Environment Health Techniques Antioxidant defense system in solvent tolerance
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Research Paper Involvement of antioxidant defense system in solvent tolerance of Pseudomonas putida BCNU 106 Hye Jung Choi1, Ju-Soon Yoo1, Yong Kee Jeong2 and Woo Hong Joo1 1
2
Interdisplinary Program for Biotechnology and Department of Biology, Changwon National University, Changwon, Korea Department of Biotechnology, Dong-A University, Busan, Korea
The highly solvent-tolerant bacterium Pseudomonas sp. BCNU 106 was investigated to elucidate the solvent tolerance under specific culture conditions with the presence of solvents and its adaptive mechanisms to those conditions with reference to the antioxidant system. When exposed to 10% toluene, Pseudomonas sp. BCNU 106 increased the generation of reactive oxygen species assessed by monitoring the oxidation of 20 ,70 -dichlorofluorescein. Typical antioxidant enzymes (viz. catalase, superoxide dismutase, and glutathione reductase) showed increased activity with prolonged incubation in 10% toluene. In addition, the levels of these antioxidant proteins were higher during exposure to 10% toluene than in toluene-free condition. The present study indicates that antioxidant defense activity is one of the adaptive and protective mechanisms developed to avoid the deleterious damage of organic solvents, especially toluene. Abbreviations: LB – Luria–Bertani; ROS – reactive oxygen species; GR – glutathione reductase; SOD – superoxide dismutase; CAT – catalase; SDS–PAGE – sodium dodecyl sulfate–polyacrylamide gel electrophoresis; RT-PCR – reverse transcriptase-polymerase chain reaction Keywords: Pseudomonas sp. / Solvent-tolerant bacterium / Antioxidant defense activity Received: March 4, 2013; accepted: July 8, 2013 DOI 10.1002/jobm.201300176
Introduction The growth of bacteria is affected by changes in the extracellular environment. Bacteria are able to adapt to various kinds of environmental changes to a limited extent, but with increasing stresses, bacterial intracellular homeostasis occurs prior to mortality, and therefore, individual bacterial strains can exist in a variety of environments, and even in extreme conditions. Extremophilic microorganisms can survive and proliferate in extreme conditions, which may refer to extremes of temperature, salinity, pressure, pH, desiccation, and radiation, and high concentrations of organic solvents. Solvent-tolerant bacteria are a type of extremophile first described by Inoue and Horikoshi [1]. These bacteria and their enzymes can serve as biocatalysts in catalyzing Correspondence: Prof. Woo Hong Joo, Department of Biology, Changwon National University, Changwondaehak-ro, Changwon 641773, Republic of Korea E-mail: [email protected] Phone: þ82 55 2133453 Fax: þ82 55 2133459 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
biotransformations of water-insoluble substances in organic-aqueous biphasic or nonaqueous systems, and such microorganisms can be used as vehicles in bioremediation and wastewater treatment, especially in the bioremediation of natural contaminated sites saturated with solvents [2, 3]. Changing or extreme environments impose stresses on bacteria, and survival under stressed conditions requires adaptive responses to stress. In the majority of cases, the bacteria alter their pattern of gene expression to produce molecules that can cope with the given physical and (or) chemical stress. Chemical stimulation with many organic solvents including toluene imposes an extremely severe stress on microbial cells, even at the very low concentration of 0.1% v/v. Organic solvent accumulation, even in solvent-tolerant bacteria, results in several response mechanisms at the level of the cell membrane, inducing the production of chaperone proteins, initiating efflux pumps, and forcing the induction of genes involved in energy synthesis [3, 4]. Several studies have demonstrated that alcohols and aromatic compounds mediate the activation of the
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J. Basic Microbiol. 2014, 54, 945–950
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response against oxidative agents and even cause oxidative damage [4]. However, whether solvent exposure induces the expression of the genes or proteins involved in antioxidant defense in solvent-tolerant bacteria is unclear. A recent study reported that several genes of the OxyR regulon are induced by toluene in solvent tolerant Pseudomonas putida KT 2440 [5]. The aim of this study was to evaluate the toluene-mediated induction of antioxidant defense as a tolerance strategy in the solvent tolerant bacterium P. putida BCNU 106.
Materials and methods Bacterial strain and culture conditions Solvent-tolerant P. putida BCNU 106 was grown for 0.5, 1, 2, 4, 8, 12, 16, and 20 h at 37 °C with shaking at 130 rpm in 500 ml Erlenmeyer flasks containing 100 ml of Luria– Bertani (LB) medium supplemented with 10 mM MgCl2 in the presence or absence of 10% toluene [6]. Cell extracts preparation and total glutathione determination Cells were collected by centrifugation (6,800 g, 4 °C, 20 min) and were suspended in two volumes of 100 mM potassium phosphate buffer (pH 7.4). The cell suspension was lysed by sonication and was then centrifuged (15,300 g, 4 °C, 30 min) to remove cell debris and obtain cell extracts. The supernatant was analyzed for total protein concentration [7], enzyme activities, and relative changes in protein levels by Western blot. The total glutathione: reduced glutathione þ glutathione disulfide, was measured spectrophotometrically using 5, 50 -dithio2-nitrobenzoic acid, as previously described [8]. Antioxidant enzyme activities Glutathione reductase (GR) activity was determined by measuring NADPH oxidation [9]. One unit was defined as the quantity of GR capable of reducing 1 mmol GSSG to GSH in 1 min at 30 °C. Total superoxide dismutase (SOD) activity was measured by the inhibition of cytochrome c reduction by superoxide radical [10]. One unit of SOD activity was defined as the amount of the enzyme producing a 50% inhibition of cytochrome c reduction during 1 min at 30 °C. Catalase (CAT) activity was determined by monitoring the absorbance of an aqueous solution of H2O2 (54 mM) at 240 nm [11]. One unit of CAT activity corresponded to decomposition of 1 mmol substrate during 1 min at 30 °C. 20 ,70 -Dichlorofluorescein oxidation P. putida BCNU 106 cells were pelleted by centrifugation (6,800 g, 4 °C, 20 min) from 2 ml aliquots of the ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
cultures, and were washed three times with 2 ml of 50 mM sodium phosphate buffer (pH 7.4). The final cell pellet was suspended in the buffer to 2% v/v and was then pre-incubated at 28 °C for 15 min. 20 ,70 -Dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR, USA) was added as a stock 1 mM ethanol solution to a final concentration of 10 mM [12–14]. After incubation (20 min, 28 °C), the cell suspension was pelleted by centrifugation (10,600 g, 10 min, 4 °C), suspended in 50 mM sodium phosphate buffer (pH 7.4), and the cells were broken by sonication. Debris was removed by centrifugation (10,600 g) for 3 min. The fluorescence of the supernatant was measured using a spectrofluorometer (Perkin–Elmer, Waltham, MA, USA) with excitation and emission wavelengths of 488 and 520 nm, respectively. Reverse transcriptase-polymerase chain reaction (RTPCR) analysis For the RT-PCR experiments, total RNA from cells grown in the presence or absence of toluene for different times was extracted using hot phenol [15]. The purified RNA was treated with DNase I and was incubated with the after-mentioned reverse primers and Superscript II reverse transcriptase (Life Technologies, Carlsbad, CA, USA) at 42 °C for 60 min. The reaction mixture (2 ml) provided the substrate for PCR (25 cycles of 94 °C for 2 min, 50 °C for 1 min, and 72 °C for 2 min in a 50 ml reaction mixture with 2 mM MgCl2) employing Amplitaq DNA polymerase (Perkin–Elmer). The gene specific oligomers (cat; forward primer; ATA AGC AAG ATT CTC ACC ACC GCC AGC GGT and reverse primer, TTA GGC GAG ATT GAT GCC CAG GCC CTT GGC: Mnsod; forward primer; ATG CCG CAT ACC TTG CCT GCT TTG and reverse primer, ACT CAC TTC AGG GCT TCG AGG TAA: Fesod; forward primer; GAA TCA GGA GAT CCA CCA TGG CTT TTG AAT T and reverse primer, AATTCA GGC CTT GAA GGT CTT GCC TT) for cat and sod in Pseudomonas sp.) were designed using DNA sequences from GenBank for use in the secondary PCR. Amplified 16S rDNA was used as the RT-PCR control. SDS–PAGE and Western blot analysis Proteins (75 mg) were separated by 15% w/v sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [16] and were transferred to a nitrocellulose membrane (0.45 nm; Schleicher & Schuell BioScience, Dassel, Germany) in a semidry system for 75 min at 0.8 mA cm2. After blocking with 5% w/v fat-free milk powder in PBS, each membrane was hybridized with a 1:5000 dilution of rabbit anti-Cu, Zn SOD antibody (antiSOD1; Stressgen, Victoria, BC, Canada), 1:5000 dilution of
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J. Basic Microbiol. 2014, 54, 945–950
Antioxidant defense system in solvent tolerance
Results Growth, glutathione content, and production of reactive oxygen species The growth of Pseudomonas sp. BCNU 106 in the presence or absence of 10% toluene was similar to the growth pattern reported in a prior study [6]. Reactive oxygen species (ROS) formation was measured by a spectrometric method based on the oxidation of 20 ,70 -dichlorofluorescein (Fig. 1A). The solvent-tolerant Pseudomonas sp. BCNU 106 challenged by 10% toluene exhibited lower total ROS levels compared with the non-challenged cells. Only after 4 h, ROS concentrations were higher than those for the control. The time course of the total glutathione concentrations, during the cultivation in the presence or absence of 10% toluene, is shown in Fig. 1B. The total glutathione concentrations were elevated after exposure
to 10% toluene and the highest values were observed after 1 h of exposure. Compared with the control, increased concentrations of total glutathione were observed in the exposed cultures after 1 h, and up to 4 h. Antioxidant enzymes of Pseudomonas sp. BCNU 106 The antioxidant enzyme response of Pseudomonas sp. BCNU 106 to 10% toluene was assessed in LB broth (Fig. 2). After challenge by exposure to 10% toluene, Pseudomonas sp. BCNU 106 maintained CAT activity similar to the levels observed in the unchallenged control up to 2 h (unpublished data). Thereafter, CAT activity increased and reached the highest levels of 50.21 U mg1 protein at 8 h. SOD activities showed a similar pattern to CAT activity with 10% toluene treatment. In the same manner, the GR activity was maintained for 4 h in
A 60 50 U /mg protein
rabbit anti-Mn SOD antibody (anti-SOD2; Stressgen), and rabbit anti-CAT antibody (Calbiochem, La Jolla, CA, USA; 1:5,000, 1:10,000, and 1:20,000 dilution). Each membrane was exposed to a 1:25,000 dilution of secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, Amersham, Buckinghamshire, UK). Bands were detected by enhanced chemiluminescence (Amersham).
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Figure 1. ROS production (A) and glutathione concentration (B) in toluene treated and untreated cultures of P. putida BCNU 106. Symbols: No toluene exposure (&), 10% toluene exposure (&); (n ¼ 3, mean SD). ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 2. Effect of toluene exposure on CAT (A), SOD (B), and GR (C) activity in P. putida BCNU 106. Symbols: No toluene exposure (&), 10% toluene exposure (&); (n ¼ 3, mean SD).
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comparison with the control, and demonstrated the highest effects with 10.43 U mg1 protein after 8 h. The control concentrations of GR, SOD, and CAT were determined to be 0.58, 6.57, and 3.36 U mg1 protein, respectively, in the unchallenged control at 0 h. The absolute values for GR, SOD, and CAT in the challenged samples reached maximal levels of 10.43, 52.67, and 50.21 U mg1 protein at 8 h, respectively. The antioxidant enzyme response to 10% toluene was increased 8-fold in SOD to 18-fold in GR in the challenged samples at 8 h. Effect of the toluene stress on mRNA levels of antioxidant genes and antioxidant proteins To confirm the above results, the expression of antioxidant genes was assessed by RT-PCR, and protein production was assessed by Western blot. RT-PCR analysis of antioxidant Cu/Zn-SOD and Mn-SOD gene proved unclear (data not shown). Exposure to toluene induced a different pattern of response for Fe-SOD mRNA levels, with a peak at 0.5 and 8 h followed by a decrease in the mRNA levels (Fig. 3A). However, the induction in CAT
Figure 3. Effect of toluene exposure on mRNA levels of CAT and FeSOD, as determined by RT-PCR analysis (A) and steady state CAT, Mn-SOD and Cu/Zn-SOD levels, as determined by Western blot (B). (B) a – toluene-treated cells, b – non-treated cells. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
mRNA expression was lower than that of Fe-SOD and clearly showed non-differential patterns (Fig. 3A). The SOD and CAT expression levels were reconfirmed by Western blot analysis (Fig. 3B). The amount of CAT did not change up to 2 h after exposure to 10% toluene after which a marked increase was observed until 20 h. For Mn-SOD, there were no observed differences between the treated and non-treated conditions, but slight differences were observed after 4 h of toluene treatment. However, the production of Cu/Zn-SOD protein was significantly increased after 0.5 h.
Discussion P. putida BCNU 106 was isolated from Korean river sediments as a solvent tolerant bacterium. This microorganism grew well, even in the presence of high concentrations of organic solvents [6]. Because of its biodegradative, tolerance, and biocatalystic capacities, this bacterium was expected to be a potential bioresource for transformation and bioremediation processes, based on the biocatalytic and bioremediation functions of its enzymes or whole cells. However, the mechanisms responsible for solvent tolerance must be understood prior to industrial applications of solvent tolerant bacteria. A wide variety of mechanisms to overcome the toxicity associated with organic solvents and to adapt to toxic organic solvents have been reported [3, 4]. However, the specific mechanisms of solvent tolerance remain unknown. The present data progress our knowledge of such systems, through demonstrating that the anti-oxidant defense system in P. putida BCNU 106 is involved with organic solvent tolerance. The addition of solvents, such as ethanol, toluene, or xylene, triggers the inducedexpression of heat stress genes [17, 18]. Aromatic compounds cause oxidative stress [4]. Several oxidative stress genes of the OxyR regulon are induced by toluene [5]. In this paper, we studied the oxidative stress status and typical oxidative stress proteins associated with toluene exposure in detail. A decreased level of ROS was observed during the first 2 h in toluene-exposed bacteria. ROS was determined to have accumulated, slightly, at 4 h. This unchanged or slightly decreased ROS level may reflect activation of antioxidant enzymes that detoxify ROS. This disrupted propagation of ROS may lead to non-inactivation of proteins, maintenance of cell membranes, and eventually, cell survival in organic solvents. These effects have also been recently demonstrated in furfural tolerant Escherichia coli [19]. The level of total intracellular glutathione increased between 1 and 4 h. This may be
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J. Basic Microbiol. 2014, 54, 945–950
Antioxidant defense system in solvent tolerance
due to the presence of CAT, which prevents ROS from penetrating into cells and does not promote GSH oxidation, causing an increase in total glutathione [20]. SOD is one of the main cellular components used in defense against oxidative stress [21]. SODs are metalloenzymes that catalyze the conversion of superoxide molecules to hydrogen peroxide and molecular oxygen. Three types of SODs – Cu/Zn-(SodC), Fe-(SodB), or Mn-type (SodA) SODs – have been described, depending on the metal cofactor. Fe-SOD and Mn-SOD are characteristically prokaryotic enzymes, but Mn-SOD is also present in the mitochondria of eukaryotes. On the other hand, Cu/ZnSOD is primarily found in the cytosol of many eukaryotic organisms [22]. However, several prokaryotes containing Cu/Zn-SOD have been reported [23, 24]. This study also revealed that P. putida BCNU 106 contains all three forms of SODs. Furthermore, SOD is inactivated by exposure to hydrogen peroxide and other peroxides, while CAT and glutathione peroxidase are inactivated by superoxide [25, 26]. However, the P. putida CAT, SOD, and GR were not inactivated, but rather increased their activity upon toluene challenge. Further studies of CAT and Fe-SOD by RT-PCR, and successive studies of CAT, Mn-SOD, and Cu/Zn-SOD by Western blot demonstrated that the antioxidant enzymes that better responded to toluene were CAT, Cu/Zn-SOD, and Fe-SOD. Our previous study reported that trehalose accumulates through the toluene-induced expression of trehalose-biosynthetic enzyme genes, after exposure to toluene in toluene-tolerant P. putida [6]. The disaccharide trehalose accumulates by various organisms in the initial adaptive response of bacteria to various environmental stresses, including heat shock, dehydration, oxidative stress, or toluene exposure [27, 28]. Convincing evidence indicates that this accumulated cytoplasmic trehalose protects proteins and membranes from denaturation caused by these stresses. Taken together, our results support the view of a synergistic cooperation between trehalose and a set of typical antioxidant enzymes, removing ROS and protecting the integrity of P. putida against toluene stress.
Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (Contract No. 2010-0009141).
References [1] Inoue, A., Horikoshi, K., 1989. A Pseudomonas thrives in high concentration of toluene. Nature, 338, 264–266. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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[2] Sardessai, Y.N., Bhosle, S., 2004. Industrial potential of organic solvent tolerant bacteria. Biotechnol. Prog., 20, 655–660. [3] Torres, S., Pandey, A., Castro, G.R., 2011. Organic solvent adaptation of Gram positive bacteria: applications and biotechnological potentials. Biotechnol. Adv., 29, 442–452. [4] Segura, A., Molina, L., Fillet, S., Krell, T. et al., 2012. Solvent tolerance in Gram-negative bacteria. Curr. Opin. Biotechnol., 23, 415–421. [5] Domínguez-Cuevas, P., González-Pastor, J.E., Marqués, S., Ramos, J.L. et al., 2006. Transcriptional tradeoff between metabolic and stress-response programs in Pseudomonas putida KT2440 cells exposed to toluene. J. Biol. Chem., 281, 11981–11991. [6] Park, H.C., Bae, Y.U., Cho, S.D., Kim, S.A. et al., 2007. Toluene-induced accumulation of trehalose by Pseudomonas sp. BCNU 106 through the expression of otsA and otsB homologues. Lett. Appl. Microbiol., 44, 50–55. [7] Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254. [8] Akerboom, T.P., Sies, H., 1981. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol., 77, 373–382. [9] Pinto, R.E., Bartley, W., 1969. The effect of age and sex on glutathione reductase and glutathione peroxidase activities and on aerobic glutathione oxidation in rat liver homogenates. Biochem. J., 112, 109–114. [10] Fridovich, I., 1985. Cytochrome c, in: Greenwald, R.A. (Ed.), CRC Handbook for Oxygen Radical Research, CRC Press, Boca Raton, FL 121–122. [11] Aebi, H., 1984. Catalase in vitro. Methods Enzymol., 105, 121–126. [12] Jakubowski, W., Bartosz, G., 1997. Estimation of oxidative stress in Saccharomyces cerevisiae with fluorescent probes. Int. J. Biochem., 29, 1297–1301. [13] Jakubowski, W., Tomasz, B., Grzegorz, B., 2000. Oxidative stress during aging of stationary cultures of the yeast Sacchromyces cerevisiae. Free Radic. Biol. Med., 28, 659–664. [14] Fortuniak, A., Jakubowski, W., Biliński, T., Bartosz, G., 1996. Lack of evidence of oxidative damage in antioxidantdeficient strains of Saccharomyces cerevisiae. Biochem. Mol. Biol., 38, 1271–1276. [15] Köhrer, K., Domdey, H., 1991. Preparation of high molecular weight RNA. Methods Enzymol., 194, 398–405. [16] Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. [17] Brynildsen, M.P., Liao, J.C., 2009. An integrated network approach identifies the isobutanol response network of Escherichia coli. Mol. Syst. Biol., 5, 277. [18] Rutherford, M.P., Dahl, R.H., Price, R.E., Szmidt, H.L. et al., 2010. Functional genomic study of exogenous n-butanol stress in Escherichia coli. Appl. Envrion. Microbiol., 76, 1935–1945. [19] Wang, J., Zhang, Y., Chen, Y., Lin, M. et al., 2012. Global regulator engineering significantly improved Escherichia
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coli tolerances toward inhibitors of lignocellulosic hydrolysates. Biotechnol. Bioeng., 109, 3133–3142.
[24] Steinman, H.M., 1985. Bacteriocuprein superoxide dismutases in pseudomonads. J. Bacteriol., 162, 1255–1260.
[20] Smirnova, G.V., Muzyka, N.G., Glukhovchenko, M.N., Oktyabrsky, O.N., 2000. Effects of menadione and hydrogen peroxide on glutathione status in growing Escherichia coli. Free Radic. Biol. Med., 28, 1009– 1016.
[25] Pigeolet, E., Corbisier, P., Houbion, A., Lambert, D. et al., 1990. Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals. Mech. Ageing Dev., 51, 283–297.
[21] McCord, J.M., Fridovich, I., 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem., 244, 6049–6055.
[26] Blum, J., Fridovich, I., 1985. Inactivation of glutathione peroxidase by superoxide radical. Arch. Biochem. Biophys., 240, 500–508.
[22] Hassan, H.M., 1989. Microbial superoxide dismutases. Adv. Genet., 26, 65–97.
[27] Arguelles, J.C., 2000. Physiological roles of trehalose in bacteria and yeasts; a comparative analysis. Arch. Microbiol., 174, 217–224.
[23] Langford, P.R., Loynds, B.M., Kroll, J.S., 1996. Cloning and molecular characterization of Cu, Zn superoxide dismutase from Actinobacillus pleuropneumoniae. Infect. Immun., 64, 5035–5041.
[28] Joo, W.H., Shin, Y.S., Lee, Y., Park, S.M. et al., 2000. Intracellular changes of trehalose content in toluene tolerant Pseudomonas sp. BCNU 171 after exposure to toluene. Biotechnol. Lett., 22, 1021–1024.
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Research Paper Involvement of antioxidant defense system in solvent tolerance of Pseudomonas putida BCNU 106 Hye Jung Choi1, Ju-Soon Yoo1, Yong Kee Jeong2 and Woo Hong Joo1 1
2
Interdisplinary Program for Biotechnology and Department of Biology, Changwon National University, Changwon, Korea Department of Biotechnology, Dong-A University, Busan, Korea
The highly solvent-tolerant bacterium Pseudomonas sp. BCNU 106 was investigated to elucidate the solvent tolerance under specific culture conditions with the presence of solvents and its adaptive mechanisms to those conditions with reference to the antioxidant system. When exposed to 10% toluene, Pseudomonas sp. BCNU 106 increased the generation of reactive oxygen species assessed by monitoring the oxidation of 20 ,70 -dichlorofluorescein. Typical antioxidant enzymes (viz. catalase, superoxide dismutase, and glutathione reductase) showed increased activity with prolonged incubation in 10% toluene. In addition, the levels of these antioxidant proteins were higher during exposure to 10% toluene than in toluene-free condition. The present study indicates that antioxidant defense activity is one of the adaptive and protective mechanisms developed to avoid the deleterious damage of organic solvents, especially toluene. Abbreviations: LB – Luria–Bertani; ROS – reactive oxygen species; GR – glutathione reductase; SOD – superoxide dismutase; CAT – catalase; SDS–PAGE – sodium dodecyl sulfate–polyacrylamide gel electrophoresis; RT-PCR – reverse transcriptase-polymerase chain reaction Keywords: Pseudomonas sp. / Solvent-tolerant bacterium / Antioxidant defense activity Received: March 4, 2013; accepted: July 8, 2013 DOI 10.1002/jobm.201300176
Introduction The growth of bacteria is affected by changes in the extracellular environment. Bacteria are able to adapt to various kinds of environmental changes to a limited extent, but with increasing stresses, bacterial intracellular homeostasis occurs prior to mortality, and therefore, individual bacterial strains can exist in a variety of environments, and even in extreme conditions. Extremophilic microorganisms can survive and proliferate in extreme conditions, which may refer to extremes of temperature, salinity, pressure, pH, desiccation, and radiation, and high concentrations of organic solvents. Solvent-tolerant bacteria are a type of extremophile first described by Inoue and Horikoshi [1]. These bacteria and their enzymes can serve as biocatalysts in catalyzing Correspondence: Prof. Woo Hong Joo, Department of Biology, Changwon National University, Changwondaehak-ro, Changwon 641773, Republic of Korea E-mail: [email protected] Phone: þ82 55 2133453 Fax: þ82 55 2133459 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
biotransformations of water-insoluble substances in organic-aqueous biphasic or nonaqueous systems, and such microorganisms can be used as vehicles in bioremediation and wastewater treatment, especially in the bioremediation of natural contaminated sites saturated with solvents [2, 3]. Changing or extreme environments impose stresses on bacteria, and survival under stressed conditions requires adaptive responses to stress. In the majority of cases, the bacteria alter their pattern of gene expression to produce molecules that can cope with the given physical and (or) chemical stress. Chemical stimulation with many organic solvents including toluene imposes an extremely severe stress on microbial cells, even at the very low concentration of 0.1% v/v. Organic solvent accumulation, even in solvent-tolerant bacteria, results in several response mechanisms at the level of the cell membrane, inducing the production of chaperone proteins, initiating efflux pumps, and forcing the induction of genes involved in energy synthesis [3, 4]. Several studies have demonstrated that alcohols and aromatic compounds mediate the activation of the
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J. Basic Microbiol. 2014, 54, 945–950
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Hye Jung Choi et al.
response against oxidative agents and even cause oxidative damage [4]. However, whether solvent exposure induces the expression of the genes or proteins involved in antioxidant defense in solvent-tolerant bacteria is unclear. A recent study reported that several genes of the OxyR regulon are induced by toluene in solvent tolerant Pseudomonas putida KT 2440 [5]. The aim of this study was to evaluate the toluene-mediated induction of antioxidant defense as a tolerance strategy in the solvent tolerant bacterium P. putida BCNU 106.
Materials and methods Bacterial strain and culture conditions Solvent-tolerant P. putida BCNU 106 was grown for 0.5, 1, 2, 4, 8, 12, 16, and 20 h at 37 °C with shaking at 130 rpm in 500 ml Erlenmeyer flasks containing 100 ml of Luria– Bertani (LB) medium supplemented with 10 mM MgCl2 in the presence or absence of 10% toluene [6]. Cell extracts preparation and total glutathione determination Cells were collected by centrifugation (6,800 g, 4 °C, 20 min) and were suspended in two volumes of 100 mM potassium phosphate buffer (pH 7.4). The cell suspension was lysed by sonication and was then centrifuged (15,300 g, 4 °C, 30 min) to remove cell debris and obtain cell extracts. The supernatant was analyzed for total protein concentration [7], enzyme activities, and relative changes in protein levels by Western blot. The total glutathione: reduced glutathione þ glutathione disulfide, was measured spectrophotometrically using 5, 50 -dithio2-nitrobenzoic acid, as previously described [8]. Antioxidant enzyme activities Glutathione reductase (GR) activity was determined by measuring NADPH oxidation [9]. One unit was defined as the quantity of GR capable of reducing 1 mmol GSSG to GSH in 1 min at 30 °C. Total superoxide dismutase (SOD) activity was measured by the inhibition of cytochrome c reduction by superoxide radical [10]. One unit of SOD activity was defined as the amount of the enzyme producing a 50% inhibition of cytochrome c reduction during 1 min at 30 °C. Catalase (CAT) activity was determined by monitoring the absorbance of an aqueous solution of H2O2 (54 mM) at 240 nm [11]. One unit of CAT activity corresponded to decomposition of 1 mmol substrate during 1 min at 30 °C. 20 ,70 -Dichlorofluorescein oxidation P. putida BCNU 106 cells were pelleted by centrifugation (6,800 g, 4 °C, 20 min) from 2 ml aliquots of the ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
cultures, and were washed three times with 2 ml of 50 mM sodium phosphate buffer (pH 7.4). The final cell pellet was suspended in the buffer to 2% v/v and was then pre-incubated at 28 °C for 15 min. 20 ,70 -Dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR, USA) was added as a stock 1 mM ethanol solution to a final concentration of 10 mM [12–14]. After incubation (20 min, 28 °C), the cell suspension was pelleted by centrifugation (10,600 g, 10 min, 4 °C), suspended in 50 mM sodium phosphate buffer (pH 7.4), and the cells were broken by sonication. Debris was removed by centrifugation (10,600 g) for 3 min. The fluorescence of the supernatant was measured using a spectrofluorometer (Perkin–Elmer, Waltham, MA, USA) with excitation and emission wavelengths of 488 and 520 nm, respectively. Reverse transcriptase-polymerase chain reaction (RTPCR) analysis For the RT-PCR experiments, total RNA from cells grown in the presence or absence of toluene for different times was extracted using hot phenol [15]. The purified RNA was treated with DNase I and was incubated with the after-mentioned reverse primers and Superscript II reverse transcriptase (Life Technologies, Carlsbad, CA, USA) at 42 °C for 60 min. The reaction mixture (2 ml) provided the substrate for PCR (25 cycles of 94 °C for 2 min, 50 °C for 1 min, and 72 °C for 2 min in a 50 ml reaction mixture with 2 mM MgCl2) employing Amplitaq DNA polymerase (Perkin–Elmer). The gene specific oligomers (cat; forward primer; ATA AGC AAG ATT CTC ACC ACC GCC AGC GGT and reverse primer, TTA GGC GAG ATT GAT GCC CAG GCC CTT GGC: Mnsod; forward primer; ATG CCG CAT ACC TTG CCT GCT TTG and reverse primer, ACT CAC TTC AGG GCT TCG AGG TAA: Fesod; forward primer; GAA TCA GGA GAT CCA CCA TGG CTT TTG AAT T and reverse primer, AATTCA GGC CTT GAA GGT CTT GCC TT) for cat and sod in Pseudomonas sp.) were designed using DNA sequences from GenBank for use in the secondary PCR. Amplified 16S rDNA was used as the RT-PCR control. SDS–PAGE and Western blot analysis Proteins (75 mg) were separated by 15% w/v sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [16] and were transferred to a nitrocellulose membrane (0.45 nm; Schleicher & Schuell BioScience, Dassel, Germany) in a semidry system for 75 min at 0.8 mA cm2. After blocking with 5% w/v fat-free milk powder in PBS, each membrane was hybridized with a 1:5000 dilution of rabbit anti-Cu, Zn SOD antibody (antiSOD1; Stressgen, Victoria, BC, Canada), 1:5000 dilution of
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Results Growth, glutathione content, and production of reactive oxygen species The growth of Pseudomonas sp. BCNU 106 in the presence or absence of 10% toluene was similar to the growth pattern reported in a prior study [6]. Reactive oxygen species (ROS) formation was measured by a spectrometric method based on the oxidation of 20 ,70 -dichlorofluorescein (Fig. 1A). The solvent-tolerant Pseudomonas sp. BCNU 106 challenged by 10% toluene exhibited lower total ROS levels compared with the non-challenged cells. Only after 4 h, ROS concentrations were higher than those for the control. The time course of the total glutathione concentrations, during the cultivation in the presence or absence of 10% toluene, is shown in Fig. 1B. The total glutathione concentrations were elevated after exposure
to 10% toluene and the highest values were observed after 1 h of exposure. Compared with the control, increased concentrations of total glutathione were observed in the exposed cultures after 1 h, and up to 4 h. Antioxidant enzymes of Pseudomonas sp. BCNU 106 The antioxidant enzyme response of Pseudomonas sp. BCNU 106 to 10% toluene was assessed in LB broth (Fig. 2). After challenge by exposure to 10% toluene, Pseudomonas sp. BCNU 106 maintained CAT activity similar to the levels observed in the unchallenged control up to 2 h (unpublished data). Thereafter, CAT activity increased and reached the highest levels of 50.21 U mg1 protein at 8 h. SOD activities showed a similar pattern to CAT activity with 10% toluene treatment. In the same manner, the GR activity was maintained for 4 h in
A 60 50 U /mg protein
rabbit anti-Mn SOD antibody (anti-SOD2; Stressgen), and rabbit anti-CAT antibody (Calbiochem, La Jolla, CA, USA; 1:5,000, 1:10,000, and 1:20,000 dilution). Each membrane was exposed to a 1:25,000 dilution of secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, Amersham, Buckinghamshire, UK). Bands were detected by enhanced chemiluminescence (Amersham).
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Figure 1. ROS production (A) and glutathione concentration (B) in toluene treated and untreated cultures of P. putida BCNU 106. Symbols: No toluene exposure (&), 10% toluene exposure (&); (n ¼ 3, mean SD). ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 2. Effect of toluene exposure on CAT (A), SOD (B), and GR (C) activity in P. putida BCNU 106. Symbols: No toluene exposure (&), 10% toluene exposure (&); (n ¼ 3, mean SD).
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comparison with the control, and demonstrated the highest effects with 10.43 U mg1 protein after 8 h. The control concentrations of GR, SOD, and CAT were determined to be 0.58, 6.57, and 3.36 U mg1 protein, respectively, in the unchallenged control at 0 h. The absolute values for GR, SOD, and CAT in the challenged samples reached maximal levels of 10.43, 52.67, and 50.21 U mg1 protein at 8 h, respectively. The antioxidant enzyme response to 10% toluene was increased 8-fold in SOD to 18-fold in GR in the challenged samples at 8 h. Effect of the toluene stress on mRNA levels of antioxidant genes and antioxidant proteins To confirm the above results, the expression of antioxidant genes was assessed by RT-PCR, and protein production was assessed by Western blot. RT-PCR analysis of antioxidant Cu/Zn-SOD and Mn-SOD gene proved unclear (data not shown). Exposure to toluene induced a different pattern of response for Fe-SOD mRNA levels, with a peak at 0.5 and 8 h followed by a decrease in the mRNA levels (Fig. 3A). However, the induction in CAT
Figure 3. Effect of toluene exposure on mRNA levels of CAT and FeSOD, as determined by RT-PCR analysis (A) and steady state CAT, Mn-SOD and Cu/Zn-SOD levels, as determined by Western blot (B). (B) a – toluene-treated cells, b – non-treated cells. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
mRNA expression was lower than that of Fe-SOD and clearly showed non-differential patterns (Fig. 3A). The SOD and CAT expression levels were reconfirmed by Western blot analysis (Fig. 3B). The amount of CAT did not change up to 2 h after exposure to 10% toluene after which a marked increase was observed until 20 h. For Mn-SOD, there were no observed differences between the treated and non-treated conditions, but slight differences were observed after 4 h of toluene treatment. However, the production of Cu/Zn-SOD protein was significantly increased after 0.5 h.
Discussion P. putida BCNU 106 was isolated from Korean river sediments as a solvent tolerant bacterium. This microorganism grew well, even in the presence of high concentrations of organic solvents [6]. Because of its biodegradative, tolerance, and biocatalystic capacities, this bacterium was expected to be a potential bioresource for transformation and bioremediation processes, based on the biocatalytic and bioremediation functions of its enzymes or whole cells. However, the mechanisms responsible for solvent tolerance must be understood prior to industrial applications of solvent tolerant bacteria. A wide variety of mechanisms to overcome the toxicity associated with organic solvents and to adapt to toxic organic solvents have been reported [3, 4]. However, the specific mechanisms of solvent tolerance remain unknown. The present data progress our knowledge of such systems, through demonstrating that the anti-oxidant defense system in P. putida BCNU 106 is involved with organic solvent tolerance. The addition of solvents, such as ethanol, toluene, or xylene, triggers the inducedexpression of heat stress genes [17, 18]. Aromatic compounds cause oxidative stress [4]. Several oxidative stress genes of the OxyR regulon are induced by toluene [5]. In this paper, we studied the oxidative stress status and typical oxidative stress proteins associated with toluene exposure in detail. A decreased level of ROS was observed during the first 2 h in toluene-exposed bacteria. ROS was determined to have accumulated, slightly, at 4 h. This unchanged or slightly decreased ROS level may reflect activation of antioxidant enzymes that detoxify ROS. This disrupted propagation of ROS may lead to non-inactivation of proteins, maintenance of cell membranes, and eventually, cell survival in organic solvents. These effects have also been recently demonstrated in furfural tolerant Escherichia coli [19]. The level of total intracellular glutathione increased between 1 and 4 h. This may be
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due to the presence of CAT, which prevents ROS from penetrating into cells and does not promote GSH oxidation, causing an increase in total glutathione [20]. SOD is one of the main cellular components used in defense against oxidative stress [21]. SODs are metalloenzymes that catalyze the conversion of superoxide molecules to hydrogen peroxide and molecular oxygen. Three types of SODs – Cu/Zn-(SodC), Fe-(SodB), or Mn-type (SodA) SODs – have been described, depending on the metal cofactor. Fe-SOD and Mn-SOD are characteristically prokaryotic enzymes, but Mn-SOD is also present in the mitochondria of eukaryotes. On the other hand, Cu/ZnSOD is primarily found in the cytosol of many eukaryotic organisms [22]. However, several prokaryotes containing Cu/Zn-SOD have been reported [23, 24]. This study also revealed that P. putida BCNU 106 contains all three forms of SODs. Furthermore, SOD is inactivated by exposure to hydrogen peroxide and other peroxides, while CAT and glutathione peroxidase are inactivated by superoxide [25, 26]. However, the P. putida CAT, SOD, and GR were not inactivated, but rather increased their activity upon toluene challenge. Further studies of CAT and Fe-SOD by RT-PCR, and successive studies of CAT, Mn-SOD, and Cu/Zn-SOD by Western blot demonstrated that the antioxidant enzymes that better responded to toluene were CAT, Cu/Zn-SOD, and Fe-SOD. Our previous study reported that trehalose accumulates through the toluene-induced expression of trehalose-biosynthetic enzyme genes, after exposure to toluene in toluene-tolerant P. putida [6]. The disaccharide trehalose accumulates by various organisms in the initial adaptive response of bacteria to various environmental stresses, including heat shock, dehydration, oxidative stress, or toluene exposure [27, 28]. Convincing evidence indicates that this accumulated cytoplasmic trehalose protects proteins and membranes from denaturation caused by these stresses. Taken together, our results support the view of a synergistic cooperation between trehalose and a set of typical antioxidant enzymes, removing ROS and protecting the integrity of P. putida against toluene stress.
Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (Contract No. 2010-0009141).
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