved growth, whereas only transgenic lines in the wild-type background showed increased efflux of arsenite into the external medium. The presence of ACR3 hardly affected tissue arsenic levels, but increased arsenic translocation to the shoot. ? Heterologous expression of yeast ACR3 endows plants with greater arsenic resistance, but does not lower significantly arsenic tissue levels.     

Validation of Arsenic Resistance in <Emphasis Type="Italic">Bacillus cereus</Emphasis> Strain AG27 by Comparative Protein Modeling of <Emphasis Type="Italic">ars</Emphasis>C Gene Product

The ars gene system provides arsenic resistance to a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. Therefore, arsC gene from Bacillus cereus strain AG27 isolated from soil was amplified, cloned and sequenced. The strain exhibited a minimum inhibitory concentration of 40 and 35 mM to sodium arsenate and sodium arsenite, respectively. Homology of the sequence, when compared with available database using BLASTn search showed that 300 bp amplicons obtained possess partial arsC gene sequence which codes for arsenate reductase, an enzyme involved in the reduction of arsenate to arsenite which is then effluxed out of the cell, thereby indicating the presence of efflux mechanism of resistance in strain. The efflux mechanism was further confirmed by atomic absorption spectroscopy and scanning electron microscopy studies. Moreover, three dimensional structure of modeled arsC from Bacillus cereus strain shares significant structural similarity with arsenate reductase protein of B.subtilis, consisting of, highly similar overall fold with single α/β domain containing a central four stranded, parallel, open-twisted β-sheet flanked by α-helices on both sides. The structure harbors the arsenic binding motif AB loop or P-loop that is highly conserved in arsenate reductase family.     

Plasmid-determined resistance to arsenic and antimony inPseudomonas aeruginosa

Resistance to arsenic salts in aPseudomonas aeruginosa clinical isolate was shown to be determined by a 100 kb transferable plasmid. The resistance pattern included arsenate, arsenite, and antimonate ions. Arsenate and arsenite resistances were inducible by previous exposure of cultures to subinhibitory amounts of either of the two ions. Phosphate ions protectedP. aeruginosa cells from the toxic effects of arsenate but did not alter arsenite toxicity.     

Molecular analysis of an anion pump: purification of the ArsC protein.

The ars operon of resistance plasmid R773 encodes an anion-translocating ATPase which catalyzes extrusion of the oxyanions arsenite, antimonite, and arsenate, thus providing resistance to the toxic compounds. Although both arsenite and arsenate contain arsenic, they have different chemical properties. In the absence of the arsC gene the pump transports arsenite and antimonite, oxyanions with the +III oxidation state of arsenic or antimony. The complex neither transports nor provides resistance to arsenate, the oxyanion of the +V oxidation state of arsenic. The arsC gene encodes a 16-kDa polypeptide, the ArsC protein, which alters the substrate specificity of the pump to allow for recognition and transport of the alternate substrate arsenate. The arsC gene was cloned behind a strong promoter and expressed at high levels. The ArsC protein was purified and crystallized.     

Genetic analysis of arsenic metabolism in <Emphasis Type="Italic">Micrococcus luteus</Emphasis> BPB1, isolated from the Bengal basin

A highly arsenic-metabolizing bacterial strain was isolated from an agricultural field known for arsenic contamination near Munshiganj (Bangladesh). Based on 16S rRNA gene analysis, the strain was identified as Micrococcus luteus and designated as strain BPB1. Arsenate and arsenite minimal inhibitory concentrations of 650 mM and 7.5 mM, respectively, were observed for strain BPB1, slightly higher than the figures observed in its close relative M. luteus DSM 20030^T. Such observations were consistent with the presence of arsenic-metabolizing genes in the genome of M. luteus. We describe this strain as having an MSH/Mrx-dependent class of arsenate reductase, and an arsenite transporter family in the ACR3(1) group. Besides an intracellular arsenic resistance mechanism, experiments carried out using field emission scanning electron microscopy-energy dispersive X-ray spectroscopy (FESEM-EDS) and Fourier transform infrared spectroscopy (FTIR) demonstrated the ability of BPB1 to sequester arsenate in extracellular polymeric substances on its cell surface.     

An arsenate-activated glutaredoxin from the arsenic hyperaccumulator fern Pteris vittata L. regulates intracellular arsenite

To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.     

arsRBOCT Arsenic Resistance System Encoded by Linear Plasmid pHZ227 in Streptomyces sp. Strain FR-008

In the arsenic resistance gene cluster from the large linear plasmid pHZ227, two novel genes, arsO (for a putative flavin-binding monooxygenase) and arsT (for a putative thioredoxin reductase), were coactivated and cotranscribed with arsR1-arsB and arsC, respectively. Deletion of the ars gene cluster on pHZ227 in Streptomyces sp. strain FR-008 resulted in sensitivity to arsenic, and heterologous expression of the ars gene cluster in the arsenic-sensitive Streptomyces strains conferred resistance on the new hosts. The pHZ227 ArsB protein showed homology to the yeast arsenite transporter Acr3p. The pHZ227 ArsC appears to be a bacterial thioredoxin-dependent ArsC-type arsenate reductase with four conserved cysteine thioredoxin-requiring motifs.     

Phylogenetic analysis of bacterial and archaeal <Emphasis Type="Italic">arsC</Emphasis> gene sequences suggests an ancient,common origin for arsenate reductase

Background  

The ars gene system provides arsenic resistance for a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. A survey of GenBank shows that arsC appears to be phylogenetically widespread both in organisms with known arsenic resistance and those organisms that have been sequenced as part of whole genome projects.     

Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli

Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI’s role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells expressing E. coli TPI. Excised frond tissue infiltrated with arsenate reduced arsenate significantly more under light than dark. This research highlights a novel role for P. vittata cTPI in arsenate reduction.     

Novel channel enzyme fusion proteins confer arsenate resistance

Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H(2)As(V)O(4)(-)/HAs(V)O(4)(2-)) to arsenite (As(III)(OH)(3)) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion.