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(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
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 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
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
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
[
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 [
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
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2427-2432
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-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.
/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).
E. coli Strains, Plasmids, and Media
The
bacterial strains and plasmids used in this study are described in Table 1. E. coli cells were grown in LB medium at 37
°C. Ampicillin (100 µg/ml), kanamycin (80 µg/ml),
tetracycline (15 µg/ml) or chloramphenicol (20 µg/ml) were
added as required. For protein expression, 0.5 mM isopropyl-1-thio--D-galactopyranoside or 20
µM sodium arsenite were used as inducer, except where
otherwise noted. Plasmid pCX1 was made by cloning the SphI and XmaIII fragment from pWPS1 (Carlin et al., 1995) into
vector plasmid pJBS633 (Broome-Smith and Spratt, 1986) that had been
digested with both SphI and XmaIII. This fragment
contained 630 bp (
)upstream of the translational initiation
point of the arsR gene, the entire arsR gene, and
part of the arsB gene. To remove a PvuII site
upstream of the ars promoter but within the insert, a XcmI fragment was deleted from plasmid pCX1, producing plasmid
pCX2. Removal of the PvuII site allowed the PvuII
site in front of the blaM` gene to be used for construction of arsR::blaM fusions (see below). To delete the tet promoter in the vector sequence, plasmid pCX2 was digested with BssHII and EcoRV, filled in with the Klenow fragment
of DNA polymerase, and intramolecularly ligated to produce plasmid
pCX3. Plasmid pCX11 was created by ligating the HindIII-EcoRV fragment, which consists of 630 bp
upstream of the translational initiation point of arsR, the
entire arsR gene, and part of the arsB gene from
plasmid pWPS1 into pJBS633 that had been digested with both HindIII and EcoRV. Plasmid pCX12 was derived from
plasmid pCX11 by cloning the EcoRI-ScaI fragment
containing the chromosomal ars promoter and arsR gene
into EcoRI-ScaI-digested plasmid pACYC184 (Chang and
Cohen, 1978).
DNA Manipulation and Sequencing
Preparation of
plasmid DNA was performed by using a Wizard DNA purification kit
(Promega). Endo- and exonuclease digestions, DNA fragments separations
and isolations, ligations, transformations, and Klenow fragment fill in
were performed according to standard procedures (Sambrook et
al., 1989) unless otherwise noted. The Sequenase kit (version 2.0,
U. S. Biochemical Corp.) was employed for sequencing of double-stranded
DNA.Polymerase Chain Reaction
Polymerase chain
reactions (PCR) (Mullis and Faloona, 1987) were run on a Stratagene
Robocycler 40 thermal cycler. The reactions contained in a final volume
of 50 µl of 2 mM Tris-HCl (pH 8), 1 mM EDTA, 0.1
mM dithiothreitol, 0.5 mg/ml bovine serum albumin, 5% (v/v)
glycerol, 1.5 mM MgCl
, 2.5 units of Taq DNA polymerase, 0.1 mM each deoxynucleoside triphosphate,
0.25 µg of each primer, and 20 µg of DNA. Each reaction was
overlaid with 20 µl of light mineral oil and cycled 30 times: 94
°C for 1 min, 60 °C for 0.5 min, 72 °C for 1 min, followed
by a single cycle at 72 °C for 10 min.Cloning of the arsR Gene
Two PCR primers were used
for PCR cloning of the arsR gene. The forward primer had the
sequence 5`-ATCAGGAGCGCCATATGTC-3`. This introduces an NdeI
site at the start codon of the arsR gene by changing the third
A before the start codon ATG in the original sequence to C. The reverse
primer had the sequence 5`-TCCCGGATAAAACACATCTG-3`, corresponding to
the 20 nucleotides immediately downstream of the coding segment of the arsR gene. The PCR product was first cloned into vector
plasmid pGEM-T (Promega), generating plasmid pCR0. The PCR product was
sequenced to confirm that no errors had occurred during amplification.
A 375-bp NdeI fragment was then isolated from pCR0 and cloned
into the expression vector pT7-7 (Tabor and Richardson, 1985).
Plasmid pCR1 was shown to have the insert in the proper orientation for
expression.Expression and Purification of ArsR
Cells of E. coli strain BL21(DE3) bearing construct pCR1 were grown
overnight in LB medium containing ampicillin at 37 °C. The culture
was diluted to 100-fold with fresh, prewarmed medium and grown at 37
°C. When the culture reached an A of 0.8,
expression of the arsR gene was induced by addition of 0.5
mM isopropyl-1-thio-
-D-galactopyranoside for an
additional 3 h. Under these growth conditions, the majority of ArsR was
found as a soluble protein in the cytosolic fraction. Induced cells
were harvested by centrifugation and washed once with buffer A (50
mM Tris-HCl, pH 7.5, 1 mM EDTA, and 14.4 mM -mercaptoethanol). The pelleted cells were suspended in 5 ml
of buffer A/g of wet cells and disrupted by a single passage through a
French pressure cell at 20,000 p.s.i. Unbroken cells and membranes were
removed by centrifugation at 150,000 
g for 1 h. The
soluble fraction was loaded onto a 2.5-cm diameter column filled to 22
cm with S-Sepharose (Pharmacia Biotech Inc.) pre-equilibrated with
buffer A. The column was eluted with 600 ml of a linear gradient of
0.1-0.8 M NaCl in the same buffer. Fractions of 6 ml
were collected and analyzed by SDS-polyacrylamide gel electrophoresis
(Laemmli, 1970). Fractions containing ArsR were pooled and
concentrated. A portion of the concentrated protein was applied to a
2-cm diameter column filled to 45 cm with Superose 12 (Pharmacia) and
eluted with buffer A containing 0.2 M NaCl. Fractions were
analyzed by SDS-polyacrylamide gel electrophoresis, and those
containing ArsR were pooled, concentrated, and stored at -70
°C until use. From the intensity of staining, ArsR was judged to be
approximately 80-90% pure.Construction of arsR::blaM` Gene Fusions
To create
a series of chromosomal arsR::blaM` gene fusions, plasmid pCX3
was linearized by digestion at the unique XmaIII site followed
by exonuclease III digestion. The digestion products were removed at
various times during ExoIII digestion and pooled. The ends
were flushed with S1 nuclease, followed by digestion with PvuII. The resulting linear fragments were self-ligated with
T4 DNA ligase, and the ligation mixture was transformed into cells of E. coli strain JM109 made competent by the method of Chung et al.(1989). Kanamycin-resistant transformants were selected.
Cells with in-frame fusions of arsR gene to the portion of the blaM` gene containing the coding region of the mature form of
the TEM -lactamase were identified by growth as patches on LB agar
containing 100 µg/ml ampicillin and 50 µM sodium
arsenite, and confirmed by digestion of BamHI and PstI. To determine the fusion site of blaM` with arsR, plasmids were sequenced using a primer with the sequence
5`-GTCGTGCACCCAACTGA-3`, which is complementary to codons 14-18
of the mature form of TEM -lactamase. The resulting arsR::blaM` fusion plasmids were named as the pCRB series,
with the numbers representing the amino acid residue number of the ArsR
protein to which the mature TEM -lactamase was fused.Expression of ArsR-
Cells of E. coli strain AW10 bearing each arsR::blaM` fusion plasmid alone or both the fusion plasmid
and plasmid pCX12, which contains a wild-type arsR gene, were
grown overnight at 37 °C in LB medium containing kanamycin or both
kanamycin and chloramphenicol, respectively. The cultures were diluted
100-fold into fresh LB medium and grown at 37 °C to an absorbance
of 0.6 at 600 nm, at which time 50 µM sodium arsenite was
added as inducer. Growth was continued at 37 °C for 1 h following
induction. The cells were collected by centrifugation and suspended in
0.1 ml of SDS sample buffer for analysis. For immunoblotting, the
proteins were transferred to a nitrocellose membrane using a transblot
apparatus (Bio-Rad) according to the manufacturer's instructions.
Proteins were electrophoretically transferred to a nitrocellulose sheet
(0.2 µm pore size) and immunoblotted as described previously (Tisa
and Rosen, 1990). A chemiluminescent assay was used to detect the
antigen-antibody reaction. The filter was incubated with 10 ml of the
enhanced chemiluminescence solution (Amersham Corp.) and exposed on
x-ray film for 1 min at room temperature.-Lactamase Fusion
ProteinsIsolation of RNA
TRI Reagent(TM)
RNA/DNA/protein isolation reagent (MRC, Inc.) was used to isolated
total cellular RNA according to the manufacturer's directions
from cells of E. coli strain JM109 carrying plasmid pWPS1
induced with 20 µM sodium arsenite. RNA was suspended in
ethanol and stored at -70 °C until use.Primer Extension
A primer,
5`-CCAGACGGGTTTCATCAGCAAGAATTTTG-3`, corresponding to a region within
the coding sequence of arsR, end-labeled with
[-
P]ATP was used for primer extension
analysis (Sambrook et al., 1989). Total RNA was mixed with
end-labeled primer, dNTPs, and Superscript II RNaseH
reverse transcriptase (Life Technologies, Inc.). The primer
extension product was loaded on an 8% polyacrylamide, 8 M urea
sequencing gel. A size ladder produced by dideoxy sequencing of plasmid
pWPS1 with the same primer used in the primer extension reaction was
used to measure the length of the primer extension product.
Gel Mobility Shift and DNase I Footprinting
Assays
Gel mobility shift and DNase I footprinting assays were
performed as described previously (Wu and Rosen, 1993). A fragment
including the regulatory region DNA and start of the arsR gene
was generated by PCR using the primer extension oligonucleotide and a
second primer with the sequence 5`-CGGAATTCCGACGCAAAGTC-3`, containing
an EcoRI site at the 5`-end. The purified PCR product was
digested with MunI, labeled with
[
-P]dATP and the Klenow fragment of DNA
polymerase, and purified using a Wizard DNA purification kit (Promega).
This 208-bp labeled fragment was prepared for gel mobility shift assay
and coding strand footprinting. For footprinting the non-coding strand,
a 203-bp fragment was prepared by digesting the purified PCR product
with TfiI and was labeled as described above. A DNA probe
carrying the plasmid R773 ars promoter was prepared as
described previously (Wu and Rosen, 1993). A mixture of two DNA probes
containing the lac promoter (211 bp) and a nonspecific DNA
fragment (111 bp) were obtained by digestion of plasmid pUC19 with BamHI and PvuII, and labeled as described above. The
nucleotide size ladders were created by dideoxy sequencing of the
plasmid pCX11 using primer 5`-AATTGGATGGGTAACAGAA-3` for DNase I
footprinting of the coding strand and primer 5`-ATTCACCTCCTTTCAAATG-3`
for footprinting the noncoding strand.
Phenylarsine Oxide Is an Inducer of the ars
Operon
Phenylarsine oxide (PAO) is an organoarsenical containing
As that interacts strongly with proteins containing
vicinal cysteine pairs (Hoffman and Lane, 1992). Its ability to induce
the chromosomal ars operon was examined using plasmid pCRBB91,
which carried a reporter construct in which the blaM` gene was
fused to the arsB gene and expression of the gene fusion was
under control of the ars promoter. Cells with this plasmid
become ampicillin resistance when induced from the ars promoter. At low concentrations PAO (up to 10 µM) was
as or more effective an inducer of the operon in vivo than
arsenite itself (Fig. 1). Higher concentrations of PAO (in
excess of 0.1 mM) were toxic, producing a zone of clearing
around the disk. It should be pointed out, however, that in vivo induction is multifactorial, reflecting a combination of uptake
and transcriptional activation. Thus efficacy in vivo may not
correspond to in vitro action. As shown below, PAO is
considerably more effective in in vitro assays than either
arsenite or antimonite. This is the first demonstration of induction of
an ars operon by an organoarsenical.
) 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.
Expression and Purification of ArsR Protein
The arsR gene was isolated from the E. coli chromosomal ars operon by PCR, and ArsR was substantially purified. From
its elution from a Superose 12 column, the apparent molecular mass of
ArsR was calculated to be 25.9 kDa (data not shown), close to the
theoretical value of 26,504 Da for a homodimer, as had been suggested
for the R773 ArsR repressor (Wu and Rosen, 1993).Construction and Expression of arsR-blaM` Fusion
Genes
A series of plasmids were constructed in which the blaM` gene was fused in-frame to the arsR gene. Eight
translational fusions were obtained, with the fusion junctions from the
20th to the 114th aminoacyl residue of the 117-residue ArsR protein (Fig. 2). To study expression of the ArsR--lactamase
chimeras, the eight fusions were individually transformed into cells of E. coli strain AW10, in which the chromosomal ars operon has been deleted (Carlin et al., 1995). Proteins
from uninduced cells or cells induced with arsenite were analyzed by
SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with
antibody to -lactamase (Fig. 3A). Chimeras in which the
fusion sites were at residues 20, 69, 74, and 79 of the ArsR proteins
were produced in both arsenite-induced and uninduced cells, indicating
that these chimeric proteins were constitutively expressed. In
contrast, the hybrid ArsR--lactamase proteins from plasmids with
fusions at codons 92, 101, 104, or 114 of the arsR gene were
visible only in arsenite-induced cells. Thus expression of the chimeric
proteins in these four fusions remained inducible by arsenite.
-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.
The ArsR Protein Is a trans-acting Repressor
To
demonstrate that ArsR is a trans-acting repressor, the arsR gene was cloned into vector plasmid pACYC184, yielding
plasmid pCX12. Four arsR::blaM` fusion plasmids, pCBR69,
pCBR79, pCBR92, and pCBR104 were individually co-transformed with
plasmid pCX12 into E. coli strain AW10, and expression of
these four chimeric proteins with or without the inducer arsenite was
monitored by immunoblotting. Immunoreactive hybrid proteins were only
observed in arsenite-induced cells (Fig. 3B). Although
the chimeric proteins in pCBR69 and pCBR79 were constitutively
expressed in the absence of an arsR gene (Fig. 3A), in the presence of an arsR gene in trans they became inducible. There was no trans effect of a wild-type arsR gene on expression of the
fusions in pCBR92 and pCBR104, in which the chimeric ArsR proteins
still functioned as repressors (Fig. 3B). These results
demonstrate that ArsR is a trans-acting regulatory protein.
Furthermore, the sequence from the 92nd aminoacyl residue to the C
terminus is not required for the regulatory activity of the protein.Localization of the Transcription Initiation Site of the
Chromosomal ars Operon
To identify the promoter responsible for
chromosomal ars transcription, total RNA was isolated from
cells containing plasmid pWPS1, which contains the entire chromosomal ars operon, grown in LB medium with sodium arsenite as
inducer. The 5`-end of the RNA was determined by primer extension. The
same primer was used in a DNA sequencing reaction with plasmid pWPS1 as
template to determine the nucleotide to which the primer extension
product mapped. Only one primer extension product was observed; it
corresponded to a cytosine nucleotide located 16 nucleotides upstream
from the ATG start codon of arsR gene (Fig. 4).
Putative -10 and -35 promoter sequences were identified as
GACACT and TTGACT.
The ArsR Protein Is a DNA Binding Protein
Gel
mobility shift assays were used to examine the DNA binding activity of
ArsR. Both the chromosomal and R773 ArsR proteins were able to retard
the migration of DNA fragments containing either the chromosomal (Fig. 5A) or the plasmid R773 (Fig. 5B) ars promoters. However, with neither protein was a mobility
shift observed with DNA fragments carrying either the lac promoter or nonspecific DNA (data not shown). The chromosomal ArsR
protein could be dissociated from its promoter by addition of inducer (Fig. 6). However, neither arsenite and antimonite were very
effective in vitro, requiring nonphysiological concentrations
of inducer to effect dissociation. Although the explanation is unknown,
a similar unresponsiveness to arsenite had been noted for the R773 and
pSX267 ArsR proteins (Wu and Rosen, 1993; Rosenstein et al.,
1994). On the other hand, the organoarsenical PAO was extremely
effective, with 1 µM PAO sufficient to completely
dissociate the repressor.
-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.
-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.
DNase I Footprint Analysis of the Binding Site for the
ArsR Repressor in the Chromosomal ars Regulatory Region
Using
purified chromosomal ArsR, the site of binding to the chromosomal ars regulatory region was analyzed by DNase I protection
assays. A protected region of approximately 33 bp (from nucleotides
-64 to -31) in both the coding strand (Fig. 7A)
and the noncoding strand (Fig. 7B) was observed. Protection
was prevented by addition of the inducer PAO (data not shown). R773
ArsR protected this same region of the DNA, and chromosomal ArsR
protected the same region of the R773 promoter DNA as did the plasmid
repressor (data not shown).
-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.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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