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.     

The ArsR protein is a trans-acting regulatory protein

The arsR gene encodes the regulatory protein of the plasmid-encoded arsenical resistance operon. A series of in-frame fusions was constructed between the C-terminally truncated arsR gene and the coding region for the mature form of beta-lactamase (blaM). Fusions containing most of the arsR gene were still inducible by arsenicals. Fusions containing less than 102 residues of the 117-residue ArsR protein were constitutive. When a wild-type arsR gene was placed in trans, the constitutive constructs were again inducible. The results demonstrate that the ArsR protein is a trans-acting regulatory protein which controls its own expression.     

Effects of carriage and expression of the Tn10 tetracycline-resistance operon on the fitness of Escherichia coli K12

We have been examining the consequences of alternative modes of regulation of plasmid-borne, Tn10-encoded tetracycline resistance for the fitness of Escherichia coli. In a tetracycline-free environment, we measured the effects on fitness that were caused by (1) maximally induced expression of the resistance operon, (2) low-level constitutive expression of the resistance protein, (3) residual expression of the repressed resistance operon, (4) carriage of the resistance operon, (5) the remainder of the plasmid genome, and (6) hyperexpression of the repressor protein. We observed large reductions in fitness that were associated with induction and with constitutive expression of the tetracycline-resistance protein, but there was no discernible effect of hyperexpression of the repressor protein. We also observed a small reduction in fitness associated with the remainder of the plasmid genome. However, any reductions in fitness that were caused by residual expression and by carriage of the repressed operon were not more than 0.3%. We conclude that tight gene regulation has eliminated antagonistic pleiotropic effects of the resistance gene on fitness, so that possession of an inducible Tn10-encoded tetracycline-resistance operon imposes essentially no burden in the absence of antibiotic.     

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.     

Binding of ArsR,the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter

arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (rs) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 by DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 by polymerase chain reaction (PCR) fragment, which mainly comprised the ?35 and ?10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the ?35 and ?10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.     

Binding of ArsR, the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter

arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (rs) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 by DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 by polymerase chain reaction (PCR) fragment, which mainly comprised the –35 and –10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the –35 and –10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.     

Alleviation of type I restriction in Escherichia coli K12 in the presence of the arsR gene from pKW301 of Acidiphilium multivorum AIU 301

A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is on pKW301. In Escherichia coli, arsR cloned under the control of Plac in a multi-copy vector alleviated restriction of nonmodified lambda DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI). Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas the motif is in the C domain in the Ard proteins. The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs. The total charge is -4 in pKW301 ArsR and +2 in R64 and R733 ArsRs. A total negative charge was assumed to contribute to the antirestriction activity.