INTRODUCTION

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Pentachlorophenol (PCP) is a polychlorinated aromatic compound that is primarily used as a wood preservative in the lumber industry in North America. Other roles for PCP include use as an herbicide, an insecticide, and a fungicide (22). The high toxicity of PCP makes cleanup of contaminated soil necessary, and bioremediation is an attractive alternative to conventional physical and chemical methods of soil reclamation (1, 22).

PCP is degraded by a number of microorganisms, including the well-studied strain Flavobacterium chlorophenolica ATCC 39723 (27). This strain has been reclassified as a member of the genus Sphingomonas (10). In Sphingomonas chlorophenolica ATCC 39723, PCP first undergoes oxygenolytic dechlorination to tetrachloro-p-hydroquinone by a type 4 monooxygenase (35), and then tetrachloro-p-hydroquinone is further reductively dechlorinated to 2,6-dichloro-p-hydroquinone by a dehalogenase belonging to the theta class of the glutathione S-transferase superfamily (23). A third enzyme, first characterized as a chlorohydrolase (17) but later shown to be an Fe²⁺-requiring dioxygenase (34), cleaves the ring of 2,6-dichloro-p-hydroquinone at an undetermined position. The resulting product is further metabolized to nontoxic end products. The evolution of this three-enzyme pathway consisting of a PCP-inducible type 4 monooxygenase (PcpB), a constitutively expressed dehalogenase (PcpC), and a PCP-inducible dioxygenase (PcpA) has recently been reviewed (6).

We previously isolated and characterized two bacterial strains from PCP-contaminated soil samples (18). These isolates, Pseudomonas sp. strains UG25 and UG30, were subsequently also reclassified as members of the genus Sphingomonas based on lipid analysis and fatty acid profiles (19). By using DNA hybridization techniques, the UG25 and UG30 isolates were shown to possess genes homologous to pcpB and pcpC in strain ATCC 39723, suggesting that similar pathways for PCP degradation are present in these three Sphingomonas strains (18). This suggestion is supported by our recent success in cloning, sequencing, and expressing all three gene products, including that of the pcpA gene (unpublished data). The UG25 and UG30 genes exhibit about 90% sequence identity with the ATCC 39723 genes.

In an initial study we determined long-term PCP mineralization thresholds for strains UG25 and UG30. Using phosphorus-31 nuclear magnetic resonance (³¹P NMR) spectroscopy, we also observed that addition of PCP to concentrated cell suspensions resulted in collapse of the transmembrane pH gradient and lower nucleoside triphosphate levels (18). However, ³¹P NMR data obtained with S. chlorophenolica ATCC 39723 showed that cells induced with 50 µg of PCP per ml during the early exponential phase were resistant to the uncoupling action of PCP (29). In the present study we extended our previous work by investigating how PCP pretreatment affected the physiology of strains UG25 and UG30. Batch culture mineralization studies using either cell suspensions or agarose-immobilized cells were performed with all three Sphingomonas strains (UG25, UG30, and ATCC 39723) to examine how pretreatment with 50 µg of PCP per ml affects mineralization thresholds. Agarose-immobilized cells were subsequently used in long-term ³¹P NMR perfusion studies in which the effects of PCP on the cellular transmembrane pH gradient and ATP levels were monitored over time. We also compared the lipid fractions of cells grown in the absence and in the presence of PCP to determine the effect of PCP on the cellular membrane composition. Finally, the effects of PCP on oxygen consumption levels in resting and glutamate-metabolizing cell suspensions were studied in an attempt to determine whether PCP is an uncoupler or inhibitor of oxidative phosphorylation in strains UG25 and UG30.

	MATERIALS AND METHODS

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Chemicals and media. Minimal salt glutamate (MSG) medium contained (per liter) 0.5 g of NaNO₃, 1.65 g of K₂HPO₄, 0.17 g of KH₂PO₄, 0.1 g of MgSO₄ · 7H₂O, 5 mg of FeSO₄ · 7H₂O, and 4 g of monosodium glutamate. The final pH was 7.3. HEPES-buffered low-phosphate MSG medium (HMSG) contained 50 mM HEPES (pH 7.3), 0.165 g of K₂HPO₄ per liter, and 0.017 g of KH₂PO₄ per liter; the other ingredients were the same as those in MSG medium. MS medium and HMS medium were variations of MSG medium and HMSG medium, respectively, but contained no glutamate.

Microrganisms and growth conditions. PCP-degrading Sphingomonas sp. strains UG25 and UG30 and S. chlorophenolica ATCC 39723 were transferred weekly to fresh MSG medium plates (MSG medium plus 15 g of agar per liter). The plates were incubated at 30°C for 2 to 3 days before they were stored at 22°C. The strains were cultured in MSG medium at 30°C with shaking at 300 rpm (see below).

PCP degradation studies with cell suspensions. Aliquots (25 ml) of mid-exponential-phase cells (optical density at 600 nm [OD₆₀₀], 0.5; 8 × 10⁷ CFU/ml) grown in MSG medium were diluted with equal volumes of fresh MSG medium containing PCP so that the final PCP concentrations varied from 0 to 350 µg/ml. Portions (50 ml) of the resulting cultures (2 × 10⁹ cells per experiment) were dispensed into sterile 250-ml Erlenmeyer flasks and incubated at 30°C with shaking at 300 nm. Samples of cultures were monitored for cell growth by monitoring changes in the OD₆₀₀, and then aliquots were each diluted with an equal volume of 1.0 N NaOH and centrifuged at 13,000 rpm in a desk top Microfuge for 2.5 min. The absorbance at 318 nm of each resulting supernatant was monitored to determine the PCP concentration. For PCP-induced cells, the protocol described above was used, except that mid-exponential-phase cultures were exposed to 50 µg of PCP per ml for 2 to 3 h before they were transferred to fresh sterile media containing PCP at the concentrations indicated above. Experiments were repeated three times, and each time a freshly prepared inoculum of cells was used.

PCP degradation studies with agarose-immobilized cells. All procedures were performed aseptically. Mid-exponential-phase cells were cooled to 4°C on ice, harvested by centrifugation, and resuspended in 20 ml of sterile HMS medium. The resulting cell suspension was subsequently mixed at 37°C with an equal volume of 4% low-temperature-gelling agarose (type VII; Sigma). Beads were generated in sterile vegetable oil as previously described (20). After cooling, the agarose beads were sized with wire nets. Beads between 0.5 and 1.0 mm in diameter were then washed so they were free of oil. For each experiment 3-g (wet weight) portions of beads containing approximately 2 × 10⁹ cells were transferred to 250-ml Erlenmeyer flasks containing 50 ml of MSG medium supplemented with PCP at concentrations ranging from 0 to 450 µg/ml. The flasks were incubated and analyzed for cell growth and PCP degradation as described above. Less than 10% free cells were observed in the medium after 24 h. For studies involving PCP-pretreated immobilized cells, the beads were first incubated for 2 h in MSG medium containing 50 µg of PCP per ml. After this the cells were sieved free of medium, cultured, and incubated as described above. These experiments were repeated three times, and each time a new preparation of immobilized cells was used.

Preparation of immobilized cells for NMR. The protocol used to prepare immobilized cells for NMR was essentially the same as that described above, except that freshly immobilized cells were incubated for 18 h in HMSG medium to increase the cell density in the beads. After this incubation period the beads were drained, and a 10-g sample was transferred aseptically to a 15-mm NMR tube. A sealed capillary containing 0.2 M methylenediphosphonic acid (MDP) was included as a chemical shift and intensity reference standard. A modified Teflon plug allowed incoming, oxygenated medium to enter at the bottom of the NMR tube and the exiting medium to be siphoned from the top of the beads. The beads were perfused for 12 h at a rate of 5 ml/min from a reservoir containing 250 ml of HMSG medium. This medium was bubbled with oxygen. After 12 h the medium in the reservoir was replaced with fresh HMSG medium containing 120 µg of PCP per ml (perfusion using nontreated cells) or 175 µg of PCP per ml (perfusion using PCP-pretreated cells). Incubation was continued for another 30 to 50 h. During this time aliquots of the medium were periodically withdrawn so that PCP degradation could be monitored as described above. Experiments were performed in duplicate with strain UG25 and with strain UG30.

Preparation of detergent-phospholipid micelles for NMR analysis. Cells were grown to an OD₆₀₀ of 1.0 in MSG medium or MSG medium containing 50 µg of PCP per ml. In the latter case, as PCP was degraded, fresh aliquots of PCP were periodically added to keep the level in the medium between 30 and 50 µg/ml. Cells were harvested by centrifugation and washed once in HMSG medium. Membrane vesicles were prepared by the method of Kaback (14). The samples used for NMR (total volume, 6 ml) contained 35 mg of vesicles per ml, 0.32% sodium deoxycholate, 3 mM EDTA, 0.35 mM MDP, and 8.3% D₂O in 50 mM Tris buffer (pH 8.4). Under these conditions deoxycholate generated micelles which had a much narrower NMR line width than vesicles. D₂O was used for lock purposes, and MDP served as an internal standard.

Extraction of cell lipids for acyl chain analysis. Cells grown under the conditions described above were harvested and immediately extracted with chloroform-methanol-H₂O as described by Bligh and Dyer (2). The lipid fraction was dried under nitrogen gas in ampoules, sealed, and sent to Microbial ID, Inc. (Newark, Del.) for commercial analysis of the fatty acid composition.

NMR conditions. ³¹P NMR spectra were obtained at 25°C with a Bruker AM-400 wide-bore spectrometer by using 15-mm NMR tubes. The parameters used to acquire spectra of the immobilized cells were the parameters described by Lohmeier-Vogel et al. (20). The parameters used to study the detergent-phospholipid micelles were the parameters used previously for analysis of yeast cell extracts (21), except that the recycle time was 5.1 s. All experiments were conducted a minimum of two times.

Oxygen uptake experiments. Sphingomonas sp. strain UG30 was grown to an OD₆₀₀ of 1.0 in 1 liter of MSG medium, harvested by centrifugation, washed once with sterile MS medium (pH 7.4), and resuspended in 100 ml of MS medium. The cells were incubated at 30°C with shaking at 200 rpm for 2 h to starve them of endogenous carbohydrate stores. After this the cells were pelleted and then resuspended in fresh MS medium at an OD₆₀₀ of 1.0. Oxygen consumption by resting cells was measured at 22°C with an oxygen electrode (YSI 5739 D.O. probe; Yellow Springs Instrument Co.). The substrate glutamate (final concentration, 4 g/liter) and/or the substrate sodium PCP (0 to 100 µg/ml) was added 10 min after oxygen measurement commenced.

	RESULTS

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Determination of PCP degradation thresholds. Figure 1 summarizes the PCP degradation thresholds for freely suspended or agarose-immobilized Sphingomonas sp. strains UG25 and UG30 and S. chlorophenolica ATCC 39723. In this study the PCP degradation threshold was defined as the level of PCP which cells could degrade by 50% during incubation for 24 h. Occasionally, the PCP in a culture containing 100 µg of PCP per ml was completely degraded by the cells but no degradation was observed in a culture containing 150 µg of PCP per ml. In such cases we estimated that the degradation threshold was the average of the two values.

View larger version (36K): [in this window] [in a new window]   FIG. 1.   PCP thresholds for batch cultures of Sphingomonas sp. strains UG30 and UG20 and S. chlorophenolica ATCC 39723. Solid bars, suspension cultures; cross-hatched bars, suspension cultures of cells pretreated with 50 µg of PCP per ml; gray bars, immobilized cultures; open bars, immobilized cultures pretreated with 50 µg of PCP per ml.

³¹P NMR studies with immobilized UG25 and UG30. Figure 2A to E show the collapse and recovery of the pH gradient and ATP levels as a function of time when immobilized UG30 cells were perfused with HMSG medium containing 120 µg of PCP per ml. The spectrum in Fig. 2A, obtained in the absence of PCP, has three peaks, which were derived mainly from the , , and  phosphates of ATP (and other nucleoside triphosphates). The  and  phosphates from nucleoside diphosphates like ADP have the same chemical shift as the ATP  and  peaks, and so only the ATP  peak at 18.2 ppm most accurately reflects the intracellular ATP content. Since this peak is well resolved in the spectrum in Fig. 2A, we concluded that the cells were energized when they were perfused with oxygenated HMSG medium.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   (A to E) 31P NMR spectra for agarose-immobilized Sphingomonas sp. strain UG30 perfused with oxygenated HMSG medium. Each spectrum represents a 10-h period. (A) No PCP in perfusion medium (0 to 9 h). (B) Addition of 120 µg of PCP per ml to perfusion medium at 10 h (10 to 19 h). (C to E) Continuation of PCP degradation with time (20 to 49 h). Peak assignments: SP, sugar phosphomonoesters; Pi(int) and Pi(ext), intracellular and extracellular inorganic phosphate, respectively; PDE, phosphodiesters from lipids and cell wall components; ATP-, contributions from nucleotide triphosphate  resonances and nucleotide diphosphate  resonances; ATP-, contributions from nucleotide triphosphate  resonances and nucleotide diphosphate  resonances; NAD and NADP resonances (reduced and oxidized); UDPG, uridine diphosphoglucose resonance; ATP-, contributions from only the nucleoside triphosphate  resonances. (F to J) 31P NMR spectra of agarose-immobilized Sphingomonas sp. strain UG30 pretreated for 2 h with 50 µg of PCP per ml and subsequently perfused with oxygenated HMSG medium. (F) No PCP in perfusion medium. (G) Addition of 175 µg of PCP per ml to perfusion medium. (H to J) Continuation of PCP degradation with time.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   PCP concentrations remaining in the perfusion medium of agarose-immobilized Sphingomonas sp. strain UG30. See the legend to Fig. 2 and the text for details. Different symbols represent different experiments. B to E correspond to spectra in Fig. 2.

Membrane phospholipid compostion of PCP-degrading strains. ³¹P NMR spectra of membrane phospholipids in deoxycholate vesicles were obtained for Sphingomonas sp. strain UG30 and S. chlorophenolica ATCC 39723 (Fig. 4). The phospholipid profiles in Fig. 4A and C are profiles for cells which had been grown in the absence of PCP and the phospholipid profiles in Fig. 4B and D are profiles for cells that had been pretreated with 50 µg of PCP per ml. Most peaks in the spectra were identified by using commercially available standards including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Since upfield shifts in phospholipid families have been reported to occur as a result of acyl chain saturation (24), we also included PC, PG, and PE standards with disaturated or diunsaturated acyl chains. Some peaks in Fig. 4 were not identified and may correspond to members of the sphingolipid family. Our chemical shift values (Table 1) are similar to previously reported values for phospholipids in nondetergent solutions (26) but on average are about 0.68 ppm downfield from the values obtained in another study performed with detergent-phospholipid micelles (9) (Table 1). Because we used sodium deoxycholate rather than potassium cholate to solubilize our lipids and the pH of our samples was 8.4 rather than 7, these systematic differences were expected.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   31P NMR spectra for Sphingomonas sp. strain UG30 phospholipid-deoxycholate micelles (A), PCP-pretreated Sphingomonas sp. strain UG30 phospholipid-deoxycholate micelles (B), S. chlorophenolica ATCC 39723 phospholipid-deoxycholate vesicles (C), and PCP-pretreated S. chlorophenolica ATCC 39723 phospholipid-deoxycholate vesicles (D). PG, CL, PE, and PC peaks were assigned by adding standard compounds. Note that the spectra in panels A and B are plotted on a different vertical scale than the spectra in panels C and D, as shown by the different signal-to-noise ratios.

Acyl chain compositions of PCP-treated and nontreated cells. Table 2 shows the types of fatty acids found in membranes of Sphingomonas sp. strains UG30 and UG25 and S. chlorophenolica ATCC 39723 cultured in the presence and in the absence of 50 µg of PCP per ml. The fatty acid profile of strain ATCC 39723 is similar to that of UG25 and UG30, except for the absence of fatty acids 14:0 and 17:0 ante (methyl group at the third carbon from the end). Our results show that there were no dramatic differences in the acyl chain contents of PCP-pretreated and control cells.

Oxygen consumption studies. Table 3 shows the results obtained when resting cells of Sphingomonas sp. strain UG30 were treated with different levels of PCP. With both PCP-pretreated cells and control cells, addition of 25 or 50 µg of PCP per ml enhanced the rate of oxygen consumption compared with the background (endogenous) rate. At PCP concentrations greater than 75 µg/ml, however, the oxygen consumption rates declined, and at a PCP concentration of 100 µg/ml the respiration rate was only slightly higher than the endogenous value.

	DISCUSSION

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In our batch culture studies with three PCP-degrading microorganisms, Sphingomonas strains UG25 and UG30 and S. chlorophenolica ATCC 39723, two observations were made. First, we observed that agarose immobilization raised the PCP degradation threshold of all three strains by 10 to 20%. Second, we observed that UG25 and UG30 cells treated for 2 h with 50 µg of PCP per ml could subsequently degrade 30 to 50% higher levels of PCP. Strain ATCC 39723, on the other hand, had the same PCP degradation threshold regardless of whether the cells had been exposed to PCP previously.

Microorganisms encapsulated in various matrices can be protected from toxins or predators in the environment (3); thus, it was not surprising to find that our cells could degrade 20% higher PCP levels after they had been immobilized in agarose beads. Even greater levels of enhancement were observed when Sphingomonas sp. strain UG30 cells were entrapped in -carrageenan-clay beads (4). Unfortunately, the presence of paramagnetic ions in the clay amendments adversely affected the resolution of the NMR spectra. For this reason an agarose matrix, which was used successfully in previous NMR study (20), was chosen instead for this study.

The ³¹P NMR spectra obtained with agarose-immobilized UG25 and UG30 cells showed that the transmembrane pH gradient and ATP levels decreased after 120 µg of PCP per ml was added to the perfusion medium. The same result was observed previously when concentrated UG30 cell suspensions were treated with PCP (18). In the present study, however, we were able to monitor recovery of the transmembrane pH gradient and ATP levels. This occurred when most of the PCP in the perfusion medium had been degraded so that the concentration was around 15 µg/ml.

When the experiment described above was repeated with PCP-pretreated UG25 and UG30 cells, the transmembrane pH gradient and ATP levels were not affected by addition of PCP to the perfusion medium. These cells were resistant to PCP deenergization, as has been reported previously for PCP-pretreated S. chlorophenolica ATCC 39723 (29). However, in the present study we found that in contrast to strains UG25 and UG30, PCP-pretreated ATCC 39723 cells did not have a higher PCP degradation threshold after PCP pretreatment. We were curious to understand why.

Bacteria vary in the ability to tolerate environmental toxins (16). Some strains can alter their membrane lipid compositions (5, 13, 25); others synthesize stress proteins (12) or actively excrete toxins (15). We focused on the membrane barrier as the locus for increased PCP tolerance in strains UG25 and UG30.

Acyl chain modification of the membrane phospholipids is one way in which bacteria can adapt to environmental pollutants. By increasing the concentration of saturated fatty acids or by trans isomerization of exisiting cis unsaturated acyl chains, membrane fluidity is decreased and compounds like PCP have a harder time penetrating the cells (5, 25). Unfortunately, commercial acyl analysis did not allow resolution of cis and trans isomers of 18:1 and 18:2 fatty acids. Thus, we were not able to determine whether cis-trans isomerization played an important role in increasing cellular resistance to PCP. In addition, the differences in the levels of saturated and unsaturated fatty acids between PCP-treated and untreated cells were minor and often inconsistent (Table 2). We did, however, observe major reproducible differences in membrane phospholipid composition between PCP-treated and control cells with strains UG25 and UG30. Specifically, PCP-pretreated cells had a higher CL content than nontreated cells. The phospholipid profile of S. chlorophenolica ATCC 39723, on the other hand, was the same regardless of whether cells had been exposed to PCP.

In the bacterium Escherichia coli, PG is converted to CL as cultures age and when compounds such as dinitrophenol, cyanide, penicillin, and colicin K are added to the medium (7). An increase in the CL content of cell membranes has also been reported for Pseudomonas putida after exposure to organic solvents like toluene (25). It has been suggested that higher levels of CL increase membrane rigidity and thus present a physical barrier for the penetration of toxic compounds (28). A higher CL level in PCP-pretreated UG25 and UG30 cells would explain why these cells were subsequently able to tolerate higher PCP concentrations, whereas strain ATCC 39723, whose phospholipid composition remained unaltered, could not.

While our data showed that PCP modifies oxygen consumption (Table 3), they did not allow us to conclude whether PCP functions mainly as an uncoupler or as a respiratory inhibitor in Sphingomonas sp. strain UG30. Previous reports concerning the effects of PCP on cell physiology are often conflicting. PCP concentrations as low as 0.01 mM (about 3 µg/ml) have been shown to uncouple ATP synthesis in rat liver mitochondria (32). On the other hand, PCP has been shown to associate specifically with the mitochondrial protein fraction in rat liver mitochondria (33), and studies with phenolic compounds related to PCP have demonstrated that inhibition of electron transport occurs at the cytochrome bc₁ complex (30). We observed that the oxygen consumption rate of resting UG30 cells depended on the PCP concentration. Similar concentration-dependent effects on respiration were observed previously when -pinene was added to yeast mitochondria (31). Mitochondrial respiratory stimulation was observed at low concentrations of -pinene, whereas inhibition of respiration occurred at higher concentrations, similar to the data in Table 3.

In conclusion, our results show that PCP-pretreated Sphingomonas sp. strains UG25 and UG30 have diminished ATP levels and reduced transmembrane pH gradients when they are exposed to PCP levels higher than 15 µg/ml. We showed that when strains UG25 and UG30 are pretreated with 50 µg of PCP per ml, they can adapt to higher PCP concentrations. The adaptation process involves increasing the levels of CL in the membranes. Interestingly, the related organism S. chlorophenolica ATCC 39723 did not show a change in CL content when it was preexposed to PCP and subsequently did not tolerate higher PCP levels.