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. 2017 Nov;106(3):469-478.
doi: 10.1111/mmi.13826. Epub 2017 Sep 13.

A Novel MAs(III)-selective ArsR Transcriptional Repressor

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Free PMC article

A Novel MAs(III)-selective ArsR Transcriptional Repressor

Jian Chen et al. Mol Microbiol. .
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Abstract

Microbial expression of genes for resistance to heavy metals and metalloids is usually transcriptionally regulated by the toxic ions themselves. Arsenic is a ubiquitous, naturally occurring toxic metalloid widely distributed in soil and groundwater. Microbes biotransform both arsenate (As(V)) and arsenite (As(III)) into more toxic methylated metabolites methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)). Environmental arsenic is sensed by members of the ArsR/SmtB family. The arsR gene is autoregulated and is typically part of an operon that contains other ars genes involved in arsenic detoxification. To date every identified ArsR is regulated by inorganic As(III). Here we described a novel ArsR from Shewanella putrefaciens selective for MAs(III). SpArsR orthologs control expression of two MAs(III) resistance genes, arsP that encodes the ArsP MAs(III) efflux permease, and arsH encoding the ArsH MAs(III) oxidase. SpArsR has two conserved cysteine residues, Cys101 and Cys102. Mutation of either resulted in loss of MAs(III) binding, indicating that they form an MAs(III) binding site. SpArsR can be converted into an As(III)-responsive repressor by introduction of an additional cysteine that allows for three-coordinate As(III) binding. Our results indicate that SpArsR evolved selectivity for MAs(III) over As(III) in order to control expression of genes for MAs(III) detoxification.

Figures

Fig. 1
Fig. 1. Genes for a novel type of arsR are linked with arsP or arsH in ars operons
Shown are representative ars operons (accession numbers in parentheses) containing arsR and arsP or arsH genes (black fill). Vibrio splendidus 12B01 (AAMR00000000.1), Yersinia enterocolitica subsp (AM286415), Plesiomonas shigelloides 302–73 (LT575468.1), Escherichia coli IAI39 (NC_002695), Aeromonas veronii B565 (CP012504.1), Citrobacter freundii CFNIH1 (CP007557.1), Desulfobulbus propionicus DSM 2032 (NC_014972.1), Marinobacterium jannaschii (JHVJ00000000.1), Shewanella woodyi ATCC 51908 (NC_010506.1), Endozoicomonas elysicola (JOJP00000000.1), Photobacterium sp. SKA34 (GCF_000153325.1), Serratia liquefaciens FK01 (NC_021741.1), Edwardsiella tarda ATCC 23685 (ADGK00000000.1), Serratia plymuthica PRI-2C (CP015613.1), Shewanella putrefaciens 200 (NC_017566.1).
Fig. 2
Fig. 2. Multiple alignment of SpArsR homologs and homology modeling
A: Representative ArsR orthologs (accession numbers in parentheses) are from: S. putrefaciens (ADV53698); Enterovibrio calviensis (WP_017007765); Sinorhizobium meliloti 1021 (NP_385183); A. ferrooxidans (ACK80311); Propionibacterium avidum (WP_063279264); C. glutamicum CgArsR1 (CAF21518); R773 ArsR (CAA34168); Synechocystis sp. PCC 7942 SmtB (CAA45872) and plasmid pI258 CadC (P20047). The multiple alignment was calculated with CLUSTAL W. B: Homology modeling of As(III)-bound AfArsR and MAs(III)-bound SpArsR were performed as described in Experimental Procedures. The cysteines are shown in sticks. The arsenic atoms are shown as spheres.
Fig. 3
Fig. 3. SparsR and SparsP are linked transcriptionally by MAs(III) induction
The SparsR and SparsP are transcribed in opposite directions. Shown is the location of the primers for RT-PCR analysis (Table S1) of the ars transcripts. Total RNA isolated from S. putrefaciens 200 in exponential phase (A600nm = 1.2) in the presence of 2 μM MAs(III) or 20 μM As(III) were used as templates in reverse transcriptase reactions to generate cDNA and then amplified with the indicated primers. PCR products were resolved on a 1% agarose gel stained with ethidium bromide. Genomic DNA from S. putrefaciens was used as a positive control for each primer set.
Fig. 4
Fig. 4. Binding of MAs(III) to SpArsR involves specific cysteine residues
Expression of the gfp reporter gene was assayed as described under Experimental Procedures. A) Mutagenesis of Cys83, Cys101 and Cys102 and their contribution to MAs(III) binding. Cells were grown in M9 medium for 14 h. Cells of E. coli strain AW3110(DE3) bearing plasmids with wild type SpArsR, C83S, C101S or the C102S mutant in trans with reporter plasmid pACYC184-ParsP-gfp were grown without arabinose (solid black bars ((left)), 0.2% arabinose (open bars (middle)), or 0.2% arabinose and 2 μM MAs(III) (solid grey bars (right)). B) Comparison of the response of the bacterial biosensor to inorganic and organic arsenicals. Additions: (●), As(III); (▼), As(V); (△), MAs(III); (■), MAs(V). C) SpArsR can be converted to an As(III)-responsive ArsR by introduction of an additional cysteine residue from AfArsR. SpArsR with addition of the C-terminus of AfArsR containing AfArsR Cys102 (SpArsRAC) responds to As(III) four-fold better than the wild type. As(III) and MAs(III) were assayed at the indicated concentrations. Fluorescence intensities of cell suspensions were quantified using a Photon Technology International spectrofluorometer with an excitation wavelength of 470 nm and emission wavelength of 510 nm. The data are the mean ± SE (n = 3).
Fig. 4
Fig. 4. Binding of MAs(III) to SpArsR involves specific cysteine residues
Expression of the gfp reporter gene was assayed as described under Experimental Procedures. A) Mutagenesis of Cys83, Cys101 and Cys102 and their contribution to MAs(III) binding. Cells were grown in M9 medium for 14 h. Cells of E. coli strain AW3110(DE3) bearing plasmids with wild type SpArsR, C83S, C101S or the C102S mutant in trans with reporter plasmid pACYC184-ParsP-gfp were grown without arabinose (solid black bars ((left)), 0.2% arabinose (open bars (middle)), or 0.2% arabinose and 2 μM MAs(III) (solid grey bars (right)). B) Comparison of the response of the bacterial biosensor to inorganic and organic arsenicals. Additions: (●), As(III); (▼), As(V); (△), MAs(III); (■), MAs(V). C) SpArsR can be converted to an As(III)-responsive ArsR by introduction of an additional cysteine residue from AfArsR. SpArsR with addition of the C-terminus of AfArsR containing AfArsR Cys102 (SpArsRAC) responds to As(III) four-fold better than the wild type. As(III) and MAs(III) were assayed at the indicated concentrations. Fluorescence intensities of cell suspensions were quantified using a Photon Technology International spectrofluorometer with an excitation wavelength of 470 nm and emission wavelength of 510 nm. The data are the mean ± SE (n = 3).
Fig. 4
Fig. 4. Binding of MAs(III) to SpArsR involves specific cysteine residues
Expression of the gfp reporter gene was assayed as described under Experimental Procedures. A) Mutagenesis of Cys83, Cys101 and Cys102 and their contribution to MAs(III) binding. Cells were grown in M9 medium for 14 h. Cells of E. coli strain AW3110(DE3) bearing plasmids with wild type SpArsR, C83S, C101S or the C102S mutant in trans with reporter plasmid pACYC184-ParsP-gfp were grown without arabinose (solid black bars ((left)), 0.2% arabinose (open bars (middle)), or 0.2% arabinose and 2 μM MAs(III) (solid grey bars (right)). B) Comparison of the response of the bacterial biosensor to inorganic and organic arsenicals. Additions: (●), As(III); (▼), As(V); (△), MAs(III); (■), MAs(V). C) SpArsR can be converted to an As(III)-responsive ArsR by introduction of an additional cysteine residue from AfArsR. SpArsR with addition of the C-terminus of AfArsR containing AfArsR Cys102 (SpArsRAC) responds to As(III) four-fold better than the wild type. As(III) and MAs(III) were assayed at the indicated concentrations. Fluorescence intensities of cell suspensions were quantified using a Photon Technology International spectrofluorometer with an excitation wavelength of 470 nm and emission wavelength of 510 nm. The data are the mean ± SE (n = 3).
Fig. 5
Fig. 5. Evolutionary relationships of SpArsR with ArsRs from members of other bacterial species
A neighbor-joining phylogenetic tree shows that there are four type of ArsRs (boxes) with different placement of As(III)- or MAs(III)-binding cysteine residues. SpArsR and AfArsR, are indicated by black triangles.
Fig. 6
Fig. 6. Location of metal(loid) binding sites in ArsR/SmtB repressors
Metal(loid) binding sites in members of ArsR/SmtB family of repressor proteins are shown on a surface model of the CadC aporepressor structure by coloring CadC residues corresponding to each binding site as identified from the structure-based alignment (Fig. 2A). The S3 As(III) binding site of the R773 ArsR (red) formed within each monomer overlaps with the corresponding S4 Cd(II) binding site of CadC (yellow) formed between the N-terminus of one subunit and the DNA binding domain of the other subunit. The S3 binding sites of CgArsR1 (green) include residues in the DNA binding site. The Zn(II) binding sites of CadC and SmtB (cyan) are formed between the antiparallel C-terminal α6 helices. The S3 As(III)-binding site of AfArsR (purple) and the S2 MAs(III)-binding site of SpArsR (blue) differ by a single cysteine residues. The variety of the location of metal(lloid) binding sites distributed over the surface of the respective repressors demonstrates the plasticity of evolutionary solutions to similar environmental stresses.

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