ABSTRACT
SmtB is a member of a family of repressors which dissociate from DNA in the
presence of metals; Zn
2+
being the most potent inducer of metallothionein gene (
smtA
) transcription
in vivo
. In
Synechococcus
PCC 7942 cells devoid of chromosomal
smtB
, four plasmid-encoded mutants of SmtB (C61S, T11S/C14S, C121S and H105R/H106R) repressed
lacZ
expression driven by the
smtA
operator-promoter. Gel retardation assays with extracts from the complemented cells
detected multiple SmtB-dependent complexes similar to those obtained with extracts from wild-type cells or with recombinant-SmtB. Elevated [Zn
2+
] alleviated repression
in vivo
by all of the mutants except H105R/H106R. These His residues (one or both) are
therefore essential for Zn
2+
-sensing while, contrary to expectations, Cys residues are not. Hence
different motifs facilitate metal-induced DNA-dissociation by SmtB and ArsR (the related oxyanion-sensing repressor), presumably generating variety in the
spectra of metals sensed. Nucleotides and amino acids involved in DNA-SmtB interaction have been further defined/inferred and we also confirm
that additional unknown factors form specific associations with the
smt
operator-promoter in elevated [Zn
2+
].
The cyanobacterial metallothionein divergon
smt
, which includes the metallothionein gene
smtA
and a divergently transcribed gene
smtB
, is required for normal tolerance to Zn
2+
and, to some extent, to Cd
2+
(
1
). The
smtB
gene encodes a
trans
-acting repressor required for Zn
2+
-responsive expression of
smtA
and
Synechococcus
PCC 7942 cells deficient in
smtB
show highly elevated constitutive expression of a reporter gene associated with
the
smtA
operator-promoter (
2
). Three complexes (designated MAC1, MAC2 and MAC3) were detected in gel
retardation assays with a 100 bp DNA fragment containing the
smt
operator-promoter and protein extracts from
Synechococcus
PCC 7942. One of these complexes (MAC1) was missing when extracts from mutants
deficient in
smtB
were used (
3
). Re-introduction of plasmid-borne
smtB
restored ability to form MAC1 and it was proposed that SmtB is the protein
component of MAC1 (
3
). MAC1 shows Zn
2+
-dependent dissociation and treatment with Zn
2+
-chelators facilitates reassociation of MAC1
in vitro
(
3
). Recombinant SmtB has more recently been expressed in
Escherichia coli
and confirmed to bind to the
smt
operator-promoter (
4
). At low protein concentrations a single complex (presumed to be analogous to
MAC1) formed with the
smt
operator-promoter and was shown by methylation interference assays to result from
SmtB binding to (either one of) a pair of sites located near the
smtA
transcription start site (
4
). Multiple complexes were detected in gel retardation assays performed with
higher concentrations of recombinant SmtB (
4
). This suggested that the MAC2 and MAC3 complexes may in fact be composed of
multimeric SmtB rather than additional unknown proteins, as previously proposed
(
3
). However, different conditions were used for the gel retardation assays in the
two studies (
3
,
4
). Gel retardation assays have therefore been performed with recombinant SmtB
and with protein extracts from both
Synechococcus
PCC 7942 and mutants deficient in
smtB
(i) to confirm that recombinant SmtB does indeed form multiple complexes with
the
smt
operator-promoter, and (ii) to test whether or not specific complexes involving
proteins other than SmtB also form with the
smt
operator-promoter.
SmtB was shown (
2
,
3
) to have sequence similarity to ArsR, the metal-oxyanion responsive repressors of the arsenic-resistance (
ars
) operons (
5
-
8
), to MerR associated with Hg
2+
resistance in
Streptomyces
lividans
(
9
; note that this is distinct from other MerR `transcriptional switch' proteins)
and to CadC, which in
Staphylococcus aureus
is known to be the Cd
2+
-responsive repressor of expression of the Cd
2+
-efflux ATPase CadA (
10
). SmtB, ArsR and CadC have been referred to as the arsR family of
metalloregulatory proteins (
11
). These proteins contain conserved Cys residues associated with the N-terminal extremity of putative DNA-binding helix-turn-helix motifs (
12
). These Cys residues were predicted to bind to metals via formation of metal-thiolate bonds, and in so doing inhibit binding to DNA via the adjacent
helix-turn-helix region (
12
). The `motifs' programme (available in the UWGCG package on the SEQNET service)
currently searches for this proposed DNA-binding, metal-sensing arsR family signature (predicted from ArsR, CadC and SmtB
sequences) within novel protein sequences. Consistent with the motif
prediction, mutants of ArsR in which these Cys residues are changed (to Phe or
Tyr) have subsequently been selected based upon their ability to bind to the
ars
promoter but inability to respond to inducer, antimonite (
11
). Sequences of the arsR family members, and additional proteins with sequence
similarity to SmtB which are also now known (
13
,
14
), are given in Figure
1
. These include a novel sequence derived from the
Synechocystis
PCC 6803 genome project (
15
) which we have designated PacR due to the location of its coding region;
transcribed divergently from a gene which encodes a deduced copper-ATPase, PacS.
Cyanobacterial strains used were R2-PIM8, a derivative of
Synechococcus
PCC 7942 which is cured of the small plasmid (
20
), and an
smt
mutant of R2-PIM8, deficient in both
smtA
and
smtB
[previously designated R2-PIM8(
smt
)] (
1
). Cells were cultured as described previously (
1
) and where appropriate dl-methionine (30 [mu]g/ml), streptomycin (5 [mu]g/ml), chloramphenicol (7.5 [mu]g/ml) and carbenicillin (50 [mu]g/ml) were added to the growth medium.
E.coli
strain JM101 was grown at 37oC in/on LB medium (
21
) supplemented with 100 [mu]g/ml
carbenicillin and 34 [mu]g/ml chloramphenicol, as necessary. Competent
E.coli
cells were prepared and used according to the methods of Chung
et al
. (
22
). Transformation of
Synechococcus
cells was performed as previously described (
1
). Restriction and modification enzymes were supplied by New England Biolabs
Inc. or Promega Corp., [[alpha]-
32
P]dCTP and [[alpha]-
32
P]dATP were obtained from Amersham International plc. Other chemicals and
antibiotics were purchased from Sigma Chemical Co.
Plasmid pJHNR49 (
2
), 1 [mu]g, which contains the
smt
divergon (Fig.
2
A) was used as template DNA in polymerase chain reactions (PCR), performed as
described previously (
2
), with an M13 Reverse primer (as a 5' primer) and mismatched 3' primers. The 3' primers were designed to anneal immediately upstream of
the
smtA
coding region and introduce specific mutations, transversions, within the
smt
operator-promoter (MUT1-4; Fig.
2
B) and a
Bam
HI site at the 3'-end. The PCR amplification products (602 bp), which contained the mutated operator-promoter regions and the entire
smtB
coding sequence, were ligated into the
Hin
dIII/
Bam
HI site of pSK+ (Stratagene Ltd) and checked by nucleotide sequence analysis
prior to subcloning into the
Sal
I/
Bam
HI site of pLACPB2 (
23
) to create transcriptional fusions with
lacZ
(Fig.
2
B). The control construct pLACPB2(
smt
-5'), referred to herein as pWT, was generated previously (
2
) and contains 602 bp from upstream of
smtA
(equivalent to those contained within the four mutant operator-promoter constructs) fused to
lacZ
in pLACPB2 (Fig.
2
B).
A 512 bp
Bsp
HI-
Hin
dIII DNA fragment from pJHNR49 (
2
) containing the entire SmtB coding region was introduced into the
Nco
I/
Hin
dIII site of pKK233-2 (Pharmacia Biotech Ltd) to create plasmid pSmtB where
smtB
is under the control of the strong
trc
promoter. Cultures of
E.coli
harbouring pSmtB (or pKK233-2) were exposed (4 h) to 1 mM isopropyl [beta]-d-thiogalactopyranoside (IPTG), crude cell extracts
prepared as described previously (
3
) and in some experiments fractionated using heparin Sepharose (Pharmacia). Heparin Sepharose columns (2 ml) were equilibrated with 20 mM HEPES buffer (pH 2.8), 50 mM NaCl, 0.5 mM dithiothrietol and 0.5 mM phenylmethylsulfonylflouride, followed by 1 ml of the same buffer containing 20% (w/v) glycerol. Cell extracts (1 ml)
were applied, allowed to react for 10 min, columns washed with 16 ml of buffer
containing 100 mM NaCl followed by 8 ml of buffer containing 200 mM NaCl.
Fractions were dialysed for 12 h against 2 l of buffer without either NaCl or phenylmethylsulfonylflouride. All manipulations were performed at 4oC.
Tricine-SDS PAGE was performed as described by Schägger and von Jagow (
24
) and proteins visualised with Coomassie Blue R-250. Proteins were transferred to ProBlott (Applied Biosystems Ltd) soaked
in 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid and 10% (v/v) methanol, by
electroblotting. The membrane was stained with Coomassie Blue R-250 to locate (then excise) the band corresponding to putative SmtB and
protein sequencing performed using an Applied Biosystems 477A Protein
Sequencer, according to the manufacturers protocols.
In the standard binding assay (10 [mu]l), protein extracts were incubated for 10 min at 0oC with 2 [mu]g poly(dI-dC)[middot]poly(dI-dC) (Pharmacia) in 20 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, 1 mM
EDTA, 3% glycerol and 0.05 mM or 0.5 mM spermidine. 1,10-phenanthroline (1 or 10 mM) or ZnCl
2
(5, 10, 50 or 100 [mu]M) were also included in some binding assays (EDTA was omitted from binding
assays supplemented with Zn
2+
). A 100 bp
Bsp
HI fragment of DNA from pJHNR49, containing the
smt
operator-promoter (
3
), was labelled with [[alpha]-
32
P]dATP and [[alpha]-
32
P]dCTP and used as probe, ~0.3 ng was added to binding assays and incubation continued for 20 min at
25oC. Samples were loaded onto 5% polyacrylamide gels (30:1 acrylamide:
bisacrylamide) and electrophoresed using TAE (6.7 mM Tris-HCl, pH 7.5, 3.3 mM sodium acetate and 2 mM EDTA) or 1* TBE (
21
) as the buffer system. Electrophoresis was at 240 V for 2 min followed by 140 V for 2-2.5 h DNA-protein complexes were visualised by direct autoradiography.
Plasmid pJSTNR5.1 was generated by subcloning a 602 bp
Bam
HI-
Hin
dIII fragment of
smtA
5' flanking region (containing the
smt
operator promoter and
smtB
) from pWT (Fig.
2
B) into the
Bam
HI/
Hin
dIII site of pSK+. PCR was performed using divergent mismatching PCR primers (
25
) to convert codon-14 of
smtB
from TGC to TCT, codon-61 from TGT to AGT, codon-121 from TGT to TCT or codons-105 and -106 from CATCAC to CGTCGC. Primary PCR reactions used
pJSTNR5.1 as template DNA and a 5' (sense) primer (5'-CGGTAGTCTCTCAAGGGACT-3' to mutate codon-14; 5'-CGATCGGAGTTAAGTGTTGGGG-3' to mutate
codon-61; 5'-CTTACAAGAGTCTAGATAGAGATGC-3' to mutate codon-121; or 5'-CAGGATCGTCGCATTGTGGCG-3' to mutate
codons-105 and -106) with an M13 forward primer and a 3' (antisense) primer (5'-AGTCCCTTGAGAGACTACCG-3' for codon 14; 5'-CCCCAACACTTAACTCCGATCG-3' for
codon-61; 5'-GCATCTCTATCTAGACTCTTGTAAG-3' for codon-121; or 5'-CGCCACAATGCGACGATCCTG-3' for codons-105 and-106)
with an M13 reverse primer. Products from each pair of divergent primary
reactions were recovered from agarose gels (
26
), combined and used as template DNA for a secondary PCR reaction using M13
forward and reverse primers. The final PCR products (containing the
smtA
operator-promoter and mutated
smtB
) were cloned into pGEM-T (Promega) or pSK+ prior to subcloning into the
Sal
I/
Bam
HI site of pLACPB2.
These assays were performed as described previously (
3
). Cyanobacterial cultures with an optical density at 595 nm of 0.08-0.15, were exposed to 0 and 2.5 [mu]M Zn
2+
for 2 h prior to assay.
Previous
in vitro
studies have shown that the
smtB
-dependent MAC1 complex forms with a region of the
smt
operator-promoter immediately upstream of
smtA
(
3
), corresponding to nucleotides -15 to +24 relative to the
smtA
transcription start site. Features within this region of DNA include a perfect
6-2-6 hyphenated direct repeat, a degenerate 6-2-6 hyphenated inverted repeat and a degenerate 12-2-12 hyphenated inverted repeat (Fig.
2
B). Methylation interference assays have since demonstrated that (at low
concentrations) recombinant SmtB interacts
in vitro
with a pair of sense/antisense (symmetrical) sites which lie within this region
(Fig.
2
C;
4
).
To examine the SmtB-DNA binding site
in vivo
, four mutant
smt
operator-promoters (MUT1-4) were generated in which either one or both halves of each of
the 6-2-6 repeats and hence, to varying degrees, the overlapping degenerate
12-2-12 repeat, were altered (Fig.
2
B). The mutated DNA fragments (containing
smtB
and the mutated operator-promoters) were fused to
lacZ
in pLACPB2 and used to transform
smt
deficient mutants of
Synechococcus
PCC 7942 to carbenicillin resistance. Elevated constitutive (in the absence of
added metal ions) expression was detected with all the operator-promoter mutations compared with expression from the wild type operator-promoter, although the increase was relatively small with MUT1
(Fig.
3
).
The second helix of the predicted helix-turn-helix is conserved within SmtB and PacR from
Synechocystis
PCC 6803 (Fig.
1
). These two proteins are therefore predicted to interact with similar
nucleotides within the promoters of their target genes. Within the promoter region of the predicted target gene (
pacS
) of PacR is a similar repeat to that contained within the SmtB-binding site (Fig.
2
C). A similar repeat is also located in the promoter of the predicted target gene,
mtnA
(which encodes metallothionein), of a sequence that we have designated MtnB
from another cyanobacterium
Synechococcus vulcanus
(Fig.
1
). The sequence of the `recognition' helix of MtnB remains to be determined.
Similar amino acid-nucleotide interactions are proposed for these three
cyanobacterial proteins.
Recombinant SmtB was expressed in
E.coli
, from plasmid pSmtB,
under the control of the strong
trc
promoter. An ~13.5 kDa protein, which corresponds to the predicted
M
r
of SmtB (13 536), was detected in extracts from
E.coli
expressing SmtB and was absent in extracts from control cells containing vector
pKK233-2 alone (data not shown). The 13.5 kDa protein was partially purified by
heparin Sepharose affinity chromatography, resolved by Tricine-SDS PAGE and, following transfer to membrane, N-terminal sequence analysis generated the partial sequence TKPVLQDGE
confirming its identity as SmtB (refer to Fig.
1
).
Gel retardation assays using extracts from
Synechococcus
PCC 7942 showed a single complex, C1 (presumed to be analogous to MAC1),
forming with the
smt
operator-promoter which was not detected with extracts from mutants deficient in
smtB
(Fig.
4
A). These assays were performed using 0.05 mM spermidine in the binding buffer
and TBE as the buffer system for electrophoresis. Dilute extracts (crude and
purified) from
E.coli
expressing SmtB retarded the mobility of the
smt
operator-promoter to the same degree as extracts from
Synechococcus
PCC 7942 (Fig.
4
A). More concentrated extracts generated slower migrating complexes (Fig.
4
A), which are known to contain multimeric SmtB (
4
). No specific complex was formed with extracts from control
E.coli
cells (containing pKK233-2 alone).
Two complexes (C2 and C3), and a third less prominent complex, were detected
using extracts from
smt
deficient mutants of
Synechococcus
PCC 7942 in gel retardation assays performed with a higher level of spermidine
(0.5 mM) in binding reactions and TAE as the buffer system for electrophoresis
(Fig.
5
). Similarly retarded complexes were observed under these conditions using
extracts from wild-type cells (data not shown). The abundance of C3 is enhanced by treatment
with Zn
2+
in vitro
(Fig.
5
), whilst all complexes are diminished by the addition of 1,10-phenanthroline to binding reactions. This is, of course, the converse of
SmtB and supports the hypothesis (
3
,
28
) that an uncharacterised activator may also contribute towards metal-responsive expression of
smtA
in
Synechococcus
PCC 7942. These complexes were not visible in the previous gel retardation
assays, examining SmtB-DNA binding (Fig.
4
A and C), presumably due to the use of different assay conditions which were
equivalent to those used by Erbe and co-workers (
4
).
Codons-14, -61 and -121 of
smtB
which encode Cys residues were converted by PCR-mediated mutagenesis to encode Ser, thereby creating three separate mutant
smtB
genes. Codons-105 and -106 were converted (together) to encode Arg rather than His. The
mutated DNA fragments (containing the mutated
smtB
genes and the
smtA
operator-promoter) were fused to
lacZ
in pLACPB2 and sequenced, confirming the mutations. The pLACPB2 clone
containing
smtB
mutated at codon-14 was found to contain an additional (spontaneous) mutation converting
codon-11 from Thr to Ser. A separate clone containing C14S was therefore also
analysed, however this contained a mutation converting codon-7 from Gln to a translational stop site. The reporter gene constructs
containing the T11S/C14S, C61S, C121S and H105R/H106R mutated fragments (Fig.
1
) were used to transform
smt
mutants of
Synechococcus
PCC 7942,
which are devoid of chromosomal
smtB
(
1
), to carbenicillin resistance.
Consistent with previous observations (
2
,
3
) [beta]-galactosidase activity was highly elevated in cells devoid of
smtB
and was several fold greater than maximum Zn
2+
-induced levels detected in cells containing plasmid-borne repressor (compare SmtB
-
with WT in Fig.
6
). Compared with cells devoid of the repressor, [beta]-galactosidase activity was low in cells containing constructs with
mutated
smtB
showing that the mutant SmtB proteins retain repressor function (Fig.
6
). In common with cells containing non-mutated SmtB (WT), activity was also stimulated by Zn
2+
in cells containing C61S or C121S revealing that the thiol groups of Cys-61 or Cys-121 are not essential for inducer recognition by SmtB (Fig.
6
). It is noted that both constitutive and Zn
2+
-induced expression is slightly elevated in cells containing the C61S
mutant compared with wild type SmtB suggesting some impairment of DNA binding
associated with this mutation. Most significantly, little [beta]-galactosidase activity was observed in cells containing H105R/H106R
in either the absence or presence of elevated concentrations of Zn
2+
, indicating that either one or both of these His residues are essential for
inducer (Zn
2+
)-recognition by SmtB. It is formally possible that amino acid substitutions
may influence SmtB function indirectly via changes in overall structure.
However, in view of the known role of His residues in Zn
2+
binding in other proteins, it is speculated that the loss of inducer
responsiveness observed with the H105R/H106R mutant is associated with loss of
metal binding at one or both of these residues. Although [beta]-galactosidase activity was stimulated by Zn
2+
in cells containing the T11S/C14S mutant, levels of activity in these cells was
consistently reduced compared with cells containing wild-type
smtB
suggesting some increased DNA binding associated with one, or both, of these
mutations.
Gel retardation assays were performed using extracts from
smt
deficient mutants of
Synechococcus
PCC 7942 containing plasmid-borne wild-type or mutated (T11S/C14S, C61S, C121S and H105R/H106R)
smtB
. These assays were performed using the lower level of spermidine (0.05 mM) in binding reactions and TBE as the
buffer system for electrophoresis. Extracts from cells expressing each of the
mutant
smtB
genes showed equivalent retardation of the
smt
operator-promoter as extracts from cells expressing wild-type
smtB
(Fig.
7
). This again confirms that the mutant SmtB proteins are being synthesised in
these cells and that these proteins have functional DNA-binding domains, since extracts from the
smtB
deficient mutants alone do not form these complexes (Fig.
4
A and C). Binding of the altered SmtB proteins to the
smt
operator-promoter was enhanced by the addition of increasing concentrations (1 and
10 mM) of 1,10-phenanthroline to binding reactions, as observed with wild-type SmtB (Fig.
7
). This suggests that some Zn
2+
associates with all of the mutant SmtB proteins, including H105R/H106R, to
impair DNA-binding.
SmtB exerts metal ion-inducible negative control of transcription of the cyanobacterial
metallothionein gene
smtA
(
2
). SmtB functions by binding to the
smt
operator-promoter and dissociating in the presence of Zn
2+
(
3
,
4
). Compared with other Zn
2+
-binding transcription factors SmtB is atypical since metal ions inhibit,
rather than promote, SmtB-DNA binding. A mutant SmtB with altered residues His-105 and His-106 was identified, following site-directed mutagenesis, as being unable to respond to
inducer
in vivo
(Fig.
6
). The introduced mutations neither impaired
smtB
-mediated repression
in vivo
nor DNA binding
in vitro
(Figs
6
and
7
).
Methylation interference assays using recombinant SmtB has previously identified
one pair of symmetrical DNA contact points adjacent to the
smtA
transcription start site (Fig.
2
C;
4
). In accordance with the mapped site
in vitro
, targeted mutations within this region of the
smt
operator-promoter conferred increased expression of an associated reporter gene
consistent with loss of repression (Fig.
3
). Cleavage at unmethylated guanines does not give an indication of the full
extent of protein-nucleotide interactions. The
in vivo
studies show that nucleotides required for functional interactions with SmtB
are not confined to either of two short (6-2-6 bp) repeat motifs as previously proposed (
3
,
4
) but are contained within a larger region which includes a degenerate 12-2-12 inverted repeat (Fig.
2
). These studies also indicate that binding to nucleotides contained within both
sides of the 12-2-12 inverted repeat is required for `normal' repression. Within the
promoter of the predicted target gene (
pacS
) of PacR is a degenerate 12-2-12 inverted repeat with similarity to that within the
smtA
operator-promoter (Fig.
2
C). Furthermore, a similar repeat is located in the promoter of
mtnA
from
Synechococcus vulcanus
. The sequence of the `recognition' helix of MtnB remains to be determined but
we predict that it will be similar to those of SmtB and PacR (Fig.
1
). The DNA-binding site for ArsR from
E.coli
has been mapped to a 24 bp region and data suggest that the repressor binds as
a dimer to two 4 bp sequences located 10 bp apart, within this region (
29
). By contrast, the DNA-binding site for ArsR from
Staphylococcus xylosus
has been mapped to five sites of close contact contained within two regions
spanning 23 and 9 bp (
30
).
The slowest migrating DNA complex detected with recombinant SmtB and the
smt
operator-promoter involves additional contacts at a second pair of sites, one on
each strand, identified within a perfect 7-2-7 inverted repeat; CT
Inducer recognition by SmtB has been proposed (
11
,
12
) to involve the formation of metal-thiolate bonds to Cys-61 which is associated with the N-terminal extremity of a predicted helix-turn-helix motif. This residue is situated within a
sequence which is (at least in part) conserved in members of the arsR family,
although it is not present in several other related proteins which are now
known (Fig.
1
). Little [beta]-lactamase activity, driven by the
ars
operator-promoter, was found in cells bearing ArsR mutants at Cys-32 (equivalent to Cys-61 in SmtB) or Cys-34, even in the presence of inducer (
11
,
31
). By contrast, conversion of Cys-61 in SmtB to Ser does not abolish inducer recognition or
smtB
-mediated repression of expression from the
smtA
operator-promoter (Fig.
6
). It is of significance that in SmtB this is a lone Cys residue while in ArsR
and CadC it forms part of a Cys-Xaa-Cys motif (where Xaa is an amino acid other than Cys) (Fig.
1
), a motif which is involved in metal-thiolate bonding in many proteins including metallothioneins. In common
with SmtB, there is also only a single Cys residue at this site in MtnB (Fig.
1
).
The H105R/H106R mutated SmtB was unable to respond to inducer (Zn
2+
)
in vivo
, indicating that either one or both of these His residues are essential for Zn
2+
sensing by SmtB. Only a single His residue is present at this location in other
members of the arsR family of regulators. The H105R/H106R mutated repressor bound to the
smtA
operator-promoter
in
vitro
(Fig.
7
). However, DNA-binding was still enhanced by treatment with 1,10-phenanthroline
in vitro
(Fig.
7
). This is analogous to results reported for mutants of ArsR, at Cys-32 or Cys-34, that did not respond to inducer
in vivo
but did (partially) respond to inducer in gel retardation assays (
11
). It was suggested that (an) other residue(s) involved in metal recognition
allow(s) some metal response in the
in vitro
assay system (
11
). In the present work, [beta]-galactosidase activity was reduced in cells containing T11S/C14S
compared with cells containing wild-type
smtB
(Fig.
6
), suggesting increased DNA-binding associated with one (or both) of these mutations. It is formally
possible that Cys-14 has some role in inducer recognition although it is clearly not
absolutely required for Zn
2+
-sensing
in vivo
. It is noted that Cys-14 is conserved in some members of the arsR family, being located within
an N-terminal domain which is present in the Zn
2+
/Cd
2+
-responsive members but absent from the ArsR proteins (Fig.
1
). Clearly, different members of the arsR family of metal-responsive transcriptional regulators have (at least some) differences in
their metal-sensing mechanisms. This indicates the intriguing possibility of a family of metal-responsive repressors possessing distinct metal-binding sites which give diversity in the spectra of metals
sensed; a preference for Cd
2+
by CadC, oxyanions by ArsR, Zn
2+
by SmtB, Hg
2+
by MerR (of
S.lividans
) and (we hypothesise) copper ions by PacR.
This work was supported by research grant GR/J37126 from the UK Biotechnology
and Biological Sciences Research Council (BBSRC). P.D.G. is supported by a
Research Studentship from the BBSRC. The authors thank A.P. Morby for his
contributions at the outset of this research. This work benefited from the use
of the UWGCG package on the SEQNET facility at the Daresbury Laboratory.
REFERENCES
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