INTRODUCTION

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The cadmium cation is toxic to most microorganisms, probably by binding to essential respiratory proteins (54) and through oxidative damage by production of reactive oxygen species (50). Cadmium enters bacterial cells by the transport systems for essential divalent cations such as Mn²⁺ (53) or Zn²⁺ (22). Microbial resistance to cadmium is usually based on energy-dependent efflux mechanisms (46).

One of the best-characterized bacterial cadmium resistance mechanisms is determined by the cadmium-transporting ATPase found initially in gram-positive bacteria (47). The cadmium-transporting ATPase is a P-type ATPase, a member of the cation-transporting ATPases found in both Bacteria and Eucarya (48). It is widespread in Staphylococcus aureus (36) and Listeria monocytogenes (23). The ATPase is encoded by cadA, which is usually plasmid-borne and associated with transposons in L. monocytogenes (23, 24). The cadmium efflux genes in S. aureus are both plasmid-borne and chromosomal. The chromosomal locus of S. aureus is similar to cadAC of the plasmid-borne genes but confers resistance to low concentrations (MIC of 128 µg/ml) of cadmium nitrate (56). CadC, encoded immediately downstream of cadA, is a regulatory protein, which is also required for cadmium resistance in gram-positive bacteria. CadC binds to the promoter-operator area of the cadA gene and works as a transcriptional repressor in vitro (12).

Another class of cadmium resistance genes in S. aureus includes cadB or the cadB-like cadD, which confers a different mechanism of resistance (11, 39). The function of CadB is not well defined, but it may protect bacterial cells by binding cadmium in the membrane (39). A positive response regulator gene, cadX, was found in the cadB-like operon on plasmid pLUG10 in Staphylococcus lugdunensis. CadX is similar to CadC of the cadA operon but acts as a positive regulator (7). CadD of S. aureus is similar to CadB of S. lugdunensis. Hydropathy analysis of the CadD from plasmid pRW001 revealed transmembrane domains with potential cadmium cation-binding motifs in the cytosolic domain (11).

A well-characterized cadmium resistance system in gram-negative bacteria is the cadmium, zinc, and cobalt (czc) resistance determinant of Alcaligenes eutrophus. The CzcC, CzcB, and CzcA proteins comprise an active efflux mechanism driven by a cation-proton antiporter, rather than a cation-transporting ATPase (35). Homologs of the czc genes, called czr, which conferred cadmium and zinc resistance, were recently identified in the chromosome of Pseudomonas aeruginosa and appear to be highly conserved in environmental isolates of that species (14). In addition, a homolog of the cadAC operon, found previously only in gram-positive bacteria, was identified in the gram-negative bacterium Stenotrophomonas maltophilia (1). The flanking insertion sequences and unusual G+C content of the locus was suggestive of its transfer from gram-positive bacteria (10).

Recently, the genome sequences of several gram-negative bacteria have revealed homologs of cadA. Functional analysis of their role in metal resistance has been conducted in Helicobacter pylori (16) and with the Escherichia coli cadA homolog, zntA (42). ZntA was originally described as a zinc-transporting ATPase, but it also confers resistance to cadmium and lead. Recent studies proposed that CadA of S. aureus and ZntA of E. coli are Pb(II)-transporting ATPases (40, 41, 45). In contrast to cadA of gram-positive bacteria, zntA expression is regulated by zntR, encoding a MerR homolog, but located in another region of the E. coli chromosome from zntA (6, 37).

In this study, and in previous reports (14, 18, 28, 43), Pseudomonas spp. from the soil and other environments have been shown to vary widely in sensitivity to cadmium. In addition to the finding of czc homologs in P. aeruginosa, one report identified cadA-encoded cadmium resistance in a Pseudomonas sp. from river sediment (61). For most pseudomonads, the basis of cadmium resistance has not been characterized, but the recent finding of cadA homologs in several bacterial genome sequences suggests that CadA may play a broader role in cadmium resistance in gram-negative bacteria. We report here homologs of CadA-ZntA and ZntR that are adjacent in the chromosome of a rhizosphere strain of Pseudomonas putida 06909 (59) and confer higher levels of cadmium resistance than ZntA of E. coli but not lead resistance. In addition, we investigated the specificity of metal ion induction of cadA and cadR transcription and the presence of cadA homologs in various Pseudomonas species.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains and plasmids. Bacterial strains and plasmids are listed in Table 1. E. coli strains were cultured at 37°C on Luria-Bertani (LB) agar or in LB broth supplemented with the appropriate antibiotics (29). Antibiotic concentrations used for E. coli strains were as follows: tetracycline, 15 µg/ml; kanamycin, 50 µg/ml; ampicillin, 100 µg/ml; or gentamicin, 20 µg/ml. E. coli strain GM2163 (dam mutant) was a host strain used for manipulation of the plasmid carrying a ClaI site that was resistant to the restriction enzyme due to an overlapping Dam methylation site. P. putida strains were grown at 28°C on mannitol-glutamate medium (MG) (20) supplemented with yeast extract (0.25 g/liter) (MGY) or in MGY broth. Antibiotic concentrations used in MGY were as follows: tetracycline, 20 µg/ml; kanamycin, 30 µg/ml; or gentamicin, 20 µg/ml. To conduct maker exchange mutagenesis, P. putida strains were cultured in LB broth under the same conditions without antibiotics.

General DNA manipulations and DNA sequencing. Standard recombinant DNA techniques were carried out for restriction endonuclease digestion, ligation, transformation of plasmid DNA, and isolation of total DNA (44). A DNA sequencing facility at the University of California, Berkeley, was used for DNA sequencing of cadmium resistance genes. The DNA sequences were analyzed with a software package from the Genetics Computer Group of the University of Wisconsin and the BLAST programs provided by the National Center for Biotechnology Information. The primers used for DNA sequencing were synthesized commercially (Genosys Biotechnologies, Inc., Woodlands, Tex). Preliminary sequence data was obtained from The Institute for Genomic Research website at http://www.tigr.org.

Plasmid construction for marker exchange mutagenesis. Insertional mutations in cadA and the gene downstream from cadA encoding a putative LysR family response regulator were constructed by cloning a kanamycin resistance gene cassette into XhoI sites in each of these genes, with the cassette in the opposite orientation relative to the transcription of the target genes (Table 1). The cadR gene was similarly mutated by insertion of a gentamicin resistance gene cassette. After subcloning of these constructs into the broad-host-range plasmid pRK415, the plasmids were introduced into the wild-type strain P. putida 06909 by triparental mating with pRK2013 as a helper plasmid. Marker exchange mutagenesis was carried out as described previously (57). Mutants sensitive to tetracycline but resistant to kanamycin or gentamicin were selected, and the correct gene replacement was confirmed by Southern blot hybridization. P. putida 06909s21x, 06909s22x, and 06909s23 were selected as LysR, CadA, and CadR mutants, respectively.

Plasmid construction to measure promoter activity. A low-copy-number broad-host-range transcriptional fusion vector, pRKL1, was constructed and used to analyze promoter activity from cadA and cadR. A promoterless lacZ gene was cloned into pRK415 in the opposite orientation to the lac promoter, and the promoter regions of divergently transcribed cadA and cadR were cloned in front of lacZ in this vector (Table 1). The resulting pRCD31 carries the cadA promoter, and pRCD32 carries the cadR promoter, as transcriptional fusions with lacZ.

MIC determination. The MICs of several metals, along with cadmium chloride, were determined on mannitol-glutamate agar (20) supplemented with yeast extract at 0.25 g/liter (MGY agar) and one of the following chemicals, at various concentrations, as described previously (9): CdCl₂ · 2.5H₂O, CuSO₄ · 5H₂O, ZnSO₄ · 7H₂O, HgCl₂, AgNO₃, CoCl₂ · 6H₂O, NiSO₄, KCl, NaCl, FeCl₃ · 6H₂O, CaCl₂ · 2H₂O, Pb(C₂H₃O₂) · 3H₂O, and MnCl₂ · 4H₂O.

-Galactosidase assays. P. putida 06909 or 06909s23 transconjugants carrying various plasmids were grown at 28°C overnight in 5 ml of MGY broth supplemented with appropriate antibiotics. A 0.3-ml sample of each culture was transferred into a fresh 5-ml portion of MGY broth supplemented with appropriate antibiotics and one of the metal salts at a subinhibitory concentration. The subinhibitory concentration chosen for each metal was the highest level that allowed the same growth rate of the strain as obtained without metals added, based on the MIC studies described above. The supplemented concentration of each metal for the induction studies was as follows: CdCl₂ · 2.5H₂O, 12.5 µM; CuSO₄ · 5H₂O, 25 µM; ZnSO₄ · 7H₂O, 50 µM; HgCl₂, 0.5 µM; CoCl₂ · 6H₂O, 5 µM; NiSO₄, 10 µM; Pb(C₂H₃O₂) · 3H₂O, 10 µM; and MnCl₂ · 4H₂O, 25 µM. The bacterial culture was further shaken under the same conditions for 6 h. Finally, the grown cells were resuspended into sterile water and lysed to measure the -galactosidase activity as described by Miller (29) with o-nitrophenyl--D-galactopyranoside (ONPG) as the substrate.

Detection of cadA homologs from Pseudomonas species. Total genomic DNA from various bacteria was isolated as described previously (25). The SphI fragment of pUIVS22 was gel purified and labeled by random primed labeling with a digoxigenin-dUTP DNA-labeling kit (Boehringer GmbH, Mannheim, Germany). Southern blot analysis of BamHI-digested genomic DNA from various bacteria was performed on nylon membranes (MSI, Westboro, Mass.). Posthybridization washes were carried out at relatively low stringency (2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate]-0.1% sodium dodecyl sulfate at 62°C). The hybridization was detected with a chemiluminescent substrate, disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo(3.3.1.1^3,7)decan}-4-yl)phenyl phosphate (CSPD; Boehringer), as described in the manufacturer's instructions.

Nucleotide sequence accession number. The DNA sequence containing the P. putida 06909 cadA and cadR genes has been assigned GenBank accession no. AF333961.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Identification and mutagenesis of a cadmium-transporting ATPase. We have previously cloned genes from P. putida 06909 induced during colonization of a plant pathogenic fungus, Phytophthora parasitica (25). One of the clones, pUIVS2, carried at its 5' end a partial sequence of a heavy-metal-transporting ATPase gene. The nucleotide sequence was determined for 1,043 bp of the pUIVS22 clone. The deduced amino acid sequence of the 1,030-bp open reading frame (ORF) was highly similar to the C-terminal half of cadmium-transporting ATPases and other heavy-metal-transporting ATPases from many bacterial species. Comparison of the deduced amino acid sequences showed that the ORF in clone pUIVS22 is not complete, missing the N-terminal sequences and its promoter. Based on the 342-amino-acid peptide sequence, our clone was the most similar to the cadmium-transporting ATPases of gram-positive bactaria. Since experiments to detect plasmids from wild-type strain P. putida 06909 were unsuccessful (data not shown), the ORF is likely to reside in the bacterial chromosome. An insertional mutation of the partial clone was made by inserting a kanamycin resistance gene cassette in the bacterial chromosome through marker exchange mutagenesis as described in Materials and Methods. Mutational analysis of the partial clone showed that the ATPase mutant, 06909s22x, was highly sensitive to cadmium and had moderately decreased zinc resistance (Table 2). The analysis of the 06909s22x strain, along with other mutants, is further described in the next section. Therefore, we designated the gene cadA, encoding a cadmium-transporting ATPase. The ATPase was not important for fungal hyphae colonization (data not shown).

Organization and nucleotide sequences of genes required for cadmium resistance. Since we had only a partial fragment of the cadmium-transporting ATPase gene, the clone carrying the full-length gene was obtained by complementing the cadmium-sensitive mutant 06909s22x with a subgenomic library of wild-type strain P. putida 06909 DNA constructed in a broad-host-range plasmid pRK415. The subgenomic library was constructed with PstI inserts of ca. 4 to 6 kb, since Southern hybridizations showed that a 4.2-kb PstI fragment of the wild-type strain 06909 hybridized with the cadA probe from pUIVS22 (data not shown). The plasmid pRCD12 complemented the cadmium sensitivity of mutant 06909s22x. When the orientation of the 4.2-kb insert of pRCD12 was reversed in pRCD22, the clone still complemented the mutation in 06909s22x. A subcloned 3-kb PstI-HindIII fragment in pRCD13 also complemented the cadmium sensitivity of 06909s22x, but a 2-kb subclone in pRCD14 did not (Fig. 1B). This indicated that the 3-kb DNA fragment was sufficient for cadmium resistance in P. putida 06909. DNA sequence analysis of the insert in pUCD12 revealed two ORFs (Fig. 1A). One was cadA, encoding the cadmium-transporting ATPase, and the other was cadR, named after sequence comparison and mutational analysis. cadR was divergently transcribed from cadA, indicating the presence of separate promoters for the two genes. Another ORF found downstream of cadA was a partial fragment of a LysR family response regulator, which may be involved in bacterium-fungus interactions (25).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Complementation of cadium-sensitive mutant 06909s22x (cadA::km). (A) Map of a 4.2-kb PstI fragment carrying cadA and cadR. (B) Complementation of cadmium sensitivity by different subclones. Plac indicates lac promoter in pRK415. Restriction endonuclease sites of the subclones are not indicated. A "+" indicates complementation of cadmium sensitivity to cadmium resistance, but a "" indicates no complementation. Abbreviations: C, ClaI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; Sp, SphI; Xh, XhoI.

Similarity to genes for heavy metal resistance. cadA encodes 737 amino acids, and the deduced amino acid sequence shared strong similarity with other known heavy-metal-transporting ATPases, especially cadmium-transporting ATPases and zinc-transporting ATPases (Fig. 2). Alignment of CadA of P. putida 06909 showed the conserved motifs and residues for P-type ATPase function, including metal binding (Fig. 2), ATP binding, and aspartyl phosphorylation sites (data not shown) (49). Initially, ZntA was identified as a zinc-transporting ATPase in E. coli. However, it was also shown that ZntA transports Cd(II) and Pb(II) (40, 41). CadA of pI258 in S. aureus, a homolog of ZntA, also transports Cd(II), Zn(II), and Pb(II) (40). In contrast, CadA of P. putida 06909 provided more specific resistance to cadmium. It was partially responsible for zinc resistance, but its contribution to lead resistance was negligible. The level of cadmium resistance in P. putida 06909 was also 17-fold higher than the cadmium resistance of E. coli K-12 carrying zntA.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Alignment of the N-terminal region and metal-binding domains of CadA or ZntA from P. putida, P. aeruginosa (51), E. coli (48), S. maltophilia (1), S. aureus (42), B. firmus (19), and L. monocytogenes (27). Identical bases are shown as white letters on a dark background. Asterisks indicate identical residues among all seven proteins.

View larger version (41K): [in this window] [in a new window]   FIG. 3.   Alignment of CadR of P. putida 06909 with the MerR family of activator-repressors, including the predicted product of gene PA3689 of P. aeruginosa (51), ZntR of E. coli (6), and MerR of Tn501 in pVS1 of P. aeruginosa (31). Identical bases are shown as white letters on a dark background. Asterisks are Hg(II)-binding cysteine residues of MerR (62), and numbers 1 to 7 are the heptad repeats that form helical structures to promote homodimer formation (6). The identities (ID) of the amino acid sequences to CadR are indicated.

Mutation of cadA and cadR and heavy metal sensitivity. While the MIC of cadmium chloride for the wild-type P. putida 06909 was 1.7 mM, the MICs of cadmium chloride for the mutant P. putida 06909s22x (cadA::km) and 06909s23 (cadR::gm) were 0.05 and 1.3 mM, respectively (Table 2). The MIC of zinc sulfate was also different among mutant strains. The 06909s22x (cadA::km) mutant showed less resistance to zinc sulfate, although the MIC (7.0 mM) was still high. The mutant 06909s23 (cadR::gm) also showed only a slight decrease in its MIC (10.5 mM) of zinc sulfate from the wild-type MIC of 11.5 mM. A trivial decrease in the MIC of lead acetate was observed from the two mutant strains. The MIC to lead acetate was 2.4 mM for the wild type and 2.3 mM for both the cadA and the cadR mutants. However, the MICs of other heavy metals tested for mutant P. putida 06909s22x and 06909s23 strains were not different from that of wild-type P. putida 06909 (Table 2). The mutational analysis was consistent with cadA encoding a cadmium-specific transporting ATPase that is partially responsible for zinc resistance in P. putida 06909. cadR is also necessary for full resistance to zinc and cadmium. The MIC of cadmium chloride for E. coli K-12 strain carrying zntA was 0.1 mM on MGY plates (data not shown), while the MIC of cadmium acetate for this strain on LB medium was 1.5 mM (42). This indicated that cadA of P. putida 06909 is responsible for much higher resistance to Cd(II) than zntA of E. coli.

Induction of the promoter region by cadmium and its specificity. The 84-bp space between the divergently transcribed cadA and cadR genes was assumed to contain two promoters with different orientations (Fig. 1A). A transcriptional fusion vector, pRKL1, with a promoterless lacZ gene was constructed. Since the vector pRKL1 alone showed a low background level of -galactosidase activity in the presence of various metals in our strain (data not shown), it was used to construct a transcriptional fusion between the cadA or cadR promoter and the promoterless lacZ.

View this table: [in this window] [in a new window]   TABLE 3.   Specificity of expression of -galactosidase activity from the promoter regions of cadA and cadR in wild-type 06909 with different metal ions

View this table: [in this window] [in a new window]   TABLE 4.   -Galactosidase activity from promoter regions of cadA and cadR in wild-type strain 06909 and CadR mutant 06909s23 backgrounds

Conservation of cadmium-transporting ATPase genes among Pseudomonas species. Southern hybridizations were carried out to determine whether the cadmium-specific P-type ATPase is widespread in various Pseudomonas species. The bacterial strains, their characteristics, and respective cadmium chloride MICs are summarized in Fig. 4. Most of the pseudomonads tested showed various degrees of hybridization signals with the cadmium-transporting ATPase of P. putida 06909 except Pseudomonas sp. strain 07887 isolated from tomato (Fig. 4). No hybridization was observed from cadmium-sensitive Xanthomonas axonopodis pv. vesicatoria, Agrobacterium radiobacter, or E. coli DH5. Southern hybridizations suggested that the CadA ATPase is conserved among many Pseudomonas species and strains. It will be interesting to investigate whether variations in the CadA sequence or in its expression contribute to the observed variation in cadmium resistance among Pseudomonas species and strains.

View larger version (65K): [in this window] [in a new window]   FIG. 4.   Southern blot hybridization of the cadA gene of P. putida 06909 to total DNA from cadmium-resistant Pseudomonas species and other gram-negative bacteria. The MICs (mM) of cadmium chloride for each strain are indicated in parentheses with the original strain reference in brackets as follows: lane 1, P. putida 06909 (1.7 [59]); lane 2, P. putida 08891 (4.0 [9]); lane 3, P. fluorescens 09906 (0.7 [58]); lane 4, P. fluorescens 2-79 (0.7 [55]); lane 5, P. fluorescens 08908 (>4.0 [8]); lane 6, P. fluorescens 0785-17 (1.0 [30]); lane 7, P. fluorescens 08892 (0.1 [9]); lane 8, P. fluorescens 513 (0.1); lane 9, P. aeruginosa PAO1 (4.0 [17]); lane 10, P. stutzeri ATCC 17588 (4.0); lane 11, P. cichorii 07881 (0.1 [9]); lane 12, P. syringae pv. syringae PS61 (0.5 [3]); lane 13, P. syringae pv. tomato (0.4 [2]); lane 14, Pseudomonas sp. strain 02894 (2.0 [8]); lane 15, Pseudomonas sp. strain 07887 (1.0 [8]); lane 16, Pseudomonas sp. strain 07888 (1.2 [8]); lane 17, Xanthomonas axonopodis pv. vesicatoria 07882 (0.2 [9]); lane 18, Agrobacterium radiobacter K84 (0.05 [34]); lane 19, E. coli DH5 (0.2 [44]); and lane 20, plasmid DNA of pUIVS22 carrying the cadA gene. The DNA in each lane was completely digested with BamHI, and a total of 2 µg of DNA per lane was loaded for each sample. A total of 100 ng of plasmid DNA was loaded for the plasmid in the lane 20.