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Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada
Correspondence
Jian-Shen Zhao
jian-shen.zhao{at}nrc.ca
Jalal Hawari
jalal.Hawari{at}nrc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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(551 nm) or 
(418–420 nm) bands during anaerobic incubation with RDX. In both cases we found that RDX biotransformation was accompanied by oxidation of reduced cytochrome. Furthermore, O2, an oxidant of reduced cytochrome, inhibited RDX transformation. The present results demonstrate that S. halifaxensis HAW-EB4 metabolizes RDX optimally under TMAO-reducing conditions, and that c-type cytochromes are involved.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Lotufo et al., 2001; Robidoux et al., 2002). Both aerobic and anaerobic bacteria have been found to be capable of biotransforming RDX (Zhao et al., 2004a; Lewis et al., 2004; Crocker et al., 2006). Previously, strains of Shewanella were isolated from an unexploded ordnance (UXO)-contaminated marine sediment site near Halifax Harbor for their potential to mineralize RDX under oxygen-limiting conditions via the intermediary formation of nitroso derivatives and ring cleavage products (Fig. 1; Zhao et al., 2004b). These strains were also found to represent novel species of Shewanella, including Shewanella halifaxensis and Shewanella sediminis (Zhao et al., 2005, 2006). Recently, the genomes of S. halifaxensis HAW-EB4 and S. sediminis HAW-EB3 were sequenced to help determine how RDX is degraded in the environment.
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). Nitrate (Kamykowski & Zentara, 1985; Kaspar, 1982; Jørgensen, 1989; Prospero & Savoie, 1989; Sayama, 2001) and TMAO (Yancey et al., 1982; Kelly & Yancey, 1999) are abundant in marine sediments; therefore, they are expected to influence in situ growth of Shewanella, including S. halifaxensis HAW-EB4 in the marine sediment from which this bacterium was isolated. Despite recent progress made toward understanding RDX degradation, little information is available on the effect of TEA on the expression of RDX metabolic activity in anaerobic bacteria (Kitts et al., 2000) such as Shewanella and on the type of enzymes involved in RDX degradation. The goal of the present study was to determine the effect of TEA on RDX metabolic activity in S. halifaxensis HAW-EB4 and the potential involvement of c-type cytochrome in RDX reduction. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Growth of S. halifaxensis HAW-EB4.
S. halifaxensis HAW-EB4 (=NCIMB 14093=DSM 17350) was routinely grown at 10 °C under aerobic conditions, either on solid Brewer anaerobic agar containing 4 % (w/v) sea salts (Sigma) or in marine broth 20 medium (MB 20) composed of Bacto peptone (1.6 %, w/v), yeast extract (0.4 %, w/v) and sea salts (4 %, w/v). MB 20 was also used as a basal medium for growth of strain HAW-EB4 under aerobic, O2-limiting, or anaerobic conditions in the presence of nitrate or TMAO, or in the absence of any TEA. Growth in liquid medium was conducted on a rotary shaker at 150 r.p.m. and 10 °C, unless otherwise noted. Aerobic growth was conducted in shaken, foam-plugged 1 l Erlenmeyer flasks containing 150 ml medium. Growth under O2-limiting conditions was conducted in static, foam-plugged 37.5 ml serum bottles containing 20 ml medium. Anaerobic growth was conducted in sealed serum bottles (37.5 ml) containing 20 ml medium in the presence of nitrate or TMAO, or in the absence of any TEA. TMAO or sodium nitrate was added to the medium from a sterile stock solution (1.1 M, prepared in MB 20 medium before use) to the desired concentrations. Prior to inoculation, the liquid media were made anaerobic by degassing, followed by charging with argon three times. The inocula (10 %, v/v) were prepared by growing strain HAW-EB4 in MB 20 medium aerobically for 24 h to reach an OD600 close to 4.
RDX metabolism by growing or resting cells of strain HAW-EB4.
RDX degradation by growing cells (initial OD600 0.1) of strain HAW-EB4 was conducted by incubating the bacterium in MB 20 medium with RDX (20 mg l–1) under aerobic, O2-limiting or anaerobic (in the presence of 5 mM sodium nitrate or TMAO, or in the absence of TEA) conditions.
RDX metabolism by resting cells was carried out using the following protocols. Briefly, cells of strain HAW-EB4 were harvested by centrifugation at 11 000 g, 4 °C for 20 min after 12.5 h incubation (initial OD600 0.38) either in 150 ml aerobic medium (in a 1 l flask) or in 650 ml (2x500 ml serum bottles, each containing 325 ml medium) O2-limiting or anaerobic medium, using the above conditions. The final OD600 and the wet weight of biomass after harvest were [condition/OD600/wet weight of cells (g)]: aerobic/1.88/1.5; O2-limiting/0.44/0.94; nitrate (50 mM)-reducing/0.54/1.7; TMAO (100 mM)/0.85/1.8; absence of any TEA/0.42/0.94. Cells were resuspended in varying volumes of anaerobic sea salt (4 %) solution to give an OD600 of 8.1 (wet weight 34±6 g l–1). RDX biotransformation mixtures were prepared by mixing 8.5 ml of the above-mentioned cell suspensions with 11.5 ml RDX (33.8 mg l–1, sterilized by filtering through a 0.22 µm pore-size membrane filter) sea salt stock solution in 20 ml serum bottles (final cell density 15±2 g wet weight l–1). Afterwards, the serum bottles were sealed and made anaerobic by briefly degassing (1 min), followed by recharging with argon three times. RDX biotransformation was conducted at 10 °C in the dark under static incubation conditions. Aerobic reactions were conducted in foam-plugged serum bottles on a rotary shaker at 150 r.p.m. and 10 °C. An abiotic control containing a sterile sea salts solution of RDX without bacteria was prepared. Sea salts (4 %, w/v) solution used for resuspending the cells was made anaerobic prior to use by repeated degassing (10–20 min), followed by recharging with argon three times.
Preparation of spheroplast and periplasmic proteins.
Spheroplasts and periplasmic proteins were prepared by the subcellular fractionation method of Birdsell & Cota-Robles (1967) as modified by Easter et al. (1983). This method has been widely used for subcellular fractionation of Shewanella, including Shewanella frigidimarina NAMB400 (previously classified as Alteromonas sp. NCMB 400; Easter et al., 1983; Reid & Gordon, 1999; Reyes-Ramirez et al., 2003) and Shewanella massilia (Dos Santos et al., 1998). Cells of strain HAW-EB4 were harvested from 266 ml aerobic culture (17 h), 820 ml nitrate-reducing culture (17 h), 410 ml TMAO-reducing culture (17 h) or 1600 ml culture incubated in the absence of any TEA (24 h). All subsequent anaerobic steps were carried out in an anaerobic glove box. Briefly, freshly prepared cell pellets were first rinsed once with 40 ml of an anaerobic buffer (10 mM Tris-HCl, 100 mM NaCl, pH 7.5) and then resuspended in an anaerobic sucrose-Tris buffer (10 mM Tris-HCl, pH 8.0, 0.5 M sucrose) with volumes ranging between 19 and 36 ml, giving cell densities ranging between 44 and 55 mg wet cells ml–1. After equilibration at 21 °C for 7 min, the cell walls were digested with lysozyme (2 mg dissolved in 0.5 ml sucrose-Tris buffer) for 20 min. Subsequently, 0.2 ml EDTA (100 mM, pH 8.0) and an equal (to that of cell suspension) volume of Tris-HCl buffer (10 mM, pH 8.0) were added and incubated for another 15 min at room temperature to allow the release of periplasmic proteins by moderate osmotic shock and the complete formation of spheroplasts. By centrifugation (in tightened centrifugal bottles) at 27 000 g and 10 °C for 30 min, the periplasmic fraction of proteins was harvested as the supernatant (slightly pinkish) and spheroplasts as pellets. Periplasmic protein solution was filtered through a 0.45 µm pore-size membrane filter (Millex HV, PVDF) (Millipore) for further use. The spheroplasts were disintegrated by sonication in an anaerobic glove box (at a 70 % output, 
22 W, setting 14, three 10 s intervals), followed by centrifugation (in tightened centrifugal bottles) at 17 000 g at 10 °C for 15 min to harvest cytoplasmic and membrane-bound proteins as the supernatant. The protein content was determined using the bicinchoninic acid method following the instructions of the BCA Protein Assay kit (Pierce). The protein content (mg ml–1) in the periplasmic fraction prepared from cells grown on O2 (air), nitrate or TMAO, or in the absence of any TEA, was 1.7, 1.4, 1.2 or 1.3, respectively. The isolated periplasmic proteins were kept at 4 °C in sealed serum bottles under N2 (3 % H2, v/v).
In vitro anaerobic RDX metabolism by periplasmic proteins in the presence of NADH.
In an anaerobic glove box, 0.5 ml RDX stock solution (40 mg l–1 in 10 mM Tris-HCl buffer, pH 8.0) was mixed with 0.5 ml of the above filtered periplasmic proteins in 10 ml vials. RDX biotransformation was initiated by the addition of 10 µl NADH stock solution (75 mM NADH, 10 mM Tris, pH 8). The vial was then sealed and incubated statically (outside the glove box) at 10 °C. Two controls were prepared, one containing RDX and NADH and the second containing RDX and the periplasmic proteins. Another reaction containing RDX, NADH and periplasmic protein was incubated in foam-plugged vials to test aerobic RDX biotransformation. All tests were run in triplicate. The reaction was stopped at 3, 9 or 25 h by aeration. The whole vial was sacrificed for analysis of substrate RDX and products, as described below.
UV–visible spectroscopy of cytochromes.
The cell suspension (OD600 0.9) and periplasmic protein solution as prepared above were placed in a quartz cuvette for UV–visible measurements. To oxidize cytochromes, cell suspension or periplasmic protein samples were aerated by gently pipetting samples three times. To reduce cytochromes, a few grains of sodium dithionite were added to the cell suspension and periplasmic protein solution and mixed for 2 min. The UV–visible spectra (300–700 nm) of the above samples were measured on a Beckman DU 640 UV–visible spectrophotometer (Beckman Instruments) (speed, 1200 nm min–1; interval, 25 s). Tris-HCl (pH 8, 10 mM) buffer was used as a blank for the periplasmic sample, whereas sea water was used as a blank for the whole-cell suspension. Reduced minus oxidized spectra were obtained by measuring spectra of the reduced sample using the oxidized sample as a blank.
Determination of cytochromes by SDS-PAGE analysis.
SDS-PAGE analyses of periplasmic proteins were conducted on an XCell SureLock Mini-Cell using a gradient gel (NuPAGE Novex Bis-Tris gels, 4–12 %, 0.1x8x8 cm) and a homogeneous buffer (NuPAGE MES SDS running buffer: MES, 2.5 mM; Tris base, 2.5 mM; SDS, 5 %, w/v; pH 7.3) according to the instructions from Invitrogen. To detect haem-containing protein, periplasmic proteins were concentrated 17-fold (12-fold for those in aerobic cells) using a Microcon centrifugal filter device (Ultracel YM-10, Millipore). Fifteen microlitres of concentrated protein samples containing 140 µg proteins (160 µg protein for cells grown on nitrate) were loaded. The SeeBlue Plus2 pre-stained standard (Invitrogen) was also loaded (10 µl) as a molecular marker that contained myosin (188 kDa), phosphorylase (98 kDa), BSA (62 kDa), glutamic dehydrogenase (49 kDa), alcohol dehydrogenase (38 kDa), carbonic anhydrase (28 kDa), myoglobin red (17 kDa), lysozyme (14 kDa), aprotinin (6 kDa) and insulin B chain (3 kDa). The gel ran at 200 V for 45 min. To detect haem proteins, the gel was stained by incubation in a solution of 3,3,5,5-tetramethylbenzidine (TMBZ) for 2 h, followed by the addition of 3 ml H2O2 (30 %) (Thomas et al. 1976; Morris et al. 1990). Blue-coloured bands that indicated haem-containing proteins appeared after 10–60 min incubation in the dark at room temperature.
RDX oxidation of dithionite-reduced cytochrome.
To prepare reduced cytochromes, 0.5 ml of periplasmic protein solution was incubated in a 1.5 ml Eppendorf tube briefly (2 min) with a few grains of sodium dithionite in an anaerobic glove box. The salts in the mixture were removed by ultrafiltration using a Microcon centrifugal filter device with an Ultracel YM-10 (molecular weight cut-off 10 kDa) membrane filter (Millipore) and a bench-top Sanyo Micro Centaur Microcentrifuge (MSB010 CX1.1; 11 600 g, 45 min) placed inside an anaerobic glove box. The protein retentate was rinsed with 0.3 ml Tris-HCl buffer (10 mM, pH 8.0) and further concentrated by centrifugation. The obtained dithionite-free protein retentate was then reconstituted with 0.5 ml anaerobic Tris buffer (10 mM, pH 8.0) and mixed with an equal volume of RDX solution (40 mg l–1 in 10 mM Tris-HCl buffer, pH 8) in a quartz cuvette. A control was prepared by mixing 0.5 ml of the above dithionite-free reduced cytochrome sample with an equal volume of Tris buffer (pH 8, 10 mM) in a quartz cuvette. Mineral oil (0.3 ml) was added to cover the media in cuvettes before incubation at 10 °C outside the glove box. The cuvettes containing the reaction medium and control were scanned periodically to measure their visible absorption spectra (400–700 nm) using Tris-HCl buffer (10 mM Tris-HCl, pH 8) as a blank. After the completion of the experiment, RDX remaining in the reaction medium and products was measured as described below.
Monitoring of cytochrome by spectrophotometry during in vitro incubation with RDX and NADH.
In an anaerobic glove box, 0.5 ml of periplasmic protein solution was mixed in a quartz cuvette with an equal volume of RDX stock solution (40 mg l–1, in Tris-HCl buffer) and 10 µl of 75 mM NADH stock solution (in Tris-HCl buffer). A control was prepared by mixing 0.5 ml of periplasmic protein solution with an equal volume of Tris-HCl buffer (pH 8, 10 mM) and 10 µl of 75 mM NADH stock solution (in 10 mM Tris-HCl buffer). Mineral oil was added to cover the reaction and control medium to maintain anaerobic conditions. All reactions and controls were then incubated statically at 10 °C in an incubator outside the anaerobic glove box. During incubation, the cuvettes containing the reaction medium and control were scanned periodically to measure their UV–visible absorption spectra (300–700 nm) using Tris buffer as a blank. NADH concentration was monitored by its absorbance at 340 nm. After the completion of the experiment, RDX remaining in the reaction medium and products was measured as described below.
Chemical analysis.
The concentrations of RDX, MNX, DNX and TNX were determined by HPLC, as described previously (Hawari et al., 2000). NDAB and MEDINA were determined on an AnionSep Ice-Ion-310 Fast organic acids HPLC column (6.5x150 mm; Cobert Associates Chromatography Products) at 225 nm and 35 °C (Zhao et al., 2004b). The methods for analysis of 
, N2O and HCHO were as described in a previous report (Zhao et al., 2002). All tests were performed in triplicate.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), with very poor growth observed under O2-limiting (data not shown) or fully anaerobic conditions in the absence of any external TEA (Fig. 2b). The addition of TMAO (Fig. 2c) significantly enhanced anaerobic growth of strain HAW-EB4. In the presence of 10 mM TMAO and with an initial OD600 of 0.1, biomass (OD600) increased fivefold to 0.5 in 30 h. In the presence of 100 mM TMAO and with an initial OD600 of 0.5, OD600 increased threefold to 1.5 in 30 h. Growth in the presence of TMAO was accompanied by an increase in pH to 9 due to the reduction of TMAO to alkaline trimethylamine.
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), but growth on nitrate was lower than that on TMAO. For example, in the presence of 20 mM nitrate and with an initial OD600 of 0.1, biomass (OD600) tripled to 0.3 in 30 h (Fig. 2d). In the presence of 50 mM nitrate and with an initial OD600 of 0.5, OD600 only increased by 50 % to 0.77 in 30 h (Fig. 2d). During growth on nitrate, N2O accumulated, indicating incomplete denitrification. Therefore, nitrate reduction is not an energy-efficient respiratory process for the growth of strain HAW-EB4. RDX did not increase the anaerobic growth of strain HAW-EB4 in MB 20 medium in comparison to the control that contained no RDX, suggesting that RDX did not serve as a TEA for the growth of this bacterium.
RDX removal kinetics in S. halifaxensis regulated by TEA
Strain HAW-EB4 degraded RDX when growing at 10 °C in the presence of TMAO or under O2-limiting conditions, or being incubated in the absence of any TEA, but failed to degrade RDX under aerobic or nitrate-reducing conditions. These results showed that the presence of TMAO, O2-limitation and the absence of TEA favoured bacterial degradation of RDX in S. halifaxensis strain HAW-EB4.
Fig. 3 shows the biotransformation of RDX by 4.1±0.3 g wet weight l–1 (OD600 3.4) of resting cells of strain HAW-EB4 obtained after 12.5 h of incubation (exponential phase) at 10 °C under TMAO-reducing conditions (Fig. 3a), in the absence of TEA (Fig. 3b), or under O2-limiting (not shown, but similar to Fig. 3b), nitrate-reducing (Fig. 3c) or aerobic conditions (Fig. 3d). Cells pre-incubated under anaerobic conditions in the presence (Fig. 3a) or absence (Fig. 3b) of TMAO, or under O2-limiting conditions (not shown, but similar to Fig. 3b), removed RDX at similar rates (9.3–11 µM h–1), much faster than cells pre-grown on nitrate (3.2 µM h–1) (Fig. 3c) or O2 (air) (0.36 µM h–1) (Fig. 3d). As reported in a previous study (Zhao et al., 2004b), aerobically grown cells of strain HAW-EB4 degrade RDX in anaerobic sea water suspension. The present results demonstrated that aerobically grown cells were about 10–30 times slower than anaerobically grown cells in removing RDX. These data further prove that TMAO-reducing and O2-limiting conditions, or the absence of TEA, enhance the removal of RDX by strain HAW-EB4. Of the three anaerobic conditions, TMAO-reducing conditions were optimal for both the growth of strain HAW-EB4 and RDX removal; therefore, the presence of TMAO would be expected to improve RDX removal in sediments in which strain HAW-EB4 is present.
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), cells harvested at mid (8 h) or late (14 h) exponential phase, or early (24 h) or late (48 h) stationary phase, displayed similar rates of RDX removal, demonstrating that RDX removal activity is constitutive and co-metabolic. On the other hand, when resting cells of strain HAW-EB4 were incubated aerobically with RDX, none of the cells harvested under the previously described five conditions removed RDX, indicating that the enzyme(s) responsible for removal of RDX in this bacterium is O2 sensitive. Because of O2 and nitrate inhibition (as found in the assays that employed growing cells) of RDX metabolism, aerobically and nitrate-grown cells must first penetrate to the anoxic and nitrate-poor layer of marine sediment, where UXO are most likely buried, for RDX removal to occur.
RDX metabolic pathways in S. halifaxensis regulated by TEA
Previously, aerobically grown cells of S. halifaxensis HAW-EB4 were shown to degrade RDX to both nitroso (MNX, DNX and TNX; Fig. 1, path b) and ring cleavage (nitrite, MEDINA, HCHO and N2O; Fig. 1, path a) products (Zhao et al., 2004b). The present results showed that cells pre-grown anaerobically on TMAO or nitrate, or pre-incubated in the absence of any TEA, also degraded RDX via both the denitration route, giving the ring cleavage products (MEDINA, HCHO and N2O) (Fig. 1, path a), and the nitroso route, giving the nitroso products (MNX, DNX and TNX) (Fig. 1, path b). However, the nitroso route (Fig. 1, path b) was predominant in cells grown on TMAO (Fig. 3a) or pre-incubated in the absence of TEA (or under O2-limiting conditions) (Fig. 3b). For example, for TMAO-grown cells, MNX (5.7 µM h–1) and DNX (1.5 µM h–1) accounted for 70 % of initial RDX removal (9.3 µM h–1). In the case of cells pre-incubated under anaerobic conditions in the absence of any TEA, MNX (8.5 µM h–1) and DNX (0.73 µM h–1) accounted for 82 % of initial RDX removal (11 µM h–1 at 5 h). In comparison, MNX formed in aerobically (Fig. 3d) or nitrate-grown (Fig. 3c) cells accounted for 20–26 % of initial RDX removal. RDX transformation by TMAO-grown cells was accompanied by the formation of nitrite, MEDINA and HCHO, at respective rates of 0.73, 0.9 and 1.9 µM h–1, clearly demonstrating ring cleavage following denitration (Fig. 1, path a) that accounted for 30 % of initial RDX removal. For cells incubated anaerobically in the absence of TEA, 18 % of initial RDX removal was found to be ring cleavage products, including nitrite (0.4 µM h–1), MEDINA (0.85 µM h–1) and HCHO (1.1 µM h–1). However, for the less active nitrate (Fig. 3c) and aerobically (Fig. 3d) grown cells, up to 76 % of initial RDX removal was due to the formation of MEDINA and HCHO, demonstrating the dominance of RDX denitration in cells grown under these two conditions. In all cases, NDAB, a product found in biodegradation of RDX by aerobic bacteria (Fournier et al., 2002; Bhushan et al., 2003) or hydrolysis of RDX or MNX (Balakrishnan et al., 2003), was only detected as a minor anaerobic RDX product with a formation rate 5–10 times lower than that of MEDINA (Fig. 3).
These results showed that RDX in marine sediment under all five possible field growth conditions should degrade to both nitroso and ring cleavage products. Better removal and more intermediary formation of nitroso derivatives should be expected if TMAO is present in anoxic environments. It should be noted that in the assays that used growing cells, none of the nitroso derivatives, including TNX, was found to persist indefinitely; rather, they disappeared to produce HCHO, CO2 and N2O.
In vitro anaerobic RDX metabolism by periplasmic proteins
To determine the cellular location of proteins that metabolized RDX, subcellular fractions including spheroplast and periplasmic fractions of cells of strain HAW-EB4 were prepared using a moderate osmotic shock-based method (Birdsell & Cota-Robles, 1967; Easter et al., 1983) to test their potential to metabolize RDX. The proteins in the periplasmic fraction of TMAO-grown cells showed NADH-dependent anaerobic RDX degradation activity (within 5 h), whereas no RDX removal was found in cytoplasmic and membrane-bound proteins or in the controls that contained NADH or periplasmic proteins alone. Similarly, RDX metabolic activity was found in periplasmic proteins of cells grown in the presence of nitrate or O2 (air), or pre-incubated in the absence of any TEA. These data indicate that the proteins responsible for RDX metabolism are located in the periplasmic space of strain HAW-EB4.
Likewise, periplasmic proteins from cells grown on TMAO (14 µM h–1; Fig. 4a) or pre-incubated in the absence of any TEA (21 µM h–1; Fig. 4b) transformed RDX at rates faster than those of periplasmic proteins from nitrate- (8.3 µM h–1; Fig. 4c) or aerobically (2.3 µM h–1; Fig. 4d) grown cells (in all cases, protein content was 0.6–0.85 mg ml–1). As for the distribution of products, periplasmic proteins of cells grown on TMAO (Fig. 4a) or pre-incubated in the absence of any TEA (Fig. 4b) gave nitrite, MEDINA and HCHO as ring cleavage products (Fig. 1, path a), and MNX, DNX and TNX as nitroso products (Fig. 1, path b). As found in whole-cell assays, periplasmic proteins of nitrate (Fig. 4c) or aerobically (Fig. 4d) grown cells metabolized RDX mainly via cleavage to MEDINA and HCHO (Fig. 1, path a). No NDAB was detected in any of the above anaerobic biotransformations of RDX with periplasmic proteins.
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Regulation by TEA of c-type cytochrome production in S. halifaxensis
Several strains of Shewanella have been reported to produce c-type cytochromes capable of reducing insoluble metal oxide (Tsapin et al., 1996, 2001; Gordon et al., 2000). In the present study, the cytochrome content of S. halifaxensis HAW-EB4 grown under both anaerobic and aerobic conditions was characterized and determined for its potential involvement in RDX metabolism. Table 1 shows that freshly prepared or dithionite-reduced periplasmic proteins from cells grown on TMAO or pre-incubated in the absence of TEA had maximal absorption bands at 418–419, 522–523 and 551 nm. Similar bands (417–418, 521–522 and 551 nm) were also found in dithionite-reduced periplasmic proteins from cells grown on nitrate and O2 (air) (Table 1). The absorption maxima at 417–419, 522 and 551 nm corresponded, respectively, to the (Soret), β and bands of c-type cytochrome in the reduced state (Poole, 1994), and are similar to those of c-type cytochromes found in other strains of Shewanella (Tsapin et al., 2001; Pitts et al., 2003; Yamada et al., 2000). Freshly prepared or air-oxidized periplasmic proteins from nitrate- or O2 (air)-grown cells showed a single maximal band at 410 or 413 nm, corresponding to the band of oxidized c-type cytochrome. A similar band of c-type cytochrome was found at 408–409 nm in oxidized (after brief exposure to air) periplasmic proteins from cells grown on TMAO or pre-incubated in the absence of TEA. Furthermore, the reduced minus oxidized difference spectra of all periplasmic proteins and whole-cell suspensions (data not shown) also clearly showed the (419–420 nm), β (522 nm) and (551 nm) bands of c-type cytochrome (Table 1), demonstrating the production of c-type cytochromes by strain HAW-EB4 grown under all four tested conditions.
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). For example, the wavelength of the band (409 nm) of air-oxidized cytochrome of TMAO-grown cells was lower than that of aerobically (413 nm) or nitrate (410 nm)-grown cells (Table 1). This suggests that strain HAW-EB4 produces different sets of c-type cytochromes in the presence of various TEA.
Using SDS-PAGE and subsequent staining with TMBZ, proteins containing covalently bound haem groups were detected in periplasmic proteins grown under all four conditions (Fig. 5). Because the haem group of the detected proteins could not be dissociated from protein by SDS, the haem group(s) must be covalently bound to the proteins, consistent with the structure of c-type cytochromes (Bartsch, 1968). Fig. 5 shows that periplasmic proteins of TMAO-grown cells had about six haem protein bands, ranging from 14 to 80 kDa, and that the band larger than 62 kDa was the predominant one. In contrast, periplasmic proteins of nitrate-grown cells had two smaller haem proteins ranging from 14 to 28 kDa, whereas those of cells pre-incubated in the absence of TEA had a band close to (but smaller than) 62 kDa. In the case of aerobically grown cells, the cytochrome content was so low that one haem protein between 14 and 28 kDa was only detected in high protein-loading (450 µg; three times the 140 µg for TMAO-grown cells) SDS-PAGE analysis, in which protein separation was rather poor (data not shown). These results demonstrated that S. halifaxensis HAW-EB4 grown on various TEA had different sets of c-type cytochromes, and produced several more c-type cytochromes in the presence of TMAO.
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). The crude cytochrome preparation also catalysed an NADH-dependent RDX biotransformation to products (Fig. 6b) similar to those found in the case of total periplasmic proteins (Fig. 4c). In both cases, the nitroso derivatives resulting from stepwise reduction of -N-NO2 to -N-NO, and the ring cleavage products (MEDINA, HCHO, N2O) resulting from initial denitration were detected. This experimental observation further suggests that c-type cytochromes are linked to RDX metabolism.
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(418–420 nm) and (551 nm) bands of reduced cytochrome (as shown in Fig. 7a1 for cytochrome in TMAO-grown cell) compared to that of the controls containing no RDX (Fig. 7a2). Table 2 shows that dithionite-reduced cytochromes prepared from cells grown on TMAO or pre-incubated in the absence of TEA were oxidized at rates (as indicated by the rate of decrease in absorbance of the or band) that were much faster (four to eight times faster by the band; two to five times faster by the band) in the presence of RDX than in the absence of RDX. Dithionite-reduced cytochromes from nitrate or aerobically grown cells were only oxidized (as indicated by the decrease in absorbance at the or band) in the presence of RDX, with little oxidation in the controls containing no RDX (Table 2). Cytochromes from cells grown on TMAO or from cells pre-incubated in the absence of TEA also appeared to be more easily (10–30 times faster by the rate of decrease in absorbance of the or band) oxidized by RDX than cytochrome from nitrate- or aerobically grown cells (Table 2), consistent with the trend of rates for RDX removal found earlier in assays using periplasmic proteins (Fig. 4).
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band at 417–418 nm and an band at 551 nm (Table 1, Fig. 7b2). Cytochrome reduction was also observed during in vitro anaerobic incubation of periplasmic proteins with both NADH and RDX, especially in the early stage (Fig. 7b1); however, after 150 min incubation, the absorbance of the or band of reduced cytochrome in the presence of RDX was approximately two to ten times lower than that in the controls containing no RDX (Table 2). Fig. 7(b1) clearly illustrates a rapid oxidation of NADH-reduced cytochrome from TMAO-grown cells by RDX compared to that in the RDX-free control, in which cytochrome remained reduced under similar conditions (Fig. 7b2). These data demonstrated that RDX oxidized reduced cytochromes during anaerobic RDX biotransformation by periplasmic proteins in the presence of NADH. Consistent with these findings, NADH oxidation rates (as measured by the rate of decrease in absorbance at 340 nm; Table 2) during RDX biotransformation were seven to 38 times faster than those found in controls that contained no RDX. The faster NADH oxidation during RDX biotransformation was attributed to c-type cytochrome, since RDX alone did not oxidize NADH in the abiotic control without periplasmic protein (cytochrome) (data not shown). These findings, together with those observed in tests with dithionite-reduced cytochromes, clearly demonstrate that S. halifaxensis HAW-EB4 employs c-type cytochromes in RDX metabolism.
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), the reduced cytochrome in each type of cell was very sensitive to O2 and could be instantly oxidized by a brief exposure to air. This is in line with RDX persistence when incubated with each type of periplasmic protein and NADH under aerobic conditions.
On the other hand, auto-oxidation of dithionite-reduced cytochromes in cells grown on TMAO or pre-incubated in the absence of TEA was much faster than that of reduced cytochrome from nitrate- or aerobically grown cells, as shown in Table 2, suggesting that cytochromes in the former two types of cells have a lower redox potential.
All the above data demonstrate that strain HAW-EB4 incubated with various TEA produces different sets of c-type cytochromes for co-metabolism of RDX. The present findings are believed to provide the first evidence for involvement of c-type cytochrome in anaerobic RDX metabolism. c-Type cytochromes have been reported for their enzymic activity to reduce sulfur (Fauque et al., 1979), U (VI) (Lovley et al., 1993), Fe (III) (Aubert et al., 1998), Cr (IV) (Lovley & Phillips, 1994) and nitrite (Poock et al., 2002; Liu & Peck, 1981; Einsle et al., 2000). Other cytochromes, such as a eukaryotic cytochrome P450 from rabbit liver (Bhushan et al., 2003) and a cytochrome P450-like protein (XplA) in the Gram-positive aerobic actinomycete Rhodococcus rhodochrous strain 11Y (Seth-Smith et al., 2002), have been reported to be involved in RDX degradation. Both cytochrome P450 and XplA in Rhodococcus catalyse aerobic degradation of RDX, which is different from the enzyme of strain HAW-EB4 in the present study, which catalysed anaerobic RDX transformation alone. A preliminary search of genomes of Shewanella (in GenBank) by BLAST showed that the cytochrome P-450 (GenBank accession no. AAA31210) and XplA (GenBank accession no. AF449421) proteins were missing from the genomes of all sequenced strains of Shewanella, including the shotgun sequences of strain HAW-EB4 (J.-S. Zhao and others, unpublished data), suggesting that the present strain does not use XplA for RDX degradation.
c-Type cytochrome is known to mediate a 1e– transfer process (Bartsch, 1968). In our case, a 1e– nitro group reduction would lead to formation of an unstable anion free radical, which upon denitration would lead to ring cleavage and formation of MEDINA (Fig. 1, path a), consistent with those proposed in RDX metabolism by diaphorase (Bhushan et al., 2002; Fig. 1). In an earlier study, an oxygen-sensitive reductase present in Escherichia coli and rat hepatic microsomes was found to reduce nitrofurazone to its nitroso and amine derivatives via stepwise 1e– transfers (Peterson et al., 1979). A similar mechanism might be used by the cytochromes in strain HAW-EB4 for the stepwise reduction of RDX to MNX, DNX and TNX (Fig. 1, path b).
In summary, the present experimental evidence shows that S. halifaxensis has a TEA-regulated co-metabolic RDX degradation activity that involves c-type cytochrome. Shewanella are known to produce diverse soluble low-redox-potential multihaem c-type cytochromes (Tsapin et al., 1996, 2001; Gordon et al., 2000), and genomes of multiple strains of Shewanella have been sequenced (http://img.jgi.doe.gov/). Shewanella is thus an ideal model organism to investigate the molecular mechanisms that underlie the TEA-regulated multiple electron-transfer pathways of anaerobic RDX biotransformation via either initial denitration or initial reduction of -N-NO2 to -N-NO. In addition to other approaches, such as the recently described microbial electron shuttling (Bhushan et al., 2006; Kwon & Finneran, 2006), the involvement of c-type cytochrome in anaerobic RDX reduction provides a basis for the development of a new strategy to improve RDX bioremediation in the anoxic environment.
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ACKNOWLEDGEMENTS |
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Edited by: D. J. Arp
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REFERENCES |
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Received 19 September 2007;
revised 20 December 2007;
accepted 3 January 2008.
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