Physiological and Transcriptional Responses of Nitrosomonas

Apr 25, 2008 - Ammonia oxidizing bacteria (AOB) are inhibited by many compounds found in wastewater treatment plant (WWTP) influent, including aromati...
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Environ. Sci. Technol. 2008, 42, 4093–4098

Physiological and Transcriptional Responses of Nitrosomonas europaea to Toluene and Benzene Inhibition TYLER S. RADNIECKI,* MARK E. DOLAN, AND LEWIS SEMPRINI School of Chemical, Biological and Environmental Engineering; 101 Gleeson Hall, Oregon State University, Corvallis, Oregon 97331

Received October 16, 2007. Revised manuscript received February 22, 2008. Accepted March 3, 2008.

Ammonia oxidizing bacteria (AOB) are inhibited by many compounds found in wastewater treatment plant (WWTP) influent, including aromatic hydrocarbons. The detection of “sentinel genes” to identify the presence of aromatic hydrocarbons could be useful to WWTP operators. In this study, the transcriptomic responses of Nitrosomonas europaea during the cometabolism of benzene to phenol and toluene to benzyl alcohol and benzaldehyde were evaluated using whole genome Affymetrix microarrays and qRT-PCR. Benzyl alcohol and benzaldehyde were found not to inhibit N. europaea. However, phenol concentrations as low as 5 µM directly inhibited ammonia oxidation. Surprisingly, there were no significant upor down-regulation of genes in N. europaea cells exposed to 20 µM toluene, which caused 50% inhibition of ammonia oxidation. Exposing N. europaea to 40 µM benzene, which caused a similar degree of inhibition, resulted in the up-regulation of seven adjacent genes, including NE 1545 (a putative pirin protein) and NE 1546 (a putative membrane protein), that appear to be involved with fatty-acid metabolism, lipid biosynthesis, and membrane protein synthesis. qRT-PCR analysis revealed that NE 1545 and NE 1546 were significantly up-regulated upon exposure to benzene and phenol, but not upon exposure to toluene. Transmission electron microscope images revealed a shift in outer cell structure in response to benzene exposure.

Introduction Ammonia oxidizing bacteria (AOB) play a critical role in the removal of ammonia from wastewater treatment plants (WWTPs) by oxidizing ammonia to nitrite. AOB have the potential for bioremediation by cometabolizing various environmental pollutants (1–4). However, AOB are often considered the most sensitive organisms in ammonia removal (5) and are vulnerable to disturbance by a wide variety of contaminants including heavy metals (6–9), pH shifts (10, 11), and organic solvents including aromatic hydrocarbons (7, 12–14). Benzene and toluene are part of the BTEX (benzene, toluene, ethlylbenzene, and xylene) classification and are common groundwater contaminants often originating from leaking underground storage tanks at gas stations or oil and gas pipeline leaks (15). The influence of benzene and toluene on Nitrosomonas europaea, the model AOB, at * Corresponding author phone: (541) 231-9351; fax: (541) 7374600; e-mail:[email protected]. 10.1021/es702623s CCC: $40.75

Published on Web 04/25/2008

 2008 American Chemical Society

the physiological and transcriptional level is of interest because these compounds can potentially inhibit nitrification activity in multiple ways; including acting as a cometabolic energy drain (2), forming cytotoxic daughter products, or disrupting vital cell processes via solvent effects on the outer membrane (16–18). This study examines the physiological and transcriptional responses of N. europaea upon exposure to toluene and benzene. N. europaea is an obligate chemolithoautotroph and derives its energy for growth solely from the oxidation of ammonia (NH3) to nitrite (NO2-) (19, 20). Ammonia-monooxygenase (AMO), a key enzyme in the oxidation of NH3, has broad substrate specificity and oxidizes a wide range of substrates including aromatic hydrocarbons, halogenated aromatic hydrocarbons, polycyclic aromatic hydrocarbons, and chlorinated aliphatic hydrocarbons (1–3, 14, 21). However, N. europaea is unable to derive electrons from the oxidation of these organic compounds resulting in a reduction in nitrifying activity. This reduction in nitrifying activity may have severe consequences in the removal of NH3 from WWTPs (5). While the transformation of aromatic hydrocarbons by N. europaea has been characterized previously (2, 22), the molecular responses to aromatic hydrocarbons have not been studied and may reveal defense mechanisms employed during times of solvent stress. The genome of N. europaea has been fully sequenced (23), and recent studies have been conducted using whole-genome Affymetrix microarrays to study the stress responses of N. europaea to starvation conditions (24) and exposure to chlorinated aliphatic hydrocarbons (12) at the transcriptional level. This study examined the global transcriptional response of batchcultured N. europaea to toluene and benzene inhibition using whole-genome Affymetrix microarrays and quantitative reverse-transcriptase-PCR (qRT-PCR). Toluene and benzene were chosen from several previously studied aromatic hydrocarbons (1, 2, 18, 22) because they represent two distinct categories of aromatic hydrocarbons, substituted and nonsubstituted, respectively, that can be cometabolized by N. europaea. These compounds are oxidized at different locations with benzene being oxidized on the ring and toluene oxidized on the methyl group (2). Aromatic hydrocarbons have been shown to alter bacterial outer-membranes (16–18) and genes associated with lipid biosynthesis, membrane proteins, and fatty acid metabolism were found to be upregulated in response to benzene inhibition. In light of this information, the outer membrane structures of benzeneexposed N. europaea cells were investigated via transmission electron microscopy (TEM).

Material and Methods Benzene, Toluene, and Phenol Inhibition Studies. The criteria used to select the proper toluene and benzene exposure concentrations for microarray analysis and phenol concentration for qRT-PCR analysis was to cause a significant change in cell physiology without being too damaging to vital cell processes. Thus, 50% inhibition in the rate of NO2production after 1 h of exposure was determined to be the desired level of inhibition for this study. The inhibition studies were performed in batch reactors with midexponential phase N. europaea cells in the presence of ammonia with and without (control) exposure to either 40 µM benzene, 20 µM toluene, or 10 µM phenol. Cell density, ammonia-monooxygenase specific oxygen uptake rate (AMO-SOUR), hydroxylamine oxidoreductase (HAO) specific oxygen uptake rate (HAO-SOUR), and NO2- production, via colorimetric assay VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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(26), were monitored at 45 min intervals for 3 h as described previously (4). Cells for total RNA extraction were harvested from both the control and treatment bottle 30, 60, and 180 min into the experiment. At the end of the experiment, cells were harvested for electron microscopy examination (0.1 mg of protein). See the Supporting Information for more details on culturing conditions and experimental procedures. AMO- and HAO-SOUR Measurements. AMO-SOUR and HAO-SOUR were measured using a 30 °C water-jacketed glass cell (Gilson Medical Electronics, Inc.) fitted with a Clark microelectrode (Yellow Springs Instrument Co model no. 5331) attached to a YSI model 5300 biological oxygen monitor (Yellow Springs Instrument Co.) and a flatbed recorder as described by Ely et al. (25). To measure HAO-SOUR, AMO activity was blocked by the addition of allylthiourea (ATU; 100 µM) and hydrazine (750 µM) was added as an alternative substrate for HAO. Daughter Product Inhibition. N. europaea cells were exposed to benzyl alcohol, benzaldehyde, or phenol. The concentrations tested were equivalent to the amounts measured after 3 h of exposure to either toluene or benzene. Cells in the midexponential phase were placed into fresh medium containing 2.5 mM (NH4)2SO4 and either 20 µM benzyl alcohol, or 1 µM benzaldehyde, or 10 µM phenol. Bottles without the addition of a daughter product were run as controls. NO2- production was monitored at 30 min intervals for 2 h. See the Supporting Information for more details on experimental procedures. Reversibility Experiments. Midexponential phase N. europaea cells were placed into fresh medium containing 2.5 mM (NH4)2SO4 and either 40 µM toluene, or 40 µM benzene, or 10 µM phenol. Cells not exposed aromatic hydrocarbon served as controls. NO2- production was monitored at 30 min intervals for 2 h. After two hours of exposure, cells were harvested, washed, and placed into fresh media. NO2- production was monitored at 30 min intervals for 2 h to determine if inhibition was reversible. See the Supporting Information for more details on experimental procedures. GC Measurement of Toluene and Benzene. Toluene and benzene were measured in the headspace gas samples from the batch reactors. Calibration curves for the compounds were developed using external standards. The total mass of toluene and benzene were determined via mass balance by applying Henry’s Law using Henry’s Law Constants obtained from the CRC Handbook of Chemistry and Physics (26). See the Supporting Information for more details on the analytical procedures. HPLC Measurements of Oxidized Aromatic Hydrocarbons. Oxidized aromatic hydrocarbons, including benzyl alcohol, benzaldehyde, and phenol, were measured with a Dionex-500 HPLC chromatograph equipped with UV/vis detector operated at 257 nm and an Alltech Platinum C18 column (Alltech Associates, Inc., Deerfield, IL). The eluent, composed of 25 mM KH2PO4 with 10% acetonitrile at pH 2.5, was metered through the column at 2 mL/min. Microarray Data Analysis. All annotated genes (2460 total) in the N. europaea genome (23) were represented on highdensity Affymetrix GeneChips (Affymetrix, Santa Clara, CA). All microarray analyses were conducted in triplicate using total RNA extracted from three independent experiments (untreated control cells and toluene- or benzene-treated cells) 60 min after exposure to toluene or benzene. The results were normalized and filtered to identify genes with statistically significant increases or decreases in ratios of transcript levels as described previously (12). The microarray data for this study are available at the Gene Expression Omnibus database (http://www.ncbi.nih.gov/geo) under accession number GSE10507. See the Supporting Information for more details on RNA extraction, labeling, hybridization, scanning, 4094

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and data analysis experimental procedures. MIAME compliance and microarray quality control data can also be found in the Supporting Information. Quantitative Reverse-Transcriptase-PCR Analysis. qRTPCR was conducted to verify the microarray results and to track the expression of NE 1545 and NE 1546 in cells exposed to toluene, benzene, or phenol. qRT-PCR was carried out in triplicate on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) using the iQ SYBR Green Supermix kit (Bio-Rad, Hercules, CA) per manufacturer’s instructions. The relative expression values for the reactions were determined using DART (Data Analysis for Real Time)PCR analysis (27). See the Supporting Information for more details on experimental procedures. Transmission Electron Microscopy Analysis. Control N. europaea cells and N. europaea cells exposed to 40 µM benzene for 4 h were pelleted and fixed for 1.5 h at room temperature in a solution containing 2.5% glutaraldehyde, 1% formaldehyde, and 1% tannic acid in 0.1 M cacodylate acid, pH 2.5. Postfixation was conducted in 1% osmium tetroxide in 0.1 M cacodylate acid, pH 7.4. Cells were dehydrated in acetone, embedded in Spurr’s resin and polymerized at 60 °C for 24 h. Thin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate. Slices were examined at 60 kV with a Philips CM12 TEM (Koninklijke Philips Electronics N.V, The Netherlands). The 4 h exposure time was chosen to give N. europaea, with a doubling time of 8–12 h, enough time to make significant changes to the membrane structure. The t test was used to measure the significance of any changes in membrane thickness.

Results and Discussion Physiological Responses to Aromatic Hydrocarbons. N. europaea demonstrated a linear 50% inhibition in NO2production when exposed to 20 µM toluene (Figure 1a) over the 180-min time course. A 50% inhibition in AMO-SOUR was also observed (Figure 1c), but no inhibition was noticed in HAO-SOUR (Figure 1d) indicating that the inhibition was directed at the AMO enzyme or electron transfer to AMO during the 180 min of exposure. Over the course of the experiment, 14.5 µM toluene was oxidized into 14 µM benzyl alcohol and 0.4 µM benzaldehyde (Figure 1b). N. europaea demonstrated a nonlinear inhibition in nitrite production when exposed to 40 µM benzene with 50% inhibition observed after 1 h and 65% inhibition noticed after 3 h (Figure 2a). AMO-SOUR measurements mimicked NO2measurements with 50% inhibition observed after 1 h and 65% inhibition noticed after 3 h (Figure 2c). HAO-SOUR measurements did not show any inhibition upon exposure to benzene (Figure 2d) once again indicating that the inhibition was directed at the AMO enzyme or electron transfer to AMO. Over the 180-min time course, 14 µM benzene was oxidized into 11 µM phenol (Figure 2b). The rate of benzene transformation decreased with time of exposure, consistent with the decreased rate of phenol production. The slowing in the rate of nitrite production and decrease in AMO-SOUR were correlated with the accumulation of phenol. The inhibitor concentrations are 5 times lower for toluene inhibition and 3 times lower for benzene inhibition than reported in previous experiments (2). However, in those previous experiments, cell concentrations were three times higher than those used here indicating there may be a cell concentration effect on toluene and benzene inhibition. In addition, N2H4 was added in the previous experiments to prevent the electron flux from being limiting and may account for the increased tolerance observed. To determine whether the daughter products of toluene and benzene oxidation were responsible for the observed

FIGURE 1. (A) Nitrite production of N. europaea control cells (9) and cells exposed to 20 µM toluene (2). (B) The oxidation of toluene (µ) into benzyl alcohol (∆) and benzaldehyde (X). (C) AMO-SOUR of control N. europaea cells (9) and cells exposed to 20 µM toluene (9). (D) HAO-SOUR of control N. europaea cell (2) and cells exposed to 20 µM toluene (∆). Error bars represent 95% confidence intervals.

FIGURE 2. (A) Nitrite production of N. europaea control cells (9) and cells exposed to 40 µM benzene (O). (B) The oxidation of benzene (O) into phenol (9). (C) AMO-SOUR of control N. europaea cells (9) and cells exposed to 40 µM benzene (0). (D) HAO-SOUR of control N. europaea cell (2) and cells exposed to 40 µM benzene (∆). Error bars represent 95% confidence intervals. inhibition, separate batch experiments were conducted with N. europaea in the presence of benzyl alcohol, benzaldehyde, and phenol. Benzyl alcohol and benzaldehyde (the daughter products of toluene oxidation) did not cause inhibition in nitrite production at concentrations measured in these batch studies (Table 1). Phenol, however, caused significant inhibition in nitrite production (Table 1) causing more severe inhibitions as concentrations were increased. The results indicate that the accumulation of phenol (Figure 2b) likely accounted for most of the inhibition observed (Figures 2a

and 2c). Note that exposure to 10 µM phenol (Table 1) can account for 67% inhibition in ammonia oxidation rate which is essentially the same amount of phenol present during benzene inhibition batch tests when the inhibition reached 61% (Figures 2a and 2b). The estimated 50% inhibition level of phenol (8 µM) is in line with previous reports of 11.7 µM (28) and 10.4 µM (29) phenol causing 50% inhibition on pure N. europaea cultures. The reversibility in ammonia oxidation inhibition was evaluated by measuring the rate of ammonia oxidation after VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Inhibition and Reversibility of Toluene, Benzene, and Daughter Productsa compound

concentration (µM)

nitrification activity

control toluene benzyl alcohol benzaldehyde benzene phenol phenol phenol phenol

40 20 1 40 5 10 15 20

100% ( 1% 16% ( 2% 100% ( 6% 100% ( 2% 13% ( 3% 83% ( 12% 33% ( 3% 9% ( 1% 6% ( 2%

reversibility 100% ( 1% 94% ( 3% 95% ( 1% 106% ( 7%

a N. europaea cells were incubated with NH4+ and benzene, toluene, or their daughter products for 2 hrs and the nitrite production rates were measured. To determine reversibility, the cells were washed 3 times in K2PO4 buffer and placed into fresh media and the nitrite production rates were measured for 2 hrs.

exposure to toluene, benzene, and phenol for 180 min and again upon washing the cells to remove the aromatic hydrocarbons and their oxidized daughter products. Toluene, benzene, and phenol inhibition was found to be completely reversible (Table 1), therefore, the inhibition observed during exposure to these compounds was apparently nontoxic. Transcriptional Responses to Aromatic Hydrocarbons. A previous transcriptional study with N. europaea showed that the most significant changes in gene expression occurred after 60 min of exposure to chloroform (12). Therefore, to help with cross-platform comparisons of exposure to different classes of chemicals and to detect early signals of inhibition, the 60 min. time-point was chosen in this study for microarray analysis. Whole genome microarray analysis revealed that although 40 µM toluene decreased nitrification activity by 50%, N. europaea did not respond to this inhibition at the transcriptional level, indicating that not all inhibition of activity, even activity of the key energetic enzymes, will necessarily result in transcriptional changes. Since toluene was not toxic and the inhibition observed was not great enough to cause severe energy shortages, N. europaea may have simply not responded with transcriptional changes to the presence of a low concentration of toluene. In the case of benzene inhibition, only seven genes were found to be up-regulated after 50% inhibition. In neither case were amoABC genes up- or down-regulated according to microarray results. This is interesting as AMO- and HAO-SOUR results show that AMO activity was inhibited by both toluene and benzene. The lack of change in amoA expression in the presence of a decline in AMO activity has been observed previously in NH3-starved N. europaea cells (30). The exposure of N. europaea to 40 µM benzene for 60 min resulted in the significant up-regulation of seven consecutive genes, NE 1545 through NE 1551 (Supporting Information, Table S.3). While five of the genes were up-regulated 4-fold, NE 1545 (a putative pirin protein) and NE 1546 (a putative membrane protein) showed the greatest apparent upregulation at 31 and 43-fold, respectively. Three of these genes, NE 1546, 1547, and 1550, encode membrane proteins and may suggest changes in membrane protein composition. A study on E. coli exposed to phenol showed an increase in the protein–lipid content causing a rigidifying effect on the outer-membrane (31). The up-regulation of membrane protein genes in N. europaea exposed to benzene may be an attempt to rigidify its outer-membrane. NE 1545 is a putative pirin protein which has been shown to be involved in the regulation of pyruvate catabolism to Acyl CoA in Serratia marcescens (32). NE 1548 is an Acyl CoA dehydrogenase which plays a role in fatty acid metabolism 4096

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FIGURE 3. (A) Relative expression of genes NE 1545 (9) and NE 1546 (0) over time in N. europaea exposed to 40 µM benzene. (B) Relative expression of genes NE 1545 (9) and NE 1546 (9) in N. europaea exposed to benzene, phenol or toluene for 60 min. Error bars represent 95% confidence intervals. through the beta-oxidation of fatty acids and NE 1549 is a long chain fatty acid CoA ligase/synthase enzyme which plays a role in lipid biosynthesis and fatty acid degradation (33, 34). The up-regulation of these genes leads to the hypothesis that this gene cluster deals with membrane protein composition, lipid biosynthesis and fatty acid metabolism. The expression of NE 1545 and NE 1546 were monitored over the course of the benzene inhibition experiments using qRT-PCR analysis. NE 1545 and NE 1546 showed 5-fold upregulation after 30 min of exposure to benzene and the expression remained high throughout the experiment (Figure 3a). While the up-regulated expression of NE 1545 and NE 1546 is significant (p ) 0.0006 and p ) 0.0002, repectively), the up-regulation was not found to be as great as that measured in the microarray results, but does fall in line with the expression measured by microarrays for the other five consecutive genes. The increased expression of NE 1545 and NE 1546 appears to be specific to the presence of phenol as greater upregulation was observed in phenol-exposed cells than the benzene-exposed cells (Figure 3b). This increase in gene expression may be due to the longer exposure time to 10 µM phenol, 1 h, compared to the exposure time and concentration of phenol generated from the transformation of benzene. A similar expression of NE 1545 and NE 1546 is seen after 3 h of exposure to benzene (Figure 3a) in which the cells were exposed to approximately 9–12 µM phenol for about 2 h (Figure 2b). As expected, no significant up-regulation was observed in the toluene-exposed cells (Figure 3b). Similar results have been seen in E. coli exposed to benzene and toluene. E. coli cells exposed to benzene showed an increase in the synthesis of lipids containing fatty acids, whereas E. coli cells exposed to toluene show virtually no change in fatty acid composition (35). Our results are in stark contrast to previous work with 50% inhibition of N. europaea by chloroform or chloromethane, which resulted in 175 and 67 genes being up-

FIGURE 4. TEM images of control N. europaea cells (A) and N. europaea cells exposed to 40 µM benzene for 180 min (B). The arrows point to the outer-membrane which appears more compact and highly structured in benzene exposed cells compared to control cells not exposed to benzene. regulated and 501 and 148 genes being significantly downregulated, respectively (12). Chloroform has been shown to be highly toxic to N. europaea, as defined by AMO recovery, with no AMO activity recovered during reversibility studies, while chloromethane has been shown to be slightly toxic to N. europaea with partial (80%) activity of AMO recovered during reversibility studies (21). Benzene and toluene, however, are essentially nontoxic toward N. europaea at 50% inhibition concentrations. Therefore, there appears to be an association between the toxicity of substrate and the quantity and extent of genes regulated in response to that substrate. Further studies will evaluate whether higher levels of inhibition by benzene or toluene can cause toxicity and result in an increase in gene expression. Also of interest is the inhibition and gene response of N. europaea upon exposure to other substituted aromatic hydrocarbons including xylene, analine, and cresols. The limited gene regulation in N. europaea significantly inhibited by toluene and benzene suggests that posttranscriptional changes may be occurring in response to the inhibitor. Future work will include the examination of proteomic changes in N. europaea inhibited by toluene and benzene with 2-dimensional protein gels. Changes in Outer-Membrane Composition and Morphology. The primary damage caused by organic solvents in gram-negative bacteria is to the outer-membrane. This damage results in the impairment of vital functions, the loss of macromolecules and ions and the inhibition of membrane proteins through the loss of the pH gradient and electrical potential (36, 37). Gram-negative bacteria respond to aromatic hydrocarbons by either metabolizing the compound, removing the aromatic hydrocarbon from the cell via efflux pumps or rigidifying the outer-membrane by increasing the membrane’s protein content and/or shifting the cis/trans ratio of their ester-linked fatty acids (38–41). The up-regulation of apparent gene cluster for fatty acid metabolism, lipid biosynthesis and membrane protein synthesis in response to exposure to benzene and phenol led us to consider whether N. europaea might be changing the composition of its outer-membrane in response to benzene or its daughter product, phenol. Cyclic hydrocarbons, such as benzene, have been shown to accumulate in the outer membrane of prokaryotes (37, 42) potentially causing the inhibition of respiratory enzymes (17, 43). Accumulation of hydrocarbons in the outer membrane may also disrupt the structural integrity of the outer membrane, allowing ions to more freely flow in and out of the cell causing a drop in the proton motive force resulting in energy drains (17, 44). TEM images were generated to determine if the outermembrane of N. europaea changed in response to benzene exposure (Figure 4). Upon exposure to 40 µM benzene, the average thickness of the outer membrane, as defined by the outermost edge of the outer cell structure to the cytoplasm, decreased from 103 ( 11 nm to 71 ( 4 nm (n ) 30). An

increase was also observed in the number of cells, from 33 to 69%, that showed a completely encompassing outer cell structure (Figure 4b) as compared to a less dense, partially indistinct outer cell structure found more often in the control culture (Figure 4a). The significant decrease in thickness (p ) 0.00001), but increase in order of the cell’s outer structure, is potentially a defense mechanism against benzene or phenol by increasing the rigidity of the outer-membrane, constricting pore size, and limiting the permeability of the outer cell structure, making diffusion of benzene more difficult, and thus keeping control of ion flow across the membrane. The benzene concentrations used for TEM imaging were 3–4 orders of magnitude less than the concentration(s) used to see membrane composition changes in benzene/phenolexposed E. coli cells (35, 45). More research using long-term exposure to benzene or phenol is needed to determine the significance of the TEM results. Future research will also include TEM images of N. europaea cells exposed to a variety of aromatic hydrocarbons to determine if changes in membrane structure correspond to the up-regulated genes observed here.

Acknowledgments We thank the Luis Sayavedra-Soto for the N. europaea culture and Barbara Gvakharia for microarray analysis assistance. Dan Arp and Peter Bottomly are thanked for providing constructive input on the work. We thank Mohammad Azizian for help with the analytical techniques used, Michael Nesson for the electron microscope scans, and the Oregon State University Center for Genome Research and Biocomputing for microarray and qRT-PCR assistance. Funding was provided by a grant from the National Science Foundation’s Division of Bioengineering and Environmental Systems Genome-Enabled Environmental Sciences and Engineering Program.

Supporting Information Available More details about the materials and methods procedures, MIAME compliance information, qRT-PCR primer sequences and detailed microarray results. This material is available free of charge via the Internet at http://pubs.acs.org.

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