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Department of Microbiology, Cornell University, Ithaca, NY 14853, USA
Correspondence
Eugene L. Madsen
elm3{at}cornell.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Xue & Warshawsky, 2005). Naphthalene is commonly used as a model for studying PAH metabolism by bacteria because it is the simplest and most soluble PAH, as well as a frequent constituent of PAH-contaminated environments (Peters et al., 1999). Studies of naphthalene degradation have shown that there are two primary pathways for the metabolism of naphthalene, which are distinguished by the conversion of salicylate to catechol or gentisate. Metabolism of naphthalene via catechol has been studied extensively in two Pseudomonas species that carry the archetypal catabolic plasmids NAH7 (in P. putida G7) and pDTG1 (in P. putida NCIB 9816-4) (Dennis & Zylstra, 2004; Yen & Serdar, 1988; Sota et al., 2006). The nah dissimilatory genes are organized into two operons: one encoding the enzymes involved in the conversion of naphthalene to salicylate (naphthalene degradation upper pathway) and another encoding the conversion of salicylate to catechol, followed by ortho- or meta-cleavage to TCA cycle intermediates (naphthalene degradation lower pathway) (Yen & Gunsalus, 1982). In contrast, the nag genes found in Ralstonia sp. strain U2 are organized in a single operon and encode the alternative gentisate pathway, which converts naphthalene to fumarate and pyruvate via salicylate and gentisate (Fuenmayor et al., 1998; Zhou et al., 2002).
Although the genetics and biochemistry of naphthalene metabolism have been studied in depth, the inhibition of naphthalene metabolism due to the toxicity of naphthalene and naphthalene metabolites has received less attention (Auger et al., 1995; Murphy & Stone, 1955; Park et al., 2004). Naphthalene has been reported to be directly toxic to P. putida G7 under oxygen- and nitrogen-limited conditions, although it is unclear if the toxicity was due to naphthalene or a naphthalene metabolite (Ahn et al., 1998). Naphthalene was also shown to be toxic to non-naphthalene-degraders, as P. putida KT2440 showed reduced viability in soil amended with naphthalene (Park et al., 2004), and a bioluminescent strain of Escherichia coli showed reduced bioluminescence in the presence of naphthalene (Lee et al., 2003).
Polaromonas naphthalenivorans CJ2 was isolated from coal-tar-contaminated freshwater sediment for its ability to use naphthalene as its sole carbon source, and was shown to be responsible for in situ naphthalene degradation by field-based stable isotope probing (Jeon et al., 2003). The naphthalene catabolic genes in strain CJ2 are homologous to the nag operon of Ralstonia sp. strain U2, but the genes are arranged in two separate clusters, each with its own regulatory protein (Jeon et al., 2006). In the present investigation, we show that strain CJ2 metabolizes naphthalene via the gentisate pathway using respirometry, GC-MS and cell-free enzyme assays. In addition, we explore the inhibitory effects of naphthalene and its metabolites on the growth of strain CJ2.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Naphthalene degraders P. putida NCIB 9816-4 (Yen & Serdar, 1988) and Ralstonia sp. strain U2 (Zhou et al., 2002) were included in this study for comparison. Strain CJN110 is a mutant of strain CJ2 in which the regulatory gene nagR has been disrupted by a suicide vector carrying a kanamycin resistance gene (Jeon et al., 2006).
Growth assays.
All growth assays were conducted with Stanier mineral salts media (MSB; Stanier et al., 1966) supplemented with pyruvate (18 mM), glucose (6 mM) and/or naphthalene crystals. The aqueous concentration of naphthalene was lowered with Amberlite XAD7 resin. Naphthalene binds to the XAD7 resin, which acts as a reservoir, and maintains the aqueous concentration of naphthalene below saturation (230 µM or 30 p.p.m.) while supplying enough naphthalene to support growth over the course of the experiment (Morasch et al., 2001). Naphthalene (6–14 mg) was added to 6 ml MSB containing 0.1 g XAD7 before the tubes were sealed with Teflon-lined stoppers and autoclaved. To grow strain CJ2 in MSB saturated in naphthalene, a large inoculum (>7 %, v/v) from a dense starter culture was required. Viability of test bacteria was measured by enumerating c.f.u. from 10 µl drops of serially diluted cultures on R2A agar. Kanamycin was added to media at 40 µg ml–1 when appropriate.
Respirometry.
Bacterial cells were grown to late exponential phase in MSB with naphthalene (0.5 % w/v) and harvested (6000 g). Cells were washed twice and resuspended in 50 mM KH2PO4 buffer (pH 7.4). Endogenous respiration was measured with an oxygen electrode (Rank Brothers) after adding 2 ml washed cell suspension to the incubation chamber, and oxygen consumption was recorded following the addition of 20 mM substrate dissolved in N,N-dimethylformamide (DMF). Respiration upon addition of DMF only was included as a control treatment.
Enzyme assays.
Bacterial cells were grown in 500 ml MSB with 18 mM pyruvate or 4 mM salicylate to late exponential phase, and cells were harvested by centrifugation (6000 g) and washed once with 50 mM KH2PO4 buffer (pH 7.4). Cells were resuspended in buffer to a concentration of 0.1 g ml–1 and sonicated three times for 30 s with 60 s cooling intervals. Cellular debris was cleared by centrifugation (25 000 g for 45 min) and protein concentrations were determined with the Bio-Rad Bradford assay. Enzyme assays were performed with a minimum of 50 µg protein. The enzyme substrates, gentisate (0.19 µmol) or catechol (0.9 µmol), were added in 1 ml volumes. Gentisate 1,2-dioxygenase activity was measured spectrophotometrically by measuring the increase of absorption at 334 nm, due to the formation of maleylpyruvate, and was calculated with an extinction coefficient of 10 800 M–1 cm–1 (Crawford et al., 1975). Catechol 1,2-dioxygenase activity was assayed by measuring the increase in absorption at 260 nm, due to the formation of cis,cis-muconate, and was calculated with an extinction coefficient of 16 800 M–1 cm–1 (Dorn & Knackmuss, 1978). Catechol 2,3-dioxygenase activity was measured by monitoring the increase in 2-hydroxy-cis,cis-muconate semialdehyde at 375 nm, and was calculated with an extinction coefficient of 33 000 M–1 cm–1 (Cerdan et al., 1994).
GC-MS detection of naphthalene pathway metabolites.
Cells (500 ml) grown on naphthalene or pyruvate were harvested, washed twice and resuspended in 10 ml 50 mM KH2PO4 buffer. Fifty microlitres of 200 mM naphthalene in DMF was added to 5 ml of the concentrated cell suspension and metabolism was allowed to proceed for 15 min. Suspensions were acidified with HCl to pH 1.5 and extracted twice with 7.5 ml ethyl acetate, which was then dried over anhydrous Na2SO4 and concentrated under an atmosphere of N2 to a volume of 300 µl. Extracts were derivatized with 25 µl BSTFA [bis(trimethylsilyl)trifluoroacetimide] for 5 min prior to GC-MS analysis and quantified using external standard calibration curves.
HPLC analysis of naphthalene and inhibitory compounds.
Naphthalene concentrations were determined by HPLC. Samples (100 µl) were taken from culture tubes and immediately fixed in an equal volume of methanol. Samples were filtered through tightly packed glass wool prior to injection, and naphthalene was separated using a PAH-Hypersil column (150x4.6 mm; Keystone Scientific) and a Waters model 590 HPLC pump with a mobile phase of methanol/water (65 : 35) at a flow rate of 1 ml min–1. Eluents were monitored by UV-visible light detection (ABI analytical absorbance detector; Spectroflow 757) at a wavelength of 270 nm and quantified using an external standard calibration curve.
Putative naphthalene metabolites were separated with a Hypersil BDS-C18 column (4.6x250 mm; Agilent) at a flow rate of 1 ml min–1 with a Spectra-Physics model SP8800 ternary HPLC pump. The mobile phase consisted of 20 % methanol and 80 % 40 mM acetic acid for 10 min, followed by a linear increase in methanol to 50 % over 10 min; after 5 min the methanol concentration was linearly increased to 90 % over 10 min and held for 15 min. Eluents were detected at 260 nm using a UV-Vis detector (SPD-10A VP; Shimadzu). Aged 1,2-naphthoquinone solution (50 µM final concn) was prepared by diluting a filter-sterilized 10 mM methanolic stock, aseptically adding it to sterile MSB and allowing the solution to shake for 48 h.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The simultaneous adaptation assays were aimed at confirming that strain CJ2 metabolizes naphthalene via gentisate as suggested by the sequence homology of the naphthalene catabolic operons in Ralstonia sp. strain U2 and strain CJ2, as well as to reveal potential physiological effects of the naphthalene catabolic gene arrangement in strain CJ2.
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). Ralstonia sp. strain U2 cells consumed oxygen when exposed to naphthalene, salicylaldehyde and salicylate. Ralstonia sp. strain U2 cells, our positive control for the gentisate pathway, did not display enhanced respiration when exposed to gentisate (Fig. 1). Strain CJ2 consumed oxygen after addition of naphthalene, salicylaldehyde and salicylate. However, similar to Ralstonia sp. strain U2, neither gentisate nor catechol led to rapid depletion of oxygen. Ralstonia sp. strain U2 is known to lack a gentisate transporter (Xu et al., 2006), and if the same is true for strain CJ2, this accounts for the absence of enhanced oxygen uptake in the presence of gentisate. Regardless, the lack of enhanced oxygen uptake in the presence of catechol by Ralstonia sp. strain U2 and strain CJ2 reveals a key distinction between these two strains and P. putida NCIB 9816-4.
Metabolite detection
We measured potential naphthalene metabolites with GC-MS to support the respirometry data and to eliminate ambiguity regarding the role of gentisate or catechol in the naphthalene metabolic pathway in strain CJ2. Again, P. putida NCIB 9816-4 and Ralstonia sp. strain U2 served as controls for the catechol and gentisate pathways, respectively. Table 1 shows the detected metabolites from washed cell suspensions that were incubated for 15 min after addition of naphthalene in DMF to a final concentration of 2 mM.
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Dioxygenase assays
To provide further evidence for naphthalene metabolism by the gentisate pathway in strain CJ2, dioxygenase assays were conducted with cell-free extract from induced (salicylate-grown) and non-induced (pyruvate-grown) strain CJ2, P. putida NCIB 9816-4 and Ralstonia sp. strain U2. Gentisate 1,2-dioxygenase activity was detected in strain Ralstonia sp. strain U2 when the cultures were induced by salicylate [0.150 µmol min–1 (mg protein)–1], but not when grown on pyruvate [0.004 µmol min–1 (mg protein)–1]. Although gentisate dioxygenase activity was not as pronounced in strain CJ2, activity was induced by salicylate [0.032 µmol min–1 (mg protein)–1] and not by pyruvate [0.003 µmol min–1 (mg protein)–1]. Neither ortho- nor meta-cleavage of catechol was observed in strain CJ2 or Ralstonia sp. strain U2. As expected, P. putida NCIB 9816-4 displayed catechol 1,2-dioxygenase activity and no gentisate dioxygenase activity.
Growth inhibition of strain CJ2 by naphthalene
Although strain CJ2 was isolated under naphthalene vapour as a carbon source, a comparison of growth on naphthalene in minimal medium (MSB) between strain CJ2, P. putida NCIB 9816-4 and Ralstonia sp. strain U2 revealed unusual growth characteristics in strain CJ2. Both P. putida NCIB 9816-4 and Ralstonia sp. strain U2 grew equally well in MSB broth supplemented with 18 mM pyruvate or saturated with naphthalene crystals (
230 µM). In contrast, strain CJ2 was unable to grow in MSB broth supplemented with naphthalene crystals (Fig. 2a), but grew well in MSB broth with 18 mM pyruvate (Fig. 2b), demonstrating that the inhibition of growth is substrate-specific. Additionally, the lack of growth in MSB supplemented with naphthalene was accompanied by the appearance of a light orange colour in the medium, indicating the possible accumulation of naphthalene metabolites. Incidentally, growth of strain CJ2 in MSB saturated with naphthalene was influenced by cell density, as growth occurred if a large inoculum (>7 %) of a dense starter culture was used (data not shown).
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). Once an equilibrium was established between XAD7-bound naphthalene and aqueous naphthalene, strain CJ2 was inoculated (initial cell density 1.56x106 c.f.u. ml–1) into media with initial aqueous concentrations of naphthalene of approximately 23, 40, 55, 78 or 100 µM, as determined by HPLC analysis of aqueous naphthalene in sterile medium. Growth was strongly inhibited when the initial naphthalene concentration in the medium was higher than 78 µM. However, strain CJ2 grew well when the initial naphthalene concentration of the medium was at or below 55 µM (Fig. 3). This indicates that growth inhibition by naphthalene is concentration-dependent, with optimum growth occurring at 40 µM naphthalene and inhibitory effects manifest above 78 µM naphthalene.
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To determine if naphthalene is directly toxic to strain CJ2, we measured the growth of CJN110, a NagR1 regulatory mutant of CJ2 that is unable to grow on naphthalene (Jeon et al., 2006), in MSB supplemented with 6 mM glucose in the presence of naphthalene at concentrations that were inhibitory or non-inhibitory to the wild-type strain. Partially inhibited growth occurred on glucose in the presence of 23 µM naphthalene. However, at all other naphthalene concentrations, growth on glucose was severely inhibited (Fig. 4). Because strain CJN110 is unable to metabolize naphthalene, inhibition of growth was not due to the production of potentially toxic naphthalene metabolites. Therefore, naphthalene has a direct inhibitory or toxic effect on strain CJN110 derived from strain CJ2.
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Growth occurred as expected in cultures containing 6 or 10 mg naphthalene (Fig. 5a), with both culture conditions reaching an OD600 near 0.68 and cultures containing 10 mg growing slightly slower than those containing 6 mg. However, in tubes containing 12 mg naphthalene, growth was severely inhibited and did not exceed an OD600 of 0.13. The aqueous naphthalene concentration dropped below 10 µM in cultures with 6 mg naphthalene (Fig. 5b), suggesting that strain CJ2 metabolized naphthalene faster than it was diffusing from the XAD7 resin. In cultures containing 10 or 12 mg naphthalene there was an initial increase in the aqueous naphthalene concentration, indicating that a new equilibrium was established upon the addition of the cells. The subsequent decrease in naphthalene concentration shows that even the inhibited cultures were metabolizing naphthalene.
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). However, after 34 h no viable cells were detected, and the loss of viability corresponded with the appearance of a light orange colour in the media, which appeared between 34 and 47 h. These results suggest that not only does naphthalene have an inhibitory effect, but also naphthalene metabolism by inhibited cells results in the accumulation of a naphthalene metabolite that is toxic to the cells. It should be noted that even though there was no visible colour accumulation prior to 34 h, it is possible that a low level of the toxic metabolite had accumulated and was responsible for growth inhibition.
Analysis of toxic accumulated metabolites
Previous reports have suggested that the accumulation of an orange metabolite in naphthalene-metabolizing cultures results from the abiotic oxidative transformation of 1,2-dihydroxynaphthalene to 1,2-naphthoquinone (Auger et al., 1995; Davies & Evans, 1964; Murphy & Stone, 1955). Spectrophotometric scans (240–400 nm) of 1,2-naphthoquinone standards and coloured media from naphthalene-inhibited cultures of strain CJ2 suggested that 1,2-naphthoquinone was present at concentrations between 50 and 100 µM (data not shown). Therefore, we used HPLC to compare 1,2-naphthoquinone standards (dissolved in MSB) with media from strain CJ2 cultures that were either successfully grown on naphthalene or cultures that were inhibited by naphthalene and had accumulated orange-coloured metabolites.
Analysis of 1,2-naphthoquinone dissolved in MSB revealed that it too was unstable in aqueous media. When 50 µM 1,2-naphthoquinone was analysed by HPLC shortly after dissolution in MSB, there was one major peak with a retention time of 20 min (peak II in Fig. 6a). A probable oxidation product with a retention time of 24 min (peak III in Fig. 6a) also appeared. However, after 50 µM 1,2-naphthoquinone was incubated for 48 h in sterile MSB, peak II was no longer detected, while new peaks (I and IV) with retention times of 15 and 26 min, respectively, were abiotically produced (Fig. 6b). Analysis of the coloured medium from a naphthalene-inhibited culture showed three peaks: one peak (II) corresponds with the primary peak of freshly dissolved 1,2-naphthoquinone, while the other two peaks, I and IV, correspond with daughter products of aged 1,2-naphthoquinone (Fig. 6c). No peaks of putative inhibitory metabolites were present in the uncoloured medium from cultures of CJ2 that successfully grew on naphthalene (Fig. 6d). Chemical instability, ineffective derivatization procedures and lack of authentic standards prevented GC-MS identification of the putative toxic compounds eluting as peaks I and IV in the inhibited culture of strain CJ2 (Fig. 6c). Support for the contribution of 1,2-naphthoquinone (peak II) to toxicity was obtained in assays showing that growth of strain CJ2 was completely inhibited in MSB-glucose medium when 1,2-naphthoquinone was present at a concentration of 50 µM (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In this study, we gained insight into the physiology of naphthalene metabolism in strain CJ2 by comparing it to two archetypal naphthalene-degrading bacteria. Our data from respirometry, metabolite detection by GC-MS and enzyme assays showed that strain CJ2 metabolizes naphthalene via the gentisate pathway. Growth assays revealed that strain CJ2 cannot grow on naphthalene at concentrations above 78 µM, and that metabolic imbalances may lead to inhibition and toxicity.
Bacteria that metabolize aromatic hydrocarbons face the challenge of acquiring carbon and energy from compounds that are potentially toxic (Ramos et al., 2002; Sikkema et al., 1995). The inability of potential biodegrading populations to tolerate aromatic hydrocarbon toxicity may contribute to the persistence of pollutants in the environment. The mechanisms of toxicity are generally believed to be disruption of biological membranes (Sikkema et al., 1995) and the production of toxic metabolites (e.g. Park et al., 2004). The lipophilic character of aromatic hydrocarbons can alter membrane fluidity, permeablize the membrane and cause swelling of the lipid bilayer. Alteration of membrane structure can disrupt energy transduction and the activity of membrane-associated proteins (Sikkema et al., 1995). Additionally, metabolites of aromatic compounds, such as catechols and quinones, can be more toxic than the parent compound due to an increase in solubility, production of reactive oxygen species, or adduct formation with DNA and proteins (Penning et al., 1999; Schweigert et al., 2001).
In the present investigation we have shown that Polaromonas naphthalenivorans CJ2 is susceptible to both (i) direct naphthalene inhibition and (ii) formation of toxic intermediate metabolites. Naphthalene inhibited the growth of strain CJ2 at concentrations of 55 µM (Fig. 4), which is well below the aqueous saturation point of naphthalene (230 µM). Naphthalene has been reported to be toxic to the archetypal naphthalene degraders P. putida G7 and P. putida NCIB 9816-4, but only under nitrogen- or oxygen-limiting conditions (Ahn et al., 1998) or during incubation in soil amended with a high concentration (0.2 % w/v) of naphthalene crystals (Park et al., 2004), respectively. We found that inhibition of strain CJ2 by naphthalene was independent of metabolism, and, based on the study of Sikkema et al. (1994), we speculate that the mechanisms of direct inhibition are likely to be related to impaired membrane function.
In addition to growth inhibition, naphthalene metabolism by strain CJ2 at inhibitory concentrations resulted in the accumulation of toxic oxidation products derived from 1,2-naphthoquinone, which resulted in a complete loss of viability (Figs 5c and 6c). Davies & Evans (1964) showed that a 25 µM solution of 1,2-dihydroxynaphthalene is converted by non-enzymic oxidation to 1,2-naphthoquinone at a rate of approximately 20 % min–1 at pH 6.5. 1,2-Naphthoquinone has been reported to accumulate and inhibit both growth and naphthalene metabolism when ferrous and magnesium salts are omitted from growth media (Murphy & Stone, 1955) or when naphthalene bioavailability is increased by the addition of surfactant (Auger et al., 1995). Our analysis by HPLC suggests that 1,2-naphthoquinone (presumably produced abiotically from 1,2-dihydroxynaphthalene) and two abiotic transformation products of 1,2-naphthoquinone accumulate when strain CJ2 is exposed to inhibitory concentrations of naphthalene.
Strain CJ2 was shown to metabolize naphthalene in situ by stable isotope probing, which suggests that strain CJ2 is an active member of the naphthalene-degrading population in the sediment (Jeon et al., 2003). Strain CJ2 has evolved, apparently successfully, to occupy a niche as a naphthalene degrader, even though naphthalene has a strong inhibitory effect and subsaturation levels of naphthalene can result in toxic metabolite accumulation. It is possible that strain CJ2 never experienced selective pressure to develop greater tolerance to naphthalene because adsorption to soil and metabolism of naphthalene by other bacteria kept naphthalene concentrations well below inhibitory levels. If this is the case, strain CJ2 may not have evolved adaptation mechanisms frequently associated with tolerance to aromatic compounds, such as membrane isomerization and efflux pumps (Ramos et al., 2002). Furthermore, the accumulation of 1,2-naphthoquinone-related oxidation products might be due to unrealistic naphthalene concentrations imposed in laboratory incubations combined with the slow growth of strain CJ2. An enzyme in the naphthalene catabolic pathway may have a low specific activity that is only problematic when the concentration of naphthalene exceeds a threshold. Another possibility is that if strain CJ2 is adapted to low naphthalene bioavailability in soil, the bacterium has evolved to accumulate as much naphthalene as possible, whether through active uptake or through modifications of the cell membrane and envelope. Thus, when presented with naphthalene at the concentrations used in this study, strain CJ2 accumulated naphthalene to inhibitory and toxic quantities.
This study also showed that strain CJ2 metabolizes naphthalene via gentisate using the nag-type pathway found in Ralstonia sp. strain U2. Metabolism of aromatics via gentisate has been studied less extensively than metabolism via catechol, and it is not clear whether one pathway has an advantage over the other. A study investigating bacteria that metabolize 3-chlorobenzoate suggested that micro-organisms using the gentisate pathway have lower maximum specific growth rates and lower apparent half-saturation constants for oxygen and 3-chlorobenzoate; thus, they may be well adapted to substrate- and/or oxygen-limited conditions (Krooneman et al., 2000). If these same characteristics are applicable to strain CJ2, they could help explain why it is successful in naphthalene-contaminated sediments despite being sensitive to inhibitory effects of naphthalene.
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ACKNOWLEDGEMENTS |
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Edited by: H. L. Drake
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 18 June 2007;
revised 13 July 2007;
accepted 18 July 2007.
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) and Ralstonia sp. strain U2 (
), which grew equally well with pyruvate and naphthalene, strain CJ2 (
) grew only in MSB with pyruvate but not in MSB saturated with naphthalene. The experimental values represent the mean±SD of triplicate experiments.
, 23 µM; 
, 100 µM naphthalene.
, 23 µM naphthalene, no glucose.
