Appl Biochem Biotechnol DOI 10.1007/s12010-014-0930-2
Influence of DMF-Induced Oxidative Stress on Membrane and Periplasmic Proteins in Paracoccus sp. SKG B. Kirankumar & Guruprasad B. Kulkarni & S. Sanjeevkumar & I. Mukram & T. B. Karegoudar
Received: 13 December 2013 / Accepted: 21 April 2014 # Springer Science+Business Media New York 2014
Abstract The present study describes the N,N-dimethylformamide (DMF)-induced oxidative stress in Paracoccus sp. SKG. The oxidative stress was evaluated by analysing membrane and periplasmic proteins and K+ efflux, as well as by monitoring the activities of antioxidant enzymes like catalase, superoxide dismutase (SOD) and glutathione S-transferase (GST). The exposure of bacterial cells to a higher concentration of DMF resulted in the modification of membrane fatty acid composition which is accompanied by K+ efflux. Further, this oxidative stress resulted in increased periplasmic protein which can be attributed to the induction of GST and methionine sulphoxide reductase (Msr) enzymes under solvent stress. Paracoccus sp. SKG is tolerant to high concentrations of DMF up to 6 % (v/v) and its toxic effects. DMF concentration-dependent induction of GST and Msr activities advocates the significant role of these enzymes in the bacterial defence system. The present study provides information which helps us to understand the ROS scavenging machinery in bacteria. The high tolerance of Paracoccus sp. SKG to DMF can be efficiently explored for various bioremediation and biotransformation applications. Keywords DMF . Periplasmic protein . Glutathione S-transferase . Methionine sulphoxide reductase . Oxidative stress . Paracoccus sp
Introduction Organic solvents are highly toxic to living organisms because of their devastating effects on biological membranes [1]. In bacteria, the toxicity is mainly due to their preferential partitioning into membranes, increasing in the fluidity of the membrane which leads to its non-specific permiabilization [2, 1]. The growth inhibition caused by solvent is concentrationdependent and follows a similar pattern in the majority of aerobic microbes [3]. N,NDimethylformamide (DMF) is a versatile and widely used organic solvent because of its potential industrial applications. After solute recovery, most of the DMF is released in the
B. Kirankumar : G. B. Kulkarni : S. Sanjeevkumar : I. Mukram : T. B. Karegoudar (*) Department of Biochemistry, Gulbarga University, Gulbarga 585106 Karnataka, India e-mail: [email protected]
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effluent, and hence, it is the most common and predominant chemical found in industrial effluents [4, 5]. Paracoccus sp. SKG which is a member of the alpha proteobacteria family is a very interesting microbe because of its versatile and unique metabolism. It is predominantly a chemoorganotroph or facultative chemolithotroph; however, it can also grow by using methanol (methanotropically). Many members belonging to this genus have been previously isolated from polluted soil, water and sewage purification units. All of these features point towards the solvent tolerant and degradation ability of these organisms which can be potentially explored for bioremediation [6]. Modifications such as morphological changes in the bacteria, changes in the outer lipid membrane composition and an enhanced K+ efflux were reported in bacterial cells under solvent stress conditions [7, 8]. The exposure of aerobic microbes to organic solvents leads to the generation of reactive oxygen species (ROS), consequently increasing oxidative stress [9]. Glutathione S-transferases (GST) and methionine sulphoxide reductase (Msr) are integral parts of the bacterial defence mechanism against the ROS produced under toxic organic substrates [9]. GST is family of multifunctional proteins involved in different stress conditions [10]. Vuilleumier (2001) suggests the probable involvement of GST in the bioremediation of xenobiotics. Msr catalyzes the reduction of both free and protein-bound methionine sulphoxide residues to methionine, restoring their specific biological properties [11]. In view of all these aspects, the present study was aimed towards the investigation of the effect of different concentrations of DMF on the membrane fatty acid composition. The influence of DMF on potassium ion efflux, periplasmic proteins, and induction of GST and Msr was tested. The levels of antioxidants generated were studied in terms of the activities of antioxidant enzymes like catalase and SOD.
Materials and Methods Chemicals DMF, methanol, acetonitrile, propionitrile, propanol, trimethylamine, methylamine, dimethylamine, butanol, hexane and chloroform were obtained from S.D. Fine Chemicals, Mumbai, India, and boron trifluoride/methanol, 1-chloro-2, 4-dinitroenzene (CDNB), glutathione (GSH), methionine sulphoxide (Met-so) and bacterial acid methyl esters (BAMEs) were procured from Sigma-Aldrich, USA. All other chemicals used in the study were of analytical grade. Bacterium and Growth Conditions Paracoccus sp. SKG used in the present study was previously isolated from chemical waste samples in our laboratory. This small rod or coccoid shaped strain is a gram-negative bacterium and is a member of Proteobacteria capable of utilizing acetonitrile as a sole source of carbon and nitrogen [6]. Paracoccus sp. SKG was grown in a mineral salt medium containing (in g l−1) K2HPO4 6.8, KH2PO4 1.2; MgSO4·7H2O 0.1; MnSO4·4H2O 0.1; CaCl2·2H2O 0.1; FeSO4·7H2O 0.1; and Na2MoO7·2H2O 0.006 supplemented with 15 mM sodium succinate and NH4NO3 as a source for carbon and nitrogen, respectively. The pH of the medium was adjusted to 7.0 before sterilizing. Cells were grown in a 250-ml Erlenmeyer flask in an incubating shaker (B. Braun Ceretomat S II, Germany) at 30 °C and 180 rpm.
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Growth was measured in terms of increase in absorbance at 600 nm using a spectrophotometer (SPECORD 50, Germany). Incubation with Toxic Compounds The effect of different organic solvents on the growth of Paracoccus sp. SKG was investigated. The toxicity of the organic solvents was measured as per Heipieper et al. [12]. The effect of DMF on growth inhibition was measured by counting the colonyforming units (CFU) formed in its presence and absence. The effects were calculated by comparing the differences in the growth rates μ (h−1) between solvent-exposed (μtoxin) and control cultures (μcontrol). Growth inhibition by organic solvents is defined as the percentage of growth rates of the solvent-exposed cultures in relation to that of the control cultures without the solvents. Inhibition of growth ð%Þ ¼
μtoxin 100 μcontrol
Analysis of Fatty Acids by GC For the analysis of fatty acid components under stress conditions, the membrane lipids were extracted with chloroform/methanol/water as described by Bligh and Dyer [13]. Fatty acid methyl esters (FAME) were prepared by incubating the chloroform extract for 15 min at 95 °C in boron trifluoride/methanol following the method of Morrison and Smith [14] and later extracted with hexane. Analysis of FAME was performed in hexane using the gas chromatography (GC) system (Agilent 7820A) equipped with split/splitless injector with a flame ionization detector (FID), DB 5 column (length 30 m, inner diameter 0.25 mm, 0.25 μm film). The GC conditions for the analysis were injector temperature 240 °C and detector temperature 270 °C. Splitless injection and carrier gas nitrogen (N) flow rates were 2 ml min−1. The temperature programme was 60– 200 °C at 20 °C min−1 and 200–250 °C at 5 °C min−1 for a hold time of 2 min. The peak areas of the FAME were measured and used to determine their relative concentrations. The extracted fatty acids were identified by GC and compared with the authentic reference compounds. The degree of membrane fatty acid saturation is defined as the ratio between the three saturated fatty acids (16:0, 17:0, 18:0) and unsaturated fatty acids (16:1Δ 9 cis, 18:1Δ 9 cis) present in the bacterium. Measurement of Cellular K+ Content K+ contents released from the control and solvent-exposed cells were measured according to the method of Neumann et al. [7]. Briefly, aliquots of 1-ml samples were withdrawn from the cultures before and after the addition of the toxin. The cell pellet was collected by centrifugation at 8,000 rpm for 15 min at 4 °C. The cell pellets were disrupted using 5 % (w/v) trichloroacetic acid, and the debris was removed by centrifugation at 8,000 rpm for 15 min. The K+ released into the supernatant was measured by an atomic absorption spectrometer (Thermo Fisher Scientific iCE 3000 series AAS) fitted with a Beckman total-consumption burner for sample atomization with SOLAAR series software. The conditions were as follows: wavelength 766.5 nm, split size 0.2 nm, burner angle 200°, and flame air-acetylene. Experiments were carried out in triplicates.
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Enzyme Extraction and Assay For enzyme extraction, different concentrations of DMF were added at mid-exponential growth of cells. Cells were harvested by centrifugation at 8,000 rpm for 10 min at 4 °C. The cells were washed twice with potassium phosphate buffer (PPB) (50 mM, pH 7.0) and resuspended in 2 ml of buffer. Cells were disrupted using a Vibracell Ultrasonicator by imparting four pulses of 20 s each at 4 °C. Cell debris was removed by centrifugation at 10,000g for 20 min at 4 °C. The resulting supernatant was used directly as the enzyme source for all enzyme assays. Catalase activity was assayed according to the method described by Roggenkamp et al. [15]. Briefly, the assay mixture (1 ml) consists of 50 mM potassium phosphate buffer (pH 7.0) and hydrogen peroxide (10 mM) as the substrate. The activity was calculated by measuring the decrease in absorbance at 240 nm. The superoxide anion (O−2) scavenging activity was determined as per Beauchamp and Fridovich [16]. Briefly, the assay mixture (3 ml) approximately consists of 50 mM methionine, 75 μM NBT, 2 μM riboflavin, 0.1 mM EDTA, diluted cell-free extract. The tubes were vertexed and placed 30 cm below two 15-W fluorescent lamps. The reaction was carried out for 20 min. Absorbance was read at 560 nm against a non-irradiated mixture used as blank. Total protein content was estimated according to Lowry et al. [17]. Periplasmic Protein Extraction Periplasmic proteins were extracted by osmotic shock according to the modified method of Neu and Heppel [18]. Different concentrations of DMF were added to the mid-exponential growth of phase cells. The cells were incubated for 3 h with DMF and harvested by centrifugation at 8,000 rpm for 10 min at 4 °C. The cells were washed twice with potassium phosphate buffer (PPB) (50 mM, pH 7.0), and the cell pellet was isolated by centrifugation. The cell pellet was suspended in 20 % sucrose containing 0.2 M Tris–HCl (pH 7.3) and 5 mM EDTA and shocked in cold water. The shocked cells were subjected to centrifugation, and the supernatant containing periplasmic proteins was collected. The protein concentration was determined according to Lowry et al. [17]. Periplasmic protein profile was studied in sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) which was carried out according to the method of Laemmli [19]. Protein was denatured at 95 °C for 5 min, and 30 μg of each protein per lane was loaded onto a 12 % (w/v) polyacrylamide gel. Gels were stained with silver according to the method of Switzer et al. [20]. GST Assay GST activity in periplasmic protein was determined spectrophotometrically according to the method described by Habig et al. [21] using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate. The assay mixture consists of 0.1 M potassium phosphate buffer, 1 mM CDNB in ethanol, and 1 mM GSH, pH 6.5, at 25 °C. The reaction was initiated by the addition of enzyme solution. Enzyme activity was calculated by measuring the increase in absorbance at 340 nm caused by the formation of thioether in the reaction mixture. Methionine Sulphoxide Reductase Assay The ability of methionine sulphoxide reductase to reduce free methionine sulphoxide was assayed according to the method described by Brot et al. [22]. Briefly, the
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reaction mixture contained 50 mM PPB (pH 7.5), 20 mM DTT, 1 mM free methionine sulphoxide and enzyme solution. The reaction was carried out at 37 °C for 2 h. The enzyme activity was calculated by measuring the amount of methionine formed, which was estimated as described by Horn et al. [23]. The presence of methionine as the reaction product in the reaction mixture was further confirmed on thin layer chromatography (TLC) plates (Merck, USA) using a solvent system (n-butanol/acetic acid/water, 4:1:5). The methionine spot on TLC using ninhydrin reagent and the Rf values were compared with that of authentic methionine (Sigma-Aldrich).
Results Growth Inhibition Studies The growth inhibitory studies were carried out in mineral salt medium supplemented with sodium succinate as a sole source of carbon and energy. Different solvents were added in different concentrations to the mid-exponential growth phase of Paracoccus sp. SKG cells. The absorbance was recorded 3 h post-exposure to solvents, and respective LD50 values were calculated. The LD50 value of each solvent reflects the respective toxicity on the growth of Paracoccus sp. SKG cells. Figure 1 shows the LD50 values of different organic solvents, with the highest value for DMF (4.2 %) followed by acetonitrile (2.2 %), propionitrile (1.6 %), propanol (1.1 %), trimethylamine (0.7 %), methylamine (0.5 %), dimethylamine (0.4 %) and, least, butanol (0.2 %). Influence of DMF on Membrane Fatty Acid Profile and K+ Efflux
Concentration of sovent (%, v/v)
A significant difference was observed in the degree of saturation between the control and DMF-exposed Paracoccus sp. SKG. Exposure to DMF resulted in the modification of saturated fatty acids (16:0, 17:0, 18:0) to unsaturated fatty acids (16:1Δ 9 cis, 18:1Δ 9 cis). Figure 2 shows that the degree of saturation of fatty acids in the DMFexposed cells (1.49) was considerably high as compared to non-solvent-treated control cells (1.03). Results of the K+ efflux studies gave further supportive evidence to the increased membrane permeability. The increased concentration of solvent resulted in
7 Maximum inhibiƟon concentra on
6 5
LD 50 value
4 3 2 1 0
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Fig. 1 Effect of different organic solvents on the maximum inhibition of growth and LD50 values of Paracoccus sp. SKG at different concentrations
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DMF (%, v/v) Fig. 2 Effect of different concentrations of DMF on the degree of saturation of membrane phospholipid fatty acids (black diamonds) potassium efflux (red up-pointing triangles) and activity of GST (blue squares) on Paracoccus sp. SKG grown on sodium succinate
increased potassium ion efflux. K+ ion efflux from the cells results in decreased K+ concentration inside the cell. The K+ concentration in the non-solvent-exposed cells is 567 nM, which considerably shows that the reduction in K+ in the cells was directly proportional to the increased DMF concentration. Influence of DMF-Induced Toxicity on SOD and Catalase Major adaptive mechanisms against stress include inductions of antioxidative enzymes like SOD and catalase. These enzymes play an important role in the formation of hydrogen peroxide with superoxides and degradation of hydrogen peroxide, respectively. The activities of both enzymes were directly proportional to the concentration of toxins exposed, with highest activities (catalase 0.288 U and SOD 37.93 U) observed in the bacterium exposed to 6 % DMF and least activities observed in non-solvent-exposed cells (catalase 0.027 U and SOD 14.09 U) (Fig. 3). Influence of DMF on Periplasmic Proteins The prominent proteins present in periplasmic membrane are GST and Msr; DMF exposure has a significant effect on the activities of these enzymes in Paracoccus sp. SKG. Solvent exposure resulted in the gradual increase in GST and Msr activities. Maximum activities were observed at 5 % of DMF exposure. Increase in DMF concentration (6 %) witnessed a slight decrease in the activities. Msr activities also showed a similar pattern of induction as that of GST, with highest activities observed in cells exposed to 6 % DMF and lowest witnessed in non-solvent-exposed cells (Fig. 4). The presence of Msr and its activities in periplasmic proteins were confirmed by detecting methionine in the reaction mixture. The extracted reaction product showed an identical retention time (Rf value 0.46) to that of authentic methionine.
Appl Biochem Biotechnol 40 0.30
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Fig. 3 Effect of different concentrations of DMF on the activity of catalase (black squares), SOD (red diamonds) and growth (blue down-pointing triangles) of Paracoccus sp. SKG
Protein Profile Modification To investigate the modification in the periplasmic protein profile induced under different concentrations of DMF in Paracoccus sp. SKG cells, SDS-PAGE was performed. An electrophoregram (Fig. 5) clearly depicts the difference among the protein profile patterns of control and solvent-exposed bacterial cells. The intensities of certain proteins increased with exposure to increased DMF. These results are well correlated with the assayed enzyme activities. The intensity of protein bands gradually increases with increase in DMF concentration up to 5 %, and at 6 %, the intensity of the bands was slightly reduced. The protein band corresponding to the 29-kDa molecular marker band showed significant increase in its intensity in higher DMF concentration.
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DMF (%, v/v) Fig. 4 Effect of different concentrations of DMF on the activity of GST (orange squares) and Msr (blue diamonds) in periplasmic protein of Paracoccus sp. SKG
Appl Biochem Biotechnol
Fig. 5 Periplasmic protein of Paracoccus sp. SKG after exposure to different concentrations of DMF. Lanes 1, 2, 3, 4, 5 and 6 are of DMF 1, 2, 3, 4, 5 and 6 %, respectively. Lane M is molecular markers, lane C is control without DMF
Discussion Organic solvents are known to be toxic to living cells, and this toxicity is mainly due to their mode of action on the membranes and membrane-active compounds [2]. In the present study, we have investigated the response and adaptation of Paracoccus sp. SKG under solvent exposure. DMF exposure resulted in a marked modification in the membrane fatty acid composition of the bacterium, the significant modification being the conversion of saturated to unsaturated fatty acids. Similar results were reported for Ochrobactrum anthropi with atrazine, phenol, 4-chlorophenol and 1-chloro-2,4-dinitrobenzene [24]. Membrane modification in turn resulted in the increased K+ efflux; a strong correlation was observed between the degree of saturation of membrane fatty acid and potassium ion efflux. Neumann et al. [7] also reported identical findings in Pseudomonas putida. The presence of toxic organic compounds causes oxidative stress, mainly by the synthesis of ROS. SOD is an important part of the defence systems against oxidative damage in aerobic organisms [25]. SOD catalyzes the scavenging of superoxide anion (O2−) to O2 and H2O2, which gets reduced to H2O by the H2O2−-scavenging enzyme catalase. In the present study, enhanced activities of SOD and catalase towards tolerance against organic solvent-induced oxidative stress may be attributed to the induction of the defence system in the presence toxin. The effect of different concentrations of DMF on the levels of GST was also investigated; GST are cytosolic or microsomal enzymes which catalyze the conjugation of electrophilic xenobiotics to GSH [26]. Favaloro et al. [24] correlated the increased GST levels in O. anthropi with the influence of toxins to the adaptive cell responses such as increase in fatty acid saturation. All these studies advocate that the concentration-dependent increased level of GST and K+ efflux along with membrane fatty acid modification are the general response mechanism of bacterial cells to any toxins. The GST level in Paracoccus sp. SKG
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increases with increase in concentration of DMF. Armstrong [27] suggested that GST is one of the important factors for determining the sensitivity of the bacterial cells to a broad spectrum of toxins. Along with GST, the levels of Msr in solvent-exposed Paracoccus sp. SKG were also studied. The Msr is an enzyme responsible for catalysis of the reduction of both free and protein-bound methionine sulphoxide residues to methionine restoring their specific biological activities [11]. Both GST and Msr play a significant role in repairing oxidative damage of biomolecules (proteins) and S-thiolated enzymes [27–29]. GST and Msr function along with antioxidant enzymes like catalase and SOD to reduce oxidative stress induced by various toxins. Tamburro et al. [9] describe Msr and GST as important parts of the “oxidative defence system” represented by catalase and SOD. DMF exposure to Paracoccus sp. SKG also yielded results which can be correlated with these observations. The induction of Msr and GST is directly proportional to the increased level of catalase and SOD. In other words, induction of Msr and GST can be attributed to increased ROS level in the cell. The results of periplasmic protein profiling were further carried out by SDS-PAGE and confirmed by the induction of GST and Msr in solvent-exposed cells. Tamburro et al. [9] studied the induction of localized GST and Msr in periplasmic space of bacteria. Among all other proteins, the bands corresponding to 27 and 72 kDa showed concentration-dependent induction in their intensities. Electrophoretic studies in DMF expressed Paracoccus sp. SKG revealed the induction proteins corresponding to 27 and 72 kDa molecular markers in the periplasmic protein profile. Henson et al. [30] purified the GST and determined its molecular weight to be 27 kDa. Similarly, Benjamin et al. [31] purified and determined the Msr molecular weight to be 72 kDa. These observations advocate that the induced protein bands corresponding to 27 and 72 kDa in Paracoccus sp. SKG can be attributed to the induction of GST and Msr in the presence of high concentration of DMF. We have correlated the solvent stress with the bacterial membrane composition modifications and K+ efflux. Additional evidence for the elevated oxidative stress status in the bacterium was provided in terms of induced antioxidative enzyme activities. The periplasmic protein profile provides evidence for the probable involvement of GST and Msr in the bacterial defence system active against organic solvent stress. Because of all these defence mechanisms, Paracoccus sp. SKG could tolerate a high concentration of DMF (6 % v/v). This property can be efficiently explored in bioremediation and biotransformation applications. Acknowledgments The authors thank the University Grants Commission (UGC), New Delhi, India, for the financial support through SAP and Basic Scientific Research (BSR) fellowship programme.
References 1. Sikkema, J., de Bont, J. A. M., & Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiological Reviews, 59, 201–222. 2. Heipieper, H. J., Weber, F. J., Sikkema, J., Keweloh, H., & de Bont, J. A. M. (1994). Mechanisms behind resistance of whole cells to toxic organic solvents. Trends in Biotechnology, 12, 409–415. 3. Isken, S., & Heipieper, H. J. (2002). Toxicity of organic solvents to microorganisms. In G. Bitton (Ed.), Encyclopedia of environmental microbiology (pp. 3147–3155). New York: Wiley.
Appl Biochem Biotechnol 4. Bromley-Challenor, K. C. A., Caggiano, N., & Knapp, S. J. (2000). Bacterial growth on N, Ndimethylformamide: implication for the biotreatment of industrial wastewater. Journal of Industrial Microbiology and Biotechnology, 25, 8–16. 5. Hasegawa, Y., Masaki, M., Yoshinori, S., & Tokuyama, T. (1997). Purification and characterization of N,Ndimethylformamidease from Alcaligenes sp. KUFA-1. Journal of Fermentation and Bioengineering, 84, 543–547. 6. Santoshkumar, M., Veeranagouda, Y., Lee, K., & Karegoudar, T. B. (2011). Utilization of aliphatic nitrile by Paracoccus sp. SKG isolated from chemical waste sample. International Biodeterioration and Biodegradation, 65, 153–159. 7. Neumann, G., Kabelitz, N., & Heipieper, H. J. (2003). The regulation of cis-trans isomerase (CTI) unsaturated fatty acids in Pseudomonas putida: correlation between CTI activity and K+-uptake systems. European Journal of Lipid Science and Technology, 105, 585–589. 8. Veeranagouda, Y., Karegoudar, T. B., Neumann, G., & Heipieper, H. J. (2006). Enterobacter sp. VKGH12 growing with n-butanol as the sole carbon source and cells to which the alcohol is added as pure toxin show considerable differences in their adaptive responses. FEMS Microbiology Letters, 254, 48–54. 9. Tamburro, A., Robuffo, I., Heipieper, H. J., Allocati, N., Rotilio, D., Di Ilio, C., et al. (2004). Expression of glutathione S-transferase and peptide methionine sulphoxide reductase in Ochrobactrum anthropi is correlated to the production of reactive oxygen species caused by aromatic substrates. FEMS Microbiology Letters, 241, 151–156. 10. Vuilleumier, S. (2001). Bacterial glutathione S-transferases and the detoxification of xenobiotics: dehalogenation through glutathione conjugation and beyond. In R. M. Zablotowicz, J. C. Hall, & R. E. Hoagland (Eds.), Pesticide biotransformations in plants and microorganisms (Vol. 777, pp. 240–252). Oxford: Oxford University. 11. Kuschel, L., Hansel, A., Schoherr, R., Weissbach, H., Brot, N., Hoshi, T., et al. (1999). Molecular cloning and functional expression of a human peptide methionine sulfoxide reductase (hMsrA). FEBS Letters, 456, 17–21. 12. Heipieper, H. J., Loffeld, B., Keweloh, H., & de Bont, J. A. M. (1995). The cis/trans isomerization of unsaturated fatty acids in Pseudomonas putida S12: an indicator for environmental stress due to organic compounds. Chemosphere, 30, 1041–1051. 13. Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917. 14. Morrison, W. R., & Smith, L. M. (1964). Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. Journal of Lipid Research, 5, 600–608. 15. Roggenkamp, R., Sahm, H., & Wagner, F. (1974). Microbial assimilation of methanol induction and function of catalase in Candida boidinii. FEBS Letters, 41, 283–286. 16. Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase: improved assay and an assay applicable to polyacrylamide gels. Analytical Biochemistry, 44, 276–287. 17. Lowry, O. H., Rose, B. N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265–275. 18. Neu, H. C., & Heppel, L. A. (1965). The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. Journal of Biological Chemistry, 240, 3685–3692. 19. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the dead of bacteriophage T4. Nature, 227, 680–685. 20. Switzer, R. C., Merril, C. R., & Shifrin, S. (1979). A highly sensitive stain for detecting proteins and peptides in polyacrylamide gels. Analytical Biochemistry, 98, 231–237. 21. Habig, W. H., Pabst, M. J., & Jakoby, W. B. (1974). Glutathione S-transferase; the first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry, 249, 7130–7139. 22. Brot, N., Werth, J., Koster, K., & Weissbach, H. (1982). Reduction of N-acetyl methionine sulfoxide: a simple assay for peptide methionine sulfoxide reductase. Analytical Biochemistry, 122, 291–294. 23. Horn, M. J., Jones, D. B., & Blum, A. E. (1946). Colorimetric determination of methionine in proteins and foods. Journal of Biological Chemistry, 166, 313–320. 24. Favaloro, B., Tamburro, A., Trofino, M. A., Bologna, L., Rotilio, D., & Heipieper, H. J. (2000). Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress. Biochemical Journal, 346, 553–559. 25. Scandalios, J. G. (1993). Oxygen stress and superoxide dismutases. Plant Physiology, 101, 7–12. 26. Bernard, K. M. G., & Margaret, O. J. (2000). Activities of affinity-isolated glutathione S-transferase (GST) from channel catfish whole intestine. Aquatic Toxicology, 49, 27–37. 27. Armstrong, R. N. (1997). Structure, catalytic mechanism, and evolution of the glutathione S-transferase. Chemical Research in Toxicology, 10, 2–18.
Appl Biochem Biotechnol 28. Sheehan, D., Meade, G., Foley, V. M., & Dowd, C. A. (2001). Structure, function and evolution of glutathione transferase: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochemical Journal, 360, 1–16. 29. Levine, R. L., Mosoni, L., Berlett, B. S., & Stadtman, E. R. (1996). Methionine residues as endogenous antioxidants in proteins. Proceedings of the National Academy of Sciences of the United States of America, 93, 15036–15040. 30. Henson, K. L., Gregory, S., & Evan, P. G. (2001). Induction of glutathione S-transferase activity and protein expression in brown bullhead (Ameiurus nebulosus) liver by ethoxyquin. Toxicological Sciences, 62, 54–60. 31. Benjamin, E., Bos, J., Frédéric, B., & Laurent, A. (2005). Methionine sulfoxide reduction and assimilation in Escherichia coli: new role for the biotin sulfoxide reductase BisC. Journal of Bacteriology, 187, 231–237.
Influence of DMF-Induced Oxidative Stress on Membrane and Periplasmic Proteins in Paracoccus sp. SKG B. Kirankumar & Guruprasad B. Kulkarni & S. Sanjeevkumar & I. Mukram & T. B. Karegoudar
Received: 13 December 2013 / Accepted: 21 April 2014 # Springer Science+Business Media New York 2014
Abstract The present study describes the N,N-dimethylformamide (DMF)-induced oxidative stress in Paracoccus sp. SKG. The oxidative stress was evaluated by analysing membrane and periplasmic proteins and K+ efflux, as well as by monitoring the activities of antioxidant enzymes like catalase, superoxide dismutase (SOD) and glutathione S-transferase (GST). The exposure of bacterial cells to a higher concentration of DMF resulted in the modification of membrane fatty acid composition which is accompanied by K+ efflux. Further, this oxidative stress resulted in increased periplasmic protein which can be attributed to the induction of GST and methionine sulphoxide reductase (Msr) enzymes under solvent stress. Paracoccus sp. SKG is tolerant to high concentrations of DMF up to 6 % (v/v) and its toxic effects. DMF concentration-dependent induction of GST and Msr activities advocates the significant role of these enzymes in the bacterial defence system. The present study provides information which helps us to understand the ROS scavenging machinery in bacteria. The high tolerance of Paracoccus sp. SKG to DMF can be efficiently explored for various bioremediation and biotransformation applications. Keywords DMF . Periplasmic protein . Glutathione S-transferase . Methionine sulphoxide reductase . Oxidative stress . Paracoccus sp
Introduction Organic solvents are highly toxic to living organisms because of their devastating effects on biological membranes [1]. In bacteria, the toxicity is mainly due to their preferential partitioning into membranes, increasing in the fluidity of the membrane which leads to its non-specific permiabilization [2, 1]. The growth inhibition caused by solvent is concentrationdependent and follows a similar pattern in the majority of aerobic microbes [3]. N,NDimethylformamide (DMF) is a versatile and widely used organic solvent because of its potential industrial applications. After solute recovery, most of the DMF is released in the
B. Kirankumar : G. B. Kulkarni : S. Sanjeevkumar : I. Mukram : T. B. Karegoudar (*) Department of Biochemistry, Gulbarga University, Gulbarga 585106 Karnataka, India e-mail: [email protected]
Appl Biochem Biotechnol
effluent, and hence, it is the most common and predominant chemical found in industrial effluents [4, 5]. Paracoccus sp. SKG which is a member of the alpha proteobacteria family is a very interesting microbe because of its versatile and unique metabolism. It is predominantly a chemoorganotroph or facultative chemolithotroph; however, it can also grow by using methanol (methanotropically). Many members belonging to this genus have been previously isolated from polluted soil, water and sewage purification units. All of these features point towards the solvent tolerant and degradation ability of these organisms which can be potentially explored for bioremediation [6]. Modifications such as morphological changes in the bacteria, changes in the outer lipid membrane composition and an enhanced K+ efflux were reported in bacterial cells under solvent stress conditions [7, 8]. The exposure of aerobic microbes to organic solvents leads to the generation of reactive oxygen species (ROS), consequently increasing oxidative stress [9]. Glutathione S-transferases (GST) and methionine sulphoxide reductase (Msr) are integral parts of the bacterial defence mechanism against the ROS produced under toxic organic substrates [9]. GST is family of multifunctional proteins involved in different stress conditions [10]. Vuilleumier (2001) suggests the probable involvement of GST in the bioremediation of xenobiotics. Msr catalyzes the reduction of both free and protein-bound methionine sulphoxide residues to methionine, restoring their specific biological properties [11]. In view of all these aspects, the present study was aimed towards the investigation of the effect of different concentrations of DMF on the membrane fatty acid composition. The influence of DMF on potassium ion efflux, periplasmic proteins, and induction of GST and Msr was tested. The levels of antioxidants generated were studied in terms of the activities of antioxidant enzymes like catalase and SOD.
Materials and Methods Chemicals DMF, methanol, acetonitrile, propionitrile, propanol, trimethylamine, methylamine, dimethylamine, butanol, hexane and chloroform were obtained from S.D. Fine Chemicals, Mumbai, India, and boron trifluoride/methanol, 1-chloro-2, 4-dinitroenzene (CDNB), glutathione (GSH), methionine sulphoxide (Met-so) and bacterial acid methyl esters (BAMEs) were procured from Sigma-Aldrich, USA. All other chemicals used in the study were of analytical grade. Bacterium and Growth Conditions Paracoccus sp. SKG used in the present study was previously isolated from chemical waste samples in our laboratory. This small rod or coccoid shaped strain is a gram-negative bacterium and is a member of Proteobacteria capable of utilizing acetonitrile as a sole source of carbon and nitrogen [6]. Paracoccus sp. SKG was grown in a mineral salt medium containing (in g l−1) K2HPO4 6.8, KH2PO4 1.2; MgSO4·7H2O 0.1; MnSO4·4H2O 0.1; CaCl2·2H2O 0.1; FeSO4·7H2O 0.1; and Na2MoO7·2H2O 0.006 supplemented with 15 mM sodium succinate and NH4NO3 as a source for carbon and nitrogen, respectively. The pH of the medium was adjusted to 7.0 before sterilizing. Cells were grown in a 250-ml Erlenmeyer flask in an incubating shaker (B. Braun Ceretomat S II, Germany) at 30 °C and 180 rpm.
Appl Biochem Biotechnol
Growth was measured in terms of increase in absorbance at 600 nm using a spectrophotometer (SPECORD 50, Germany). Incubation with Toxic Compounds The effect of different organic solvents on the growth of Paracoccus sp. SKG was investigated. The toxicity of the organic solvents was measured as per Heipieper et al. [12]. The effect of DMF on growth inhibition was measured by counting the colonyforming units (CFU) formed in its presence and absence. The effects were calculated by comparing the differences in the growth rates μ (h−1) between solvent-exposed (μtoxin) and control cultures (μcontrol). Growth inhibition by organic solvents is defined as the percentage of growth rates of the solvent-exposed cultures in relation to that of the control cultures without the solvents. Inhibition of growth ð%Þ ¼
μtoxin 100 μcontrol
Analysis of Fatty Acids by GC For the analysis of fatty acid components under stress conditions, the membrane lipids were extracted with chloroform/methanol/water as described by Bligh and Dyer [13]. Fatty acid methyl esters (FAME) were prepared by incubating the chloroform extract for 15 min at 95 °C in boron trifluoride/methanol following the method of Morrison and Smith [14] and later extracted with hexane. Analysis of FAME was performed in hexane using the gas chromatography (GC) system (Agilent 7820A) equipped with split/splitless injector with a flame ionization detector (FID), DB 5 column (length 30 m, inner diameter 0.25 mm, 0.25 μm film). The GC conditions for the analysis were injector temperature 240 °C and detector temperature 270 °C. Splitless injection and carrier gas nitrogen (N) flow rates were 2 ml min−1. The temperature programme was 60– 200 °C at 20 °C min−1 and 200–250 °C at 5 °C min−1 for a hold time of 2 min. The peak areas of the FAME were measured and used to determine their relative concentrations. The extracted fatty acids were identified by GC and compared with the authentic reference compounds. The degree of membrane fatty acid saturation is defined as the ratio between the three saturated fatty acids (16:0, 17:0, 18:0) and unsaturated fatty acids (16:1Δ 9 cis, 18:1Δ 9 cis) present in the bacterium. Measurement of Cellular K+ Content K+ contents released from the control and solvent-exposed cells were measured according to the method of Neumann et al. [7]. Briefly, aliquots of 1-ml samples were withdrawn from the cultures before and after the addition of the toxin. The cell pellet was collected by centrifugation at 8,000 rpm for 15 min at 4 °C. The cell pellets were disrupted using 5 % (w/v) trichloroacetic acid, and the debris was removed by centrifugation at 8,000 rpm for 15 min. The K+ released into the supernatant was measured by an atomic absorption spectrometer (Thermo Fisher Scientific iCE 3000 series AAS) fitted with a Beckman total-consumption burner for sample atomization with SOLAAR series software. The conditions were as follows: wavelength 766.5 nm, split size 0.2 nm, burner angle 200°, and flame air-acetylene. Experiments were carried out in triplicates.
Appl Biochem Biotechnol
Enzyme Extraction and Assay For enzyme extraction, different concentrations of DMF were added at mid-exponential growth of cells. Cells were harvested by centrifugation at 8,000 rpm for 10 min at 4 °C. The cells were washed twice with potassium phosphate buffer (PPB) (50 mM, pH 7.0) and resuspended in 2 ml of buffer. Cells were disrupted using a Vibracell Ultrasonicator by imparting four pulses of 20 s each at 4 °C. Cell debris was removed by centrifugation at 10,000g for 20 min at 4 °C. The resulting supernatant was used directly as the enzyme source for all enzyme assays. Catalase activity was assayed according to the method described by Roggenkamp et al. [15]. Briefly, the assay mixture (1 ml) consists of 50 mM potassium phosphate buffer (pH 7.0) and hydrogen peroxide (10 mM) as the substrate. The activity was calculated by measuring the decrease in absorbance at 240 nm. The superoxide anion (O−2) scavenging activity was determined as per Beauchamp and Fridovich [16]. Briefly, the assay mixture (3 ml) approximately consists of 50 mM methionine, 75 μM NBT, 2 μM riboflavin, 0.1 mM EDTA, diluted cell-free extract. The tubes were vertexed and placed 30 cm below two 15-W fluorescent lamps. The reaction was carried out for 20 min. Absorbance was read at 560 nm against a non-irradiated mixture used as blank. Total protein content was estimated according to Lowry et al. [17]. Periplasmic Protein Extraction Periplasmic proteins were extracted by osmotic shock according to the modified method of Neu and Heppel [18]. Different concentrations of DMF were added to the mid-exponential growth of phase cells. The cells were incubated for 3 h with DMF and harvested by centrifugation at 8,000 rpm for 10 min at 4 °C. The cells were washed twice with potassium phosphate buffer (PPB) (50 mM, pH 7.0), and the cell pellet was isolated by centrifugation. The cell pellet was suspended in 20 % sucrose containing 0.2 M Tris–HCl (pH 7.3) and 5 mM EDTA and shocked in cold water. The shocked cells were subjected to centrifugation, and the supernatant containing periplasmic proteins was collected. The protein concentration was determined according to Lowry et al. [17]. Periplasmic protein profile was studied in sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) which was carried out according to the method of Laemmli [19]. Protein was denatured at 95 °C for 5 min, and 30 μg of each protein per lane was loaded onto a 12 % (w/v) polyacrylamide gel. Gels were stained with silver according to the method of Switzer et al. [20]. GST Assay GST activity in periplasmic protein was determined spectrophotometrically according to the method described by Habig et al. [21] using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate. The assay mixture consists of 0.1 M potassium phosphate buffer, 1 mM CDNB in ethanol, and 1 mM GSH, pH 6.5, at 25 °C. The reaction was initiated by the addition of enzyme solution. Enzyme activity was calculated by measuring the increase in absorbance at 340 nm caused by the formation of thioether in the reaction mixture. Methionine Sulphoxide Reductase Assay The ability of methionine sulphoxide reductase to reduce free methionine sulphoxide was assayed according to the method described by Brot et al. [22]. Briefly, the
Appl Biochem Biotechnol
reaction mixture contained 50 mM PPB (pH 7.5), 20 mM DTT, 1 mM free methionine sulphoxide and enzyme solution. The reaction was carried out at 37 °C for 2 h. The enzyme activity was calculated by measuring the amount of methionine formed, which was estimated as described by Horn et al. [23]. The presence of methionine as the reaction product in the reaction mixture was further confirmed on thin layer chromatography (TLC) plates (Merck, USA) using a solvent system (n-butanol/acetic acid/water, 4:1:5). The methionine spot on TLC using ninhydrin reagent and the Rf values were compared with that of authentic methionine (Sigma-Aldrich).
Results Growth Inhibition Studies The growth inhibitory studies were carried out in mineral salt medium supplemented with sodium succinate as a sole source of carbon and energy. Different solvents were added in different concentrations to the mid-exponential growth phase of Paracoccus sp. SKG cells. The absorbance was recorded 3 h post-exposure to solvents, and respective LD50 values were calculated. The LD50 value of each solvent reflects the respective toxicity on the growth of Paracoccus sp. SKG cells. Figure 1 shows the LD50 values of different organic solvents, with the highest value for DMF (4.2 %) followed by acetonitrile (2.2 %), propionitrile (1.6 %), propanol (1.1 %), trimethylamine (0.7 %), methylamine (0.5 %), dimethylamine (0.4 %) and, least, butanol (0.2 %). Influence of DMF on Membrane Fatty Acid Profile and K+ Efflux
Concentration of sovent (%, v/v)
A significant difference was observed in the degree of saturation between the control and DMF-exposed Paracoccus sp. SKG. Exposure to DMF resulted in the modification of saturated fatty acids (16:0, 17:0, 18:0) to unsaturated fatty acids (16:1Δ 9 cis, 18:1Δ 9 cis). Figure 2 shows that the degree of saturation of fatty acids in the DMFexposed cells (1.49) was considerably high as compared to non-solvent-treated control cells (1.03). Results of the K+ efflux studies gave further supportive evidence to the increased membrane permeability. The increased concentration of solvent resulted in
7 Maximum inhibiƟon concentra on
6 5
LD 50 value
4 3 2 1 0
Organic solvent
Fig. 1 Effect of different organic solvents on the maximum inhibition of growth and LD50 values of Paracoccus sp. SKG at different concentrations
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DMF (%, v/v) Fig. 2 Effect of different concentrations of DMF on the degree of saturation of membrane phospholipid fatty acids (black diamonds) potassium efflux (red up-pointing triangles) and activity of GST (blue squares) on Paracoccus sp. SKG grown on sodium succinate
increased potassium ion efflux. K+ ion efflux from the cells results in decreased K+ concentration inside the cell. The K+ concentration in the non-solvent-exposed cells is 567 nM, which considerably shows that the reduction in K+ in the cells was directly proportional to the increased DMF concentration. Influence of DMF-Induced Toxicity on SOD and Catalase Major adaptive mechanisms against stress include inductions of antioxidative enzymes like SOD and catalase. These enzymes play an important role in the formation of hydrogen peroxide with superoxides and degradation of hydrogen peroxide, respectively. The activities of both enzymes were directly proportional to the concentration of toxins exposed, with highest activities (catalase 0.288 U and SOD 37.93 U) observed in the bacterium exposed to 6 % DMF and least activities observed in non-solvent-exposed cells (catalase 0.027 U and SOD 14.09 U) (Fig. 3). Influence of DMF on Periplasmic Proteins The prominent proteins present in periplasmic membrane are GST and Msr; DMF exposure has a significant effect on the activities of these enzymes in Paracoccus sp. SKG. Solvent exposure resulted in the gradual increase in GST and Msr activities. Maximum activities were observed at 5 % of DMF exposure. Increase in DMF concentration (6 %) witnessed a slight decrease in the activities. Msr activities also showed a similar pattern of induction as that of GST, with highest activities observed in cells exposed to 6 % DMF and lowest witnessed in non-solvent-exposed cells (Fig. 4). The presence of Msr and its activities in periplasmic proteins were confirmed by detecting methionine in the reaction mixture. The extracted reaction product showed an identical retention time (Rf value 0.46) to that of authentic methionine.
Appl Biochem Biotechnol 40 0.30
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Fig. 3 Effect of different concentrations of DMF on the activity of catalase (black squares), SOD (red diamonds) and growth (blue down-pointing triangles) of Paracoccus sp. SKG
Protein Profile Modification To investigate the modification in the periplasmic protein profile induced under different concentrations of DMF in Paracoccus sp. SKG cells, SDS-PAGE was performed. An electrophoregram (Fig. 5) clearly depicts the difference among the protein profile patterns of control and solvent-exposed bacterial cells. The intensities of certain proteins increased with exposure to increased DMF. These results are well correlated with the assayed enzyme activities. The intensity of protein bands gradually increases with increase in DMF concentration up to 5 %, and at 6 %, the intensity of the bands was slightly reduced. The protein band corresponding to the 29-kDa molecular marker band showed significant increase in its intensity in higher DMF concentration.
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DMF (%, v/v) Fig. 4 Effect of different concentrations of DMF on the activity of GST (orange squares) and Msr (blue diamonds) in periplasmic protein of Paracoccus sp. SKG
Appl Biochem Biotechnol
Fig. 5 Periplasmic protein of Paracoccus sp. SKG after exposure to different concentrations of DMF. Lanes 1, 2, 3, 4, 5 and 6 are of DMF 1, 2, 3, 4, 5 and 6 %, respectively. Lane M is molecular markers, lane C is control without DMF
Discussion Organic solvents are known to be toxic to living cells, and this toxicity is mainly due to their mode of action on the membranes and membrane-active compounds [2]. In the present study, we have investigated the response and adaptation of Paracoccus sp. SKG under solvent exposure. DMF exposure resulted in a marked modification in the membrane fatty acid composition of the bacterium, the significant modification being the conversion of saturated to unsaturated fatty acids. Similar results were reported for Ochrobactrum anthropi with atrazine, phenol, 4-chlorophenol and 1-chloro-2,4-dinitrobenzene [24]. Membrane modification in turn resulted in the increased K+ efflux; a strong correlation was observed between the degree of saturation of membrane fatty acid and potassium ion efflux. Neumann et al. [7] also reported identical findings in Pseudomonas putida. The presence of toxic organic compounds causes oxidative stress, mainly by the synthesis of ROS. SOD is an important part of the defence systems against oxidative damage in aerobic organisms [25]. SOD catalyzes the scavenging of superoxide anion (O2−) to O2 and H2O2, which gets reduced to H2O by the H2O2−-scavenging enzyme catalase. In the present study, enhanced activities of SOD and catalase towards tolerance against organic solvent-induced oxidative stress may be attributed to the induction of the defence system in the presence toxin. The effect of different concentrations of DMF on the levels of GST was also investigated; GST are cytosolic or microsomal enzymes which catalyze the conjugation of electrophilic xenobiotics to GSH [26]. Favaloro et al. [24] correlated the increased GST levels in O. anthropi with the influence of toxins to the adaptive cell responses such as increase in fatty acid saturation. All these studies advocate that the concentration-dependent increased level of GST and K+ efflux along with membrane fatty acid modification are the general response mechanism of bacterial cells to any toxins. The GST level in Paracoccus sp. SKG
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increases with increase in concentration of DMF. Armstrong [27] suggested that GST is one of the important factors for determining the sensitivity of the bacterial cells to a broad spectrum of toxins. Along with GST, the levels of Msr in solvent-exposed Paracoccus sp. SKG were also studied. The Msr is an enzyme responsible for catalysis of the reduction of both free and protein-bound methionine sulphoxide residues to methionine restoring their specific biological activities [11]. Both GST and Msr play a significant role in repairing oxidative damage of biomolecules (proteins) and S-thiolated enzymes [27–29]. GST and Msr function along with antioxidant enzymes like catalase and SOD to reduce oxidative stress induced by various toxins. Tamburro et al. [9] describe Msr and GST as important parts of the “oxidative defence system” represented by catalase and SOD. DMF exposure to Paracoccus sp. SKG also yielded results which can be correlated with these observations. The induction of Msr and GST is directly proportional to the increased level of catalase and SOD. In other words, induction of Msr and GST can be attributed to increased ROS level in the cell. The results of periplasmic protein profiling were further carried out by SDS-PAGE and confirmed by the induction of GST and Msr in solvent-exposed cells. Tamburro et al. [9] studied the induction of localized GST and Msr in periplasmic space of bacteria. Among all other proteins, the bands corresponding to 27 and 72 kDa showed concentration-dependent induction in their intensities. Electrophoretic studies in DMF expressed Paracoccus sp. SKG revealed the induction proteins corresponding to 27 and 72 kDa molecular markers in the periplasmic protein profile. Henson et al. [30] purified the GST and determined its molecular weight to be 27 kDa. Similarly, Benjamin et al. [31] purified and determined the Msr molecular weight to be 72 kDa. These observations advocate that the induced protein bands corresponding to 27 and 72 kDa in Paracoccus sp. SKG can be attributed to the induction of GST and Msr in the presence of high concentration of DMF. We have correlated the solvent stress with the bacterial membrane composition modifications and K+ efflux. Additional evidence for the elevated oxidative stress status in the bacterium was provided in terms of induced antioxidative enzyme activities. The periplasmic protein profile provides evidence for the probable involvement of GST and Msr in the bacterial defence system active against organic solvent stress. Because of all these defence mechanisms, Paracoccus sp. SKG could tolerate a high concentration of DMF (6 % v/v). This property can be efficiently explored in bioremediation and biotransformation applications. Acknowledgments The authors thank the University Grants Commission (UGC), New Delhi, India, for the financial support through SAP and Basic Scientific Research (BSR) fellowship programme.
References 1. Sikkema, J., de Bont, J. A. M., & Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiological Reviews, 59, 201–222. 2. Heipieper, H. J., Weber, F. J., Sikkema, J., Keweloh, H., & de Bont, J. A. M. (1994). Mechanisms behind resistance of whole cells to toxic organic solvents. Trends in Biotechnology, 12, 409–415. 3. Isken, S., & Heipieper, H. J. (2002). Toxicity of organic solvents to microorganisms. In G. Bitton (Ed.), Encyclopedia of environmental microbiology (pp. 3147–3155). New York: Wiley.
Appl Biochem Biotechnol 4. Bromley-Challenor, K. C. A., Caggiano, N., & Knapp, S. J. (2000). Bacterial growth on N, Ndimethylformamide: implication for the biotreatment of industrial wastewater. Journal of Industrial Microbiology and Biotechnology, 25, 8–16. 5. Hasegawa, Y., Masaki, M., Yoshinori, S., & Tokuyama, T. (1997). Purification and characterization of N,Ndimethylformamidease from Alcaligenes sp. KUFA-1. Journal of Fermentation and Bioengineering, 84, 543–547. 6. Santoshkumar, M., Veeranagouda, Y., Lee, K., & Karegoudar, T. B. (2011). Utilization of aliphatic nitrile by Paracoccus sp. SKG isolated from chemical waste sample. International Biodeterioration and Biodegradation, 65, 153–159. 7. Neumann, G., Kabelitz, N., & Heipieper, H. J. (2003). The regulation of cis-trans isomerase (CTI) unsaturated fatty acids in Pseudomonas putida: correlation between CTI activity and K+-uptake systems. European Journal of Lipid Science and Technology, 105, 585–589. 8. Veeranagouda, Y., Karegoudar, T. B., Neumann, G., & Heipieper, H. J. (2006). Enterobacter sp. VKGH12 growing with n-butanol as the sole carbon source and cells to which the alcohol is added as pure toxin show considerable differences in their adaptive responses. FEMS Microbiology Letters, 254, 48–54. 9. Tamburro, A., Robuffo, I., Heipieper, H. J., Allocati, N., Rotilio, D., Di Ilio, C., et al. (2004). Expression of glutathione S-transferase and peptide methionine sulphoxide reductase in Ochrobactrum anthropi is correlated to the production of reactive oxygen species caused by aromatic substrates. FEMS Microbiology Letters, 241, 151–156. 10. Vuilleumier, S. (2001). Bacterial glutathione S-transferases and the detoxification of xenobiotics: dehalogenation through glutathione conjugation and beyond. In R. M. Zablotowicz, J. C. Hall, & R. E. Hoagland (Eds.), Pesticide biotransformations in plants and microorganisms (Vol. 777, pp. 240–252). Oxford: Oxford University. 11. Kuschel, L., Hansel, A., Schoherr, R., Weissbach, H., Brot, N., Hoshi, T., et al. (1999). Molecular cloning and functional expression of a human peptide methionine sulfoxide reductase (hMsrA). FEBS Letters, 456, 17–21. 12. Heipieper, H. J., Loffeld, B., Keweloh, H., & de Bont, J. A. M. (1995). The cis/trans isomerization of unsaturated fatty acids in Pseudomonas putida S12: an indicator for environmental stress due to organic compounds. Chemosphere, 30, 1041–1051. 13. Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917. 14. Morrison, W. R., & Smith, L. M. (1964). Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. Journal of Lipid Research, 5, 600–608. 15. Roggenkamp, R., Sahm, H., & Wagner, F. (1974). Microbial assimilation of methanol induction and function of catalase in Candida boidinii. FEBS Letters, 41, 283–286. 16. Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase: improved assay and an assay applicable to polyacrylamide gels. Analytical Biochemistry, 44, 276–287. 17. Lowry, O. H., Rose, B. N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265–275. 18. Neu, H. C., & Heppel, L. A. (1965). The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. Journal of Biological Chemistry, 240, 3685–3692. 19. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the dead of bacteriophage T4. Nature, 227, 680–685. 20. Switzer, R. C., Merril, C. R., & Shifrin, S. (1979). A highly sensitive stain for detecting proteins and peptides in polyacrylamide gels. Analytical Biochemistry, 98, 231–237. 21. Habig, W. H., Pabst, M. J., & Jakoby, W. B. (1974). Glutathione S-transferase; the first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry, 249, 7130–7139. 22. Brot, N., Werth, J., Koster, K., & Weissbach, H. (1982). Reduction of N-acetyl methionine sulfoxide: a simple assay for peptide methionine sulfoxide reductase. Analytical Biochemistry, 122, 291–294. 23. Horn, M. J., Jones, D. B., & Blum, A. E. (1946). Colorimetric determination of methionine in proteins and foods. Journal of Biological Chemistry, 166, 313–320. 24. Favaloro, B., Tamburro, A., Trofino, M. A., Bologna, L., Rotilio, D., & Heipieper, H. J. (2000). Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress. Biochemical Journal, 346, 553–559. 25. Scandalios, J. G. (1993). Oxygen stress and superoxide dismutases. Plant Physiology, 101, 7–12. 26. Bernard, K. M. G., & Margaret, O. J. (2000). Activities of affinity-isolated glutathione S-transferase (GST) from channel catfish whole intestine. Aquatic Toxicology, 49, 27–37. 27. Armstrong, R. N. (1997). Structure, catalytic mechanism, and evolution of the glutathione S-transferase. Chemical Research in Toxicology, 10, 2–18.
Appl Biochem Biotechnol 28. Sheehan, D., Meade, G., Foley, V. M., & Dowd, C. A. (2001). Structure, function and evolution of glutathione transferase: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochemical Journal, 360, 1–16. 29. Levine, R. L., Mosoni, L., Berlett, B. S., & Stadtman, E. R. (1996). Methionine residues as endogenous antioxidants in proteins. Proceedings of the National Academy of Sciences of the United States of America, 93, 15036–15040. 30. Henson, K. L., Gregory, S., & Evan, P. G. (2001). Induction of glutathione S-transferase activity and protein expression in brown bullhead (Ameiurus nebulosus) liver by ethoxyquin. Toxicological Sciences, 62, 54–60. 31. Benjamin, E., Bos, J., Frédéric, B., & Laurent, A. (2005). Methionine sulfoxide reduction and assimilation in Escherichia coli: new role for the biotin sulfoxide reductase BisC. Journal of Bacteriology, 187, 231–237.
