| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK-Rosario, Argentina
Correspondence
Fernando C. Soncini
soncini{at}ibr.gov.ar
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
copA strain, a second Salmonella-specific P-type ATPase, GolT, can substitute the copper transporter, diminishing the effect of its deletion. The overall results highlight the importance of the cue system for controlling intracellular copper stress. The observed differences between Salmonella and E. coli in handling copper excess may contribute to our understanding of the distinct capability of these related pathogenic bacteria to survive outside the host.
These authors contributed equally to this work.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Macomber et al., 2007). In order to prevent copper damage, the cytoplasmic concentration of free copper must be negligible. The strategies used to eliminate copper excess are diverse, including different regulatory systems (MerR-like regulators, repressors and two-component systems) that modulate the expression of factors involved in active extrusion and sequestration, as well as oxidation of the metal ion in the periplasmic space (Magnani & Solioz, 2005; Moore & Helmann, 2005; Rensing & Grass, 2003). Among these factors, there is a broad conservation of CPx/P1-type ATPases, in addition to small copper-binding proteins (copper chaperones) and the sporadic presence in certain species of an RND ancillary copper-efflux system. In Escherichia coli, for instance, copper homeostasis is maintained by the coordinated action of two regulatory systems, CueR, a MerR-like protein, and the two-component system CusR/CusS (Outten et al., 2001; Rensing & Grass, 2003). CueR directly stimulates the transcription of copA and cueO, coding for a P-type ATPase and a multicopper oxidase, respectively. CopA is predicted to translocate Cu(I) from the cytoplasm to the periplasmic space, where it is converted to the less toxic form Cu(II) by CueO (Outten et al., 2000; Petersen & Moller, 2000; Rensing & Grass, 2003).
The E. coli cus regulon was found to play a role in maintaining copper homeostasis under anaerobic conditions, when the oxidase CueO is inactive (Outten et al., 2001). The CusR/CusS system is predicted to monitor the periplasmic concentration of the metal ion, to modulate the expression of an RND-type copper efflux pump, encoded by the cusCFBA operon (Franke et al., 2003; Munson et al., 2000). Recently, it was shown that transcription of a second, uncharacterized, two-component system, encoded by the yedWV operon, is activated by copper ions in a CusR-dependent manner (Yamamoto & Ishihama, 2005), although its role in copper tolerance remains unclear.
Other stress-response regulatory systems, such as CpxR/CpxA and SoxR/SoxS, have been found to be induced by the addition of copper (Kershaw et al., 2005; Yamamoto & Ishihama, 2005), probably as a result of cellular damage caused by the metal ion (Macomber, 2007)
In this work, we have characterized the Salmonella enterica serovar Typhimurium (S. Typhimurium) response to copper. Previous reports revealed the importance of the multicopper oxidase (named CuiD in Salmonella) and the transcriptional regulator CueR/SctR for copper tolerance (Kim et al., 2002; Lim et al., 2002). Here, we demonstrate that expression of CopA in Salmonella depends on CueR, and that this transporter and the multicopper oxidase CuiD are essential for full copper tolerance under both aerobic and anaerobic conditions. We also provide evidence that, in the absence of a functional CopA, the Salmonella-specific P-type ATPase GolT compensates for the deficiency directing active efflux of copper.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Bacterial strains were grown overnight at 37 °C in Luria broth (LB) or LB-agar plates (Checa et al., 2007). Ampicillin, kanamycin and chloramphenicol were used at 100, 25 and 10 µg ml–1, respectively. When necessary, CuSO4 was added to the cultures or plates at the indicated concentration. Cell culture medium reagents and chemicals were from Sigma. Oligonucleotides were purchased from Bio-Synthesis. Primer sequences are available on request.
|
; Ellermeier et al., 2002) in strain LB5010 (Bullas & Ryu, 1983). All constructions were transferred to the wild-type strain 14028s by P22 transduction (Davis et al., 1980). When necessary, the antibiotic-resistance cassette inserted at the deletion point was removed using the temperature-sensitive plasmid pCP20 carrying the FLP recombinase (Cherepanov & Wackernagel, 1995). All mutated DNA fragments were sequenced to confirm the required mutation and to screen against undesired mutations. The cueR locus was PCR-amplified from the Salmonella chromosome using the primers cueR-ORF-F (5'-GAGGATCCATATGAATATTAGCG-3') and cueR-ORF-R (5'-ACCCAAGCTTCAACGTGGCTTTTGC-3'). The amplified fragment was cloned into pUH21-2 laqIq to generate the cueR-expression plasmid pCUER (pPB1205). Plasmid DNA was introduced into bacterial strains by electroporation using a Bio-Rad apparatus, following the manufacturer's recommendations.
Copper induction and inhibition assays.
β-Galactosidase assays were carried out essentially as described by Miller (1972). For metal-sensitivity assays, a 5x10–7 dilution from overnight culture of the wild-type or each mutant strain was done in PBS. A 30 µl aliquot was applied on LB plates containing increasing concentrations of CuSO4. Plates were incubated at 37 °C for 40 h under aerobic conditions or 64 h under anaerobic conditions. Anaerobic environments were generated in a Gaspak jar system using AnaeroGen sachets (Oxoid). Anaerobic indicators (Oxoid) were employed to verify oxygen consumption, following the manufacturer's recommendations. After incubation, c.f.u. per ml were calculated and the percentage survival was estimated based on the count of the corresponding strain grown in the absence of metal added (Checa et al., 2007).
CueR purification.
Salmonella CueR was overproduced and purified from the wild-type strain carrying plasmid pCUER grown in the presence of 1 mM IPTG essentially as described previously (Outten et al., 2000). All procedures were carried out at 4 °C. The protein profile of the purified proteins was determined by SDS-PAGE. Protein concentration was determined by Bradford assay, using BSA as standard.
RNA isolation and primer extension.
Total RNA was extracted from mid-exponential-phase cultures (OD600 0.4–0.6) of wild-type S. Typhimurium and its isogenic cueR mutant strain grown in LB medium with or without the addition of 1 mM CuSO4 as previously described (Aguirre et al., 2000). cDNA synthesis was performed using 2 pmol of the 32P-end-labelled primer PROM-copA-R (5'-CCCAAGCTTCGCCAGCTCAACATC-3'), with 100 µg total RNA and 1 U SuperScript II RNaseH2 reverse transcriptase (Life Technologies). The extension products were analysed by electrophoresis on a 6 % polyacrylamide-8 M urea gels and compared with sequence ladders initiated with the same 32P-labelled primer that was used for primer extension.
Protein–DNA interaction analysis.
Electrophoretic gel mobility shift assays (EMSAs) were performed essentially as previously described (Lejona et al., 2003). DNA fragments (343 bp for the copA promoter region) were PCR-amplified using the primers PROM-copA-F (5'-CCGGAATTCGGTGCGATAACCATT-3') and PROM-copA-R (5'-CCCAAGCTTCGCCAGCTCAACATC-3'). Labelled DNA was incubated at room temperature for 20 min with purified CueR in the amounts indicated in the figure. The binding buffer used for protein–DNA interactions contained 25 mM Tris/HCl (pH 8.0), 50 mM NaCl, 5 mM MgCl2, 5 mM DTT and 10 % glycerol. Samples were run on an 8 % non-denaturing Tris/glycine polyacrylamide gel at room temperature. After electrophoresis, the gel was dried and autoradiographed.
DNase I footprinting assay.
DNase I protection assays were done for both DNA strands essentially as previously described (Aguirre et al., 2000; Lejona et al., 2003). Binding reactions with different amounts of purified CueR protein and 6 fmol labelled DNA were performed as described for the EMSAs. Then 0.05 U DNase I (Life Technologies) was added in a final volume of 100 µl. After incubation for 1 min at room temperature, the reaction was stopped by adding 90 µl of 20 mM EDTA (pH 8), 200 mM NaCl and 100 µg tRNA ml–1 . DNA fragments were purified by phenol/chloroform extraction and resuspended in 5 µl H2O. Samples (5 µl) were analysed by denaturing polyacrylamide (6 %) gel electrophoresis by comparison with a DNA sequence ladder generated with the appropriate primer.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). This screening revealed some important differences between these related enterobacteria. Both S. enterica serovars harbour orthologues to all components of the cue regulon, including the previously identified MerR-like regulator CueR/SctR, the periplasmic copper oxidase CuiD/CueO, as well as a close homologue to the inner-membrane P-type transporter CopA (Table 2). Interestingly, no orthologues to the components of the cus system were detected, except for the YedW/YedV two-component system that in E. coli was recently shown to be under transcriptional control of CusR (Yamamoto & Ishihama, 2005). In addition, S. Typhimurium harbours a second cue-like regulon, gol. We have recently shown that this regulon, which includes a P-type ATPase (GolT), a putative metal-binding protein (GolB) and a transcriptional regulator (GolS), endows S. Typhimurium with resistance to gold salts (Checa et al., 2007). The golTS operon is present in most Salmonella subspecies and in Salmonella bongori (http://www.sanger.ac.uk/Projects/Salmonella/), but absent in S. Typhi (Table 2) and S. enterica serovar Paratyphi A. On the other hand, golB is present in all sequenced salmonellas.
|
). To probe if expression of copA depends on CueR, we first constructed a strain carrying a chromosomal lacZ reporter fusion to the promoter of the transporter gene (see Methods for details) and assayed its expression in the presence or absence of copper ions. The lacZ insertion was generated as a copA operon fusion, without disrupting the transporter gene. This will ensure proper copper efflux. Addition of 1 mM CuSO4 to the culture medium induced copA expression by 13-fold in LB (Fig. 1b) and by eightfold in SM9 minimal medium (Checa et al., 2007). This copper-dependent activation of copA was eliminated in a cueR null mutant, but not affected in a golS strain, confirming that CueR controls the expression of the Cu(I) transporter CopA in Salmonella. A similar result was obtained using a cuiD-lacZ reporter whose expression also depends on CueR (Fig. 1c). Interestingly, deletion of cueR increased rather than decreased copper-induced expression of the GolS-controlled gene golB (Fig. 1d). This increase probably reflects accumulation of cytoplasmic copper ions, because of low-level expression of CopA in the cueR strain (Fig. 1b, see also below).
|
cueR mutant cells grown in the presence or absence of CuSO4. A single primer extension product, corresponding to a G residue located 26 nt upstream of the copA start codon, was observed only in samples obtained from the wild-type strain grown in the presence of copper ions (Fig. 2a). The Salmonella copA transcription start site differs in one base from the one determined previously in E. coli by Outten et al., (2000), Petersen & Moller (2000) and Stoyanov et al. (2001).
|
). Both the extension of the protected region (from nt –39 to –13 relative to the transcription start site in the coding strand and from nt –15 to –40 in the non-coding strand) and the presence of hypersensitive bands (at nt –31 and –19 and at –33 and –21 in the coding and non-coding strands, respectively) are common features of the protein–DNA interaction described for the MerR family (Ansari et al., 1995; O'Halloran et al., 1989; Outten et al., 1999). The protected sequence of the copA promoter encompasses the sequence 5'-TTGACCTTAACCTTGCTGGAAGGTTTA-3', which includes an imperfect ACCTTCC inverted repeat sequence located between the predicted –35 and –10 elements in the copA promoter region, is similar to the predicted E. coli CueR-binding site (Fig. 2c, d; Outten et al., 2000; Stoyanov et al., 2001; Yamamoto & Ishihama, 2005).
CopA and CuiD are essential for copper tolerance under both aerobic and anaerobic conditions
We performed copper-sensitivity assays under both aerobic and anaerobic conditions using different mutant strains in the CueR-regulated genes. In the presence of oxygen, copper tolerance decreased in strains carrying mutations in either cueR, copA or cuiD, although the latter strain showed the most severe phenotype (Table 3). Copper susceptibility increased even more in the cuiD copA double mutant strain, supporting the relevant role of both proteins in maintaining copper homeostasis. The marked copper susceptibility of the single cuiD mutant compared with the cueR or the copA strains suggests that even basal levels of CuiD are enough to guarantee survival in copper-rich medium, and supports the crucial role assigned to this enzyme for copper tolerance under aerobic conditions in Salmonella (Lim et al., 2002).
|
), when the multicopper oxidase CueO is predicted to be inactive (Outten et al., 2001). Unexpectedly, we observed that survival of a cuiD mutant strain in the presence of copper was affected even in cells grown under anaerobic conditions (Table 3), suggesting that the encoded enzyme could play an additional role in copper homeostasis in Salmonella. Periplasmic multicopper oxidases are involved in the conversion of the harmful Cu(I) to Cu(II) in the presence of oxygen (Singh et al., 2004; Tree et al., 2005).
We observed that under anaerobic conditions copper inhibited the bacterial growth even more strongly than under aerobic conditions (Table 3), as was previously observed in E. coli (Beswick et al., 1976; Outten et al., 2001; Rensing & Grass, 2003). This supports the notion that copper injury to bacterial cells cannot be mediated exclusively by oxidative DNA damage (Macomber et al., 2007).
The Salmonella YedW/YedV system is not involved in copper homeostasis
Salmonella lacks the ancillary copper-detoxification cus system, but conserves genes homologous to the E. coli yedWV operon, STM1096 and STM1095 (Table 1). The yedWV operon encodes a two-component system, transcription of which in E. coli is activated by copper ions (Yamamoto & Ishihama, 2005).
We analysed whether YedW/YedV contributed to maintaining copper homeostasis in Salmonella, testing survival of the yedWV mutant strain in the presence of CuSO4 (Table 3). yedWV expression is not induced by addition of up to 2 mM CuSO4 (data not shown). Moreover, deletion of yedWV does not affect copper tolerance of the wild-type strain or of the cueR or the cueR golS mutants (see below), under either aerobic or anaerobic conditions, arguing against a role of this operon in copper homeostasis in Salmonella.
The gold transporter GolT can contribute to copper tolerance in the absence of CopA
We have recently shown that Salmonella has a second CueR homologue highly sensitive to gold ions, GolS, which induces the expression of a CopA-homologous protein, GolT, and a putative metal-binding protein, GolB (Checa et al., 2007). We found that this Salmonella-specific regulon is required for gold resistance, but not for copper tolerance, except in a strain in which the main copper transporter CopA has been deleted (Checa et al., 2007; see also Table 3). These results prompted us to investigate whether some of the GolS-controlled genes, including golS, would acquire relevance in copper homeostasis when the ancestral copper-detoxification system cue is inactive or absent. We constructed a series of mutant strains in which copA and the different genes coding for components of the gol regulon were deleted. As seen in Table 3, only the deletion of the P-type metal transporter gene golT rendered a marked reduction in copper tolerance in a copA strain. Moreover, the simultaneous deletion of the two transporter genes, copA and golT, further increased the susceptibility of a copA cuiD mutant strain. The contribution of GolT to copper detoxification in the absence of a functional CopA was also observed in cells grown under anaerobic conditions (Table 3). Unlike golT, deletion of golB did not affect metal tolerance of a copA strain, but slightly reduced survival of a strain with both CopA and GolT transporters deleted (Table 3). Neither single mutants in GolS-regulated genes, nor a mutant with the whole gol locus deleted, altered Salmonella copper susceptibility of a cuiD or a wild-type background (Table 3). In accordance, deletion of the gold-sensor gene golS had only a minor effect on copper tolerance in a cueR mutant strain (Table 3), which was only evident in liquid media and under aerobic conditions (Fig. 3a).
|
). We analysed the copper-induced expression of golB as a GolS-dependent gene, in wild-type and copA backgrounds (Fig. 3b). Copper-induced golB expression increased up to 24-fold in the copA strain compared with the levels obtained in the wild-type strain. This induction was dependent on the intactness of golS (Fig. 3b), suggesting that, in the absence of CopA, there is a rise of intracellular copper concentration that can induce the expression of the gol regulon. In this condition, GolT will contribute to transporting the excess copper ions out of the cytoplasm, reducing the toxicity of the cation. In support of these observations, we found an increase in copper susceptibility of the double copA golS mutant when compared with the single mutants (Table 3; see also Fig. 3c). Altogether, these results indicate that, in the absence of the main copper transporter, at least some of the factors controlled by GolS can assist in copper tolerance. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Moore & Helmann, 2005; Nies, 2003; Rensing & Grass, 2003). Among these systems, transporters of the P-type ATPases family (Arguello et al., 2007; Kuhlbrandt, 2004) are usually involved. We have demonstrated here that expression of the Salmonella P-type ATPase CopA is induced by copper ions in a CueR-dependent manner (Figs 1 and 2) and that deletion of the copA gene affects copper tolerance under both aerobic and anaerobic conditions (Table 3). These observations, in addition to the previously reported role of CueR and CuiD in copper homeostasis (Kim et al., 2002; Lim et al., 2002), indicate that this pathogenic enterobacterium possesses a complete and functional cue system, similar to the E. coli counterpart (Outten et al., 2000; Rensing & Grass, 2003). Nevertheless, the contribution of CopA and CuiD to copper tolerance in Salmonella differs in some aspects from E. coli. First, the main mechanism apparently used by Salmonella to eliminate excess of copper under aerobic conditions is the conversion of Cu(I) to Cu(II) by CuiD (Table 3). Second, CuiD is also required for copper tolerance under anaerobic conditions (Table 3), differing from E. coli (Outten et al., 2001). A similar effect was reported previously for the Rhodobacter capsulatus multicopper oxidase (Wiethaus et al., 2006). It has been proposed that CueO from E. coli can also contribute to copper tolerance by loading of the folded protein with copper ions in the cytoplasm prior to its subsequent transport into the periplasmic space by the Tat system (Rensing & Grass, 2003). Our results suggest that this detoxification mechanism would acquire relevance in Salmonella, which lacks the ancillary cus system.
The E. coli yedWV orthologous genes, although present in Salmonella (Table 2), are neither required for copper tolerance (Table 3) nor induced under excess of copper (data not shown). The above observations, in addition to the absence of E. coli cus homologues in all sequenced Salmonella serovars (Table 2 and http://www.sanger.ac.uk/Projects/Salmonella/), highlight differences between these closely related enterobacteria in the approach used to control copper excess, in particular, in conditions where the cue system is overloaded.
In a previous report, we characterized the gol regulon that confers resistance to gold ions and demonstrated that, in the absence of the native copper transporter CopA, survival of a strain with the whole gol locus deleted is impaired in the presence of CuSO4 (Checa et al., 2007). Nevertheless, deletion of the gol locus did not affect copper tolerance of a wild-type or a cuiD mutant strain. In this work, we characterized the role of the gol regulon in copper detoxification in more detail. We found that among the GolS-regulated factors, the P-type ATPase GolT is mainly responsible for alleviating copper toxicity in a copA mutant strain under both aerobic and anaerobic conditions (Table 3), probably by directing active efflux of the metal ions from the cytoplasm. Our results suggest that in the absence of the main copper transporter CopA, the intracellular copper concentration increases, as was previously suggested to occur in E. coli (Stoyanov et al., 2003). This leads to the copper-mediated activation of GolS, enhancing the expression of its target genes (Fig. 3), including golT, which would extrude the excess of copper, mimicking the action of CopA. A number of observations indicate, however, that the contribution of the gol system to copper homeostasis in nature would be incidental and lacks physiological relevance. Copper-dependent activation of the gol regulon was only observed when the major copper transporter CopA was deleted (Fig. 3b). In addition, golT and golS are absent in S. Typhi (Table 2) and S. Paratyphi A. The lack of part of the gol regulon in these two serovars of S. enterica subspecies I, which are well-known human-adapted pathogens (Parkhill & Thomson, 2003), supports the notion that this regulon allows Salmonella to gain access to different environmental niches. On the other hand, the two metal transporters CopA and GolT are structural and functional homologues: they share 42 % identity at protein level (Table 2) and both are able to mediate either copper or gold resistance under certain conditions (Table 3; Checa et al., 2007). Therefore, it is highly unlikely that GolT could physiologically replace the absent cus system in Salmonella.
The periplasmic space of Gram-negative bacteria has been proposed to be an important target for copper toxicity, because two of the three copper-resistance systems from E. coli, CueO and CusCFBA, work by removing Cu(I) from this compartment (Franke et al., 2003; Outten & O'Halloran, 2001; Rensing & Grass, 2003). Therefore, it will be interesting to know how Salmonella, which lacks the cus system, can avoid periplasmic copper stress under anaerobic conditions when the multicopper oxidase is inactive. The complete elucidation of the mechanisms employed by Salmonella to eliminate the excess of copper and the differences from those previously described in E. coli will contribute to better understanding of the distinct lifestyles of these related bacteria.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: J Green
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ansari, A. Z., Bradner, J. E. & O'Halloran, T. V. (1995). DNA-bend modulation in a repressor-to-activator switching mechanism. Nature 374, 371–375.[Medline]
Arguello, J. M., Eren, E. & Gonzalez-Guerrero, M. (2007). The structure and function of heavy metal transport P(1B)-ATPases. Biometals 20, 233–248.[CrossRef][Medline]
Beswick, P. H., Hall, G. H., Hook, A. J., Little, K., McBrien, D. C. & Lott, K. A. (1976). Copper toxicity: evidence for the conversion of cupric to cuprous copper in vivo under anaerobic conditions. Chem Biol Interact 14, 347–356.[CrossRef][Medline]
Borkow, G. & Gabbay, J. (2005). Copper as a biocidal tool. Curr Med Chem 12, 2163–2175.[CrossRef][Medline]
Bullas, L. R. & Ryu, J. I. (1983). Salmonella typhimurium LT2 strains which are r– m+ for all three chromosomally located systems of DNA restriction and modification. J Bacteriol 156, 471–474.
Checa, S. K., Espariz, M., Pérez Audero, M. E., Botta, P. E., Spinelli, S. V. & Soncini, F. C. (2007). Bacterial sensing of and resistance to gold salts. Mol Microbiol 63, 1307–1318.[CrossRef][Medline]
Cherepanov, P. P. & Wackernagel, W. (1995). Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9–14.[CrossRef][Medline]
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.
Davis, R. W., Bolstein, D. & Roth, J. R. (1980). Advanced Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Ellermeier, C. D., Janakiraman, A. & Slauch, J. M. (2002). Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria. Gene 290, 153–161.[CrossRef][Medline]
Franke, S., Grass, G., Rensing, C. & Nies, D. H. (2003). Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. J Bacteriol 185, 3804–3812.
Kershaw, C. J., Brown, N. L., Constantinidou, C., Patel, M. D. & Hobman, J. L. (2005). The expression profile of Escherichia coli K-12 in response to minimal, optimal and excess copper concentrations. Microbiology 151, 1187–1198.
Kim, J. S., Kim, M. H., Joe, M. H., Song, S. S., Lee, I. S. & Choi, S. Y. (2002). The sctR of Salmonella enterica serovar Typhimurium encoding a homologue of MerR protein is involved in the copper-responsive regulation of cuiD. FEMS Microbiol Lett 210, 99–103.[CrossRef][Medline]
Kuhlbrandt, W. (2004). Biology, structure and mechanism of P-type ATPases. Nat Rev Mol Cell Biol 5, 282–295.[CrossRef][Medline]
Lejona, S., Aguirre, A., Cabeza, M. L., García Véscovi, E. & Soncini, F. C. (2003). Molecular characterization of the Mg2+-responsive PhoP-PhoQ regulon in Salmonella enterica. J Bacteriol 185, 6287–6294.
Lim, S. Y., Joe, M. H., Song, S. S., Lee, M. H., Foster, J. W., Park, Y. K., Choi, S. Y. & Lee, I. S. (2002). CuiD is a crucial gene for survival at high copper environment in Salmonella enterica serovar Typhimurium. Mol Cells 14, 177–184.[Medline]
Macomber, L., Rensing, C. & Imlay, J. A. (2007). Intracellular copper does not catalyze the formation of oxidative DNA damage in Escherichia coli. J Bacteriol 189, 1616–1626.
Magnani, D. & Solioz, M. (2005). Copper chaperone cycling and degradation in the regulation of the cop operon of Enterococcus hirae. Biometals 18, 407–412.[CrossRef][Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Moore, C. M. & Helmann, J. D. (2005). Metal ion homeostasis in Bacillus subtilis. Curr Opin Microbiol 8, 188–195.[CrossRef][Medline]
Munson, G. P., Lam, D. L., Outten, F. W. & O'Halloran, T. V. (2000). Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12. J Bacteriol 182, 5864–5871.
Nies, D. H. (2003). Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27, 313–339.[CrossRef][Medline]
O'Halloran, T. V., Frantz, B., Shin, M. K., Ralston, D. M. & Wright, J. G. (1989). The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell 56, 119–129.[CrossRef][Medline]
Outten, C. E. & O'Halloran, T. V. (2001). Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science 292, 2488–2492.
Outten, C. E., Outten, F. W. & O'Halloran, T. V. (1999). DNA distortion mechanism for transcriptional activation by ZntR, a Zn(II)-responsive MerR homologue in Escherichia coli. J Biol Chem 274, 37517–37524.
Outten, F. W., Outten, C. E., Hale, J. & O'Halloran, T. V. (2000). Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, CueR. J Biol Chem 275, 31024–31029.
Outten, F. W., Huffman, D. L., Hale, J. A. & O'Halloran, T. V. (2001). The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli. J Biol Chem 276, 30670–30677.
Parkhill, J. & Thomson, N. (2003). Evolutionary strategies of human pathogens. Cold Spring Harb Symp Quant Biol 68, 151–158.[CrossRef][Medline]
Petersen, C. & Moller, L. B. (2000). Control of copper homeostasis in Escherichia coli by a P-type ATPase, CopA, and a MerR-like transcriptional activator, CopR. Gene 261, 289–298.[CrossRef][Medline]
Rensing, C. & Grass, G. (2003). Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS Microbiol Rev 27, 197–213.[CrossRef][Medline]
Singh, S. K., Grass, G., Rensing, C. & Montfort, W. R. (2004). Cuprous oxidase activity of CueO from Escherichia coli. J Bacteriol 186, 7815–7817.
Stoyanov, J. V., Hobman, J. L. & Brown, N. L. (2001). CueR (YbbI) of Escherichia coli is a MerR family regulator controlling expression of the copper exporter CopA. Mol Microbiol 39, 502–511.[CrossRef][Medline]
Stoyanov, J. V., Magnani, D. & Solioz, M. (2003). Measurement of cytoplasmic copper, silver, and gold with a lux biosensor shows copper and silver, but not gold, efflux by the CopA ATPase of Escherichia coli. FEBS Lett 546, 391–394.[CrossRef][Medline]
Tree, J. J., Kidd, S. P., Jennings, M. P. & McEwan, A. G. (2005). Copper sensitivity of cueO mutants of Escherichia coli K-12 and the biochemical suppression of this phenotype. Biochem Biophys Res Commun 328, 1205–1210.[CrossRef][Medline]
Wiethaus, J., Wildner, G. F. & Masepohl, B. (2006). The multicopper oxidase CutO confers copper tolerance to Rhodobacter capsulatus. FEMS Microbiol Lett 256, 67–74.[CrossRef][Medline]
Yamamoto, K. & Ishihama, A. (2005). Transcriptional response of Escherichia coli to external copper. Mol Microbiol 56, 215–227.[CrossRef][Medline]
Received 29 January 2007;
revised 8 May 2007;
accepted 15 May 2007.
This article has been cited by other articles:
|
|
 |
|
  S. K. Ward, E. A. Hoye, and A. M. Talaat The Global Responses of Mycobacterium tuberculosis to Physiological Levels of Copper J. Bacteriol., April 15, 2008; 190(8): 2939 - 2946. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
), 
), 
) or 
) Salmonella strains grown on LB broth containing CuSO4, at the specified concentrations. (b) β-Galactosidase activity (Miller units) from a golB : : lacZ transcriptional fusion expressed by wild-type (
) or 
) mutant cells, grown overnight in LB broth with or without the addition of the indicated amounts of CuSO4. (c) Final OD630 reached by the cultures used in (b). The data correspond to means±SD of three independent experiments done in duplicate.