Two MerR homologues that affect copper induction of the Bacillus subtilis copZA operon

Ahmed Gaballa, Min Cao and John D. Helmann

Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA

Correspondence
John D. Helmann
jdh9{at}cornell.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Copper ions induce expression of the Bacillus subtilis copZA operon encoding a metallochaperone, CopZ, and a CPx-type ATPase efflux protein, CopA. The copZA promoter region contains an inverted repeat sequence similar to that recognized by the mercury-sensing MerR protein. To investigate the possible involvement of MerR homologues in copZA regulation, null mutations were engineered affecting each of four putative MerR-type regulators: yyaN, yraB, yfmP and yhdQ. Two of these genes affected copper regulation. Mutation of yhdQ (hereafter renamed cueR) dramatically reduced copper induction of copZA, and purified CueR bound with high affinity to the copZA promoter region. These results suggest that CueR is a direct regulator of copZA transcription that mediates copper induction. Surprisingly, a yfmP mutation also reduced copper induction of copZA. Sequence analysis suggested that yfmP was cotranscribed with yfmO, encoding a putative multidrug efflux protein. The yfmPO operon is autoregulated: a yfmP mutation derepressed the yfmP promoter and purified YfmP bound the yfmP promoter region, but not the copZA promoter region. Since the yfmP mutant strain was predicted to express elevated levels of the YfmO efflux pump, it was hypothesized that copper efflux might be responsible for the reduced copZA induction. Consistent with this model, in a yfmP yfmO double mutant copper induction of copZA was normal. The results demonstrate the direct regulation of the B. subtilis copper efflux system by CueR, and indirect regulation by a putative multidrug efflux system.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transition metals such as copper are essential cofactors for numerous enzymes. However, when redox-active metal ions are in surplus, they pose a threat of oxidative damage due to the production of free radicals that damage DNA, lipids and proteins (Imlay, 2002; Touati, 2000). As a result, defects in metal homeostasis contribute to the aetiology of numerous diseases (Bush & Tanzi, 2002; Harris, 2000; Llanos & Mercer, 2002). Metal ion homeostasis is critically dependent on metalloregulatory proteins that sense intracellular metal ion levels and regulate the appropriate homeostasis mechanisms that serve to maintain intracellular metal ions within an optimal concentration range (Hantke, 2001; Herbig & Helmann, 2002; Finney & O'Halloran, 2003).

Bacterial copper homeostasis is best understood in Enterococcus hirae and Escherichia coli. In E. hirae the cop operon encodes a copper-responsive repressor (CopY), a copper chaperone (CopZ), and two CPx-type ATPases (CopA and CopB) that are involved in copper uptake and efflux, respectively (reviewed by Solioz & Stoyanov, 2003). CopY, with a bound zinc ion, binds to the promoter region of the cop operon, repressing its expression. However, when intracellular copper levels increase, copper displaces the bound zinc and causes dissociation of the repressor (Cobine et al., 2002). E. coli possesses two inducible systems for copper resistance (reviewed by Rensing & Grass, 2003). The CueR regulon consists of CopA, a CPx-type ATPase, and CueO, a multicopper oxidase (Outten et al., 2000; Stoyanov et al., 2001; Petersen & Møller, 2000). The cus efflux system is preferentially expressed under anaerobic conditions and is hypothesized to extrude copper ions past the outer membrane (Outten et al., 2001; Franke et al., 2003).

In Bacillus subtilis, copper resistance is mediated by a copper-inducible CPx-type ATPase, CopA, and a copper chaperone, CopZ (Banci et al., 2001, 2002; Gaballa & Helmann, 2003; Radford et al., 2003). These proteins have been the subject of extensive biophysical characterization (reviewed by Banci & Rosato, 2003). Recent results indicate that in vitro CopZ transfers Cu(I) to CopA (Banci et al., 2003), consistent with a role in efflux as inferred from genetic studies (Gaballa & Helmann, 2003; Radford et al., 2003). The copZA regulatory region contains a candidate {sigma}A-type promoter element with an inverted repeat sequence between the -35 and -10 promoter elements. Sequence comparisons suggest that this inverted repeat may be recognized by a MerR-type regulator (Gaballa & Helmann, 2003).

MerR-type transcriptional regulators are found in a large number of Gram-positive and Gram-negative bacteria (reviewed by Brown et al., 2003). Most characterized MerR family members bind to atypical promoter regions with unusually long (19–20 bp) spacer regions between the -10 and -35 boxes. Typically, the regulator binds to its operator in both the presence and absence of inducer. Inducer binding triggers a protein conformation change that remodels the promoter region DNA to facilitate transcription initiation (Ansari et al., 1992, 1995; Schumacher & Brennan, 2002). Bacterial copper resistance systems under the control of MerR-type regulators are found in Pseudomonas putida (Lee et al., 2001; Adaikkalam & Swarup, 2002), Rhizobium leguminosarum (Reeve et al., 2002), E. coli (Outten et al., 2000; Stoyanov et al., 2001; Petersen & Møller, 2000) and Salmonella enterica (Kim et al., 2002). In addition, MerR homologues regulate lead resistance in Ralstonia metallidurans (Borremans et al., 2001), cadmium resistance in P. putida (Brocklehurst et al., 2003), cobalt resistance in Synechocystis PCC 6803 (Rutherford et al., 1999) and zinc resistance in E. coli (Brocklehurst et al., 1999; Outten et al., 1999).

In this study we demonstrate that two different MerR homologues affect copper induction of the copZA operon. Full induction requires CueR, which binds directly to the copZA promoter region. Induction by copper is also greatly reduced in a yfmP mutant background, probably due to derepression of a downstream multidrug efflux system (YfmO) that may act to efflux copper or a copper complex.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, media and growth conditions.
Bacterial strains used in this study are listed in Table 1. B. subtilis CU1065 was grown on LB and metals were added from filter-sterilized stocks prior to inoculation. E. coli DH5{alpha} was used for routine DNA cloning (Sambrook et al., 1989). Unless otherwise indicated, liquid media were inoculated from an overnight pre-culture and incubated at 37 °C with shaking at 200 r.p.m. Erythromycin (1 µg ml-1) and lincomycin (25 µg ml-1) [for testing macrolide-lincosamide-streptogramin B resistance (MLSR)], spectinomycin (100 µg ml-1), kanamycin (10 µg ml-1), neomycin (10 µg ml-1) and chloramphenicol (5 µg ml-1) were used for the selection of various B. subtilis strains.


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Table 1. Bacterial strains used in this study

 
DNA manipulations.
Routine molecular biology procedures were performed according to Sambrook et al. (1989). Isolation of B. subtilis chromosomal DNA, transformation and specialized SP{beta} transduction were performed as described by Cutting & Vander Horn (1990). Restriction enzymes, DNA ligase and Klenow fragment of DNA polymerase were used according to the manufacturer's instructions (New England Biolabs).

Construction of cueR, yraB, yyaN, yfmP, yfmO and yfmPO mutants.
Long-flanking homology (LFH) PCR was used as described by Wach (1996) to generate allelic replacement mutants for each gene or group of genes. In brief, approximately 1000 bp genomic regions flanking the gene(s) to be deleted were amplified from CU1065 chromosomal DNA by PCR. Primers used are summarized in Table 2. The kanamycin-resistance cassette was amplified by PCR from pDG780(kan) (Guérout-Fleury et al., 1995). For each mutant construction, equal amounts (~200–300 ng) of purified upstream and downstream flanking fragments and the corresponding drug-resistance cassette were used in a joint PCR procedure as described by Wach (1996), using the HotStarTaq Master Mix Kit (Qiagen). The resulting PCR products were purified and then directly transformed into B. subtilis wild-type strain CU1065, selecting for kanamycin resistance. The resultant mutant strains are listed in Table 1.


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Table 2. Oligonucleotides used for LFH-PCR

 
{beta}-Galactosidase assays.
Overnight cultures were diluted 1 : 100 in LB liquid medium containing different concentrations of metal ions (as indicated) and grown to mid-exponential phase. Cells were collected and assayed for {beta}-galactosidase as described by Miller (1972).

Construction of yfmP and cueR transcriptional fusions.
Promoter regions were amplified from the B. subtilis genome by PCR using primers 5'-GCGAAGCTTCAGCCTTGCACCTTCAT-3' and 5'-GCGGGATCCCCATCTATATCCCTCCCA-3' for cueR; and primers 5'-GCGAAGCTTGCACGCCTCTGGTGATAGA-3' and 5'-GCGGGATCCGCTTTGTCAGCCCGCT-3' for yfmP. The resulting products were cloned as HindIII–BamHI fragments (sites underlined) into pJPM122 (Slack et al., 1993) to generate the corresponding (promoter)–cat–lacZ operon fusions. The resulting plasmids were linearized with ScaI and transformed into ZB307A (Table 1; Zuber & Losick, 1987) with selection for neomycin resistance. SP{beta} transducing lysates were prepared by heat induction and transduced to B. subtilis CU1065.

Overproduction and purification of YfmP.
The coding region of yfmP was amplified from the B. subtilis genome using primers 5'-GATTTACCATGGAATGGATGAAGATTGA-3' and 5'-GCGAGATCTTTTCATTCGATTGATTGAATG-3'. The resulting fragment was digested with NcoI and BglII and cloned into pET16X (Novagen) and transformed into E. coli DH5{alpha}. The DNA sequence of the cloned fragment was checked by DNA sequencing in both orientations and the clone was transformed into E. coli BL21(DE3)(pLysS) (Studier et al., 1990). A single colony was grown overnight in 5 ml LB containing ampicillin (100 µg ml-1). The overnight culture was used to inoculate 500 ml LB containing 0·4 % (w/v) glucose. Cells were incubated at 37 °C with vigorous shaking to an OD600 of 0·6; IPTG was added to 1 mM (final concentration), and the cells were harvested after further incubation for 2·5 h. The cell pellet was resuspended in buffer A (50 mM Tris/HCl pH 8·0, 50 mM NaCl, 0·1 mM EDTA, 2 mM DTT, 5 %, v/v, glycerol) containing 2 % sodium deoxycholate and lysed by sonication. Inclusion bodies were recovered by centrifugation and washed twice with buffer A containing 2 % sodium deoxycholate. The inclusion bodies were dissolved in buffer A containing 0·4 % Sarkosyl and incubated at 20 °C for 30 min. The protein was diluted 10-fold by gradual addition of buffer A at 4 °C, and dialysed overnight against buffer A at 4 °C. The renatured protein was applied to a heparin-Sepharose column and bound proteins were eluted with a gradient of NaCl (0·05–1 M) in elution buffer (50 mM Tris/HCl pH 8·0, 2 mM EDTA, 0·1 mM DTT, 1 mM PMSF, 5 %, v/v, glycerol). Samples were analysed by 12 % SDS-PAGE to identify YfmP. Proteins were concentrated using a Centricon-10 microconcentrator (Amicon) and loaded on a Bio-Rad Q2 ion-exchange column via FPLC (Pharmacia). A linear gradient of 0·05–1 M NaCl was used to elute the protein and fractions containing YfmP were concentrated by a Centricon 10 microconcentrator (Amicon) prior to gel exclusion chromatography on a Superdex-75 column (Pharmacia). Purified YfmP was stored at -20 °C in buffer A containing 50 % glycerol.

Overproduction and purification of CueR.
The coding region of cueR was amplified from the B. subtilis genome using primers 5'-ACTATAACGCCATGGTTGGGAGGGA-3' and 5'-GCGGGATCCTCTAGCAATGAGAATGAAGGT-3'. The resulting fragment was digested with NcoI and BamHI and cloned into pET16X (Novagen) and transformed to E. coli DH5{alpha}. The DNA sequence of the cloned fragment was checked by DNA sequencing in both orientations and the plasmid was transformed into E. coli BL21(DE3)(pLysS).

Cells were grown in 1 litre of LB broth with vigorous shaking at 37 °C and induced with 400 µM (final concentration) IPTG at OD600 ~0·5. The cells were harvested by centrifugation 3 h after IPTG induction and stored at -80 °C. The cell pellet was resuspended in 30 ml TEDG buffer (50 mM Tris/HCl pH 8·0, 1 mM EDTA, 5 mM DTT, 5 %, v/v, glycerol) and lysed in a French pressure cell at 10 000 p.s.i. The lysate was clarified by centrifugation and applied to a heparin column (25 ml) equilibrated with TEDG buffer, and washed with 2 vols TEDG buffer. The CueR protein was eluted with a 120 ml linear gradient from 0·0 to 1·0 M NaCl in TEDG buffer. CueR was eluted with 0·5–0·6 M NaCl. Peak fractions were pooled, concentrated and desalted with a Centricon-10 microconcentrator (Amicon). The protein was further purified by separation on an FPLC Superdex-75 (25 ml) column equilibrated with TEDG buffer and 0·1 M NaCl. The CueR fractions were concentrated to ~1 ml and were >90 % pure as estimated by a 12 % SDS-PAGE gel. The protein was stored at -80 °C in TEDG buffer with 50 % glycerol.

Electrophoretic mobility shift assays.
PCR fragments containing the promoter regions of yfmP, cueR or a control fragment (the dhbA promoter region) were purified and labelled by a 3' fill-in reaction using the Klenow fragment of DNA polymerase I (New England BioLabs) and [{alpha}-32P]dATP [NEN, 3000 Ci (370 GBq) mmol-1, 10 mCi (370 MBq) µl-1]. The reactions were carried out in 20 µl gel-shift buffer (20 mM Tris/HCl pH 8·0, 100 mM NaCl, 1 mM DTT, 5 % glycerol, 5 µg salmon sperm DNA ml-1). Cu(II) was added as 50 µM CuSO4 (final concentration) where appropriate. Labelled DNA fragments and YfmP or CueR protein were added to the reaction mixture and incubated for 20 min at room temperature. Samples were loaded on a 6 % polyacrylamide gel and electrophoresed in 40 mM Tris/acetate buffer (without EDTA), pH 8·0. The gel was dried and exposed to a phosphorimager screen.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
copZA is controlled by a MerR-type regulator
The B. subtilis copZA promoter (Fig. 1) contains an inverted repeat element strikingly similar to those in MerR-regulated mercury resistance operons from Bacillus sp. RC607 (Helmann et al., 1989) and Tn501 (Misra et al., 1984). Thus, copZA may also be regulated by a MerR-type regulator. The B. subtilis genome encodes 10 MerR homologues, including four known or predicted regulators of multidrug efflux systems (BmrR, BltR, Mta, YdfL) and two regulators of nitrogen metabolism (TnrA, GlnR) (Fisher, 1999; Schumacher & Brennan, 2002). The remaining four predicted proteins are of unknown function: YyaN, YraB, YfmP and YhdQ.



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Fig. 1. Alignment of the B. subtilis copZ, Bacillus sp. RC607 mer and Tn501 mer promoter regions. The -35 and -10 regions (underlined) and transcription start site (A residue) are illustrated. The inverted repeat sequences for binding of MerR homologues are indicated. Binding of CueR blocks cleavage with the restriction endonuclease BslI (CCnnnnnnnGG; site indicated by asterisks).

 
To determine whether any of these four MerR homologues regulate copZA, mutants were constructed in the corresponding genes by allelic replacement with a KmR cassette. A PcopZ–cat–lacZ operon fusion was introduced into each mutant and the ability of copper to induce expression of {beta}-galactosidase was measured. While mutation in yyaN or yraB had no significant effect on the ability of Cu(II) to induce copZA expression, a yhdQ (henceforth renamed cueR) mutation nearly abolished induction (Fig. 2). Unexpectedly, a yfmP mutation also greatly reduced the magnitude of induction (Fig. 2).



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Fig. 2. Induction of the PcopZ–cat–lacZ fusion by Cu(II) in wild-type, yfmP, cueR, yyaN and yraB mutant cells. Cells were grown to mid-exponential phase in LB medium with (black bars) or without (white bars) 0·5 mM CuSO4 and {beta}-galactosidase was determined. Data are the means of three independent measurements; error bars represent SD.

 
CueR binds in vitro to the promoter region of copZA
To determine if either of the corresponding regulatory proteins interacted directly with the copZA regulatory region we purified both CueR and YfmP after overproduction in E. coli (Fig. 3). Purified CueR bound to the copZA promoter region with high affinity, and binding was observed both with and without copper addition (Fig. 4a). Binding is likely to be due to interaction with the noted inverted repeat element (Fig. 1) since CueR blocks cleavage of DNA with the BslI restriction enzyme (data not shown). In contrast, YfmP did not bind to the copZA promoter region even at high concentrations (Fig. 4b), suggesting that the effects of a yfmP mutation on copZA regulation might be indirect. Purified YfmP does bind to its own promoter region (Fig. 4c), demonstrating that the protein is active as purified and suggesting that the yfmPO operon is autoregulated. Binding of YfmP protects the DNA against cleavage with restriction endonuclease HpyCH4 IV (data not shown), consistent with a binding site located at or near the indicated inverted repeat (Fig. 5c).



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Fig. 3. (a) SDS-PAGE analysis of YfmP protein purified from E. coli. Lanes: 1, uninduced cell extract; 2, induced cell extract; 3, fraction from Superdex-75; 4, pooled fractions from heparin column. (b) SDS-PAGE analysis of B. subtilis CueR protein purified from E. coli. Lanes: M, molecular mass marker; 1, uninduced cell extract; 2, induced cell extract; 3, pooled fractions from heparin column; 4, fraction from Superdex-75.

 


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Fig. 4. Electrophoretic mobility shift analysis of the CueR (a) and YfmP (b, c) proteins in the presence of copZ promoter region (a, b) or yfmP promoter region (c). Protein and Cu(II) (at 50 µM) were added as indicated.

 


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Fig. 5. (a) Expression of PyfmP–cat–lacZ and PcueR–cat–lacZ fusions in wild-type (white bars), yfmP (black bars) and cueR (grey bars) mutants. Cells were grown to mid-exponential phase in LB medium with 0·5 mM CuSO4, and {beta}-galactosidase was determined. Data are the means of three independent measurements; error bars represent SD. (b) Primer extension mapping of the yfmPO transcriptional start site using RNA isolated from wild-type (lanes 1 and 2) or yfmP mutant (lanes 3 and 4) strains grown without (lanes 1 and 3) or with Cu(II) (lanes 2 and 4). (c) DNA sequence of the yfmPO operon regulatory region showing the {sigma}A-type promoter element, mapped transcriptional start site, and candidate YfmP binding site (arrows). YfmP protects the DNA against cleavage with HpyCH4 IV (ACGT) at the two sites indicated (asterisks).

 
Regulation of yfmP
The yfmP gene ends 68 bases upstream of yfmO, which encodes a putative multidrug efflux system. This suggests that these two genes constitute a single transcription unit. To characterize the regulation of yfmP and cueR, cat–lacZ transcriptional fusions were constructed. The PyfmP–cat–lacZ fusion was expressed at a low level in both wild-type and cueR mutant cells yet was derepressed in a yfmP mutant background (Fig. 5a). On the other hand, the PcueR–cat–lacZ fusion was expressed at similar levels in the wild-type and cueR mutant background, indicating that this gene is not autoregulated. Expression was reduced about twofold in a yfmP mutant. In all strains tested, addition of copper to the medium led to an approximately twofold increase in {beta}-galactosidase accumulation (Fig. 5a). These results demonstrate that YfmP is autoregulated and acts as a repressor of the yfmPO operon. Promoter mapping studies (Fig. 5b) indicate that transcription of the yfmPO operon initiates from a {sigma}A-type promoter element. Consistent with the reporter fusion studies, expression is detected in the yfmP mutant strain in both the presence and absence of copper. The predicted -35 element of this promoter is flanked by an inverted repeat element that may represent a YfmP binding site (Fig. 5c).

The effect of yfmP on copZ expression requires yfmO
We hypothesized that the observed effect of the yfmP mutation on copper induction of copZA was due to a decreased accumulation of copper in the cell caused by overexpression of the YfmO multidrug efflux protein. To test this idea, a yfmO mutant and a yfmPO double mutant were constructed. Mutation of yfmO by itself had little effect on the copper responsiveness of the PcopZ–cat–lacZ fusion, but it did suppress the effect of the yfmP mutation on copper induction (Fig. 6). This indicates that the effect of the yfmP mutation on the expression of copZA requires the product of the yfmO gene.



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Fig. 6. (a) Construction of a yfmP : : kan disruption mutation. Note that the kanamycin cassette is codirectional with the downstream yfmO gene. (b) Effect of yfmP and yfmO mutations on the expression of PcopZ–cat–lacZ fusion. Cells were grown to mid-exponential phase in LB medium with (black bars) or without (white bars) 0·5 mM CuSO4, and {beta}-galactosidase was determined. Data are the means of three independent measurements; error bars represent SD.

 
If YfmO acts to efflux copper or a copper complex, it is possible that YfmO could contribute to copper resistance. However, the yfmO mutant displayed little if any increase in sensitivity to copper in either wild-type or copA mutant backgrounds (data not shown). Indeed, of the metal ions tested, the yfmO mutation had the most dramatic effect on Cd(II) sensitivity. Whereas the MIC of Cd(II) for wild-type cells was 25 µM, this was reduced to ~8 µM in the yfmO mutant strain.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Intracellular concentrations of metal ions are tightly controlled to balance their metabolic use as cofactors and limit their toxic effect (Finney & O'Halloran, 2003). The cell achieves this delicate balance by tightly regulating the expression of metal uptake, storage and efflux systems. In this study, we have focused on the mechanisms acting to control the copper-inducible expression of a copper efflux system in B. subtilis (Gaballa & Helmann, 2003; Radford et al., 2003). The B. subtilis copper efflux system comprises a single operon that encodes CopA, a P-type ATPase that acts as efflux pump, and CopZ, a Cu(I)-binding metallochaperone (Banci et al., 2003; Gaballa & Helmann, 2003; Radford et al., 2003).

The copZA promoter sequence (Fig. 1) suggested a possible involvement of a MerR homologue in the observed copper regulation (Gaballa & Helmann, 2003). Hence, we analysed the role of four uncharacterized merR homologues on copZA expression. Our results identify yhdQ as the structural gene for CueR, a MerR-type regulator required for copper induction. The copZA promoter region is unusual in that it contains an 18 bp (rather than the expected 19 or 20 bp) spacer region. Further studies will be needed to determine if activation of this promoter proceeds via a DNA-distortion mechanism, as documented for other MerR homologues (Ansari et al., 1992, 1995; Outten et al., 1999; Heldwein & Brennan, 2001), or by another mechanism.

B. subtilis CueR is not closely related to CueR from E. coli (27 % identity over 85 amino acids). Indeed, searching the B. subtilis genome with the E. coli CueR protein as query (BLASTP) identifies all nine other MerR homologues as closer relatives. Thus, the role of this protein in copper regulation could not have been easily predicted by database searches alone. It is intriguing to note that B. subtilis CueR does not contain the two Cys residues conserved in other CueR proteins (Adaikkalam & Swarup, 2002) and shown to be important for copper-sensing in E. coli (Stoyanov & Brown, 2003). B. subtilis CueR does contain two Cys residues: one near the helix–turn–helix DNA-binding motif (C43) and one in the putative ligand-recognition domain (C63) (Fig. 7). The role of these residues in ligand recognition is not yet clear, but we note that a Cys residue at the position corresponding to C43 is also present in the lead-sensing MerR homologue PbrR (Borremans et al., 2001) and the cadmium-sensing CadR (Lee et al., 2001). Furthermore, in the zinc sensor ZntR, His residues in very similar positions appear to play a role in metal sensing (Khan et al., 2002). Significant differences between the metal recognition domains of E. coli and B. subtilis CueR may also be reflected in the metal selectivity: whereas E. coli CueR can respond to Ag(I) and Au(I) (Stoyanov & Brown, 2003), the B. subtilis CueR protein does not appear to respond to Ag(I) (Radford et al., 2003). Alternatively, this difference in metal responsiveness may reflect differences in the intracellular accumulation of the various ions (Cavet et al., 2002; Guedon & Helmann, 2003).



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Fig. 7. Sequence alignment of B. subtilis CueR and selected MerR homologues. The positions of Cys residues that are thought to serve as Cu(I) ligands in E. coli CueR are indicated by (*), those that are candidates for Cu(I) ligands in B. subtilis CueR are indicated by (+).

 
Unexpectedly, mutation of another gene encoding a MerR homologue, yfmP, also led to decreased copper induction. The corresponding protein, YfmP, autoregulates its own expression and that of the downstream gene, yfmO, encoding a member of the multidrug efflux family of proteins. The yfmP : : km insertion mutation probably led to elevated expression of yfmO due to derepression of the yfmPO promoter (Fig. 5a), readthrough transcription from the kanamycin-resistance cassette (Fig. 6a), or both. Since a yfmPO double mutant strain displays normal regulation of the copZA operon, we hypothesize that YfmO acts to efflux copper or a copper complex, thereby preventing induction of the copZA operon by CueR. Consistent with this idea, a yfmO mutant leads to a threefold decrease in the MIC of cadmium, although it has little if any effect on copper resistance. The yfmPO operon was not induced by copper or other metals (including zinc, cadmium, cobalt and nickel) or by several antibiotics (data not shown), nor are yfmPO part of the copper stimulon as determined using DNA microarrays (our unpublished data).

Several multidrug efflux systems have been shown to efflux either free metal ion or drug–metal complexes. Notably, the Listeria monocytogenes MdrL protein is a close homologue of YfmO (37 % amino acid identity) and contributes to heavy metal resistance: an mdrL mutant has twofold reductions in the MIC for zinc and cobalt, but not for cadmium or copper (Mata et al., 2000). Efflux of toxic compounds that bind to divalent cations may, in general, contribute to metal ion resistance. For example, the B. subtilis TetA efflux pump contributes to Co(II) resistance (Wang et al., 2000), and members of the multidrug and toxin efflux family in plants contribute to metal ion resistance (Rogers & Guerinot, 2002).

In conclusion, we have identified two MerR homologues that affect copper induction of the CopZA copper efflux system. The cueR (formerly yhdQ) gene encodes CueR, an activator that binds in vitro to the copZA promoter region and is needed in vivo for induction of the copZA promoter. The copZA promoter is unusual in that the spacer region is 18 bp, whereas promoters activated by MerR homologues typically have a 19 or 20 bp spacer region (Brown et al., 2003). The yfmP mutation appears to affect copZA regulation indirectly by overexpression of a putative multidrug efflux system, YfmO. We speculate that this system may act to restrict the intracellular accumulation of copper.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the National Institutes of Health (GM59323).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 25 August 2003; accepted 3 September 2003.



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