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Applied and Environmental Microbiology, April 2001, p. 1437-1444, Vol. 67, No. 4
Department of Plant Pathology, University of
California, Riverside, California 92521-0122
Received 10 October 2000/Accepted 9 January 2001
Pseudomonads from environmental sources vary widely in their
sensitivity to cadmium, but the basis for this resistance is largely
uncharactarized. A chromosomal fragment encoding cadmium resistance was
cloned from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes, cadA and cadR. CadA was similar to
cadmium-transporting ATPases known mostly from gram-positive
bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase
from Escherichia coli. CadR was related to the MerR family
of response regulators that normally control mercury detoxification in
other bacterial systems. A related gene, zntR, regulates
zntA in E. coli, but it is not contiguous with
zntA in the E. coli genome as cadA
and cadR were in P. putida. In addition, unlike
ZntA and other CadA homologs, but similar to the predicted product of
gene PA3690 in the P. aeruginosa genome, the P. putida CadA sequence had a histidine-rich
N-terminal extension. CadR and the product of PA3689 of P. aeruginosa also had histidine-rich C-terminal extensions not found in other MerR family response regulators. Mutational analysis
indicated that cadA and cadR are fully
responsible for cadmium resistance and partially for zinc resistance.
However, unlike zntA, they did not confer significant
levels of lead resistance. The cadA promoter was responsive
to Cd(II), Pb(II), and Zn(II), while the cadR promoter was
only induced by Cd(II). CadR apparently represses its own expression at
the transcriptional level. However, CadR apparently does not repress
cadA. Homologs of the cadmium-transporting ATPase were
detected in many other Pseudomonas species.
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.
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.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1437-1444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Chromosomal Locus for Cadmium Resistance in Pseudomonas
putida Consisting of a Cadmium-Transporting ATPase and a
MerR Family Response Regulator
and
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
TABLE 1.
Bacterial strains and plasmids used
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(3.3.1.13,7)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.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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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).
|
" 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.
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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.
A LysR family response regulator was located immediately downstream from cadA with nine bases between the start codon of the LysR family response regulator and the stop codon of CadA. The Shine-Dalgarno box of the LysR regulator overlaps with the stop codon of cadA, which suggests that cadA-lysR may be part of a single transcriptional unit. However, disruption of the response regulator in the wild-type chromosome (06909s21x) did not affect cadmium resistance and showed the same resistance to all of the tested metals at the level of the wild-type strain P. putida 06909 (Table 2). Thus, the LysR response regulator does not appear to be involved in cadmium resistance in P. putida 06909, but the loss of LysR did impair bacterial growth rate and colony morphology (26).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.
|
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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.
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ACKNOWLEDGMENTS |
|---|
We thank Caroline S. Harwood (University of Iowa) for providing plasmids and helpful information. Preliminary sequence data was obtained from The Institute for Genomic Research website at http://www.tigr.org.
This work was supported by U.S. Department of Agriculture/National Research Initiative competitive grant 93-37303-9227 to D. A. Cooksey.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Plant Pathology-071, 237 Webber Hall, University of California, Riverside, CA 92521-0122. Phone: (909) 787-3516. Fax: (909) 787-4294. E-mail: Cooksey{at}citrus.ucr.edu.
Present address: Korea Research Institute of Chemical Technology,
Yusong, Taejon 305-600, Korea.
Present address: IPK (Institut für Pflanzengenetik und
Kulturpflanzenforschung), Molecular Plant Physiology group,
Gatersleben, Germany.
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Applied and Environmental Microbiology, April 2001, p. 1437-1444, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1437-1444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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