Adaptation of Pseudomonas putida S12 to Ethanol and Toluene at the Level of Fatty Acid Composition of Membranes

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1994, p. 444-4444 99-224/94/$4.+ Copyright ) 1994, American Society for Microbiology Vol. 6, No.

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1994, p /94/$4.+ Copyright ) 1994, American Society for Microbiology Vol. 6, No. 12 Adaptation of Pseudomonas putida S12 to Ethanol and Toluene at the Level of Fatty Acid Composition of Membranes HERMANN J. HEIPIEPER* AND JAN A. M. DE BONT Division of Industrial Microbiology, Department of Food Science, Wageningen Agricultural University, 67 EV Wageningen, The Netherlands Received 5 August 1994/Accepted 26 September 1994 Pseudomonasputida S12 was more tolerant to ethanol when preadapted to supersaturating concentrations of toluene. Cellular reactions at the membrane level to the toxicities of both compounds were different. In growing cells of P. putida S12, sublethal concentrations of toluene resulted in an increase in the degree of saturation of the membrane fatty acids, whereas toxically equivalent concentrations of ethanol led to a decrease in this value. Contrary to this, cells also reacted to both substances with a strong increase of the trans unsaturated fatty acids and a corresponding decrease of the cis unsaturated fatty acids under conditions where growth and other cellular membrane reactions were totally inhibited. While the isomerization of cis to trans unsaturated fatty acids compensates for the fluidizing effect caused by ethanol, a decrease in the degree of saturation is antagonistic with respect to the chemo-physical properties of the membrane. Consequently, the results support the hypothesis that the decrease in the degree of saturation induced by ethanol is not an adaptation mechanism but is caused by an inhibitory effect of the compound on the biosynthesis of saturated fatty acids. Many organic solvents are toxic to organisms because they partition preferentially in membranes, causing an increase in membrane fluidity that leads to an aspecific permeabilization (12, 13, 31, 32). However, microorganisms can adapt to different organic substances, including alcohols (14) and toluene (18, 34). They react to externally dictated (and occurring) changes in their environment by modifying their membranes to keep them in the same fluidity condition. This mechanism is called homeoviscosic adaptation (25, 3). Changes in the fatty acid composition of membrane lipids are the most important reaction of bacteria against membraneactive substances (14, 15, 19). Most bacteria synthesize fatty acids by the well-established anaerobic pathway (24) and are able to change their membrane fluidity only by de novo synthesis of membrane lipids with a different ratio of saturated to cis unsaturated fatty acids during growth (5). As a result, bacteria are not able to perform a postbiosynthetic modification of their membrane fluidity. One exception to this restriction is the recently observed isomerization of cis to trans unsaturated fatty acids in strains of the genera Pseudomonas and Jlibrio (8, 11, 27, 33). The isomerization of the double bond is possibly a special mechanism of these bacteria to react to high concentrations of toxins and increases in temperature, respectively, under conditions which do not allow growth and de novo synthesis of lipids. The benefit of this isomerization is based on the steric differences between cis and trans unsaturated fatty acids. The cis configuration effects a strong increase in membrane fluidity by its bent steric structure, while the trans configuration inserts into the membrane in a fashion similar to that of saturated fatty acids. Therefore, the conversion of cis to trans unsaturated fatty acids reduces the membrane fluidity (23, 27). Both the de novo synthesis of various mixtures of saturated fatty acids to cis unsaturated fatty acids and the cis to trans * Corresponding author. Mailing address: Division of Industrial Microbiology, Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, 67 EV Wageningen, The Netherlands. Phone: Fax: Electronic mail address: Hermann.Heipieper@algemeen.im.wau.nl. isomerization of unsaturated fatty acids are possible in the strain Pseudomonas putida S12 (33). In this paper, we have investigated the effects of both ethanol and toluene on these adaptation mechanisms and demonstrated that isomerization allows a swifter adaptation than the growth-dependent system, which relies on changes in the degree of saturation. MATERUILS AND METHODS Microorganism and culture conditions. P. putida S12 was isolated previously as a styrene-degrading organism (1), but it is not able to degrade toluene. The strain was cultivated in a minimal medium as described by Hartmans et al. (9), with 15 mm glucose as the sole carbon source. Cells were grown in 6-ml shake cultures in a horizontally shaking water bath at 3 C. Adaptation of cells to supersaturating concentrations of toluene (solubility in water at 3 C ca. 6 mg/liter) was done by using cells grown on 6 mm acetate semicontinuously in three batch cultures with increasing concentrations of toluene (1 and 4 mg/liter and 1% [vol/vol]). Measurement of growth and growth inhibition. An inoculum from an overnight culture was transferred to fresh medium. After 3 h of exponential growth, the toxins were added. Cell growth was measured by monitoring the turbidity (optical density at 56 nm) of cell suspensions in a spectrophotometer. Growth inhibition caused by toxins was measured by comparing the differences in the growth rates,,u (hour-1), of poisoned cultures with that of a control culture as described by Keweloh et al. (2). The average results of three identical experiments are shown. The standard derivation was less than 5%. Preparation of resting cells. Sixty milliliters of exponentially growing cells was harvested by centrifugation and suspended in the same volume of sodium phosphate buffer (5 mm, ph 7.) with 15 mm glucose. Experiments were started 45 min after suspension of cells, by which time growth had stopped completely. Inhibition of the fatty acid biosynthesis. To inhibit the de novo synthesis of lipids, 1,ug of cerulenin (Sigma Chemical Co., St. Louis, Mo.) per ml was added to cells harvested in the log phase as described by Diefenbach et al. (6). 444

$.1 A A~~~~~~~~~-- * ;----A a B o "~~~~~o \\o~~' I A 1 2 3 4 5 6 7 8 9 ethanol (% v/v) 1 2 3 4 5 toluene (mg/1) FIG. 1. Effect of ethanol (A) and toluene (B) on the growth rate of P. putida S12.$

2 VOL. 6, 1994 MEMBRANE ADAPTATION OF P. PUTIDA TO ETHANOL (a L- If. 5~~~~~~..1 A A~~~~~~~~~-- * ;----A a B o "~~~~~o \\o~~' I A ethanol (% v/v) toluene (mg/1) FIG. 1. Effect of ethanol (A) and toluene (B) on the growth rate of P. putida S12. Toluene-adapted cells (A, A) had been grown on acetate semicontinuously in three batch cultures with increasing concentrations of toluene (1 and 4 mg/liter and 1% [vol/vol], respectively); nonadapted cells (, *) had been grown on glucose before the addition of ethanol and toluene. Lipid extraction and transesterification. Cells of 45-ml suspensions (about 3 x 11 cells) were centrifuged 3 h after addition of the toxic agents and washed with phosphate buffer (5 mm; ph 7). The lipids were extracted with chloroformmethanol-water as described by Bligh and Dyer (1). Fatty acid methyl esters were prepared by a 15-min incubation at 95 C in boron trifluoride-methanol (26). The fatty acid methyl esters were extracted with hexane. Determination of fatty acid composition. Fatty acid analysis was performed by gas chromatography (with capillary column model CP-Sil 88 [5 m long], a temperature program of 16 to 22 C, and a flame ionization detector). The instrument used was a CP-9 gas chromatograph (Chrompack-Packard). The fatty acids were identified with the aid of standards. The relative amounts of the fatty acids were determined from the peak areas of the methyl esters with a Chromatopac C-R6A integrator (Shimadsu, Kyoto, Japan). Replicate determinations indicated that the relative error [(standard deviation/ mean) x 1%] of the values was 2 to 5%. The average results of three identical experiments are presented. The standard derivation was less than 5%. Even smaller changes in the fatty acid composition, e.g., at lower concentrations of the toxic compounds, were absolutely reproducible. The degree of saturation of the membrane fatty acids was defined as the ratio between the two saturated fatty acids (16: and 18:) and the unsaturated fatty acids (16:1trans, 16:1cis, 18:1trans, and 18:1cis) of the extracts. RESULTS AND DISCUSSION Growth inhibition caused by ethanol and toluene. Cells of P. putida S12 were grown on glucose, and during the exponential growth phase, either ethanol or toluene was added at different concentrations. The organisms continued to grow exponentially but at reduced growth rates (Fig. 1). Compared with nonadapted cells, cells previously adapted to saturated concentrations (1% [vol/vol]) of toluene (34) showed an increased tolerance for all applied concentrations of ethanol (Fig. 1A). Total inhibition of growth (expressed as MIC) occurred at 9% (vol/vol) rather than at 7% (vol/vol) for the unadapted cells. Effect of ethanol and toluene on the membrane composition of growing cells. Cells adapted to supersaturating concentrations of toluene had an increased tolerance for ethanol, and the effect of adaptation to toluene and ethanol at the level of fatty acid composition was investigated. The main fatty acids of this strain when grown on glucose were 16:, 16:1trans, 16:1cis, 18:1trans, and 18:1cis, while 14:, 18:, and the cyclopropane fatty acids 17cyc and l9cyc were present in trace amounts of between.5 and 2%. Cells of P. putida S12 reacted to additions of ethanol and toluene by a conversion of their cis (C16:1i C18:1) to trans unsaturated fatty acids. These ratios of trans to cis unsaturated fatty acids are plotted versus toluene- and ethanol-affected growth rates in Fig. 2A. The degree of saturation of the membrane fatty acids was also affected by toluene, especially at concentrations which were not very toxic (Fig. 2B). The effect of ethanol was contrary to that of toluene. The degree of saturation decreased in the presence of nonlethal concentrations of ethanol. The fatty acid saturation degree was not influenced by either ethanol or toluene at concentrations which totally inhibited growth. A third value showing modifications of the cellular fatty acid composition was the ratio of C18 to C16 fatty acids as shown in Fig. 2C. In the presence of ethanol, the ratio of C18 to C16 fatty acids increased, whereas it decreased after the addition of toluene. Both toluene and ethanol increase the fluidity of the membrane and, consequently, should have the same effect on membranes (16, 17, 22, 31). However, it has been observed that bacteria react to the presence of aromatic compounds with an increase in their degree of saturation (15, 21), while in the presence of ethanol, the degree of saturation decreases (14). Similar reactions have now been found for P. putida S12. This behavior towards ethanol in both Escherichia coli (14) and P. putida S12 is very puzzling. In E. coli, an increase in membrane fluidity due to a higher growth temperature is counterbalanced by decreasing the amounts of 18:1cis and increasing that of 16: fatty acids in the membrane phospholipids (24, 29). The amount of 16:1cis, the third major fatty acid of this bacterium, remains unchanged. This can be explained by the positions in the phospholipids, whereas 16:1cis is found only in position 2. As the temperature is raised, only the fatty acids in position 1 change to 16: fatty acids in the newly synthesized phospholipids, whereas nothing changes in position 2 (24, 29). The shift

$4442 HEIPIEPER AND DE BONT 1.2 1.1 1. o.9.;_ X.8..7 1-.6 cn r_.5.4.3.2 C m(a 4- Co a).8.75.7.65.6.55.5.42.4.38.36 Cs.34 co.32.3 co o.28.26.24.22 I. B A!,~~~~~~~~~~.- c v * * l l s l Xs * a v \\/. 6.5.4.3.2.1. growth rate p (hi) FIG.$

3 4442 HEIPIEPER AND DE BONT o.9.;_ X cn r_ C m(a 4- Co a) Cs.34 co.32.3 co o I. B A!,~~~~~~~~~~.- c v * * l l s l Xs * a v \\/ growth rate p (hi) FIG. 2. Effect of ethanol and toluene on the fatty acid composition of P. putida S12. Different concentrations of ethanol (open symbols) and toluene (closed symbols) were added to glucose-growing cells in the exponential growth phase, causing reduced growth rates. Cells were harvested 3 h after addition of the toxins. (A) Ratio of trans to cis unsaturated fatty acids; (B) degree of saturation of membrane fatty acids; (C) ratio of C18 to C16 fatty acids. from 18:1cis to 16: at higher temperatures can be envisaged since phospholipids containing 18:1cis fatty acids have a transition temperature which is about 64 C lower than those containing 16: fatty acids (4, 27). An opposite reaction is found when the membrane fluidity increases because of ethanol. Now, the amount of 18:1cis does not decrease but rather increases. This behavior is difficult to understand. Possibly, the inhibition of the soluble enzymes of biosynthesis of saturated fatty acids in E. coli caused by ethanol may offer an explanation (2, 5 * 4 3 > ethanol (% v/v) APPL. ENVIRON. MICROBIOL. FIG. 3. Effect of ethanol on the fatty acid composition of resting cells of P. putida S12 in the presence of cerulenin. Ethanol was added to cells 3 h before the lipids were extracted to analyze the total amount of unsaturated fatty acids (A) and of cis () and trans (-) unsaturated fatty acids. 3). Because of this inhibition, the organism would be forced to synthesize unsaturated fatty acids. Otherwise, it seems unlikely that ethanol acts on the two different membrane-located acyl transferases which normally are responsible for adaptive changes in the fatty acid composition (2, 3, 29). Other mechanisms clearly must be in operation to overcome the increased membrane fluidity due not only to the presence of ethanol but also to the resulting shift to the unsaturated fatty acids. Dombek and Ingram (7) found that liposomes, consisting only of phospholipids, from cells grown in the presence of ethanol and therefore with a high content of unsaturated fatty acid showed an increased fluidity. However, the authors also found that intact membranes of ethanol-grown cells had a reduced fluidity (7). This observation would hint at an important role for the protein content of membranes in regulating fluidity. Proteins appear to compensate for the fluidizing effect of the increased unsaturated fatty acid content. Changes in the lipid-to-protein ratio of the membrane have also been found in E. coli cells adapted to phenol (21). Effect of ethanol and toluene on the membrane composition of cells in the presence of cerulenin. Cerulenin is an irreversible inhibitor of,-ketoacyl-acyl carrier protein synthase I and II activities and consequently totally inhibits the de novo biosynthesis of fatty acids (28). More specifically, in P. putida, 1,ug of this compound per ml completely inhibited the de novo biosynthesis of fatty acids, measured as the incorporation of "C-labelled acetate in the lipid fraction (6). The fatty acid composition as affected by ethanol and toluene was investigated in the presence of this inhibitor. No cellular growth occurred in the presence of cerulenin, but nevertheless, a concentration-dependent isomerization of cis to trans unsaturated fatty acids took place (Fig. 3). The sum of the amounts of both the four unsaturated fatty acids (16:1cis, 16:1trans, 18:1cis, and 18:1trans) and all other fatty acids remained constant at all applied ethanol concentrations. Therefore, neither the degree of saturation nor the C18-to-C16 ratio changed under these conditions. The same results could be observed with toluene (data not shown). These observations can be explained by the fact that most bacteria are able to change their membrane composition only by de novo synthesis of membrane lipids during growth (5). They also show that the conversion of cis to

VOL. 6, 1994 MEMBRANE ADAPTATION OF P. PUTIDA TO ETHANOL 4443. co Co@ C- Co 1.2 1.*.8'.6.4 -.2-15 3 45 6 75 9 15 12 time (min) FIG. 4. Kinetics of the conversion of cis to trans unsaturated fatty acids in resting cells of P.

4 VOL. 6, 1994 MEMBRANE ADAPTATION OF P. PUTIDA TO ETHANOL co C- Co *.8' time (min) FIG. 4. Kinetics of the conversion of cis to trans unsaturated fatty acids in resting cells of P. putidas12 as affected by ethanol and toluene. Glucose (15 mm) was supplied as the energy source. Ethanol (8% [vol/vol]) (A) and toluene (5 mg/liter) () were added to totally inhibit cellular growth. trans is not dependent on reactions associated with fatty acid biosynthesis. Kinetics of the isomerization of cis to trans unsaturated fatty acids caused by toluene and ethanol. The kinetics of the cis to trans isomerization as affected by ethanol and toluene were studied with resting cells from glucose-grown cells. Upon the addition of either ethanol or toluene, cells reacted by converting their cis unsaturated fatty acids into the corresponding trans configuration. Figure 4 shows the time course of the conversion of cis to trans unsaturated fatty acids effected by toxic concentrations of toluene (5 mg/liter) and ethanol (8% [vol/vol]). During the incubations, the content of all other fatty acids and the degree of saturation remained constant (data not shown). The reactions of the cells in response to the addition of toluene and ethanol occurred at nearly the same rates. The conversion reached its final trans-to-cis ratio 3 min after the addition of the toxins. The rate of the reaction was not influenced by the concentrations of the compounds, but the final trans-to-cis ratios depended on the dose of the toxins (data not shown). The dose dependency of this isomerization due to toxicity of compounds has also been found for several phenols (6, 11). From results presented in this paper and obtained by other authors (33), it can be concluded that the isomerization of trans to cis unsaturated fatty acids is a mechanism by which P. putida cells can react very quickly against modifications in their environment. REFERENCES 1. Bligh, E. G., and W. J. Dyer A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: Buttke, T. M., and L.. Ingram Effects of ethanol on the Escherichia coli plasma membrane. Mechanism of ethanol-induced changes in lipid composition of Escherichia coli: inhibition of saturated fatty acid synthesis in vivo. Biochemistry 17: Buttke, T. M., and L.. Ingram Mechanism of ethanolinduced changes in lipid composition of Escherichia coli: inhibition of saturated fatty acid synthesis in vitro. Arch. Biochem. Biophys. 23: Cronan, J. E., Jr., and E. P. Gelman Physical properties of membrane lipids: biological relevance and regulation. Bacteriol. Rev. 39: Cronan, J. E., Jr., and C.. Rock Biosynthesis of membrane lipids, p In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 1. American Society for Microbiology, Washington, D.C. 6. Diefenbach, R., H. J. Heipieper, and H. Keweloh The conversion of cis- into trans-unsaturated fatty acids in Pseudomonas putida P8: evidence for a role in the regulation of membrane fluidity. Appl. Microbiol. Biotechnol. 38: Dombek, K. M., and L.. Ingram Effects of ethanol on the Escherichia coli plasma membrane. J. Bacteriol. 157: Guckert, J. B., D. B. Ringelberg, and D. C. White Biosynthesis of trans fatty acids from acetate in the bacterium Pseudomonas atlantica. Can. J. Microbiol. 33: Hartmans, S., J. P. Smits, M. J. van der Werf, F. Volkering, and J. A. M. de Bont Metabolism of styrene oxide and 2-phenylethanol in the styrene-degrading Xanthobacter strain 124X. Appl. Environ. 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Environ. Microbiol. 33: Ingram, L Microbial tolerance to alcohols: role of the cell membrane. Trends Biotechnol. February: Ingram, L Ethanol tolerance in bacteria. Crit. Rev. Biotechnol. 9: Inoue, A., and K. Horikoshi A Pseudomonas thrives in high concentrations of toluene. Nature (London) 338: Keweloh, H., R. Diefenbach, and H. J. Rehm Increase of phenol tolerance of Escherichia coli by alterations of the fatty acid composition of the membrane lipids. Arch. Microbiol. 157: Keweloh, H., H. J. Heipieper, and H. J. Rehm Protection of bacteria against toxicity of phenol by immobilization in calcium alginate. Appl. Microbiol. Biotechnol. 31: Keweloh, H., G. Weyrauch, and H. J. Rehm Phenol induced membrane changes in free and immobilized Escherichia coli. Appl. Microbiol. Biotechnol. 33: Kitagawa, S., F. Kametani, K. Tsuchiya, and H. Sakurai ESR analysis with long-chain alkyl spin labels in bovine blood platelets. Relationships between the increase in membrane fluidity by alcohols and phenolic compounds and their inhibitory effects on aggregation. Biochim. Biophys. Acta 127: MacDonald, P. M., B. D. Sykes, and R N. McElhaney Fluorine-19 nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. A direct comparison of the effects of cis and trans cyclopropane ring and double-bond substituents on orientational order. Biochemistry 24: Magnuson, K., S. Jackowski, C.. Rick, and J. E. Cronan, Jr Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol. Rev. 57: Melchior, D. L Lipid phase transitions and regulation of membrane fluidity in prokaryotes, p In S. Razin and S. Rottem (ed.), Current topics in membrane and transport, vol. 17. Academic Press, Inc., New York. 26. Morrison, W. R, and L. M. Smith Preparation of fatty acid

4444 HEIPIEPER AND DE BONT methyl esters and dimethylacetals from lipids with boron fluoridemethanol. J. Lipid Res. 5:6-68. 27. Okuyama, H., N. Okajima, S. Sasaki, S. Higashi, and N. Murata. 1991.

5 4444 HEIPIEPER AND DE BONT methyl esters and dimethylacetals from lipids with boron fluoridemethanol. J. Lipid Res. 5: Okuyama, H., N. Okajima, S. Sasaki, S. Higashi, and N. Murata The cis/trans isomerization of the double bond of a fatty acid as a strategy for adaptation to changes in ambient temperature in the psychrophilic bacterium Vibrio sp. strain ABE-1. Biochim. Biophys. Acta 184: Omura, S Cerulenin. Methods Enzymol. 72: Rock, C Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli. J. Biol. Chem. 259: Shinitzky, M Physiology of membrane fluidity, vol. II, p CRC Press, Inc., Boca Raton, Fla. 31. Sikkema, J., J. A. M. de Bont, and B. Poolman Interactions APPL. ENVIRON. MICROBIOL. of cyclic hydrocarbons with biological membranes. J. Biol. Chem. 269: Sikkema, J., B. Poolman, W. N. Konings, and J. A. M. de Bont Effects of the membrane action of tetralin on the functional and structural properties of artificial and bacterial membranes. J. Bacteriol. 174: Weber, F. J., S. Isken, and J. A. M. de Bont Cis/trans isomerization of fatty acids as a defense mechanism of Pseudomonas putida strains to toxic concentrations of toluene. Microbiology 14: Weber, F. J., L. P. Ooijkaas, R. M. W. Schemen, S. Hartmans, and J. A. M. de Bont Adaptation of Pseudomonas putida to high concentrations of styrene and other organic compounds. Appl. Environ. Microbiol. 59:

Effect of Environmental Factors on the trans/cis Ratio of Unsaturated Fatty Acids in Pseudomonas putida S12

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1996, p. 2773 2777 Vol. 62, No. 8 0099-2240/96/$04.00 0 Copyright 1996, American Society for Microbiology Effect of Environmental Factors on the trans/cis Ratio