The ars operon of Escherichia coli confers arsenical and antimonial resistance.

The chromosomally encoded arsenical resistance (ars) operon subcloned into a multicopy plasmid was found to confer a moderate level of resistance to arsenite and antimonite in Escherichia coli. When the operon was deleted from the chromosome, the cells exhibited hypersensitivity to arsenite, antimonite, and arsenate. Expression of the ars genes was inducible by arsenite. By Southern hybridization, the operon was found in all strains of E. coli examined but not in Salmonella typhimurium, Pseudomonas aeruginosa, or Bacillus subtilis.     

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.     

Corynebacterium glutamicum as a model bacterium for the bioremediation of arsenic.

Arsenic is an extremely toxic metalloid that, when present in high concentrations, severely threatens the biota and human health. Arsenic contamination of soil, water, and air is a global growing environmental problem due to leaching from geological formations, the burning of fossil fuels, wastes generated by the gold mining industry present in uncontrolled landfills, and improper agriculture or medical uses. Unlike organic contaminants, which are degraded into harmless chemical species, metals and metalloids cannot be destroyed, but they can be immobilized or transformed into less toxic forms. The ubiquity of arsenic in the environment has led to the evolution in microbes of arsenic defense mechanisms. The most common of these mechanisms is based on the presence of the arsenic resistance operon (ars), which codes for: (i) a regulatory protein, ArsR; (ii) an arsenite permease, ArsB; and (iii) an enzyme involved in arsenate reduction, ArsC. Corynebacterium glutamicum, which is used for the industrial production of amino acids and nucleotides, is one of the most arsenic-resistant microorganisms described to date (up to 12 mM arsenite and >400 mM arseniate). Analysis of the C. glutamicum genome revealed the presence of two complete ars operons (ars1 and ars2) comprising the typical three-gene structure arsRBC, with an extra arsC1 located downstream from arsC1 (ars1 operon), and two orphan genes (arsB3 and arsC4). The involvement of both ars operons in arsenic resistance in C. glutamicum was confirmed by disruption and amplification of those genes. The strains obtained were resistant to up to 60 mM arsenite, one of the highest levels of bacterial resistance to arsenite so far described. Using tools for the genetic manipulation of C. glutamicum that were developed in our laboratory, we are attempting to obtain C. glutamicum mutant strains able to remove arsenic from contaminated water.     

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.     

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.     

Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258.

The arsenic resistance operon of Staphylococcus aureus plasmid pI258 determined lowered net cellular uptake of 73As by an active efflux mechanism. Arsenite was exported from the cells; intracellular arsenate was first reduced to arsenite and then transported out of the cells. Resistant cells showed lower accumulation of 73As originating from both arsenate and arsenite. Active efflux from cells loaded with arsenite required the presence of the plasmid-determined arsB gene. Efflux of arsenic originating as arsenate required the presence of the arsC gene and occurred more rapidly with the addition of arsB. Inhibitor studies with S. aureus loaded with arsenite showed that arsenite efflux was energy dependent and appeared to be driven by the membrane potential. With cells loaded with 73AsO4(3-), a requirement for ATP for energy was observed, leading to the conclusion that ATP was required for arsenate reduction. When the staphylococcal arsenic resistance determinant was cloned into Escherichia coli, lowered accumulation of arsenate and arsenite and 73As efflux from cells loaded with arsenate were also found. Cloning of the E. coli plasmid R773 arsA gene (the determinant of the arsenite-dependent ATPase) in trans to the S. aureus gene arsB resulted in increased resistance to arsenite.     

Arsenical resistance of growth and phosphate control of antibiotic biosynthesis in Streptomyces

Twenty-six wild-type Streptomyces strains tested for resistance to arsenate, arsenite and antimony(III) could be divided into four groups: those resistant only to arsenite (3) or to arsenate (2) and those resistant (8) or sensitive (13) to both heavy metals. All strains were sensitive to antimony. The structural genes for the ars operon of Escherichia coli were subcloned into various Streptomyces plasmid vectors. The expression of the whole ars operon in streptomycetes may be strain-specific and occurred only from low-copy-number plasmids. The arsC gene product could be expressed from high-copy plasmids and conferred arsenate resistance to both E. coli and Streptomyces species. The ars operon expressed in S. lividans and the arsC gene expressed in S. noursei did not render the synthesis of undecylprodigiosin and nourseothricin, respectively, phosphate-resistant. In addition in wild-type strains of Streptomyces phosphate sensitivity of antibiotic biosynthesis did not show strong correlation with resistance of growth to arsenicals.     

The Contribution of ArsB to Arsenic Resistance in Campylobacter jejuni

Arsenic, a toxic metalloid, exists in the natural environment and its organic form is approved for use as a feed additive for animal production. As a major foodborne pathogen of animal origin, Campylobacter is exposed to arsenic selection pressure in the food animal production environments. Previous studies showed that Campylobacter isolates from poultry were highly resistant to arsenic compounds and a 4-gene operon (containing arsP, arsR, arsC, and acr3) was associated with arsenic resistance in Campylobacter. However, this 4-gene operon is only present in some Campylobacter isolates and other arsenic resistance mechanisms in C. jejuni have not been characterized. In this study, we determined the role of several putative arsenic resistance genes including arsB, arsC2, and arsR3 in arsenic resistance in C. jejuni and found that arsB, but not the other two genes, contributes to the resistance to arsenite and arsenate. Inactivation of arsB in C. jejuni resulted in 8- and 4-fold reduction in the MICs of arsenite and arsenate, respectively, and complementation of the arsB mutant restored the MIC of arsenite. Additionally, overexpression of arsB in C. jejuni 11168 resulted in a 16-fold increase in the MIC of arsenite. PCR analysis of C. jejuni isolates from different animals hosts indicated that arsB and acr3 (the 4-gene operon) are widely distributed in various C. jejuni strains, suggesting that Campylobacter requires at least one of the two genes for adaptation to arsenic-containing environments. These results identify ArsB as an alternative mechanism for arsenic resistance in C. jejuni and provide new insights into the adaptive mechanisms of Campylobacter in animal food production environments.     

Plasmid-borne resistance to arsenate, arsenite, cadmium, and chloramphenicol in a Rhodococcus species

Summary A primarily genetic approach was employed to obtain plasmids in Rhodococcus erythropolis ATCC 12674 which carried genes conferring increased resistance to sodium arsenate and arsenite, cadmium chloride, and chloramphenicol. The plasmids were large, migrating more slowly than chromosomal DNA in agarose gels, and were made up of resistance determinants from the host organism together with part of the genome of nocardiophage Q4. Purified plasmid was used to transform a suitable recipient to increased resistance to sodium arsenate, sodium arsenite, and cadmium chloride.     

Inducible plasmid-determined resistance to arsenate, arsenite, and antimony (III) in escherichia coli and Staphylococcus aureus.

Plasmids in both Escherichia coli and Staphylococcus aureus contain an "operon" that confers resistance to arsenate, arsenite, and antimony(III) salts. The systems were always inducible. All three salts, arsenate, arsenite, and antimony(III), were inducers. Mutants and a cloned deoxyribonucleic acid fragment from plasmid pI258 in S. aureus have lost arsenate resistance but retained resistances to arsenite and antimony, demonstrating that separate genes are involved. Arsenate-resistant arsenite-sensitive S. aureus plasmid mutants were also isolated. In E. coli, plasmid-determined arsenate resistance and reduced uptake were additive to that found with chromosomal arsenate resistance mutants. Arsenate resistance was due to reduced uptake of arsenate by the induced plasmid-containing cells. Under conditions of high arsenate, when some uptake could be demonstrated with the induced resistant cells, the arsenate was rapidly lost by the cells in the absence of extracellular phosphate. Sensitive cells retained arsenate under these conditions. When phosphate was added, phosphate-arsenate exchange occurred. High phosphate in the growth medium protected cells from arsenate, but not from arsenite or antimony(III) toxicity. We do not know the mechanisms of arsenite or antimony resistance. However, arsenite was not oxidized to less toxic arsenate. Since cell-free medium "conditioned" by prior growth to induced resistant cells with toxic levels of arsenite or antimony(III) retained the ability to inhibit the growth of sensitive cells, the mechanism of arsenite and antimony resistance does not involve conversion of AsO2- or SbO+ to less toxic forms or binding by soluble thiols excreted by resistant cells.     

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.     

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.