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J Bacteriol. Oct 2006; 188(20): 7242–7256.
PMCID: PMC1636237

Survival and Growth in the Presence of Elevated Copper: Transcriptional Profiling of Copper-Stressed Pseudomonas aeruginosa

Abstract

Transcriptional profiles of Pseudomonas aeruginosa exposed to two separate copper stress conditions were determined. Actively growing bacteria subjected to a pulse of elevated copper for a short period of time was defined as a “copper-shocked” culture. Conversely, copper-adapted populations were defined as cells actively growing in the presence of elevated copper. Expression of 405 genes changed in the copper-shocked culture, compared to 331 genes for the copper-adapted cultures. Not surprisingly, there were genes identified in common to both conditions. For example, both stress conditions resulted in up-regulation of genes encoding several active transport functions. However, there were some interesting differences between the two types of stress. Only copper-adapted cells significantly altered expression of passive transport functions, down-regulating expression of several porins belonging to the OprD family. Copper shock produced expression profiles suggestive of an oxidative stress response, probably due to the participation of copper in Fenton-like chemistry. Copper-adapted populations did not show such a response. Transcriptional profiles also indicated that iron acquisition is fine-tuned in the presence of copper. Several genes induced under iron-limiting conditions, such as the siderophore pyoverdine, were up-regulated in copper-adapted populations. Interesting exceptions were the genes involved in the production of the siderophore pyochelin, which were down-regulated. Analysis of the copper sensitivity of select mutant strains confirmed the array data. These studies suggest that two resistance nodulation division efflux systems, a P-type ATPase, and a two-component regulator were particularly important for copper tolerance in P. aeruginosa.

Human activity has produced elevated levels of heavy metals in the environment (13). The U.S. Environmental Protection Agency has placed 13 heavy metals, including copper (Cu), on its priority pollutants list of 129 compounds (33). Unlike some organic contaminants, heavy metals cannot be degraded by microorganisms and, thus, are some of the most persistent pollutants.

Many heavy metals, such as Cu, iron (Fe), and zinc (Zn), are required in trace amounts for bacterial growth yet are toxic when present in excess. Toxicity in biological systems occurs through a variety of mechanisms. Heavy metals bind to free thiol groups, disrupting protein structure or function (8, 19). They can also displace essential metal cofactors in proteins (e.g., Fe in cytochromes). Some heavy metals, such as Cu, also generate reactive oxygen species (ROS) through auto-oxidation or Fenton-like reactions (52, 61). Hydrogen peroxide can be produced intracellularly through the oxidation of NADPH (equation 1) and subsequent activity of the enzyme superoxide dismutase (equation 2). Hydrogen peroxide can then interact with copper to produce ROS (equations 3 and 4).

equation M1
(1)

equation M2
(2)

equation M3
(3)

equation M4
(4)

Cu-induced oxidative stress can damage the cell membrane through lipid peroxidation, leading to membrane permeability and cell death (4, 36).

Bacteria have several mechanisms for coping with heavy metal stress, allowing them to thrive in ecosystems contaminated with toxic levels of heavy metals (49, 58, 68). Active efflux is a key aspect of resistance, transporting metal cations out of the cytosol and periplasmic space of gram-negative species (43, 56). Previous studies indicate that a network of different families of transporters coordinates efflux (38, 43, 56). One family of transporters is soft metal P-type ATPases (55). ATP hydrolysis drives transport of heavy metal cations from the cytoplasm into the periplasmic space, such as the well-characterized CopA of Escherichia coli (which effluxes Cu+1) (54). Also contributing to metal efflux across the inner membrane are cation diffusion facilitator (CDF) transporters (25, 43, 51). The CDF family has been primarily implicated in resistance to heavy metals such as Zn, Cd, and Co (3). Finally, some members of the CBA family mediate proton-driven efflux of heavy metals from the periplasm (and possibly cytoplasm) (20, 22, 43), such as the CusABCF system in E. coli (16, 22, 42, 48). Many CBA systems consist of an inner-membrane-associated resistance nodulation cell division (RND) transporter, a membrane fusion protein, and an outer membrane protein, forming a complex that spans the inner and outer membranes.

Some species can also reduce or oxidize heavy metals to less toxic forms. Pseudomonas aeruginosa reduces cytosolic mercury (Hg) to Hg0, which then passively diffuses out of cells (71, 73). The periplasmic multicopper oxidase of E. coli, CueO, has been hypothesized to protect the cell by converting Cu+1 to its less toxic form Cu+2 in an oxygen-dependent reaction (21). Alternatively (or additionally), CueO may protect E. coli by oxidizing catecholate siderophores, such as enterobactin, which can then sequester free Cu (23). The plasmid-encoded Pco system of E. coli has a multi-Cu oxidase, PcoA, that may function in a similar manner (30).

Several recent studies have explored the global responses of the paradigm enteric species, E. coli, to elevated levels of required heavy metals using transcriptional profiling (12, 34, 37, 74). Different approaches were taken to subject bacterial populations to the stress. In some cases, the heavy metal was applied to growing cultures as a “shock,” and transcriptional profiles were assayed after a short period of exposure. In other cases, profiles were determined after longer periods of exposure, i.e., after bacterial populations had adapted to the stress and were actively growing. Both approaches were informative, indicating how E. coli responds and ultimately adapts to a heavy metal stress.

However, little is known in general about the global transcriptional responses of environmental bacterial species to heavy metal stress. Other than a recent report on the transcriptional response of Bacillus subtilis to different heavy metals, little has been published on environmental species (41). Unlike E. coli, P. aeruginosa is primarily an environmental organism commonly found in soil and water. Its genome has been sequenced, and commercially available DNA microarrays make it an excellent model organism for studying global transcriptional responses to environmental stimuli (64). Indeed, this approach has been successfully used to define transcriptional profiles of P. aeruginosa cultured under conditions ranging from elevated oxidative stress to denitrifying metabolism (14, 46, 50, 59, 60, 70).

In this study we examine the transcriptional profiles of P. aeruginosa exposed to a brief pulse of excess Cu (Cu shock) and cells growing continuously in the presence of excess Cu (Cu-adapted culture). Cu shock up-regulated expression of several genes involved in active transport functions as well as genes involved in oxidative and general stress responses. In contrast, transcriptional profiles of Cu-adapted populations suggested a Cu-specific physiology, with a much narrower range of genes encoding putative Cu-specific transport functions differentially expressed. An oxidative stress response was no longer apparent in the Cu-adapted population. Continuous Cu exposure also resulted in a general down-regulation of porins belonging to the OprD family. Iron homeostasis appeared to be particularly fine-tuned in Cu-adapted cultures. Genes encoding biosynthesis and transport of the iron-scavenging siderophore pyochelin were down-regulated in Cu-adapted populations, while genes encoding production of another siderophore, pyoverdin, were up-regulated. Strains bearing mutations in key copper-responsive genes identified in our study were then analyzed for Cu and Zn sensitivity. Our results suggest that two gene clusters homologous to RND family members, a P-type ATPase, a two component regulatory system, and a periplasmic system involved in proper disulfide bond formation all play important roles in tolerating elevated levels of Cu.

MATERIALS AND METHODS

Bacterial strains, strain construction, and media.

A wild-type laboratory strain of P. aeruginosa (PAO1) was used in this study. PAO1 was grown in LBM, which consisted of Luria-Bertani (LB) broth (Fisher Scientific, Hampton, NH) amended with 100 mM MOPS [3(4-morpholino)propanesulfonic acid]. Other growth media used in this study were minimal salts vitamin medium with glucose (MSVG), MOPSO-buffered saline solution, Jensens medium (32), and a MOPS minimal medium (53, 65, 72). The form of copper used in this study was CuSO4, silver was AgNO3, and zinc was ZnSO4 (Fisher Scientific, Hampton, NH, and Sigma-Aldrich Co., St. Louis, MO). Antibiotic concentrations used for P. aeruginosa in this study were 60 μg/ml for tetracycline and 300 μg/ml for carbenicillin. Ampicillin was used for selection in E. coli at 100 μg/ml.

Standard methods were used to manipulate plasmids and DNA fragments. Restriction endonucleases and DNA modification enzymes were purchased from New England Biolabs (Beverly, MA). Chromosomal DNA was isolated using DNeasy Tissue kits (QIAGEN, Valencia, CA), and plasmid isolations were performed using QIAprep spin miniprep kits (QIAGEN). DNA fragments were purified using QIAquick mini-elute PCR purification kits (QIAGEN), and PCR was performed using the Expand Long Template PCR System (Roche, Indianapolis, IN). DNA sequencing was performed by automated sequencing technology either using the University of Oklahoma Health Science Center sequencing core facility or the University of Iowa DNA sequencing core.

A selected group of transposon mutants in PAO1 were obtained from the University of Washington Genome Center and screened for Cu sensitivity (31). The following strains were obtained from the Washington collection: PTL361, PTL396, PTL1518, PTL3663, PTL4456, PTL4590, PTL4823, PTL5121, PTL5506, PTL5729, PTL7641, PTL7760, PTL7968, PTL8385, PTL8750, PTL9314, PTL9648, PTL13061, PTL14395, PTL16055, PTL16683, PTL17602, PTL17732, PTL18229, PTL19549, PTL20046, PTL21569, PTL22097, PTL22200, PTL30399, PTL35346, PTL36065, PTL36283, PTL47603, PTL12403, PTL44858, PTL48465, PTL20694, PTL9334, PTL14744, PTL6615, PTL8802, PTL17142, PTL31953, PTL7848, PTL42174, PTL8844, and PTL12392. There are several strains that have transposon insertions in the same gene. See Table Table44 for data from representative strains that have been confirmed by PCR.

TABLE 4.
Sensitivity of mutant strains to Cu and Zn

Selected mutants were complemented by introducing the native gene on a broad-host-range plasmid. The PA3920 and PA2809 genes were cloned into the broad-host-range vector, pEX1.8. The PA3920 gene was amplified using PCR and the primers PA3920-for (5′-CGGAATTCATGAACAG CGCCACGCTAACCGAG-3′) and PA3920-rev (5′-CGGGATCCCCATATGCCTGGCGTGCTGGCTCA-3′) (restriction sites are underlined). The resulting 2.4-kb PCR product was digested with EcoRI/BamHI and cloned into pEX1.8. The resulting plasmid, pEX3920, contains PA3920 under the control of the Tac promoter. The PA2809 gene was amplified with PCR and the primers PA2809-F (5′-GGGAATTCATGAAACTGCTGATCGTCG-3′) and PA2809rev2 (5′-CGGAAGCTTTCATTCGTCGCGCTCTTCG-3′) (restriction sites are underlined). The resulting PCR product was digested with EcoRI/HindIII and cloned into pEX1.8 to create pEX2809.

For construction of the reporter plasmid pAG100, a 496-bp DNA fragment containing the PA3920 promoter was amplified by PCR with the primers PA3920promoter-for (5′-CGGGATCCCGA TCACCTTCTGCTAAGGGCCTG-3′) and PA3920promoter-rev (5′-GGGGTACCCC TCCTTGGAATGGACAGAGTGG-3′) (restriction sites are underlined). The resulting PCR product was digested with the restriction enzymes BamHI/KpnI and then ligated into the BamHI/KpnI digested lacZ reporter plasmid pQF50, creating a transcriptional fusion of the PA3920 promoter with lacZ.

PCR validation of mutant strains.

Strains harboring transposon mutations were verified using PCR. A DNA segment was amplified from the chromosome of mutant strains using primers internal to the transposon (either Hah-138, 5′-CGGGTGCAGTAATATCGCCCT, or LacZ-148, 5′-GGGTAACGCCAGGGTTTTCC) paired with a primer complementary to native chromosomal sequence close to the transposon insertion point. An amplified band of DNA of the correct size indicated the presence of the transposon at the expected location. The following primers were used to confirm mutant strains: for PA2809, 5′-CCTGCTTCGCCAGTTTCTCC; for PA3920, 5′-GGCAGCATCCAGTGCAG; for PA3521, 5′-CCTGTAGCTGGAACCAGGACATG; for PA0397, 5′-TCGACCACACATGCAGGTCG; for PA1435, 5′-GTTGGCGTGACACCGCACGC; for PA1436, 5′-TGAACTGCAGGGTCAGGGTGG; for PA1549, 5′-ATGGACCGCATCCTGCGCTGG; for PA2064, 5′-CGATCCAGCCGCTGGCGTCC; for PA2065, 5′-CGCCGTGGCCGAGACCTACG; for PA2476, 5′-GCAACCTGATCACCCTTGGC; for PA2477, 5′-AGGTAGCGCGCCAGGCTGCC; for PA2505, 5′-GTAGCGTCGACCTGCTGCCC; for PA2521, 5′-TGCGCTGGTCGGACAACTGC; for PA2522, 5′-GGCCAGCGCGGGGAAACGC; for PA2807, 5′-GAAGGTCCAGGTCAGCTCGG; for PA2810, 5′-CGACCATATGGCGATGTCCG; for PA3690, 5′-GATGGACTGCCCGACCGAG. The PCR protocol is available at the University of Washington website (http://www.genome.washington.edu/UWGC/pseudomonas/).

To further verify the accuracy of transposon insertion strains, PCR products generated from the reactions described above were subjected to sequencing to verify the location of the transposon insertion. Either the Hah-138 or the LacZ-148 primers were used in sequencing reactions. The following strains were confirmed in this manner: PTL12403, PTL16055, PTL20694, PTL17142, PTL12392, and PTL6615.

Transcriptional profiling experiments.

For Cu shock conditions, PAO1 was inoculated at an optical density at 600 nm (OD600) of 0.05 in 50 ml of LBM in 250-ml flasks and cultured at 37°C until early logarithmic phase, corresponding to an OD600 of 0.2, at which point 10 mM CuSO4 was added to half of the flasks. The other half of the flasks were treated with a small amount of 1 M HCl to lower the pH to a similar level to that of the Cu-treated flasks (6.95 ± 0.04 for Cu-treated versus 7.01 ± 0.07 for the pH control flasks). After 45 min of treatment, cells were harvested and added to the RNA stabilization solution RNAlater (Ambion Inc., Austin, TX). Cu-adapted cells were subjected to 10 mM CuSO4 at inoculation. These cultures were grown at 37°C until early logarithmic phase (OD600 of 0.2), and then cells were harvested and added to RNAlater. The control flasks were grown at 37°C until early log phase (OD600 of 0.2). For each condition, all samples were grown in triplicate, and RNA from the different flasks was combined. In addition, biological replicates of each condition were performed on a separate day and run on a separate microarray chip.

RNA was isolated from cells using RNeasy columns (QIAGEN). An additional, off-column DNase treatment with RQ1 RNase-free DNase (Promega Corp., Madison, WI) was performed and then subsequently purified on the RNeasy column. RNA quality was assessed by agarose gel electrophoresis. Synthesis of cDNA and application to the microarray were performed following the instructions of the manufacturer of the GeneChip P. aeruginosa Genome Array (Affymetrix Inc., Santa Clara, CA) with some modifications. In the cDNA synthesis, 12 μg of RNA and semirandom hexamer primers with an average G+C content of 75% were used instead. S. Lory provided the spike transcript of control transcripts from six B. subtilis genes to monitor the labeling, hybridization, and staining efficiency of cDNA.

Analysis of DNA microarray data was first performed using Affymetrix Suite version 5.0, and further analysis was done using the online program CyberT (http://visitor.ics.uci.edu/genex/cybert/). CyberT utilizes a Bayesian statistical framework to calculate a more robust estimate of variance for a small number of experiments. The Bayesian prior estimate takes into consideration the background variance of the genes with similar expression levels as the gene of interest within a certain range, based on the theory that genes with similar expression levels should also have similar variance (27, 39). A sliding window size of 100 genes and a Bayesian prior estimate of 10 were used to calculate the P value. The posterior probability of differential expression [PPDE(<P)] was also calculated. PPDE(<P) ranges from zero to 1, and a low P value corresponds to a PPDE(<P) closer to 1. Genes were considered differentially regulated if the P value was <0.001, PPDE(<P) was >0.996, and the absolute change (n-fold) was >3.

Real-time reverse transcription-PCR (RT-PCR).

Cultures of PAO1 were grown in 250-ml baffled flasks containing 20 ml of LBM at 37°C and 150 rpm. Triplicate cultures were grown for the control (no Cu addition), Cu shock (10 mM CuSO4 addition when the OD600 reached 0.2), and Cu-adapted (10 mM CuSO4 addition at inoculation) experiments. The pH was consistent among the three experimental conditions. RNA was isolated from the control experiments when the OD600 was approximately 0.5. The Cu-shocked cells were treated with copper for 45 min; the OD600 was approximately 0.5 at the end of the treatment, and RNA was isolated immediately. The Cu-adapted cells also were grown to an OD600 of approximately 0.5, at which time the RNA was isolated immediately. One RNA extraction was performed for each flask, and the RNA was pooled for each condition (control, Cu-shocked, and Cu-adapted cultures). RNA was isolated with an RNeasy kit (QIAGEN), and residual DNA was removed with DNase I treatment (RiboPureTM-Bacteria kit, Ambion, Austin, TX).

Duplicate cDNA synthesis reactions were performed with random hexamer primers and avian myeloblastosis virus reverse transcriptase (Roche, Germany). Four micrograms of RNA was mixed with 2 μl of the primers (10 μM) and nuclease-free water (Ambion) to a final volume of 10 μl. The RNA mixture was denatured at 70°C for 5 min and placed on ice. Four microliters of 5× First-Strand Buffer, 2 μl of deoxynucleoside triphosphate mix (a 10 mM concentration of each nucleoside; New England Biolabs, Beverly, MA), 2 μl of water, and 2 μl of avian myeloblastosis virus reverse transcriptase were added to each reaction on ice. Each tube was incubated at 42°C for 1.5 h in a Primus 25 Personal Cycler with a heated lid (MWG Biotech, High Point, NC). Negative controls for RT-PCR were prepared by omitting the reverse transcriptase in the cDNA synthesis reactions and were used to verify that contaminating genomic DNA was absent from the RNA preparations. Duplicate cDNA reactions were pooled and used in triplicate real-time PCRs. Real-time PCRs were run on an Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA) using Power Sybr Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions. The final concentration of each primer was 0.3 μM for all reactions. The following thermocycler program was used: 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Real-time PCR product dissociation curves were used to verify the specificity of the amplified product. Fivefold serial dilutions of cDNA, prepared from a mixture of equal aliquots of cDNA from each experimental condition, were used to construct a standard curve for each gene tested (PA2477, PA2505, PA2522, PA3521, PA3920, PA4067, and PA4946). The cycle number at which fluorescence crossed a selected threshold value during exponential amplification was correlated to a relative quantity. The relative quantity of a gene transcript was normalized to the relative quantity of the housekeeping gene (PA4946) transcript for the same experimental condition. The relative change in expression (n-fold) was calculated as the relative quantity of the target gene transcript under Cu shock or Cu-adapted conditions (normalized to PA4946), divided by the relative quantity of the target gene transcript under the control condition (normalized to PA4946).

Pyoverdine and pyochelin measurements.

Cultures were grown in a modified Jensens medium at 37°C with glucose as the sole carbon source. Pyoverdine concentrations were calculated from the absorbance at 403 nm of cell-free, diluted culture supernatants as previously described (46). Purified pyoverdine was used to generate a standard curve. A modification of a previous protocol was used to measure pyochelin levels. The dried organic fraction of ethyl acetate-extracted cell-free culture supernatants was resuspended at 10× concentration, and pyochelin levels were determined as previously described (69). Purified pyochelin was used to generate a standard curve.

MIC determination for mutant strains.

The MIC of Cu was the lowest concentration at which there was no growth after 16 h of exposure. In a 96-well microtiter plate format, PAO1 and mutant strains were subjected to an array of heavy metal concentrations in LBM. Each well contained 100 μl of LBM plus CuSO4 concentrations ranging from 1 mM to 16 mM. Each well was inoculated with a 10-μl aliquot of cells from an actively growing culture at an OD600 of 0.1. The microtiter plate was incubated at 37°C with gentle shaking for 15 s every 30 min. Growth was monitored by reading the OD600 every 30 min using a microtiter plate spectrophotometer (Genios microplate reader [Tecan Research Triangle Park, NC] or Synergy HT [Bio-Tek Instruments, Winooski, VT]), and the MIC was determined after 16 h. Each experiment was performed in triplicate.

Disk sensitivity assay.

Tested strains were grown to late logarithmic phase in LB at 37°C, and then 500 μl of cell suspension was added to a cooled, molten soft LB agar (0.5% agar). The agar was then poured into petri plates and allowed to solidify. A sterilized Whatman filter disk (catalog no. 2017013) incubated overnight in either 500 mM CuSO4 or 500 mM ZnSO4 was added to the center of the agar plate after it had solidified. The plate was then incubated at 37°C overnight, and the zone of clearance surrounding the disk was measured.

Copper viability analysis.

Tested strains were grown in 250-ml baffled flasks containing 50 ml of LBM until mid-logarithmic phase (OD600 of 0.5). Cells were harvested, centrifuged, and resuspended and washed in the minimal medium MSVG. Aliquots were subjected to an array of Cu concentrations in MSVG and then placed at 37°C. After 5 h, the samples were sonicated for 30 s (3 s on at an output of 7 and 1 s off for 10 cycles) (Ultrasonic liquid processor XL; Misonix Inc., Farmingdale, NY), and viable plate counts were performed on serial dilutions.

β-Galactosidase assays.

β-Galactosidase activity was monitored colorimetrically as described by Zubay et al. (77). To examine the expression of PA3920 in response to increasing copper levels, overnight cultures of P. aeruginosa containing pAG100 were diluted in Trypticase soy broth to an OD600 of 0.05 and incubated with shaking (250 rpm) at 37°C for 1 h. Increasing concentrations of CuSO4 (50 μM to 1.5 mM) were then added to individual cultures; cultures were incubated for 45 min, and β-galactosidase activities were determined.

Microarray accession number.

Array data were submitted to the Gene Expression Omnibus website (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE4152.

RESULTS AND DISCUSSION

Transcriptional profiles of Cu-adapted and Cu-shocked P. aeruginosa populations.

In this study, P. aeruginosa was subjected to two types of Cu stress. Cu-shocked cultures were defined as cultures actively growing under nonstressed conditions that are subjected to a brief pulse of elevated Cu. Transcriptional profiles of early log cultures were assessed shortly after Cu addition (45 min). Cu-adapted populations were defined as cells actively growing in the presence of elevated Cu (approximately 6 h after inoculation of the culture). Cu was present in the growth medium at inoculation, and early log cultures were harvested for transcriptional profiling. The rationale for testing these two conditions was to compare and contrast transcriptional profiles of a sudden Cu stress response to adapted populations growing in the presence of excess Cu.

A range of CuSO4 was added to actively growing P. aeruginosa cultures in order to define acute stress conditions. A buffered complex medium (LBM) was used to maximize biomass yield. A Cu concentration was chosen (10 mM) that impacted P. aeruginosa growth rate but was not lethal, as determined by viable plate counts. Forty-five minutes after the addition of 10 mM Cu, only minimal growth had occurred in the treated culture, while growth had occurred in the control culture (OD600 of 0.310 versus 0.396, respectively) (Fig. (Fig.11).

FIG. 1.
Growth curves of cultures used for transcriptional profiling. For Cu-shocked cultures, PAO1 was grown in 250-ml flasks containing 50 ml of LBM at 37°C until early logarithmic phase, corresponding to an OD600 of 0.2. At this point, 10 mM CuSO4 ...

If present in the growth medium at inoculation, 10 mM Cu resulted in a greater initial lag in growth and a lower growth rate than the untreated culture (Fig. (Fig.1).1). This was chosen to represent a Cu-adapted population. In all cases (control, shocked, and adapted conditions), approximately the same amount of biomass, as determined by viable plate counts and OD600 values, was harvested for transcriptional profiling.

Figure Figure2,2, Tables Tables11 to to3,3, and Table S1 in the supplemental material feature genes that were up- and down-regulated in shocked and adapted populations. Genes were considered differentially regulated if the relative change (n-fold) was >3.0, the P value was <0.001, and the PPDE(<P) was >0.996. Cu shock resulted in differential expression of 405 genes. Most of these genes were up-regulated. Although more genes would have qualified as repressed, they did not meet the P value and PPDE stringency cutoffs. In the case of Cu-adapted cultures, 331 genes were either up- or down-regulated. To further validate the array data, real-time RT-PCR was used to determine the relative quantities of selected transcripts under control and test culturing conditions. These are indicated in a separate column in Tables Tables11 and and33.

FIG. 2.
A schematic depicting genes transcriptionally regulated by copper and some of the predicted/putative functions they encode. Gene names are labeled above the arrows. Arrows are color-coded to reflect relative changes in expression (n-fold). Arrows are ...
TABLE 1.
Regulated genes involved in transport and membrane permeability and associated regulatory proteins
TABLE 3.
Other select genes differentially regulated by Cu

There were 41 genes that were differentially regulated for both types of Cu stress. These genes represented a common core response of P. aeruginosa to Cu, including the up-regulation of transporters (czrCB, PA3920, and PA3521 to PA3523), a system containing a potential Cu oxidase (pcoAB), genes encoding periplasmic thiol-disulfide interchange proteins (dsbG and PA2477 to PA2478), putative lyases (PA3516 and PA3517), and transcriptional regulators (PA2479, PA2809, PA2523, and PA3410). Since both the shocked and adapted stress conditions impact growth rate (Fig. (Fig.1),1), subsequent discussion of transcriptional profiling data places little emphasis on features pertaining to general metabolism.

Active efflux is a key aspect of copper tolerance.

RND, CDF, and P-type ATPase efflux transporters have been suggested to constitute an efficient, integrated network controlling heavy metal levels in the cytoplasm and periplasm for some gram-negative bacterial species. Several studies have shown that transporters belonging to these families are induced in the presence of elevated levels of their heavy metal substrates.

Cu stress up-regulated expression of genes encoding predicted active transport functions (Table (Table1).1). The P. aeruginosa chromosome has seven genes homologous to P-type ATPases (43). One, PA3920, was up-regulated in both shocked and adapted populations. In addition, a few RND family transporters were up-regulated under both conditions. The P. aeruginosa chromosome has 12 loci harboring genes homologous to RND family members (35, 43). Three were up-regulated in response to Cu exposure (PA1436, PA2520, and PA3522). The most prominently up-regulated locus is PA2520 to PA2522, which encodes the czrCBA transport system, previously shown to be important for Cd, Zn, and Co tolerance (26, 57). An adjacent two-component regulatory system (encoded by PA2523 and PA2524) was also up-regulated in Cu-stressed cultures. The PA3521 to PA3523 and PA1435 to PA1436 genes have no known efflux substrates (62). These loci also have no linked regulatory proteins.

A gene encoding a CDF family member, PA0397, was up-regulated in Cu-adapted populations. A homologous gene in Ralstonia metallidurans is thought to confer resistance to Cd, Zn, and Co (2). Nies suggested that the Cu ion may be too small to serve as a substrate for transport for this family (43), although Moore et al., recently reported that a CDF family member of B. subtilis conferred protection to Cu (41). The PAO1 chromosome has two other genes belonging to the CDF family, PA1297 and PA3693. PA1297 is 50% similar to the zinc transporter, ZntA, of Staphylococcus aureus and was up-regulated under Cu shock conditions.

Copper shock appears to produce an oxidative stress response.

Two previously published studies characterized the transcriptional profiles of P. aeruginosa exposed to reactive oxygen species (50, 59). Comparing our data with the data of Palma et al. and Salunhke et al. suggests that Cu shock results in an oxidative stress response. Several genes identified in these studies were also induced in the Cu-shocked cultures, such as superoxide dismutase (sodM), catalase (katB), genes involved in organic hydroperoxide resistance (ohr and ahpF), and genes involved in the DNA SOS response/DNA damage and repair (dinP, recA, and recN) (Table (Table2;2; see Table S1 in the supplemental material). Comparing their data sets with ours allowed us to filter the Cu shock data set such that none of the genes presented in Tables Tables11 and and33 are related to an oxidative stress response. This finding is in agreement with the studies of Kershaw et al. and Egler et al., where the addition of either copper sulfate or copper-glycine to E. coli cultures was shown to produce an oxidative stress response (12, 34). In contrast, Cu-adapted populations no longer showed elevated expression of these genes, possibly due to decreased intracellular Cu following adaptation of the organism to the stress.

TABLE 2.
Genes involved in an oxidative stress response

Dealing with excess copper in the periplasm.

Genes encoding periplasmic folding functions were up-regulated in response to Cu stress (Fig. (Fig.22 and Table Table3).3). One locus, PA2476 to PA2478, has three genes homologous to dsbG, dsbE, and dsbD, respectively. In E. coli, this system is thought to repair nonnative disulfide bonds in the periplasm (7). These genes were up-regulated in both shocked and adapted populations. Hiniker et al. demonstrated that a member of the dsb system, DsbC, is an important Cu resistance determinant in E. coli (28). Since free thiols interact with Cu, this system may ensure proper folding of proteins newly imported into the periplasm.

An operon encoding putative metal-binding proteins and Cu oxidases was also up-regulated in both Cu treatments (Fig. (Fig.22 and Table Table3).3). Cu stress induced expression of the pcoAB genes (PA2065 and PA2064) which are homologous (81% and 75% similarity, respectively) to the copAB genes of Pseudomonas syringae. CopA and CopB have been demonstrated to bind Cu in the periplasm (CopA) and outer membrane (CopB) (6). PcoA has a multicopper oxidase domain (SufI domain; COG2132 [http://www.ncbi.nlm.nih.gov/COG]), similar to CueO of E. coli. This enzyme might participate in the oxidation of Cu+1 to the less toxic form Cu+2 or the oxidation of catecholate-like siderophores (although P. aeruginosa is not known to produce its own catecholate siderophore). Like Pseudomonas putida, P. aeruginosa lacks homologs of the copCD genes that are present in P. syringae (5).

Another gene, PA3520, was up-regulated by Cu and is associated with a cluster of genes that are also Cu responsive. This gene is predicted to encode a small protein that contains a metal binding motif, MXCXXC. This motif is found in a variety of proteins that bind heavy metals, such as CopZ. CopZ in Enterococcus hirae is thought to be a periplasmic Cu chaperone (40). However, the localization of the PA3520 gene product to the periplasm is uncertain. Homologous genes are found on the P. putida (PP0588) and P. syringae (PSPTO0752) chromosomes.

Regulation of outer membrane permeability appears to be a feature of adaptation to copper.

Some passive transport functions were down-regulated in Cu-adapted populations. Eight different porins or predicted porins were down-regulated (Table (Table1).1). One porin, OprD, binds to basic amino acids or basic dipeptides. Interestingly, reduced OprD expression has been linked to resistance to carbapenem antibiotics, and addition of low micromolar levels of Zn to P. aeruginosa led to its reduced expression (24). Four more Cu-repressed porins, OprQ, OpdC, OpdN, and OpdP, belong to the OprD family but have no assigned uptake function (24). OprC is TonB dependent and is thought to be Cu specific. Elevated extracellular Cu was previously reported to repress its expression (76). OprG and OprE were also repressed. OprG has been suggested to be involved in low-affinity Fe uptake, and OprE is an anaerobically induced porin (18, 75). The porin encoded by PA2505 was the only porin induced by long-term Cu exposure. This gene encodes opdT, a porin that also belongs to the OprD family. Perhaps induction of opdT compensates for repression of the other OprD family members while not compromising the cell in terms of Cu permeability.

Increased porin expression has been previously reported as a feature of Cu resistance in E. coli. Egler et al. reported that OmpC expression was increased in response to Cu and controlled through the extracytoplasmic sigma factor, RpoE (12). Mutations in OmpC rendered E. coli sensitive to Cu, although the underlying mechanism was unclear.

Effects of copper stress on iron homeostasis.

A distinct group of genes involved in Fe homeostasis were induced in Cu-stressed cultures. This group overlapped with genes identified in P. aeruginosa exposed to Fe-limiting conditions (46). Of the 30 genes with the highest relative induction (n-fold) in response to Fe limitation identified by Ochsner et al., 20 were also up-regulated in response to Cu stress. Many of these 20 genes have consensus binding sequences in their promoter regions for the Fe-responsive regulators, Fur and PvdS (10, 45). In line with this observation is the up-regulation of pvdS (PA2426) under both stress conditions. Another interesting similarity with the Ochsner study is that, of the eight genes homologous to extracytoplasmic function family sigma factors they reported to be induced by Fe limitation (PA2387, PA3899, PA4896, PA2468, PA3410, PA1300, PA0472, and pvdS), seven are also up-regulated in Cu-shocked populations (all of the above except for PA2387). The heavy metal induction of Fe siderophores has previously been reported for E. coli (12, 34). The siderophore enterobactin not only may play a role in Fe transport but also may protect the cell by sequestering heavy metals in the cytoplasm (23).

Many Fe-regulated genes, including pvdS, are under control of the regulatory protein Fur, which complexes Fe and represses expression of target genes under Fe-replete conditions. Heavy metals could potentially interact with Fur, displacing Fe and thus influencing its binding to target DNA sequences (9). Another possibility is that ROS generated by Cu stress could oxidize Fur-bound Fe2+, releasing Fe from Fur and relieving Fur-mediated repression.

The major difference between this study and that of Ochsner et al. is that Cu-adapted populations repressed expression of genes involved in synthesis of the siderophore pyochelin, whereas these genes were induced under Fe-limiting conditions. This is consistent with Visca et al., who previously reported that several transition metals, including Cu, repress pyochelin synthesis (67).

In contrast, several genes involved in the biosynthesis of pyoverdine were up-regulated in Cu-adapted populations (Table (Table33 and Fig. Fig.2).2). Pyoverdine has been previously shown to be up-regulated in response to Cd and Zn (11, 29, 67). Pyoverdine might provide the bacterium with an iron-specific acquisition system, whereas pyochelin is down-regulated since its broader specificity for metal cations might allow Cu to enter the cell. The regulation of Fe acquisition appears to be carefully fine-tuned in a Cu-stressed environment.

To functionally verify transcriptional profiling data, P. aeruginosa was exposed to different levels of CuSO4, and pyoverdine and pyochelin levels were measured. Pyoverdine levels were seen to increase more than 10-fold in cultures treated with CuSO4 for a long period of time (Fig. (Fig.3).3). As suggested by the array data, pyoverdine levels did not increase after short-term exposure. A mutant strain unable to biosynthesize pyoverdine, PAO1-pvdA, did not show induced pyoverdine production in the presence of Cu. The array data also predict that long-term copper exposure would result in reduced pyochelin levels. However, even in untreated cultures, pyochelin levels were below detectable limits (data not shown).

FIG. 3.
Long-term exposure to copper induces pyoverdine production in P. aeruginosa. (A) Batch cultures were treated with different amounts of CuSO4 for different periods of time. Pyoverdine production was measured for cultures in triplicate. Long-term copper ...

The susceptibility of select mutant strains to copper and zinc supports transcriptional profiling data.

Transcriptional profiling suggested that a number of functions are important for Cu tolerance. To both validate the array data and explore the relative contribution of different genes to Cu tolerance, strains harboring transposon insertions in select genes were obtained from the mutant library at the University of Washington at Seattle. When available, multiple different mutant strains were obtained for a single gene. Data presented in Table Table44 and Fig. Fig.44 and and55 indicate the specific strain used for that experiment. The location of all transposon insertions was verified by PCR. Select PCR products were sequenced to verify the location of the transposon insertion. Polar effects caused by the transposon insertion or secondary mutations not related to the disrupted gene cannot be ruled out here. Due to the number of strains analyzed, a select few mutants with distinct phenotypes were complemented in trans, ruling out secondary mutations.

FIG. 4.
Growth curves of mutant and wild-type strains of P. aeruginosa in the presence of different CuSO4 levels. Optical density of the culture was measured as a function of time. Levels of added Cu are indicated by the symbol as it corresponds to the legend ...
FIG. 5.
Growth curves of mutant strains of P. aeruginosa in the presence of different CuSO4 levels. These strains displayed the most severe Cu sensitivity phenotype of tested strains. (A) PA3920, a strain harboring a mutation in a soft metal P-type ATPase. (B) ...

Mutant strains were evaluated for Cu sensitivity using two separate methods. The first was MIC determination for the strain in liquid culture. To be consistent with the profiling experiments, strains were grown in buffered LB and a variety of CuSO4 concentrations, and growth was measured at 16 h. The MIC of CuSO4 for the wild-type strain was 16 mM. The MICs for most of the mutant strains tested were similar to that for the wild type (Table (Table4).4). The few exceptions were strains with mutations in PA3920 (a P-type ATPase), PA2809 (a two-component system response regulator), PA1435 (a membrane fusion protein), PA2477 (a periplasmic thiol-disulfide interchange protein), and PA2522 (czrC; an outer membrane protein). In particular, PA3920 and PA2809 displayed significant Cu-sensitive phenotypes. The failure of the other strains to exhibit Cu sensitivity in this assay may be due to the ability of the organism to compensate with other Cu tolerance mechanisms.

Several strains for which the Cu MIC was comparable to the wild-type level exhibited a lag in growth in the presence of Cu compared to the wild-type strain (Table (Table4).4). Examples of such a lag are depicted in the growth curves presented in Fig. Fig.4.4. The growth of PAO1 is unaffected by added CuSO4, until MIC levels are reached (Fig. (Fig.4A).4A). However, the mutant strains show a delay in growth and a reduced growth rate compared to PAO1 at near MIC levels of CuSO4 (e.g., 8 mM in Fig. Fig.44 B, C, and D). In particular, the PA1435 mutant strain showed a delay in growth at a few CuSO4 concentrations. Other mutant strains that produced a lag in growth in the presence of Cu were PA2476 and PA2478 (both belong to the same operon as PA2477 and show homology to periplasmic thiol-disulfide folding proteins), PA2810 (a homolog of a two-component sensor), and PA3521 (an outer membrane protein).

The two strains judged to be most sensitive to Cu by MIC analysis, PA2809 and PA3920, were extremely sensitive to CuSO4 (Fig. 5A and B). To verify that the transposon insertion was responsible for the Cu-sensitive phenotype, these strains were complemented in trans by supplying a functional copy of the gene on the broad-host-range vector pEX1.8. The complemented mutant strains displayed almost wild-type levels of Cu-tolerance (Fig. 5C and D and Table Table44).

A disk sensitivity assay was used as a complementary means to evaluate Cu sensitivity. This assay uses a Cu-impregnated disk, which is set on the surface of soft LB agar. Zones of clearance in a lawn of bacteria were measured around the disk. The wild-type strain showed a clear demarcation between copious growth and no growth on the agar plate. However, some strains produced a halo of very light growth adjacent to the zone of clearance (Table (Table44).

The Cu disk assay results correlated with MIC data, with PA2809 and PA3920 displaying the largest zone of clearance (Table (Table4).4). In some cases, strains showed Cu sensitivity in the disk assay and not the MIC assay. This usually occurred with mutations in a putative operon already identified as important for Cu tolerance in the MIC analysis (e.g., PA2521). There were three exceptions. A strain with a mutation in pcoA (PA2065) showed significant Cu sensitivity in the disk assay but not in the MIC analysis. A strain with a mutation in PA0397 (a CDF family homolog) and PA2807 (a gene encoding a member of the plastocyanin/azurin Cu-binding family) were also sensitive in the disk assay. Why these strains have a sensitive phenotype in one assay and not the other is unclear.

Several genes induced by Cu stress showed homology to genes involved in responses to other cations, such as Zn. In particular, the czr system (PA2520 to PA2522) is known to be important for Zn tolerance in P. aeruginosa (26). Zinc is also a required metal for growth and in excess can present the same problems as Cu to the cell. Therefore, the mutant strains were also subjected to a zinc disk sensitivity assay. This assay demonstrated the metal specificity of certain systems. A strain with a mutation in PA3920, encoding a P-type ATPase, was very sensitive to Cu but not to Zn (Table (Table4).4). On the other hand, strains with mutations in two other P-type ATPases, PA3690 (homologous to zntA, a Zn P-type ATPase in E. coli) and PA1549, were Zn sensitive yet showed no Cu phenotype. As Hassan et al. previously demonstrated, the czrABC operon (with the exception of PA2521) appeared to be important for Zn tolerance (26). Also implicated in Zn tolerance by this assay were the pcoAB genes.

Our initial analysis suggested that PA3920 and PA2809 contributed significantly to Cu tolerance in P. aeruginosa. Therefore, these genes were subjected to further analysis.

A two-component regulator is important for growth and survival under Cu stress.

The two-component response regulator PA2809 was up-regulated in both Cu-shocked and Cu-adapted populations and may be involved in pcoAB regulation. This gene is highly homologous to copR in P. syringae (82% similar), P. putida, and Pseudomonas fluorescens and to pcoR in E. coli. The regulators in these systems control expression of the Cu-binding proteins CopAB (copR) and PcoAC (pcoR) (5, 15, 44). Due to this homology, we propose naming PA2809 copR and its adjacent sensor kinase, PA2810, copS. Two neighboring genes, PA2807 and PA2808, were also induced by Cu stress. The hypothetical protein PA2807 harbors a Cu-binding motif of the plastocyanin/azurin family. PA2807 encodes a protein similar to the Cu-binding protein Cot from P. fluorescens DF57. Cot was identified as a Cu tolerance protein that has elevated expression in response to Cu (66).

Since a mutation in PA2809 significantly influenced Cu tolerance (Table (Table4),4), this strain was examined further. The mutant strain was also tested for Zn and silver (Ag) sensitivity and found to be identical to the wild-type strain (data not shown). The toxicity of Cu (as opposed to inhibition of growth) was then tested on the PA2809 mutant and wild-type strain. Cells were exposed to a 5-h treatment of various Cu concentrations. Since complex medium has many components with a high capacity to complex Cu, this assay was performed in a minimal medium designed to minimize complexation. The PA2809 mutant strain was killed at lower Cu concentrations than wild-type (Fig. (Fig.6).6). Resistance to Cu was regained when the mutant strain was complemented in trans with a plasmid bearing the PA2809 gene (Table (Table44 and data not shown). Since PA2809 is up-regulated during both Cu treatments, it appears to be a key regulator involved in Cu tolerance in P. aeruginosa.

FIG. 6.
Cu toxicity assay. The viable plate counts of PAO1 and a strain with a mutation in PA2809 after exposure to Cu in MSVG. Cells were first grown in LBM until mid-logarithmic phase and then spun down and resuspended in MSVG. Planktonic cells were exposed ...

A soft metal P-type ATPase is important for copper tolerance.

Heavy metal resistance in bacteria is often mediated by energy-dependent protein efflux pumps of the P-type ATPase family (1, 43, 63). P. aeruginosa possesses seven ORFs with this family signature, many of which, including PA3920, possess putative metal binding motifs. PA3920 is a predicted cytoplasmic membrane protein with a molecular weight of 83,438 and possesses significant homology to several members of the P-type ATPase family including the copper transporter copA (54% similarity) from E. coli (54) and the Cu transporter yvgX (65% similarity) from B. subtilis (17). Three conserved domains are present in PA3920 including two heavy-metal-associated domains (amino acids 10 to 70 and 76 to 138) and an E1-E2 ATPase domain (amino acids 258 to 477). Within these domains PA3920 possesses all the characteristics of a soft metal ion-translocating P1-type ATPase, including two Cys-X2-Cys motifs in the C terminus (amino acids 18 to 21 and 84 to 87) and a Cys-Pro-Cys motif (amino acids 444 to 446) in a putative transmembrane segment.

The MICs of the heavy metals Cu, Co, Zn, and Hg were assayed for a strain harboring a mutation in PA3920 and compared to PAO1. In complex and defined media, the mutant exhibited increased susceptibility to copper compared to PAO1 while no differences were observed for the other heavy metals (Table (Table44 and data not shown). Cu susceptibility was tested in a complex and a minimal medium, and although the MICs were significantly affected by the medium composition, the mutant was hypersusceptible to copper in all media tested (data not shown).

PA3920 expression responds to external copper.

Genes encoding metal-transporting ATPases are often induced by the presence of their metal substrates (1, 47, 54). Thus, a transcriptional fusion of the PA3920 promoter region was cloned upstream of the promoterless reporter gene lacZ in the plasmid pAG101. In complex medium, transcription of PA3920 exhibits a linear response to increasing concentrations of copper up to 1.2 mM (Fig. (Fig.7).7). The transcriptional response is immediate, occurring within 45 min after the addition of copper.

FIG. 7.
Impact of increasing copper concentrations on PA3920 transcription. PAO1 carrying a PA3920-lacZ transcriptional fusion on the plasmid pAG100 was grown in the presence of increasing amounts of Cu. β-Galactosidase activity was determined 45 min ...

Summary.

A comparison of shocked and adapted transcriptional profiles revealed interesting insight into the physiology of Cu-stressed P. aeruginosa. The response to Cu shock appears to encompass both Cu-specific and general stress responses. Since general stress responses have global effects on physiology, determining what genes were regulated directly in response to Cu is impossible. Perhaps as different stress regulons eventually are defined at the transcriptome level, separating direct and indirect responses to Cu may be possible. The Cu-adapted profiles might provide more insight into the adaptations necessary for elevated environmental Cu. Most of the signature genes associated with general stress responses were no longer up-regulated.

Transcriptional profiling of Cu-stressed P. aeruginosa produced both expected and unexpected results. Not surprisingly, many active transport functions were up-regulated in response to Cu. Previous studies have shown that RND efflux systems, P-type ATPases, and CDF transporters play an important role for metal tolerance in other organisms. In particular, the P-type ATPase encoded by PA3920 appeared to be important for Cu tolerance. The expression of several porins appeared to be down-regulated in adapted populations. Most of these porins belonged to the OprD family, which may be involved the in passive transport of positively charged molecules.

Iron homeostasis functions were also regulated under Cu stress conditions. In general, the cells produced a response indicative of iron-limiting conditions. An interesting observation involved the two iron siderophore systems, pyoverdine and pyochelin. Pyoverdine biosynthetic genes were up-regulated in Cu-adapted populations, while pyochelin genes were down-regulated. The explanation for this observation may lie with the specificity of the siderophores. Pyoverdine is highly iron specific, while pyochelin has been shown to complex a variety of divalent metal cations, including Cu.

Finally, a variety of genes not generally associated with metal tolerance were up-regulated in the Cu-adapted population. The dsb periplasmic thiol-disulfide folding system was up-regulated and appeared to be functionally important for growth in the presence of excess Cu. Several operons containing genes of unknown functions were also up-regulated in adapted populations. Particularly interesting were the genes PA3515 to PA3520, which constitute an operon adjacent to an induced RND efflux system (PA3521 to PA3523). A few of these genes have homology to lyases, but the function of this operon is a mystery. Determining how genes such as these contribute to Cu adaptation may produce new insight into metal tolerance of microorganisms.

Supplementary Material

[Supplemental material]

Acknowledgments

G.T. was supported by grant CHE 9810378 from the Institute of Environmental Catalysis and the National Science Foundation.

We thank Charlie Cox for the gift of purified pyoverdine and pyochelin and Blaise Boles for the PAO1-pvdA strain. We thank Ivonne Granados for help with experiments and Thomas V. O'Halloran, Mike Vasil, and Hilary Godwin for helpful discussions.

Footnotes

Supplemental material for this article may be found at http://jb.asm.org/.

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