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Applied and Environmental Microbiology, July 2007, p. 4550-4558, Vol. 73, No. 14
0099-2240/07/$08.00+0     doi:10.1128/AEM.00169-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transcriptome Analysis Reveals that Multidrug Efflux Genes Are Upregulated To Protect Pseudomonas aeruginosa from Pentachlorophenol Stress{triangledown}

Jocelyn Fraga Muller,1,{dagger} Ann M. Stevens,2 Johanna Craig,3 and Nancy G. Love1,2*

Department of Civil and Environmental Engineering,1 Department of Biological Sciences,2 Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240613

Received 23 January 2007/ Accepted 20 May 2007


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ABSTRACT
 
Through chemical contamination of natural environments, microbial communities are exposed to many different types of chemical stressors; however, research on whole-genome responses to this contaminant stress is limited. This study examined the transcriptome response of a common soil bacterium, Pseudomonas aeruginosa, to the common environmental contaminant pentachlorophenol (PCP). Cells were grown in chemostats at a low growth rate to obtain substrate-limited, steady-state, balanced-growth conditions. The PCP stress was administered as a continuous increase in concentration, and samples taken over time were examined for physiological function changes with whole-cell acetate uptake rates (WAURs) and cell viability and for gene expression changes by Affymetrix GeneChip technology and real-time reverse transcriptase PCR. Cell viability, measured by heterotrophic plate counts, showed a moderately steady decrease after exposure to the stressor, but WAURs did not change in response to PCP. In contrast to the physiological data, the microarray data showed significant changes in the expression of several genes. In particular, genes coding for multidrug efflux pumps, including MexAB-OprM, were strongly upregulated. The upregulation of these efflux pumps protected the cells from the potentially toxic effects of PCP, allowing the physiological whole-cell function to remain constant.


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INTRODUCTION
 
Genetic-level responses of bacteria to chemical stress drive ecosystem level changes in both structure and function within contaminated environments. Therefore, the focus of much research has shifted to understanding the relationship between microbial community structure and bacterial function through gene expression (13, 16, 53). As a result, there are studies that have identified specific stress responses to chemical contamination within a mixed community, such as heat shock protein induction (2) and upregulation of various cellular transporters (26). Although these studies give valuable information, they are limited in scope by the specific responses chosen for analysis. Recently, more comprehensive approaches such as genomics and proteomics have contributed greatly to understanding chemical stress responses.

The pseudomonads are species of microorganisms that are found in many different contaminated environments and are known to adapt well to chemical contamination. Recent studies have examined mechanisms responsible for the adaptive ability of Pseudomonas putida, a common soil organism that is able to degrade a range of organic contaminants (7, 38, 46). These studies, looking at proteome and transcriptome responses to aromatic hydrocarbons easily degraded by P. putida, showed a trade-off in regulation between stress responses and metabolic capabilities. In each of these cases, during exponential growth, multiple stress genes were upregulated prior to or in addition to degradation genes.

Pseudomonas aeruginosa is also a soil organism that is ubiquitous throughout the environment and, like P. putida, is able to survive in many different chemically contaminated ecosystems (15, 19, 31, 50). Unlike P. putida, it is an opportunistic pathogen, with an epidemic population structure, as clonal diversity is similar throughout different environments (32). For this reason, the effects of environmental contamination on the functional responses of this bacterium are of great interest. The stress response of bacteria depends upon physiological growth conditions; therefore, it was important to study the response under conditions most closely resembling the natural environment. In the environment, bacteria are often found under low-nutrient, low growth rate conditions. Although the stationary phase of batch growth has been used in the past to simulate "starvation" conditions, this unbalanced growth phase is most likely not similar to growth experienced in the natural environment (8, 37). The chemostat more closely simulates growth in the natural environment by achieving balanced-growth, low growth rate conditions.

The objectives of this study were to investigate expression changes over the entire genome as cells were exposed to pentachlorophenol (PCP) and to correlate these changes with whole-cell physiological changes. Affymetrix GeneChips were used to study gene expression changes in P. aeruginosa as it responded to PCP. PCP was chosen as the chemical of study because of its widespread contamination of the environment and recalcitrance. The main toxic effect of PCP is thought to be on the cell membrane, where it is embedded as a monomer and acts as an indiscriminant proton shuttle, uncoupling oxidative phosphorylation (10). A chemostat was used to achieve substrate-limited conditions, and PCP was added to mimic a groundwater contaminant plume. RpoS is a sigma factor reported to be important under low growth rate conditions and to the response of many species to many various stressors. Therefore, the effect of RpoS on the response of P. aeruginosa to PCP was also examined by using an rpoS deletion mutant strain. With many transcriptome studies published to date, this is the first to examine the stress response to contamination under ecologically relevant conditions.


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MATERIALS AND METHODS
 
Chemicals.
All of the chemicals used for medium preparation were laboratory or higher grade (Fisher Scientific, Atlanta, GA). Technical-grade PCP was purchased from Sigma-Aldrich, Inc., St. Louis, MO. Hexane solvent for PCP analysis was analytical grade quality (Fisher Scientific).

Strains and batch culture conditions.
Wild-type (WT) P. aeruginosa PAO1 (WT1) and an RpoS-deficient mutant (RpoS–), obtained from E. P. Greenberg (40), were used in chemostat experiments and MIC assays with PCP. A second WT strain of PAO1 (WT2) and the MexAB-OprM mutant (PAO200) of this strain were obtained from H. Schweizer for batch growth experiments (41). Batch cultures were grown at 37°C in a minimal medium (MM) containing (in grams per liter) KH2PO4 (3.4), K2HPO4 (4.35), NH4Cl (1.0), and CH3COONa (1.65) along with (in milligrams per liter) EDTA (186), MgSO4 · 7H2O (150), MnSO4 · 4H2O (4.5), NaMoO4 · 2H2O (0.5), H3BO3 (0.15), CaCl2 (20), ZnCl2 (1.5), CuCl2 · 2H2O (0.5), CoCl2 · 6H2O (1.5), and FeCl2 · 4H2O (11). The final pH of this medium was 6.9. Cultures were grown in 250-ml Erlenmeyer flasks in a volume of 100 ml. The chemostat growth conditions are described below.

Determining the MIC of PCP.
Cells were grown in MM overnight and diluted to 106 cells/ml in either Luria-Bertani (LB) broth or MM. PCP in LB or MM was twofold serially diluted starting with a concentration of 2 g/liter in 96-well polystyrene plates (Costar, Corning, NY) to a final volume of 50 µl. LB was used, as well as MM, to be consistent with MIC assays for chemicals reported in the literature. Fifty microliters of diluted cells was added to the wells containing the PCP dilution series. The LB plate was incubated at 37°C, and the MM plate was incubated at 20°C. After 18 h (LB) or 48 h (MM), growth in the wells was recorded qualitatively by eye and confirmed by measuring absorbance (600 nm) with a microplate reader. The MIC was the lowest concentration of PCP that did not permit growth.

Chemostat growth startup and conditions.
Chemostat cultures were grown in a 5-liter vessel with a 4.8-liter working volume with stirring at 300 rpm (B. Braun Biotech International, Allentown, PA). Culture stocks were prepared to ensure that the physiological state of the cells was adapted to the MM and 20°C growth conditions. Cultures were sequentially transferred in batch (MM medium, 20°C) every day for 7 days, and the resulting culture was frozen in glycerol for storage at –50°C. Starting cultures for each chemostat were taken from plated frozen stocks, and a single colony was transferred to MM batch medium and grown at 20°C for two consecutive transfers. One hundred milliliters of batch culture was inoculated into the 4.8-liter volume of chemostat MM medium. The chemostat growth medium contained one-half of the concentration of nutrients used in the batch culture without EDTA in order to maintain a yield of approximately 2 x 108 cells/ml, because greater cell numbers caused problems with foaming and biofilm growth in the reactor. The culture was allowed to grow to 80% of the maximum optical density in batch growth before the pumps were turned on and chemostat growth was started. MM was fed to the reactor with a peristaltic pump set at 0.29 ± 0.01 liters/h, which kept the dilution rate (and therefore the growth rate) at 0.06 h–1 and the generation time at 11.5 h. Effluent was controlled by a peristaltic pump connected to a Y tube within the reactor so that the effluent was only pumped out when the volume in the reactor was at or above the 4.8-liter volume. The MM was acidified (20 mM H2SO4) to keep metals in solution; therefore, the pH was monitored with an autoclavable pH probe coupled with a pH controller (05997-20; Cole-Parmer Instrument Company, Vernon Hills, IL) linked to a peristaltic pump for addition of NaOH to maintain the pH between 6.8 and 6.9. Oxygen levels were maintained at approximately 6 mg/liter with a controlled low flow of pure oxygen. Dissolved-oxygen levels in the chemostat were monitored daily with an external dissolved-oxygen probe (YSI 5905 BOD probe, YSI model 58 dissolved-oxygen meter; Yellow Springs Instrument Co., Yellow Springs, OH).

PCP stress conditions in chemostat cultures.
PCP was added to the reactor as a continuous, slow increase in concentration up to 40 mg/liter, as might be experienced by organisms being exposed to a moving contaminated plume. A stock PCP solution (feed) was made to a final concentration of 3.5 g/liter in deionized sterile water and solubilized by the addition of 36 mM NaOH. PCP was added to the chemostat with a separate peristaltic pump at a flow rate of 4.75 ml/h. Viton and Tygon tubes were used for the PCP feed to minimize adsorption to the tube walls. Samples were collected to measure the chemostat PCP concentrations over time as explained below.

Sample timing was designed to capture significant changes in expression as a result of the changing PCP concentrations. In order to determine sample timing, preliminary experiments examined catechol-1,2-dioxygenase (C12O) induction when the sole carbon source was switched from acetate to an equal concentration of benzoate to induce C12O. C12O activity was determined immediately from cell extracts of samples removed from the reactor over time. Briefly, 75-ml samples were removed from the chemostat and cells were pelleted, washed, and resuspended in 10 ml of cold 0.01 M sodium phosphate buffer (pH 7.0, 4°C). The final suspension was placed on ice and lysed with six rounds of 30 s of sonication (40% output) with a Branson Sonifier 250 (Branson Ultrasonics Corp., Danbury, CT). Cell extracts were collected as the supernatant of centrifuged lysed cells (37,500 x g, 10 min, 4°C). These extracts were immediately tested for C12O activity with a previously described assay (28). Protein levels were determined with the Pierce BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL). Time points of 6.5, 13, and 26 h after addition of benzoate captured 50-fold, 500-fold, and 1,000-fold increases in C12O expression levels over the background.

To choose effective PCP concentrations, preliminary short-term (10 min) batch experiments were performed on chemostat-grown cells to determine the effect of PCP on respiratory inhibition as previously described (3). With this information, the chemostat experiments were designed to target 15, 30, and 40 mg/liter PCP at time points 6.5, 13, and 26 h, respectively. These concentrations, which correspond to approximately 10, 25, and 30% respiration inhibition, reflect sublethal chemical stress conditions.

To examine the stress response, samples were taken from the reactor to measure the stress response just prior to PCP addition, and 6.5, 13, and 26 h after PCP addition was started and used for RNA isolation, cell counts, whole-cell acetate uptake rate (WAUR), and PCP concentration. The WT1 chemostat experiment was performed four times with RNA from two experiments used for microarray studies and RNA from two experiments used for reverse transcriptase PCR (RT-PCR) analysis. The RpoS– mutant chemostat experiment was run in triplicate, with samples from two experiments used for RT-PCR and one for microarray analysis.

Physiological-stress analysis.
The physiological state of cells was determined by measuring optical density, cell counts, and substrate uptake rates. Optical density was measured at 600 nm; heterotrophic plate counts (HPCs) and direct microscopic counts were done to determine cell numbers. The HPCs were determined at each time point by using duplicate dilution series to 10–5 in 0.01 M potassium phosphate buffer and triplicate platings of 50 µl on LB agar for each series. HPCs are reported as viable cell concentrations. Substrate uptake rates were determined by removing samples from the chemostat and performing short-term batch experiments in triplicate. Briefly, cells were taken from the chemostat and immediately mixed with 200 mg/liter chloramphenicol to inhibit protein synthesis (45). The sample was split into three sterile flasks, and 100 mg/liter acetate was added. Every 15 min, a subsample was taken from each flask and filtered into a sterile sample vial, and the filtrate was analyzed for acetate concentrations. A DX-120 ion chromatograph with an AS9-HC IonPac column (4 by 250 mm) and an AG9-HC guard column was used to measure the acetate concentration (Dionex Corporation, Sunnyvale, CA). The eluent consisted of 12 mM Na2CO3 and 5 mM NaHCO3, at a flow rate of 1.0 ml/min. By this method, the linear range of acetate detection was between 25 and 150 mg/liter acetate. Duplicate acetate measurements were taken for technical replication. The uptake rate was determined as the average of the triplicate biological samples and normalized to viable cell number to determine the WAUR. Initial experiments determined that, based on optical density and WAUR, steady-state growth was reached in the chemostat after approximately 70 h and remained constant until at least 120 h. The initial, unstressed time point of all experiments was taken at approximately 80 h of chemostat growth in all experiments. To determine significant changes in cell viability and WAUR, t tests were performed. When comparing time points within an experiment and within a strain, a two-sided, paired t test was performed. When comparing the WT1 and RpoS– mutant experiments, a two-sided, unpaired t test (Student's t test) was performed. Stated statistical significance indicates a P value of less than 0.05.

Gene expression analysis.
Cells were removed from the chemostat in two aliquots of 75 ml and immediately centrifuged at 4°C, 37,500 x g, for 10 min. The large volume was necessary to obtain sufficient quantities of RNA. Each pellet was resuspended in 1 ml of a mixture of 0.5 mM phosphate buffer and RNAprotect Bacteria Reagent (QIAGEN, Valencia, CA) as directed by the supplier. After the cells were pelleted a second time, all supernatant was removed and pellets were stored at –50°C until they were analyzed. For extraction, the cells were thawed on ice and RNA was extracted with the RNeasy kit (QIAGEN), with on-column DNase digestion. For cell lysis, 1 mg/ml lysozyme was incubated with the cells for 10 min at room temperature. Following the addition of Buffer RLT (QIAGEN), an additional mechanical homogenization with a sterile syringe and a 23-gauge needle was performed to ensure sufficient lysis. RNA was used for either microarray analysis or RT-PCR.

Microarray analysis was done with Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the Virginia Bioinformatics Institute Core Laboratory Facility (Virginia Tech, Blacksburg, VA). RNA quality, quantity, and DNA contamination were determined with an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). cDNA preparation and hybridization were carried out as described in the Affymetrix GeneChip Expression Analysis manual for P. aeruginosa RNA samples. The GeneChip Operating System (GCOS) was used for control of hybridization and scanning of fluorescent intensities. Intensities of hybridized RNA at all time points were globally scaled to 100. This value was chosen after examining the average intensity of each chip throughout an experiment. For the P. aeruginosa array, 13 probe sets were used to determine the expression of one gene. GeneSpring software (Agilent Technologies) was used to organize the data and find genes whose expression was changing because of PCP exposure. n-Fold changes were then determined with the Comparison Analysis function of GCOS, with the pre-PCP (time zero) RNA sample as the baseline GeneChip. GCOS directly compares results between two GeneChips for each of the 13 probe sets and performs a signed-rank analysis to determine changes in expression. Changes are positive (increasing) if P is ≤0.0045 and negative (decreasing) if P is ≥0.9955. We report here average twofold or greater increases or decreases at any time point compared to time zero.

RT-PCR was used to validate microarray results of the mexAB-oprM operon with mexB. Two housekeeping genes were used as controls, proC (39) and nadB (35), and expression levels were normalized to each control. cDNA was made from 1 µg of total RNA with random hexamer primers and SuperScript II RT (Invitrogen Life Technologies, Carlsbad, CA). The resulting cDNA was diluted 1:10 and used as template DNA in RT-PCR with SYBR green detection with a Bio-Rad iCycle iQ real-time PCR detection system (Bio-Rad, Hercules, CA). The starting quantity of template DNA for each sample was determined by using a six-point standard curve. Melting curves were analyzed at the end of each PCR run, and controls included PCRs without total RNA and without SuperScript II RT. Table 1 shows the primers used for RT-PCRs.


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  		TABLE 1. Primers used for RT-PCR

PCP analysis.
PCP was measured by gas chromatography with electron capture detection after acetylation and extraction with hexanes (21). Briefly, in triplicate at each time point, 400-µl samples were mixed with 100 µl acetic anhydride, vortexed for 1 min, and allowed to react at room temperature for 1 h. The acetylated PCP was then solvent extracted overnight at 20°C in 3 ml hexanes. Samples were centrifuged at 1,000 x g and 4°C to separate the solvent and aqueous layers, and the hexane layer was removed into gas chromatograph (GC) vials crimped with Teflon-lined septa. PCP was analyzed with a Hewlett-Packard 5890 GC (Agilent Technologies) with a DB-1701 column (30 m by 25 mm [inside diameter], 0.25-µm film thickness; J+W Scientific, Agilent Technologies). One microliter was injected with a Hewlett-Packard 7673 GC/SFC autosampler. The column was held at 60°C for 1 min, ramped to 240°C at 25.7°C/min, and held at 240°C for 8 min. Helium, the carrier gas, flowed at 1.2 ml/min, and make-up nitrogen gas flowed at 20 ml/min. The inlet and electron capture detection temperatures were 225°C and 350°C, respectively.

Microarray data accession number.
The complete microarray data set used in this study is available under accession number GSE5604 in the Gene Expression Omnibus database.


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RESULTS AND DISCUSSION
 
P. aeruginosa responds to PCP with minimal stress.
In slow-growing chemostat cultures, P. aeruginosa was able to respond to PCP with minimal physiological stress. This experiment was designed to mimic contamination of an oligotrophic groundwater sediment or soil. Therefore, cells were grown under steady-state nutrient-limited conditions in a chemostat and the PCP was added gradually (Fig. 1A) to mimic concentration increases in a contaminant plume. PCP exerts its toxic effect as an uncoupler of oxidative phosphorylation (10) but has also been shown to cause oxidative stress and cytotoxicity (47, 51) and induce expression of general stress proteins (1). At the whole-cell level, an oxidative uncoupler is expected to cause an increase in the substrate uptake rate to compensate for disruption of the proton motive force, thereby shifting cellular resources away from growth and toward more critical metabolisms, such as protective stress responses.


Figure 1
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  		FIG. 1. Physiological changes as chemostat-grown cells are exposed to increasing concentrations of PCP. (A) WAUR does not change as cells are exposed to increasing PCP concentrations. (B) Cell viability of P. aeruginosa in chemostat culture exposed to PCP at each time point. PCP: {blacktriangleup}, WT1; {triangleup}, RpoS– mutant. WAUR (A) and CFU (B): •, WT1; {circ}, RpoS– mutant. Error bars indicate standard deviations for triplicate samples and at least duplicate reactor runs for each strain.

The ability of P. aeruginosa to respond to PCP on the whole-cell level was examined by determining whole-cell substrate uptake rates and cell viability. As acetate was the sole carbon source for growth in this system, WAURs were used to determine the catabolic potential of the cell. The WAUR did not change in response to PCP for WT1 (Fig. 1A). In contrast to the WAUR results, cell viability decreased slightly to 78% and 54% of the prestress levels at 13 and 26 h, respectively, in the WT1 strain upon exposure to PCP (Fig. 1B) (P ≤ 0.004 relative to the initial time point). Chemostats without PCP exposure were run for the same length of time and did not show any changes in WAUR or cell viability (data not shown; P ≥ 0.26). These data indicate that P. aeruginosa is fairly resistant to PCP stress.

RpoS is not involved in the protective mechanism.
As the sigma factor RpoS has been shown to regulate increased resistance to different types of stressors in P. aeruginosa and many other species under conditions of starvation (9, 14, 30, 36, 44), the effects of an rpoS mutation on stress resistance to PCP were also examined. Although starvation growth is different from the nutrient-limited balanced growth state achieved here, the dilution rate of the chemostat was kept low to maintain a physiology approaching a starvation state (11). Comparison of the whole-cell data for the WT and its RpoS– mutant shows that RpoS does not play a major role in protecting P. aeruginosa from PCP stress. The indicator of catabolic potential, WAUR, only increased slightly at the last time point for the RpoS– mutant (P = 0.02) (Fig. 1A). Interestingly, the cell viability of the RpoS– mutant was affected to a lesser extent than WT1 with only a slight decrease with exposure to PCP at the 26 h time point to 88% of prestress levels (12% reduction, P value = 0.0009, relative to the initial time point) (Fig. 1B). This result indicates that loss of RpoS– results in slightly greater protection of the cells. A similar phenotype has been shown recently by other researchers whereby RpoS– mutants survive either as well as or better than their WT counterparts in stress-induced environments (17, 48).

Transcriptome response to PCP.
In contrast to results from the whole-cell tests, the gene expression data show distinct physiological changes as the cells respond to PCP. In total, 60 genes showed a twofold or greater increase and 53 genes showed a twofold or greater decrease at any post-PCP time point in comparison with the pre-PCP time point in WT PAO1. The microarray data sets for WT1 and the RpoS mutant were very similar; therefore, all of the results discussed are based on the WT1 strain. Summarizing the data based on functional categories shows distinct differences in up- and downregulated genes (Fig. 2). Genes coding for transport and membrane proteins comprise more than 30% of the upregulated transcripts, while metabolism genes make up greater than 20% of the downregulated transcripts. Interestingly, genes coding for adaptation and protection proteins represent a large percentage of the downregulated transcripts.


Figure 2
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  		FIG. 2. Transcripts increasing or decreasing by at least twofold at any time point after PCP stress, grouped by functional category. The categories, as described in the P. aeruginosa Community Annotation Project (www.pseudomonas.com) (49), have been grouped to clarify differences between increasing and decreasing transcripts. Increasing transcripts, {blacksquare}; decreasing transcripts, blk12.

Multiple multidrug efflux transporters are upregulated in response to PCP.
Examination of the transport and membrane genes that changed in response to the PCP stress shows that multiple multidrug efflux operons increased (Table 2). Specifically, two operons coding for resistance-nodulation-cell division (RND) pumps (PA0425-PA0426-PA0427 and PA3676-PA3677-PA3678) and one operon (PA3720-PA3719) coding for proteins that positively regulate the RND pump MexAB-OprM are upregulated strongly at all time points in response to PCP. mexAB-oprM (PA0425-PA0426-PA0427) are genes which code for the well-characterized MexAB-OprM RND transporter (29, 33). This efflux pump is responsible for resistance to a wide variety of antibiotics and has also been shown to cause the efflux of solvents (22, 23, 33). The RND efflux operon PA3676-PA3677-PA3678 (mexJKL) encodes the system responsible for the efflux of a few different antibiotics and the antimicrobial compound triclosan (5, 6). Interestingly, the operon PA3719-PA3720 was also upregulated strongly. Although the functions of these genes are unknown, the increased expression of PA3719 has been reported to increase expression of mexAB-oprM (4).


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  		TABLE 2. Efflux, transport, and membrane transcripts increasinga in response to PCP

This is the first report of anthropogenic chemical induction of mexAB-oprM expression. The MexAB-OprM system is expressed at constitutive basal levels in WT strains and overproduced in cells containing mutations in either MexR or PA3721, transcriptional repressors of mexAB-oprM and PA3720-PA3719 transcription, respectively (4, 43). Low-iron growth conditions are the only conditions reported to induce increased levels of MexAB-OprM (27, 34).

The increase in mexAB-oprM transcription by PCP was verified by RT-PCR showing increased mexB expression during independent chemostat reactor experiments for both the WT and RpoS– strains (Fig. 3). Figure 3 shows that levels of mexB were significantly upregulated to their full extent at 13 and 26 h (P < 0.05). Consistent with the whole-cell data which indicated that the RpoS– strain had slightly greater protection from PCP, the levels of mexB were more highly expressed at these time points in the mutant compared with the WT (P = 0.04 at both 13 and 26 h). This increased expression is contrary to results that examine the transcriptome of RpoS-regulated stationary-phase genes in PAO1, in which mexAB-oprM expression was decreased in the rpoS mutant in comparison with the WT (40). As the strains for both studies are identical, the discrepancy may be accounted for by the different growth conditions.


Figure 3
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  		FIG. 3. Microarray results were validated by quantitative RT-PCR of mexB. Shown is the average fold change in proC and nadB normalized data. WT1, {square}; RpoS– mutant, {blacksquare}. Results are reported as averages and standard errors.

Transcripts coding for probable major facilitator superfamily (MFS) efflux proteins were also upregulated strongly at 13 and 26 h post-PCP stress (PA3136-PA3137 and PA5157-PA5158-PA5159-PA5160) (Table 2). These genes have not been previously described in the literature but are similar to the emrA and emrB genes of Escherichia coli, which code for components of an MFS pump that is able to cause the efflux of, among other compounds, hydrophobic uncouplers (24, 33). MFS pumps have been shown to function with only the drug efflux transporter or as a three-component pump, coupling the transporter with a membrane fusion protein and outer membrane protein (18). Both operons induced by PCP contain genes coding for the membrane fusion protein and drug efflux transporter; however, one operon, PA5157-PA5158-PA5159-PA5160, also contains genes coding for the outer membrane protein and putative regulator. A third gene coding for a putative MFS transporter, PA0246, increased only at the first time point.

Other transcripts increasing in response to PCP include those involved in different transport mechanisms and iron uptake (Tables 2 and 3). The transcripts PA0204, PA4037, and PA5216 code for probable components of different ATP-binding cassette (ABC) transporters. Transporters belonging to this ABC family use the hydrolysis of ATP to drive the transport of a compound and are involved in both uptake and efflux (18). Genes associated with iron uptake, pvdN, pvdE, and fpvA (Tables 2 and 3), code for proteins involved in pyoverdine and ferripyoverdine receptor synthesis (20, 25, 42). Interestingly, pyoverdine production and ferripyoverdine synthesis are known to be coregulated with the mexAB-oprM efflux system (34).


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  		TABLE 3. Other transcripts increasing in response to PCPa

Metabolism genes show slight, transient downregulation in response to PCP.
Transcripts downregulated (Table 4) in response to PCP include genes associated with central metabolism (cysD, rpsT, tpiA, rpmH), fatty acid metabolism (accB), and energy metabolism (sucC, etfA). With no change in whole-cell metabolism as measured by WAUR, this may be partly unexpected, but a closer examination of the timing of gene expression shows that this decrease in metabolism genes is transient and only occurs at the first time point. This transient response may be a way for the cells to temporarily shift metabolism away from less-essential metabolisms and focus the energy from sustained acetate uptake toward the response to PCP. Two of the most recent studies looking at the response of P. putida to phenol and toluene suggest that this is the result of organic solvent stress as well (7, 38). In the present study with PCP, the increase in efflux pumps was sufficiently upregulated after 13 h (one generation) for P. aeruginosa to resume the preshock metabolic state. After at least 13 h of exposure to PCP, metabolic gene expression was back to unstressed levels.


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  		TABLE 4. Transcripts decreasinga in response to PCP, grouped by functional category

PCP exposure caused a decrease in adaptation and protection genes.
Interestingly, the second most represented functional category for downregulated transcripts was adaptation/protection (Fig. 2 and Table 4). A few of these adaptation/protection genes, ohr and oprH, are downregulated consistently in response to PCP. The transcript ohr codes for the organic hydroperoxide resistance protein known to protect the cell from oxidative damage induced by reactive oxygen species. As with the metabolism genes, some of these adaptation/protection genes decrease only transiently at the first time point and are back to prestress levels. Three of these genes are involved in general stress resistance mechanisms: groES codes for a stress-induced chaperone, capB codes for cold acclimation protein B, and PA0962 codes for a probable DNA-binding stress protein. Again, this transient change might be expected if the cultures were initially stressed because of the low growth rate conditions. Upon exposure to the chemical stressor, a reorganization of cellular metabolism may have caused a temporary decrease in these stress response genes, ensuring that sufficient metabolic resources were available to allow a quick response to the PCP.

The RND pump MexAB-OprM is required for optimal growth in PCP.
To validate the results of the microarray experiments, which indicated that efflux is the major response of P. aeruginosa to PCP stress, the effect of the MexAB-OprM efflux pump on growth in the presence of PCP was examined. With a MexAB-OprM mutant (PAO200) along with its isogenic WT strain (WT2), cells were grown in batch cultures with MM with acetate as the sole carbon source. The cultures were grown at 37°C with and without 30 mg/liter PCP added. PAO200 grew normally in medium without PCP; however, growth was significantly reduced in the presence of PCP (Fig. 4). To further investigate the effect of MexAB-OprM, an MIC assay was performed on these cultures to assess their ability to grow on PCP. In rich broth (LB) at 37°C, conditions commonly used in the literature to determine antimicrobial toxicity, the PCP MIC for the WT was 1,000 mg/liter, whereas the MIC for the mutant was fourfold lower at 250 mg/liter. The test was repeated in MM at 20°C to be consistent with the chemostat studies, and although the MIC for the WT was lower than in LB at 250 mg/liter, the MIC for the mutant was still twofold lower at 125 mg/liter. These results validate the microarray data, showing that MexAB-OprM-mediated efflux of PCP is required for P. aeruginosa to maintain normal physiological status when grown in the presence of PCP.


Figure 4
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  		FIG. 4. Growth of MexAB-OprM mutant strain PAO200 is reduced in 30 mg/liter PCP in comparison with that of the WT P. aeruginosa strain PAO1. WT2, •; WT2 plus PCP, {circ}; PAO200, {blacktriangleup}; PAO200 plus PCP, {triangleup}. The experiment was repeated three times, and the results of a single representative experiment are shown.

Conclusion.
Populations of slow-growing P. aeruginosa actively respond to and resist the potentially toxic effects of PCP by upregulating multiple multidrug efflux pumps. Further analysis revealed that MexAB-OprM, in particular, is upregulated in response to the PCP and is necessary for optimal growth in the presence of the compound. This is not entirely surprising, as the MexAB-OprM system has been shown to cause the efflux of many different compounds, including a wide variety of antibiotics and a few different organic solvents (12, 29, 33). Upregulation of this pump by an anthropogenic chemical inducer, however, has not been demonstrated before now. Revealed through the analysis of genome-wide expression changes relative to whole-cell physiological function, this study emphasizes the great importance efflux expression plays in the metabolic capability and resistance properties of P. aeruginosa.

Previous research on the response of E. coli to PCP indicated that general stress response proteins were upregulated as part of the stress response (1). Similarly, recent transcriptome studies of the response of P. putida to aromatic solvent contaminants show that genes upregulated include those coding for proteins involved in oxidative stress response, glutathione-mediated stress response, and the general protective heat shock response (7, 38, 46). These studies all examined stress responses under batch growth conditions. The present study examined the transcriptome level response to a contaminant under steady-state, low growth rate conditions, and the results suggest that stress response patterns may be very different from those experienced during batch growth. As similar nutrient-limiting conditions are far more prevalent in the natural environment, future research should focus on understanding this difference in stress responses.


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ACKNOWLEDGMENTS
 
The funding for this project and for J. Fraga Muller came from the following graduate fellowships: the Marion Via Fellowship, Charles E. Via, Jr., Department of Civil and Environmental Engineering, Virginia Tech; the GAANN Fellowship, U.S. Department of Education; the P.E.O. National Scholar Award; and the AAUW Selected Professions Fellowship. Additional funding was provided by the Paul L. Busch Award for Innovation in Applied Water Quality Research.

P. aeruginosa strains were kindly provided by E. P. Greenberg, University of Washington, and H. Schweizer, Colorado State University.


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FOOTNOTES
 
* Corresponding author. Mailing address: Environmental and Water Resources Engineering, 418 Durham Hall, Virginia Tech, Blacksburg, VA 24061. Phone: (540) 231-3980. Fax: (540) 231-7916. E-mail: nlove{at}vt.edu Back

{triangledown} Published ahead of print on 25 May 2007. Back

{dagger} Present address: Department of Microbiology, University of Washington, Seattle, WA 98105. Back


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Applied and Environmental Microbiology, July 2007, p. 4550-4558, Vol. 73, No. 14
0099-2240/07/$08.00+0     doi:10.1128/AEM.00169-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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