Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA
Correspondence
John D. Helmann
jdh9{at}cornell.edu
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ABSTRACT |
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INTRODUCTION |
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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
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 (1920 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.
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METHODS |
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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 (
200300 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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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 HindIIIBamHI fragments (sites underlined) into pJPM122 (Slack et al., 1993) to generate the corresponding (promoter)catlacZ operon fusions. The resulting plasmids were linearized with ScaI and transformed into ZB307A (Table 1
; Zuber & Losick, 1987
) with selection for neomycin resistance. SP
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. 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 (0·051 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 0·051 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. 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 0·50·6 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 [-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.
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RESULTS |
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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 PcopZcatlacZ 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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DISCUSSION |
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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 helixturnhelix 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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Several multidrug efflux systems have been shown to efflux either free metal ion or drugmetal 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.
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ACKNOWLEDGEMENTS |
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Received 25 August 2003;
accepted 3 September 2003.
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