(Received for publication, August 23, 1995; and in revised form, November 20, 1995)
From the
Plasmid-encoded arsenical resistance (ars) operons
confer high level resistance to arsenicals and antimonials, while the
chromosomally encoded ars operon of Escherichia coli bestows low level resistance. The transcriptional start site of
the chromosomal ars mRNA was mapped by primer extension, and
putative -10 and -35 promoter recognition sites were
identified. The arsR gene, the first gene in this operon, was
cloned using polymerase chain reaction. The arsR gene product,
the ArsR repressor, was expressed and purified. The results of gel
mobility shift assays indicated that the repressor is a DNA binding
protein that binds to a fragment of DNA containing the chromosomal ars promoter. The specific binding site, as determined by
DNase I footprint analysis, spans 33 nucleotides in the promoter
region, including the putative -35 promoter element. By
construction and expression of a series of in-frame fusions between
truncated arsR genes and the coding region for the mature form
of -lactamase (blaM`), it was shown that ArsR is a trans-acting repressor that regulates expression of the
chromosomal ars operon. In addition, the chromosomally-encoded
repressor can regulate expression of the ars operon of plasmid
R773, and the R773 repressor can cross-regulate expression from the
chromosomal operon.
The Escherichia coli chromosomal ars operon
was identified first by analysis of the E. coli genome (Sofia et al., 1994) and later by examination of metal-responsive
gene fusions (Diorio et al., 1995). It was shown to have three
open reading frames, originally termed arsEFG that were
subsequently renamed arsRBC (Carlin et al., 1995)
because of their high degree of sequence similarity to the plasmid R773
homologues (Chen et al., 1986; San Francisco et al.,
1990). The chromosomal operon was shown to confer resistance to
arsenite and antimonite in E. coli, with resistance
correlating with increased extrusion of arsenite (Carlin et
al., 1995), as has been shown for the plasmid-encoded resistances
(for reviews, see Dey and Rosen(1995) and Rosen et al.(1995)).
The level of resistance conferred by the chromosomal operon was
considerably less than the high level of resistance produced by the
operons of the staphylococcal plasmids pI258 and pSX267 (Ji and Silver,
1992; Rosenstein et al., 1992) or the E. coli plasmids R773 or R46 (Hedges and Baumberg, 1973; Silver et
al., 1981; Mobley et al., 1984). In all plasmid-borne ars operons, transcription was controlled by the ArsR
repressor, the product of the first gene of each operon. These are all
members of the ArsR family of regulatory proteins (Shi et al.,
1994). Other members of the ArsR family include
Cd/Zn
regulatory proteins (Yoon et al., 1991; Ivey et al., 1992; Morby et
al., 1993). All are believed to be metal-inducible repressor
proteins that control the basal level expression of their respective
operons (Wu and Rosen, 1991, 1993; Morby et al., 1993;
Rosenstein et al., 1994).
The E. coli chromosomal arsR gene encodes a 13-kDa protein, ArsR, in which 75% of the residues (88 of 117) are identical to those of the plasmid R773 repressor in primary amino acid sequence but only 26% (34 of 117) are identical to the staphylococcal plasmids pI258 or pSX267 ArsR proteins (Carlin et al., 1995). In this study, the chromosomal protein was shown to be a trans-acting regulator of both the E. coli chromosomal and the R773 plasmid ars operons. The gene was expressed at a high level, and ArsR was purified. The purified protein eluted from a gel filtration at a position corresponding with that of a 26-kDa homodimer. In gel shift DNA binding assays, the purified protein retarded the migration of DNA fragments containing either the chromosomal or R773 plasmid ars promoters. From DNase I footprint analysis, the ArsR binding site was found to span nucleotides -64 to -31 of the chromosomal operon.
Figure 1:
Induction of ampicillin resistance by
arsenite and phenylarsine oxide. Cells (10) of E. coli strain AW3110 bearing plasmid pCRBB91 (arsB::blaM`) were
spread onto an LB plate containing 150 µg/ml ampicillin. Potential
inducers (8 µl) were added onto filter disks, and the plate was
incubated overnight at 37 °C. Inducer strength was estimated from
the diameter of ap
cells. 1, no inducer; 2, 1 µM PAO; 3, 10 µM PAO; 4, 50 µM PAO; 5, 10 µM sodium arsenite; 6, 50 µM sodium arsenite; 7, 150 µM sodium arsenite. The internal ring of
nongrowth with 50 µM PAO reflects PAO
toxicity.
Figure 2: Regulatory region of the chromosomal ars operon. A, promoter region of the R773 ars operon. The contact points between the R773 ArsR repressor and DNA are enclosed in boxes (Wu and Rosen, 1993). B, promoter region of the chromosomal ars operon. The deduced amino acid sequence for the open reading frame corresponding to the ArsR protein is given below the coding strand. The shaded sequence indicates the binding site for ArsR as defined by DNase I footprinting. The boxed sequences are identical to those identified to the contact points between the R773 ArsR repressor and DNA. The locations of fusion sites between arsR and blaM` are indicated below the sequence by the arsR codon numbers at the fusion sites. For both A and B, +1 indicates the start site of ars transcription, with the presumed -10 and -35 promoter elements and the most likely Shine-Dalgarno sequence sites underlined.
Figure 3:
Expression of the arsR::blaM` fusion genes and their regulation by arsR in trans. Equal amounts of total cell protein from cells of E. coli strain AW3110 harboring the indicated plasmids were
separated on 10% SDS-polyacrylamide gels followed by immunoblotting
with antiserum against TEM -lactamase. A, to measure
expression of the fusions, cells bore plasmids pCRB20 (lanes 1 and 2), pCRB69 (lanes 3 and 4), pCRB74 (lanes 5 and 6), pCRB79 (lanes 7 and 8), pCRB92 (lanes 9 and 10), pCRB101 (lanes 11 and 12), pCRB104 (lanes 13 and 14), and pCRB114 (lanes 15 and 16). Cells
were grown in the absence (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or
presence (lanes 2, 4, 6, 8, 10, 12, 14, and 16) of 50
µM sodium arsenite. For reference, the arrow indicates the predicted mass of the ArsR-BlaM chimera from plasmid
pCRB20. B, to examine regulation by fusions cells bore both
plasmid pCX12 and arsR::blaM` fusion plasmids pCRB69 (lanes 1 and 2), pCRB79 (lanes 3 and 4), pCRB92 (lanes 5 and 6) or pCRB114 (lanes 7 and 8). Cells were cultured without (lanes 1, 3, 5, and 7) or with (lanes 2, 4, 6, and 8) 50
µM sodium arsenite. For reference, the arrow indicates the predicted mass of the ArsR-BlaM chimera from plasmid
pCRB69.
Figure 4: Primer extension analysis of the transcription start site of the chromosomal ars operon. Primer extension and nucleotide sequencing assays were performed as described under ``Materials and Methods.'' Lane 1 shows the primer extension product; lanes 2-5 show the nucleotide sequence ladders generated with the same primer. The arrow indicates the position of the transcriptional initiation site. The corresponding DNA sequence of the coding strand is shown on the left. The start codon of the arsR gene, the Shine-Dalgarno sequence, and the first nucleotide of the chromosomal ars transcript are indicated.
Figure 5:
ArsR protein-DNA interaction. Mobility
shift assays were performed as described under ``Materials and
Methods.'' Two different DNA fragments were radiolabeled with
[-
P]dATP and incubated with the indicated
repressor proteins. The binding mixtures were analyzed on 6%
polyacrylamide gels. A, the radiolabeled DNA was the PCR
product containing the chromosomal ars promoter region
digested with MunI. B, the probe was a 153-bp EcoRI-DraI DNA fragment containing the R773 ars promoter region. The middle lanes contained DNA incubated
with 3 µg of purified chromosomal ArsR. The right lanes contained DNA incubated with 3 µg of purified R773
ArsR.
Figure 6:
Effect
of inducers of the chromosomal ars operon on ArsR-DNA complex
formation. Mobility shift assays were performed as described under
``Materials and Methods.'' The PCR product containing the
chromosomal ars promoter region was digested with MunI, radiolabeled with [-
P]dATP
and incubated with 3 µg of purified ArsR. Potential inducers were
added individually to the binding mixtures. The binding mixtures were
analyzed on 6% polyacrylamide gel. All lanes contained probe DNA; lanes 2-13 also contained ArsR; lane 3, 0.5
mM potassium antimonial tartrate; lane 4, 5 mM potassium antimonial tartrate; lane 6, 0.5 mM sodium arsenite; lane 7, 5 mM sodium arsenite; lane 9, 0.1 µM PAO; lane 10, 1
µM PAO; lane 12, 0.5 mM sodium arsenate; lane 13, 5 mM sodium
arsenate.
Figure 7: Identification of the binding site of ArsR on the chromosomal ars promoter. DNA fragments of the chromosomal ars promoter were labeled at nucleotide +42 (coding strand, panel A) and -128 (noncoding strand, panel B) and subjected to DNase I footprint analysis using purified chromsomal ArsR, as described under ``Materials and Methods.'' Regions protected by ArsR are indicated by filled boxes. In each panel, lanes 1 and 2 contain the A and C reactions from nucleotide sequencing of the same fragment; lanes 3-8 contain DNase I-treated DNA; lanes 3 and 8, no ArsR protein; lane 4, 0.5 µg of ArsR; lane 5, 2 µg of ArsR; lane 6, 4 µg of ArsR; lane 7, 8 µg of ArsR. The transcriptional initiation start site is indicated in B.
The chromosomal ars operon of E. coli was originally identified by sequencing of the E. coli genome (Sofia et al., 1994). This operon is responsible for the basal level resistance to arsenite, antimonite, and arsenate in plasmidless strains of E. coli (Carlin et al., 1995). The operon has three genes, arsR, arsB, and arsC. From the 75% sequence similarity of chromosomal ArsR with the ArsR repressor encoded by the ars operon of plasmid R773 (San Francisco et al., 1990), a regulatory function was proposed for the chromosomal ArsR (Sofia et al., 1994; Carlin et al., 1995).
In this study, the chromosomal arsR gene was shown to regulate expression of reporter arsR::blaM` genes in trans (Fig. 3B). Purified chromosomal ArsR, which was found to elute from a gel filtration column at a size corresponding to that of a homodimer, bound to promoter DNA (Fig. 5). Arsenite and antimonite did not dissociate the complex in concentrations at which they induce in vivo, which may suggest that dissociation is not required for induction. On the other hand, phenylarsine oxide, the only organoarsenical found thus far to induce, prevented retardation of the promoter DNA (Fig. 6) and reversed protection from DNase I digestion at 1 µM (data not shown), the same concentration at which PAO is an inducer in vivo (Fig. 1). These results indicate that induction results when the repressor dissociates from the DNA.
The region of the DNA protected from DNase I digestion by ArsR overlaps with the putative -35 element of the chromosomal ars promoter and covers the region from nucleotides -64 to -31 ( Fig. 2and Fig. 4). The plasmid R773 ArsR repressor has been shown to bind to the R773 ars promoter at a region of imperfect dyad symmetry just upstream of the -35 site (Wu and Rosen, 1993). Although the overall sequences of the two promoters and the location of the protected sequences are different between the two ars operons, higher resolution analysis of the R773 ArsR binding site revealed that only two small regions of 4 bp each (TCAT and TTTG of the coding strand) are protected separated by 7 bp (Wu and Rosen, 1993). Since the chromosomal ars sequence that was protected from DNase I by chromosomal ArsR also contained the TCAT and TTTG elements separated by 7 bp, it was possible that the two repressors could bind to the each other's promoter. As shown in Fig. 5, the chromosomal ArsR repressor retarded the migration of DNA containing the R773 ars promoter, and R773 ArsR retarded the corresponding chromosomal ars DNA. Both proteins protected the same regions of both promoters from DNase I digestion (data not shown). Thus the sequence TCATNNNNNNNTTTG appears to represent a consensus binding site for the two ArsR repressors. It is interesting that the chromosomal and plasmid R773-encoded ArsR proteins from E. coli are essentially interchangeable, even though the two proteins are 25% dissimilar, and their promoter regions contain significant differences in sequence and placement of the regulatory elements. On the other hand, the homologous ars repressor from the staphylococcal plasmid pSX267 protects two regions within the promoter region from DNase I digestion (Rosenstein et al., 1994), but this region does not contain TCATNNNNNNNTTTG, suggesting that it is a Gram-negative consensus sequence. The binding of the ArsR repressors to each other promoters may have physiological significance; in vivo when the chromosomal arsR gene was carried on a compatible plasmid with lacZ gene fused to the R773 ars promoter, expression of the reporter gene became arsenite inducible (data not shown).
We would propose that all members of the ArsR family of metalloregulatory proteins contain at least four domains. First, we have shown that a putative DNA binding domain in the R773 repressor is required for repression (Shi et al., 1994). Second, we have shown that Cys-32 and Cys-34 of R773 ArsR are part of a metal binding domain involved in induction (Shi et al., 1994). However, in the ArsR family of transcriptional repressors, both arsenic/antimony and cadmium/zinc responsive repressors have this cysteine pair. To account for differential recognition of metals, we would propose the existence of an additional metal discrimination domain in the cadmium/zinc responsive regulatory proteins. In those repressors, there is an additional N-terminal sequence with two cysteinyl residues that might provide this function.
Finally, the ArsR repressors are most
likely functional homodimers, which indicates the existence of a
dimerization domain. When the mature form of -lactamase was fused
to the ArsR protein at residue 92, 101, 104, or 114, expression of the
chimeric protein was still inducible, indicating that residues 92 to
the C terminus are not required for ArsR function. Similar results were
obtained for C-terminal chimeras of R773 ArsR (Wu and Rosen, 1991), and
those chimeras were shown to be bind to the promoter DNA as dimers (Wu
and Rosen, 1993). These results demonstrate that information required
for dimerization is not contained in residues from 92 to the C
terminus. On the other hand, chimeras with fusions at residues 79 or
closer to the N terminus were constitutively expressed. Similar
chimeras in R773 ArsR were unable to bind to DNA. These results suggest
that residues in the region of 79-92 may be involved in
dimerization. In conclusion, members of the ArsR family of repressor
proteins are postulated to have a metal binding domain, followed by a
DNA binding domain. In some members there may also be an N-terminal
metal discrimination domain. Finally, there is most likely a
dimerization domain that may require residues C-terminal to the DNA
binding domain. A more detailed analysis of these proteins will be
necessary to identify this domain.