Changes in Membrane Fluidity and Fatty Acid Composition of Pseudomonas putida CN-T19 in Response to Toluene

Transcription

1 Biosci. Biotechnol. Biochem., 66 (9), , 2002 Note Changes in Membrane Fluidity and Fatty Acid Composition of Pseudomonas putida CN-T19 in Response to Toluene In Seon KIM, 1, Jae Han SHIM, 2 andyongtacksuh 2 1 Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2G6, Canada 2 Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, College of Agriculture and Life Science, Chonnam National University, Kwang-Ju , South Korea Received Junuary 28, 2002; Accepted April 1, 2002 A bacterial isolate, Pseudomonas putida CN-T19, could grow in a two-phase medium with toluene up to 50% (v W v). Changes in fatty acid composition and membrane uidity of the isolate were investigated to understand how this microorganism responds toluene. The changes in the ratios of unsaturated to saturated fatty acids were insigniˆcant between cells grown with and without toluene. The changes in the ratio of cis- to trans-fatty acids of C 16:1 and C 18:1 was, however, signiˆcantly lower in cells grown with toluene than cells grown without toluene, giving approximately 1.3 and 9.7, respectively. Toluene had a uidizing ešect on the membrane of cells grown without toluene, resulting in decrease in membrane polarization ratio. Less uidizing ešect of toluene on the membrane of cells grown with toluene was observed, giving 11% of polarization percentage, which was signiˆcantly lower than 53% in cells grown without toluene. These results suggest that ciswtrans isomeration of C 16:1 and C 18:1 makes cell membranes more rigid to respond toluene, and is an adaptive strategy allowing P. putida CN-T19 to grow in the presence of organic solvent. Key words: Pseudomonas; solvent tolerance; fatty acid; cell membrane Microorganisms that could grow in a two-phase medium with organic solvent have been studied extensively due to their potential for bioremediation of contaminated sites and biotransformation of industrial wastes. Most water-immiscible organic solvents with low logp ow (n-octanol-water partition value) are toxic to microorganisms, 1) which has been well documented for toluene. 2) The toxic ešects of organic solvents on microorganisms are loss of cytoplasmic membrane integrity, loss of membrane functions, and cell death. 3) Mechanisms of solvent-tolerant microorganisms to counteract such toxic action of solvent are the changes in membrane lipid composition 4) and cell envelope 5) to modify cell components against solvent, and active eœux system 6) and membrane vehicle formation 7) to eliminate solvent from cells. These mechanisms allow microorganisms to maintain their physiological functions and growth in the presence of solvent. We isolated a toluene-tolerant bacterial strain from an industrial sewage eœuent with the method as described earlier. 8) The isolate is a gram-negative and motile rod, and utilized glucose, manitol, citrate, and N-acetylglucosamine as the carbon source, as determined by standard procedures. 9,10) MIDI fatty acid analysis 11) showed a similar index to Pseudomonas putida strain and, therefore, the isolate was named P. putida CN-T19. This microorganism could grow in a two-phase medium with toluene up to 50z (v W v). P. putida CN-T19couldalsogrowinLuria-Bertani medium (LB) containing cyclohexane or p-xylene, but did not grow in the presence of benzene or butanol. The isolate did not use the solvents as the sole carbon source. P. putida CN-T19 can grow in LB containing an antibiotic at following concentrations: 50 mg W ml choramphenicol, 70 mg Wml rifampicin, 120 mgwml ampicillin, 30 mgwml gentamycin, 50 mg W ml nalidixic acid, and 5 mg Wml tetracyclin. Changes in membrane uidity and fatty acid composition were investigated to understand how P. putida CN-T19 responds toluene. For membrane uidity assay and fatty acid analysis, P. putida CN- T19 was grown to a mid-exponential phase at 309Cin Luria-Bertani medium (LB) with or without toluene by shaking at 200 rpm. Cells were harvested by centrifugation at 6,400 g for 10 min and washed twice with 50 mm potassium phosphate bušer, ph 7.0, (`bušer' hereafter) before assay. Membrane uidity was investigated by measuring the uorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH, Molecular Probes, Eugene, OR, USA) inserted into the membranes with the method as described previously. 12) Brie y, cells grown on LB with or without toluene were harvested as described To whom correspondence should be addressed. Current address: Department of Environmental Science and Engineering, Kwangju Institute of Science and Technology (K-JIST) 1 Oryong Dong, Puk-Gu, Kwangju , South Korea

2 1946 I.S.KIM et al. Fig. 1. Temperature-dependent Fluorescence Polarization of DPH in Pseudomonas putida CN-T19 Cells Grown in Luria- Bertani Medium at 309C. Cells were harvested, washed, and incubated in the absence ( ) or presence ( ) of toluene before DPH was added. Results are the means of three independent determinations±sd. Fig. 2. Temperature-dependent Fluorescence Polarization of DPH in Pseudomonas putida CN-T19 Cells Grown in Luria- Bertani Medium with Toluene (10z, vwv) at 309C. Cells were harvested, washed, and then incubated in the absence ( ) orpresence( ) of toluene before DPH was added. Results are the means of three independent determinations± SD. above, and resuspended in the same bušer to give an optical density of 0.4 at 610 nm (OD 600). To assess ešects of toluene on the DPH uorescence polarization in vitro, toluene was added to cell suspension before DPH probe was added. The concentration of toluene used for DPH uorescence polarization assay was within aqueous soluble range. The suspension was then incubated for 10 min with shaking at the same temperature used for the membrane uidity assay and DPH in tetrahydrofuran (1 ml) was added to the cell suspension to give a ˆnal concentration of 3 mm. The cell suspension was mixed by magnetic stirring at 200 rpm in the dark for 20 min at the same temperature used for the membrane uidity assay. Toluene decreased in vitro the DPH uorescence polarization ratios of P. putida CN-T19 cells grown without toluene (Fig. 1). When the phase transition temperature was determined by estimating midpoint of temperature, 3 mm toluene lowered the temperature from approximately 24 to 209C. DPH partitions within the membrane hydrophobic core, where it provides information on the uid state of this region. 12,13) Therefore, decrease in polarization ratios by toluene suggested that toluene had a uidizing ešect on the hydrophobic core region of cell membranes. Toluene had less uidizing ešect on the membranes of P. putida CN-T19 cells grown with toluene (Fig. 2). The polarization pattern of cells grown with toluene was dišerent to that of cells grown without toluene, representing higher transition temperature (approximately 289C) than 249C in cells grown without toluene. When toluene was added to the suspension of cells grown with toluene and then incubated for polarization assay as described above, the membrane transition temperature was approximately 269C, giving higher temperature than 209C in cells grown without toluene. These ˆndings indicate that the membranes of cells grown with toluene are less uid as compared to those of cells grown without toluene. The ešect of toxic compounds on bacterial membranes has been demonstrated in many other studies in term of changes in membrane lipid composition, with the hypothesis that these changes would ašect the membrane uidity. We assessed the ešect of toluene on membranes by membrane uidity assay using spectro uorometery. Our results suggested in more detail that toluene had a considerable uidizing ešect on bacterial membranes. The uorescence polarization assay showed the dišerences in membrane status between cells grown with and without toluene. These dišerences were suggested to be the result of an adaptive mechanism of P. putida CN- T19 to respond toluene. The addition of membrane-active phenols to P. putida P8 changes the membrane uidity. 14,15) This could kill the cells by causing eœux of cell metabolites and ions from the cell interior. The phenoladapted cells of P. putida P8 showed less eœux of K + ion than non-adapted cells, which was due to the change in the membrane structure to respond phenol. 14,15) Toluene has a high partitioning action into cell membranes, because this solvent is very lipophilic. The hydrophobic membrane probe DPH would partition more into cells once membrane structures become more uid by addition of toluene, giving the lower uorescence value in vitro (Fig. 1). Cells

3 Solvent-tolerant Bacteria grown with toluene had higher polarization ratio than cells grown with toluene, which suggested that the membranes of cells grown with toluene were more rigid, making them more resistant to toluene. By observations together with other results, our results suggested that less uidizing ešect of toluene and increased membrane transition temperature in cells grown with toluene were due to an adaptive change in membranes. We examined the change in the fatty acid composition of membrane of cells grown with or without toluene. Membranes of P. putida CN-T19 cells grown with or without toluene were isolated and obtained by passing them twice through a French press and ultra-centrifugation with a sucrose density gradient as described previously. 16,17) Membrane lipids were extracted with a mixture of chloroform:methanol (2:1, v Wv) by the method of Bligh and Dyer, 18) and back-washed twice with 0.9z NaCl. The chloroform-soluble fraction was removed and concentrated to approximately 0.5 ml under a gentle stream of nitrogen gas. The fatty acids were then methyl-esteriˆed by heating them in Te on-lined screw-cap tube with 2 ml of 14z BF 3-methanol at 809C for 1.5 h as described previously. 19) The resulting fatty acid methyl esters (FAMEs) were extracted twice with two volumes of a mixture of hexane:methyl-tert-butyl ether (1:1, v Wv), and then backwashed with 0.3 M NaOH. The organic phase was removed and concentrated under a gentle stream of nitrogen gas. The fatty acid esters were analyzed by gas chromatograph (Hewlett-Packard model 5890A), comparing to the standard. The oven temperature was programmed to increase from 100 to 2709C at 109C per minute, and the injector and ame ionizationdetector(fid)werekeptat250and3009c, respectively. The analytical column was HP-5 capillary column (0.25 mm i.d. 25 m length, Hewlett-Packard, Avondale, PA) and the ow rate of helium as the carrier gas was 1.0 ml Wmin. Fatty acids were further analyzed by gas chromatography-mass spectrometry (GC-MS). The major fatty acids identiˆed by GC and GC-MS analysis were C 16:0, cis-c 16:1, trans-c 16:1, C 18:0, cis- C 18:1,andtrans-C 18:1 (Table 1). Hexadecanoic acid (C 16:0) was main saturated fatty acid, accounting for almost 40z of the total fatty acids in cells grown without toluene and up to about 43z of the total fatty acid in cells grown with toluene. Slight higher composition of C 16:0 was observed cells grown with toluene than that in cells grown without toluene. The changes in the ratios of the saturated fatty acids to unsaturated fatty acids were insigniˆcant between cells grown with and without toluene, giving 0.7 or 0.8 of the ratio, respectively. However, the changes in the ratios of cis- totrans-fatty acids of C 16:1 and C 18:1 were signiˆcantly dišerent between cells grown with and without. The ratio of the cis- totrans-fatty 1947 Table 1. Lipid Composition of Pseudomonas putida CN-T19 Grown in Luria-Bertani Medium with or without Toluene Fatty acid z (vwv) of toluene added Growth temperature Fatty acid content (z) 16: cis-16: trans-16: : :2 ND* ND ND ND 6.9 cis-18: trans-18:1 ND ND satwunsat** ciswtrans 9.7 (5.2)*** 1.2 (0.8)*** 1.3 (0.8)*** 1.3 (0.9)*** * Not detectable. ** sat, saturated fatty acid; unsat, unsaturated fatty acid. *** cis-c 16:1 to trans-c 16: (2.6)*** acids in cell grown with toluene was up to 1.3, considerably lower than the ratio of 9.7 of cells grown without toluene. These results indicate that P. putida CN-T19 cells grown with toluene have more transfatty acids in their membranes. The C 17:0,cyclopropane is one of fatty acid responding toluene in other solventtolerant P. putida strains. 4,20) In our study, the greatest changes in fatty acid composition of P. putida CN-T19 cells grown with toluene were transforming of cis-c 16:1 and -C 18:1 to their trans isomers instead of incorporation of C 17:0,cyclopropane into their membrane lipids. We investigated if the changes in fatty acid composition of cells grown with toluene at 309C resembled those of cells grown without toluene at a higher temperature, because toluene induced a uidizing ešect on membranes. For this, cells were grown without toluene at 379C and their membrane fatty acids were assessed as described above. The main fatty acid of cells grown on without toluene at 379C wasc 16:0, accounting for almost 40z of the total fatty acids. Increase in growth temperature decreased the total ratio of cis- totrans-fatty acid. The ratio of cis- to trans-fatty acid in cells grown without toluene at 379C was 4.6, which was lower than 9.7 in cells grown without toluene at 309C and higher than 1.3 of cells grown with toluene at 309C. Additional unsaturated fatty acid of C 18:2 in cells grown without toluene at 379C was observed. A particularly, large change in fatty acid composition of cis- W trans-fatty acid of C 16:1 was observed in cells grown without toluene at 379C. The ratio of cis- totrans-fatty acid of C 16:1 in cells grown without toluene at 379C was 2.6, which was lower than 5.2 of cells grown without toluene at 309C and higher than 0.9 of cells grown with

4 1948 I.S.KIM et al. toluene at 309C. The changes in the ratios of saturated to unsaturated fatty acids in cells grown without toluene at 379C were insigniˆcant. The change in the composition of C 18:0 in cells grown without toluene at 379C was higher than the others examined. Microorganisms that can respond an increase in growth temperature show the changes in the lipid composition of membranes in order to compensate for a uidizing ešect of the increased temperature. The uidizing ešect of the increased temperature is likely similar to that of lipophilic compounds. Therefore, the pattern of changes in the fatty acid compositions in response to the increased temperature would resemble those of cells grown with toluene. When P. putida CN-T19 cells were grown without toluene at 379C, however, the changes in fatty acid proˆle did not resemble those of cells grown with toluene at 309C. Compared with the number of studies on the changes in the fatty acid composition to respond a uidizing ešect, there are few study that examines toluene-tolerant P. putida strains for their adaptive mechanism to the increased growth temperature and to toluene. It has been reported that dišerent adaptive mechanism allows dišerent strains to survive in a variety environments. 21) The data in our study show the P. putida CN-T19 able to adapt to the increased growth temperature by increasing the compositions of trans-fatty acid of C 16:1 and saturated fatty acid C 18:0. The adaptive response of P. putida CN-T19 by cis W trans isomerization of C 16:1 would be unspeciˆc mechanism, because the isomerization of this fatty acid could be activated by not only toluene but also by the increased temperature. Nevertheless, our ˆndings suggest that P. putida CN-T19 can react against a uidizing ešect of the increased temperature by dišerent mechanism from adaptive reaction to the uidizing ešect of toluene. The cis W trans isomerization of double-bond unsaturated fatty acids is a strategy of microorganisms for adaptation mechanism to toxic solvents. 3) Such isomerization is rapid and short-term adaptive response for solvent-tolerant bacteria reacting to solvent. 4) This mechanism makes membranes more rigid, resulting in the increased microbial resistance to the partition action of the solvent. 14,15,22) P. putida IH-2000 mutant unable to transform cis-totrans-fatty acids had more hydrophobic cell envelope, resulting in more sensitivity to solvent. 23) The changes in the cell envelop by the mutation of ciswtrans isomerization also resulted in OprL protein being lacking so that the cells were killed by a very low concentration of solvent. 4) The ciswtrans isomerase is encoded by cti gene in P. putida DOT-T1E. 24) The cti-null mutant strain of P. putida DOT-T1E showed the increased solvent mortality due to the deˆciency of trans-fatty acid. Cis W trans isomerization is mediated by a cytochrome c type protein. The mutant lacking the enzyme activity had the remarkable reduction in ciswtrans isomerization. 25) From all of these studies, ciswtrans isomerization is seems to be the most important mechanism that allows microorganisms to survive in toxic environments. P. putida CN-T19 cells can grow in a twophase medium with a membrane toxic-solvent such as toluene, p-xylene, and cyclohexane. The adaptive mechanism by cis W trans isomerization was a good explanation for how this microorganism can survive such solvent environments. The cis-isomer has a kink in the acyl-chain of the fatty acids, which causes steric hinderance in membrane lipids, giving high membrane uidity. In contrast, the long and extended steric structure of the trans-isomer has a similarity to saturated fatty acids that cause membranes to be less uid. The adaptation mechanism of P. putida CN- T19 is believed to prevent toluene from accumulating in membranes, allowing cells to grow in a high toluene environment. The data of the changes in fatty acid proˆles suggest dišerent uidizing ešects with toluene or with the increased temperature. One question about solvent-tolerant bacteria is whether they can return adaptive changes back to their original growth condition. To answer this, we examined readaptation of P. putida CN-T19 cells to the absence of toluene by measuring uorescence polarization and the fatty acid composition. Cells grown with toluene at 309C to a mid-exponential phase were transferred (1z, v Wv, for inoculum) to LB without toluene. Cells were grown to a mid-exponential phase and transferred again (1z, vwv, for inoculum) to fresh LB without toluene. This transfer was repeated until cells were grown without toluene for about 95 generations. The number of generations was calculated by the number of doublings in the cell numbers assessed by enumerating colony-forming units of serially diluted cultures on LB plates with ampicillin (50 mg Wml). Periodically, membrane uorescence polarization and fatty acid composition were also measured for comparison. These examinations were also done in cells grown without toluene at 379C. Toluene ašected the membranes of cells grown without toluene, resulting in a large initial polarization percentage of 53z (Fig. 1). The percentage was calculated by the equation: [(control-sample) W control] 100, where control and sample denote the polarization ratios of P. putida CN-T19 cells incubated without toluene and with 3 mm toluene at the same temperature used for growing cells, respectively, before DPH was added to the cell suspensions. Therefore, high percentages indicate high membrane uidity, and vice versa. The membranes from cells grown with toluene had low starting value of 11z (Table 2). When cells grown with toluene were transferred into LB without toluene, the low percentage was maintained for at least 71 generations. After 87 generations, the percentage returned to the same

5 Solvent-tolerant Bacteria 1949 Table 2. Changes in the Ratios of ciswtrans Isomerization of C 16:1 and C 18:1 Fatty Acids and Membrane Polarization Percentage of Peudomonas putida CN-T19 During Readaptation Experiment Ratios Number of generations Polarization percentage* ciswtrans * The ratios were calculated by the equation [(control-sample) W control] 100, where control and sample denote polarization ratios of P. putida CN-T19 cells incubated without toluene and with 3 mm toluene at the same temperature used for growing cells, respectively, before DPH was added. value with that of cells grown originally without toluene. When cells grown with toluene were ˆrst transferred into LB without toluene, the ratio of cis- to trans-fatty acids was 1.3. This ratio increased steadily with the increased number of generations and returned to the ratio of 8.2 by 71 generations, similar to 9.7 of cells grown originally without toluene. It took 71 to 87 generations for cells grown with toluene to complete readaptation. In contrast, the readaptation of cells grown without toluene at 379C took only about six generations (data not shown). This dišerent in the number of generations needed for readaptation suggests that growth in the presence of toluene results in much more prolonged changes on membrane structure than does the growth temperature change. In this experiment, we studied the adaptive mechanism of P. putida CN-T19 to counteract toluene. The results are consistent with previous observations that the change in the ratio of cis- totransfatty acids is an adaptive mechanism in microorganisms that can grow in the presence of solvent. 4,14,15,20) Our results show that toluene ašects bacterial cells in more ways than its simply having uidizing ešects on membranes. References 1) Inoue, A., and Horikoshi, K., A Pseudomonas thrives in high concentration of toluene. Nature (London), 338, (1989). 2) DeSmet,M.J.,Kingma,J.,andWitholt,B.,The ešect of toluene on the structure and permeability of the outer and cytoplasmic membranes of Escherichia coli. Biochim. Biophys. Acta, 506, (1978). 3) Sikkema, J., De Bont, J. A. M., and Poolman, B., Mechanisms of membrane toxicity of hydrocarbons. Microbiol. Rev., 59, (1995). 4) Ramos, J., Duque, E., Rodriguez-Herva, J. J., Godoy, P., and Fernandez-Barrero, A., Mechanisms for solvent tolerance in bacteria. J. Biol. Chem., 272, (1997). 5) Aono, R., and Kobayashi, H., Cell surface properties of organic solvent-tolerant mutant of Escherichia coli K-12. Appl. Environ. Microbiol., 63, (1997). 6) Li,X.Z.,Zhang,L.,andPoole,K.,Roleofthe multidrug eœux system of Pseudomonas aeruginosa in organic solvent tolerance. J. Bacteriol., 180, (1998). 7) Kobayashi, H., Uematsu, K., Hirayama, H., and Horikoshi, K., Novel toluene elimination system in a toluene-tolerant microorganism. J. Bacteriol., 182, (2000). 8) Park, H. S., Lim, S. J., Chang, Y. K., Livingston, A. G., and Kim, H. S., Degradation of chloronitrobenzenes by a coculture of Pseudomonas putida and a Rhodococcus sp. Appl. Environ. Microbiol., 65, (1999). 9) Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T., and Williams, S. T., In ``Bergey's manual of determinative bacteriology'', The Williams & Wilkins Co., Baltimore, MD (1994). 10) Smibert, R. M., and Krieg, N. R., Phenotype characterization, p In Gerhardt, P., Murray, R. G. E., Wood, W. A., and Krieg, N. R. (ed.), Methods of general and molecular bacteriology. American Society for Microbiology, Washington, D. C. (1994). 11) Sasser, M., Technical note 102: tracking a strain using the microbial identiˆcation system. MIS Inc., North Newark, Del. (1990). 12) Kim, I. S., Lee, H., and Trevors, J. T., EŠects of 2,2?,5,5?-tetrachlorobiphenyl and biphenyl on cell membranes of Ralstonia eutropha H850. FEMS Microbiol. Lett., 200, (2001). 13) Sikkema, J., de Bont, J. A. M., and Poolman, B., Interaction of cyclic hydrocarbon with biological membranes. J. Biol. Chem., 18, (1994). 14) Heipieper, H. J., Diefenbach, R., and Keweloh, H., Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenoldegrading Pseudomonas putida P8 from substrate toxicity. Appl. Environ. Microbiol., 58, (1992). 15) Diefenbach, R., Heipieper, H. J., and Keweloh, H., The conversion of cis in trans unsaturated fatty acids in Pseudomonas putida P8: evidence for a role in the regulation of membrane uidity. Appl. Microbiol. Biotechnol., 38, (1992). 16) Herring,F.G.,Krisman,A.,Sedgwick,E.G.,and Bragg, P. D., Electron spin resonance studies of lipid uidity changes in membranes of an uncouplerresistant mutant of Escherichia coli. Biochim. Biophys. Acta, 819, (1985). 17) Souzu, H., Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of dišerent growth phases. Biochim. Biophys. Acta, 861, (1986). 18) Bligh, E. G., and Dyer, W. J., A rapid method of total lipid extraction and puriˆcation. Can. J. Biochem. Physiol., 37, (1959). 19) Morrison, W. R., and Smith, L. M., Separation of fatty acid methyl esters and dimethylacetals from lipids with boron uoride-methanol. J. Lipid Res., 5, (1964). 20) Weber, F. J., Isken, S., and de Bont, J. A. M., Cis W trans insomerization of fatty acid as a defense mechanism of Pseudomonas putida strains to toxic

6 1950 I.S.KIM et al. concentrations of toluene. Microbiology, 140, (1994). 21) Fang, J., Barcelona, M. J., and Alvarez, P. J. J., Phospholipid compositional changes of ˆve pseudomonad archetypes grown with and without toluene. App. Microbiol. Biotechnol., 54, (2000). 22) Isken, S., and de Bont, J. A. M., Active eœux of toluene in a solvent-resistant bacterium. J. Bacteriol., 178, (1996). 23) Kobayashi, H., Takami, H., Hirayama, H., Kobata, K., Usami, R., and Horikoshi, K., Outer membrane changes in a toluene-sensitive mutant of toluenetolerant Pseudomonas putida IH J. Bacteriol., 181, (1999). 24) Junker, F., and Ramos, J. L., Involvement of the cisw trans isomerase Cti in solvent resistance of Pseudomonas putida DOT-T1E. J. Bacteriol., 181, (1999). 25) Holtwick, R., Keweloh, H., and Meinhardt, F., CisW trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: Evidence for a heme protein of the cytochrome c type. Appl. Environ. Microbiol., 65, (1999).