Department of Biological Sciences, Science Drive 4, National University of Singapore, Singapore1175431
Author for correspondence: Sanjay Swarup. Tel: +65 874 7933. Fax: +65 779 2486. e-mail: dbsss{at}nus.edu.sg
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ABSTRACT |
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Keywords: copper tolerance, CPx-type ATPase, mini-Tn5::gfp mutagenesis
Abbreviations: GFP, green fluorescent protein; MIC, minimal inhibitory concentration
The GenBank accession number for the sequence reported in this paper is AF390440.
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INTRODUCTION |
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Eukaryotic and prokaryotic systems possess several mechanisms for copper homeostasis, such as reduced influx, facilitated efflux, sequestration and modification (Camakaris et al., 1999 ; Dameron & Harrison, 1998
). In eukaryotes, many transition metals, including copper, are generally sequestered by small cysteine-rich proteins termed metallothioneins. Apart from metallothioneins, eukaryotes also possess transporters such as the P-type ATPases for the facilitated efflux of these metals. P-type ATPases form a phospho-aspartate intermediate in the ATP-driven cation-transport cycle and contain conserved domains for phosphorylation, ATP binding and phosphatase activity (Lutsenko & Kaplan, 1995
). P-type ATPases involved in metal-ion transport contain additional motifs such as a conserved HP dipeptide, a transmembrane CPx motif and one or more amino-terminal metal-binding domains (Lutsenko & Kaplan, 1995
; Solioz & Vulpe, 1996
). These P-type ATPases are called P1-type ATPases or CPx-type ATPases. Examples of eukaryotic copper-transport P1-type ATPases include CaCRP1 of Candida albicans (Weissman et al., 2000
) and the Menkes disease and Wilson disease proteins of human origin (Bull & Cox, 1994
).
Prokaryotic copper-transport systems generally use copper-binding proteins and/or P1-type ATPases that are encoded by plasmids or the chromosome. P1-type ATPases have been described for bacterial copper-transport systems in many genera, including Enterococcus (Odermatt et al., 1993 ), Helicobacter (Bayle et al., 1998
) and Escherichia (Rensing et al., 2000
). Previous studies on Pseudomonas spp. have focused mainly on the high levels of copper resistance seen in strains recovered from agricultural soils with a history of copper fungicide applications. One of the best characterized determinants of copper resistance in prokaryotes is the copABCD operon system that resides on a 35 kb plasmid of the copper-resistant strain of Pseudomonas syringae pv. tomato PT23.2 isolated from Californian tomato fields (Mellano & Cooksey, 1988
). This operon, encoding copper-binding periplasmic and membrane proteins, is similar in structure and function to the other known plasmid-encoded determinant of copper resistance, the pco operon identified on a plasmid from Escherichia coli (Tetaz & Luke, 1983
). Similarly, chromosomal determinants of copper tolerance have been cloned from many Pseudomonas strains and these have been found to have similarity either to the plasmid-encoded cop operon or to the genes involved in cytochrome c biogenesis in E. coli (Cooksey et al., 1990
; Vargas et al., 1995
; Yang et al., 1996
). Recently, a copper-inducible gene was identified in the chromosome of Pseudomonas fluorescens and it was shown to affect the copper tolerance of this bacterium (Tom-Petersen et al., 2001
). A partial sequence of this chromosomal gene showed similarity to putative P-type ATPases.
Here, we show the involvement of a P1-type ATPase and a MerR-type regulatory protein encoded by a single operon in copper homeostasis in Pseudomonas putida. Further evidence shows that this chromosomally located operon, cueAR, is regulated by copper and the regulator, and that both members of this operon are partially functionally redundant.
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METHODS |
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Transposon mutagenesis and screening of mutants.
For miniTn5::gfp mutagenesis, biparental mating was performed between the donor E. coli strain and the recipient P. putida strain. Patch-matings of 4 h duration were harvested in saline and spread onto TSA plates that contained chloramphenicol, gentamicin and kanamycin. A conjugation frequency of 2x10-3 (recipient cell)-1 was obtained while the spontaneous mutation frequency was 1x10-7 (recipient cell)-1. Insertion of mini-Tn5::gfp was checked by colony PCR, in which transconjugant colonies that had been grown for 36 h were used as templates together with primers for the amplification of gfp (forward, 5'-TAGATGGTGATGTTAATGGGC-3'; reverse, 5'-GCCATGTGTGTAATCCCAGCAG-3'). The PCR protocol used was: 30 cycles of denaturation at 95 °C for 40 s, annealing at 58 °C for 40 s and extension at 72 °C for 1 min. Taq DNA polymerase (Promega) was used to catalyse the reaction. Of the 120 randomly chosen colonies that were tested, all were positive for amplification of gfp (data not shown). Approximately 9000 transconjugants carrying gfp transcriptional fusions were screened by replica-plating for increased green fluorescence when grown in the presence of transition metals on SM medium (Stanier et al., 1966 ). One of the eight putative mutants that showed the highest response to transition metals in our preliminary studies was chosen for further analyses. This mutant was designated CEM1 (copper export mutant), based on the results shown later.
Metal-sensitivity assays.
The minimal inhibitory concentration (MIC) of each of the transition metals tested was estimated by streaking single colonies from MGY plates onto plates containing varying concentrations of transition metal ions, namely, Hg2+, Ag+, Zn2+, Fe2+, Cu2+, Ni2+, Co2+ and Cd2+. The lowest metal concentration that inhibited growth after incubation for 60 h at 30 °C was recorded as the MIC. Sensitivity of the cells to copper in MGY broth was checked as described below in Complementation studies.
DNA manipulations and analyses.
Routine recombinant DNA techniques were performed as described by Sambrook et al. (1989) . Genomic DNA was isolated from the Pseudomonas cells as described previously (Syn & Swarup, 2000
). The genomic DNA of CEM1 cells was digested with BglII and KpnI, and Southern-blot analysis was done. Full-length DNA probes for the gfp and gentamicin-resistance genes were obtained from pAG408 (Suarez et al., 1997
). Digoxigenin-labelling was done according to the manufacturers protocol (Boehringer Mannheim). BglII and KpnI cut the transposon, leaving the kanamycin- and gentamicin-resistance genes intact (Suarez et al., 1997
). The transposon-flanking upstream genomic DNA from the mutant strain was cloned using BglII, and downstream genomic DNA was cloned using KpnI. After cloning of these flanking DNA fragments into the vectors pUC18 and pBSIISK+, further subcloning of the transposon-flanking DNA was done to sequence 3 kb of the upstream and 3·3 kb of the downstream region. Vector primers and gene-specific primers were used to sequence the cloned fragments. Automated DNA sequencing was performed with the BigDye Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit (Applied Biosystems). Primers containing an XbaI site (cueAR-F, 5'-CGTCTAGAGCGATGGCAAGGTCAAGTTC-3', and cueAR-R, 5'-CCTCTAGAGCTGTTCCACGGCATTCCC-3'; restriction site shown in bold) were designed to amplify a 3·6 kb fragment that encoded the entire cueAR operon along with its promoter. A proof-reading-type Vent DNA polymerase (New England Biolabs) was used to catalyse the reaction. The PCR protocol used was: 30 cycles of denaturation at 95 °C for 40 s, annealing at 60 °C for 40 s and extension at 76 °C for 3 min 40 s. Plasmid pGBcueAR was constructed by subcloning the 3·6 kb fragment into the XbaI site of the shuttle vector pGB1 (Bloemberg et al., 1997
). Using this construct, pGBcueA was generated by excising a 0·2 kb DNA fragment from the 3' end of cueR by using SacI, thereby truncating cueR. The second gene of the cueAR operon, cueR, was amplified with an upstream primer containing an EcoRI site (CueR-F, 5'-AGAATTCCTGatgGCGCTGTCGAG-3'; restriction site in bold, ATG of cueR in lower-case) and the downstream primer cueAR-R that was used for amplification of cueAR. The cueR gene fragment was cloned into the EcoRI and XbaI sites of pGB1 to construct pGBcueR, resulting in cueR expression from the lacZ promoter. In Pseudomonas, the shuttle vector pGB3 (a derivative of pGB1) allows the expression of genes under the control of the lac promoter without the need for any exogenous inducer (Bloemberg et al., 1997
). Hence, no inducer was added in the expression studies with cells containing pGBcueR. The secondary structure of a 50 bp RNA sequence of the cueAcueR junction was predicted by using the Vienna RNA package (Hofacker et al., 1994
).
Complementation studies.
The pGBcueAR, pGBcueA and pGBcueR constructs were introduced into cells of the copper-sensitive P. putida CEM1 strain by electroporation, as described by Bloemberg et al. (1997) . Cells of the wild-type strain, the CEM1 strain and the CEM1(pGBcueAR) strain were grown in MGY broth to an OD600 of 0·32, before the addition of the appropriate amounts of copper sulphate from a 100 mM stock solution to the medium. Functional complementation was studied by monitoring the growth of the cells for another 12 h at 30 °C with constant shaking (240 r.p.m.). For spectrophotometric measurements, the cells that had been grown in MGY broth were pelleted by centrifugation and resuspended in PBS (Sambrook et al., 1989
). For each experiment four replicates were maintained. For the CEM1 cells carrying pGBcueA and pGBcueR, complementation was checked by MIC analysis on MGY agar plates, as described in Metal-sensitivity assays.
Gene-expression analysis.
The cueAR transcriptional fusion with gfp (cueA::gfp) was used to study gene expression. Two methods were employed to study the production of green fluorescent protein (GFP). In the first method, the CEM1 cells were grown in MGY broth supplemented with varying levels of copper; GFP production by these cells was visualized under a confocal laser microscope (Olympus Fluoview 300 LSM). In the second method, the CEM1 strain and the CEM1(pGBcueR) strain were grown in MGY broth to an OD600 of about 0·32, before the addition of various amounts of copper sulphate to the medium. Based on the results from our preliminary studies, the cells were incubated for 4 h at 30 °C with constant shaking (240 r.p.m.). The cells were pelleted by centrifugation and resuspended in filter-sterilized PBS for measurement of their GFP fluorescence by flow cytometry (Coulter Epics Elite; Beckman Coulter). For each experiment, data from 30000 events were collected and three replicates were maintained.
Copper-accumulation analysis.
Copper sulphate was added at different concentrations to the bacterial cultures when they had reached an OD600 value of 0·32 in MGY medium. The cells were then incubated again and samples were taken from the cultures at various time intervals (0, 2, 4, 6 and 8 h). These samples were pelleted, and the copper content of the pelleted cells was analysed using an Atomic Absorption Spectrophotometer (model A6800F; Shimadzu) as described by Beard et al. (1997) . The dry weights of the cells were obtained after the pellets had been freeze-dried.
PFGE.
To ascertain the location of the cueAR region, Southern-blot analysis with the transposon-specific probe was performed on the mutant strain DNA after analysis of total DNA by PFGE. Alkaline lysis (Sambrook et al., 1989 ) and a mega-plasmid preparation (Casse et al., 1979
) were done to check for the presence of any indigenous plasmid(s). Later, PFGE was carried out to resolve the intact total DNA. Sample preparation was done based on the method described by Sambrook et al. (1989)
. In brief, agarose plugs were prepared by mixing 1 ml of Pseudomonas cells suspended in TE (10 mM Tris/HCl, 1 mM EDTA) with 1 ml of 1% low-gelling-temperature agarose prepared in TE containing 50 µg lysozyme ml-1. The plugs were treated with proteinase K before they were loaded into the wells of the PFGE gel prepared with ultra-pure DNA-grade agarose (Bio-Rad). Gel electrophoresis was carried out in 0·5xTBE at 140 V for 36 h at 1/25 with a 1:1 pulse ratio on a CHEF-DR-II apparatus (Bio-Rad). Commercially available yeast chromosomal DNA plugs (Sigma) were used for sizing the DNA fragments. Following electrophoresis, the gel was stained with ethidium bromide, visualized under a UV light and photographed. The gel was then used for Southern-blot analysis. A probe for gfp was prepared by using the DIG High-Prime Labelling and Detection Kit (Boehringer Mannheim), as per the manufacturers protocol.
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RESULTS |
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Characterization of the cueAR region
Southern-blot analysis of CEM1 genomic DNA that had been digested with BglII and KpnI showed single bands of 13·0 and 12·5 kb (data not shown) in size, respectively, indicating a single insertion of the transposon. Sequencing of the 6·3 kb DNA region surrounding the transposon insertion site revealed six ORFs and also showed that the transposon had inserted at nucleotide position 79 of a 2·4 kb ORF (Fig. 1a). The insertion site had a signature 9 bp duplication of the sequence 5'-GGCATGACC-3'. Results of a BLAST search of the 2·4 kb ORF product against the GenBank database showed that the predicted protein of 797 aa had a high degree of similarity to several functionally characterized copper-transport ATPases, including CopA of E. coli (Rensing et al., 2000
), CopA of Enterococcus hirae (Odermatt et al., 1993
) and the Wilson and Menkes disease proteins of human origin (Bull & Cox, 1994
). Based on sequence similarity and functional characteristics (shown later) similar to previously characterized copper-transport proteins, the 2·4 kb ORF of P. putida was named cueA (for Cu export ATPase A). The percent identities of the P. putida CueA amino-acid sequence with CopA of Escherichia coli, CopA of Enterococcus hirae, the human Wilson disease protein ATP7B, PA3920 of P. aeruginosa (accession no. AE004809), plasmid pPaCu1-OrfG from P. syringae (accession no. AB044355) and ActP of Sinorhizobium meliloti (accession no. AF129004) were 36, 39, 32, 68, 60 and 38, respectively. P. putida CueA is predicted to contain at least eight potential transmembrane regions, based on its hydropathy plot and its similarity to other characterized bacterial copper ATPases (Fig. 2
). The CPx motif (CPC) and other domains conserved in all P-type ATPases are present in CueA (Fig. 2
).
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Chromosomal localization of the cueAR region
Plasmid analysis was performed on the wild-type strain and on the CEM1 strain, to check for the presence of any indigenous plasmid(s) that could harbour the cueAR locus. Neither the alkaline lysis nor the mega-plasmid preparations yielded any plasmid DNA that was detectable in ethidium-bromide-stained agarose gels (data not shown). Intact genomic DNA from the wild-type strain and from the CEM1 strain was resolved by PFGE. The ethidium-bromide-stained gel showed the intact chromosomal DNA ( 2·2 Mb in size) for both strains; no signs of detectable plasmid were observed even when a higher amount of total DNA was added to the wells (Fig. 5a). Southern-blot analysis using the transposon-specific probe revealed a strong hybridizing signal only for the chromosomal DNA of P. putida CEM1 (Fig. 5b
). Thus, the transposon-tagged cueAR operon was identified as being located within the chromosome.
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Regulation of cueAR expression
Accumulation of GFP was used to study the expression of the cueAR operon, which was facilitated by the transcriptional fusion created by the fusion of mini-Tn5::gfp with cueA (cueA::gfp). Visualization of GFP expression in strain CEM1 under confocal laser microscopy showed increasing amounts of GFP fluorescence produced when the strain was grown in medium supplemented with 010 µM copper (Fig. 8a). At higher concentrations of copper (up until 50 µM), no further increase in fluorescence was visible (data not shown). No auto-fluorescence of the wild-type cells was observed (Fig. 8a
). It is noteworthy that a basal level of fluorescence was seen even when copper was not added to the growth medium. The relative amounts of fluorescence of GFP from whole cells of CEM1 and CEM1(pGBcueR) grown in the presence of varying levels of copper (060 µM) was measured by flow cytometry. Accumulation of GFP in CEM1 was dose-dependent from 0 to 30 µM copper (Fig. 8b
). At the point of maximal induction the mean level of cueA::gfp expression was twofold higher (11·59) than the background level when the cells were grown without copper (5·28). In comparison to the CEM1 cells, GFP production in the CEM1(pGBcueR) cells showed a higher response to copper. The above results clearly indicate that the cueAR operon is regulated by copper and that CueR is a positive regulator of this operon.
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DISCUSSION |
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Many Pseudomonas strains have been reported to be tolerant to varying levels of copper (Cooksey et al., 1990 ; Vargas et al., 1995
; Bender & Cooksey, 1986
). In one report, based on the screening of 20 Pseudomonas strains for copper resistance, eight strains of P. syringae pv. tomato were placed into a copper-sensitive group because of their lower tolerance to copper sulphate when grown in MGY medium supplemented with this compound (MICs of 0·40·6 mM). All eight of these strains contained plasmids (Bender & Cooksey, 1986
). Although the genes responsible for copper tolerance, and their localization, were not characterized for the eight strains, the range of their copper tolerance would place the wild-type strain studied here in the copper-sensitive group (MIC=0·4 mM). Furthermore, in those strains that are resistant to higher levels of copper, the determinants of copper tolerance have been shown to encode either copper-binding and sequestering proteins or copper oxidases (Mellano & Cooksey, 1988
; Cooksey et al., 1900
; Cervantes & Gutierrez-Corona, 1994
). Therefore, the present study is a report of the involvement of P1-type ATPase in conferring copper tolerance to a copper-sensitive group of Pseudomonas strains. Additionally, the transporter-encoding cueAR operon proved to be located in the chromosome, in contrast to the plasmid localization of many of the copper tolerance determinants of other pseudomonads (Mellano & Cooksey, 1988
; Stall et al., 1986
; Bender & Cooksey, 1986
).
cueAR is likely to play a significant role in copper homeostasis. Firstly, the wild-type strain has a relatively low level of tolerance (MIC=0·4 mM) in the context of copper resistance in bacteria. Secondly, as shown by growth studies, the loss of cueAR leads to reduced tolerance that is apparent at levels as low as 3 µM extracellular copper (Fig. 6). This observation is in agreement with the earlier understanding that micro-organisms usually satisfy their copper requirement in the 110 µM range (Cervantes & Gutierrez-Corona, 1994
); thus, cueAR putatively functions even within the range of copper levels required by the cells. Therefore, we argue that the cueAR operon in P. putida is likely to be involved in copper homeostasis, rather than conferring resistance to high levels of copper. Furthermore, the increase followed by a decline in the intracellular copper contents observed for both the wild-type and the CEM1 strain at their sub-MIC (3 µM) level (Fig. 7
) suggests more than one mechanism to be involved in copper homeostasis in P. putida. Although CueA-mediated copper transport is one mechanism involved in copper homeostasis in P. putida, the nature of other copper-homeostasis mechanisms operating in this strain is as yet unknown. In the genus Pseudomonas, and its related genus Xanthomonas, cop genes and their homologues encoded by the chromosome have been reported (Cooksey et al., 1990
; Lim & Cooksey, 1993
; Lee et al., 1994
; Vargas et al., 1995
). These genes impart relatively low levels of copper tolerance upon strains when compared to those in a P. syringae strain carrying plasmid-borne cop genes (Mellano & Cooksey, 1988
). It is noteworthy that unlike the cueA gene reported here, these plasmid-borne cop genes do not encode ATPases. In the wild-type strain studied here, which has a low level of copper tolerance, one or more of the above chromosomal genes could, therefore, possibly be present. In our study, the MIC results confirm that the loss of cueAR results in a sixfold reduction in copper tolerance; this intolerance is only to copper and not to the other metals tested (namely mercury, silver, zinc, iron, nickel, cobalt and cadmium). Moreover, it is evident that the sensitivity of strain CEM1 to copper is due to increased accumulation of this metal. Therefore, these results suggest that cueAR is specific to copper transport and CueA functions as an exporter of copper.
Gene-expression analyses, based on the cueA::gfp transcriptional fusion, indicate that the cueAR operon is autoregulated, as well as being regulated by copper. The cueAR promoter carries cis-signals (a cop box and other inverted repeats) known to bind MerR-like helixturnhelix proteins such as CueR. Moreover, CueR contains putative metal-binding residues at its C terminus. When cueR is expressed in a plasmid with the lacZ promoter that is non-responsive to copper, the expression of the native chromosomal cueAR promoter in strain CEM1 gets upregulated in a copper-dependent manner. CueR is highly similar to the Tn501 mercury-operon regulator MerR, and proteins of this class are known to become competent transcriptional activators upon metal binding (Lund & Brown, 1987 ; Outten et al., 2000
; Stoyanov et al., 2001
). Therefore, it is clear that CueR positively regulates the function of its own promoter. Additionally, as was shown in the cueA::gfp expression studies with the CEM1 strain and the cueR-complemented strain, the expression of GFP varies with respect to the extracellular concentrations of copper. Thus, it is evident that the cueAR operon is regulated by extracellular copper levels via CueR.
There may be partial functional redundancy of both cueR and cueA. The increased copper tolerance of the CEM1(pGBcueR) strain indicates that CueR alone is able to partially alleviate copper toxicity. In E. coli, it is known that the copA regulator CueR regulates the expression of another gene, cueO, which is also involved in copper tolerance (Outten et al., 2000 ; Grass & Rensing, 2001
). Perhaps an, as yet, undiscovered orthologue of CueO might exist in P. putida that may explain the partial redundancy of cueA function. With regard to P. putida CueR, the increasing GFP fluorescence obtained when CEM1 was grown in the presence of increasing levels of copper shows that the cueAR promoter responds to other transcriptional activators in the absence of CueR. Additionally, pGBcueA introduced into strain CEM1, in the absence of intact CueR, is able to restore copper tolerance levels to nearly equal those of the wild-type strain. To our knowledge, such regulation of a P1-type ATPase operon by multiple activators needs to be studied in more detail.
In addition to being under transcriptional control, cueAR function may also be under post-transcriptional control. The cueAcueR junction shows features characteristic of translational coupling (McCarthy & Gualerzi, 1990 ), with a -1 frameshift at the cueAcueR junction and the placement of a ShineDalgarno sequence as part of a stable RNA hairpin loop structure (Fig. 4
) that could prevent independent translation. Examples of tightly controlled systems created by the formation of a secondary RNA structure in the inter-cistronic boundary have been demonstrated in many cases, including that of the nifLA genes of Klebsiella pneumoniae, where inappropriate levels of NifA synthesis are avoided (Goventes et al., 1996
). It is, therefore, tempting to speculate that translational coupling could be involved in cueAR regulation, which makes this operon an interesting candidate for future studies.
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ACKNOWLEDGEMENTS |
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Received 15 February 2002;
revised 4 May 2002;
accepted 24 May 2002.