APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1992, p. 1847-18520099-2240/92/061847-06$02.00/0Copyright ©) 1992, American Society for Microbiology

Conversion of cis Unsaturated Fatty Acids to trans, a PossibleMechanism for the Protection of Phenol-DegradingPseudomonas putida P8 from Substrate Toxicity

HERMANN-JOSEF HEIPIEPER, RUTH DIEFENBACH, AND HERIBERT KEWELOH*

Department of Microbiology, University of Muenster, Corrensstrasse 3, D-4400 Muenster, Gernany

Received 10 October 1991/Accepted 16 March 1992

A trans unsaturated fatty acid was found as a major constituent in the lipids ofPseudomonas putida P8. Thefatty acid was identified as 9-trans-hexadecenoic acid by gas chromatography, argentation thin-layerchromatography, and infrared absorption spectrometry. Growing cells of P. putida P8 reacted to the presence

of sublethal concentrations of phenol in the medium with changes in the fatty acid composition of the lipids,thereby increasing the degree of saturation. At phenol concentrations which completely inhibited the growth ofP. putida, the cells were still able to increase the content of the trans unsaturated fatty acid and simultaneouslyto decrease the proportion of the corresponding 9-cis-hexadecenoic acid. This conversion of fatty acids was alsoinduced by 4-chlorophenol in nongrowing cells in which the de novo synthesis of lipids had stopped, as shownby incorporation experiments with labeled acetate. The isomerization of the double bond in the presence ofchloramphenicol indicates a constitutively operating enzyme system. The cis-to-trans modification of the fattyacids studied here apparently is a new way of adapting the membrane fluidity to the presence of phenols,thereby compensating for the elevation of membrane permeability induced by these toxic substances.

The biodegradation of hazardous substances in wastewa-ters or contaminated soils is often limited by the antimicro-bial action of the pollutants. An increase in cell toleranceagainst toxic substrates can crucially improve the degrada-tion capabilities of the concerned microorganisms. Manystudies demonstrate that degradation rates are diminished athigh concentrations of the toxic substrates and lag phases ofa considerable length precede the removal of these sub-stances (2, 3, 20). The degradation process can often bestrongly accelerated by an adaptation to the toxin, e.g., tophenol (28). Explanations for this phenomenon are theinduction of degradative enzymes and growth-related bio-mass changes; alterations of the structure and dynamics ofthe cells themselves are also possible. It is known that manypollutants like phenolic substances affect the integrity andfunctioning of the cell membrane (10, 13). The tolerance ofEscherichia coli cells to phenols can be increased by struc-tural modifications of the membrane like the fatty acidcomposition of lipids (17). The influence of phenols on thisessential cell component has never been studied with organ-isms like Pseudomonasputida P8, which have the capabilityof degrading these compounds. The dual character of phenolfor this bacterium, as a source of growth and as a growth-inhibiting toxin, leads apparently to the acquisition of specialmechanisms of cell protection as shown in this report.

MATERIALS AND METHODS

Microorganism and culture conditions. P. putida P8 was

isolated from phenol-contaminated wastewater (3). Thestrain was cultivated in a minimal medium which contained(per liter): sodium succinate (4 g), (NH4)2SO4 (1.06 g),MgSO4 (0.1 g), KCl (0.74 g), K2HPO4 (2 g), FeSO4 (1.6 mg),and 1 ml of a trace element solution (27). The pH was set to6.0 before sterilization.

Cells were grown in 50-ml shake cultures at 30°C. In the

* Corresponding author.

case of adapted cells, strain cultivation was done on agarplates with minimal medium containing 0.5 g of phenol perliter as the sole carbon source instead of succinate. For themain cultures, the minimal medium with succinate was

always used. Growing cells were harvested in the late-logphase. For the measurement of K+ efflux, potassium hydro-gen phosphate was replaced by equimolar amounts of thesodium salt and the KCl concentration was reduced to 1mM.

Preparation of nongrowing cells. Fifty milliliters of expo-nentially growing cells was harvested by centrifugation andsuspended in the same volume of sodium phosphate buffer(50 mM, pH 7.0) with or without 4 g of sodium succinate perliter. The cell suspensions were gently shaken in a gyratorywater bath shaker for 45 min before the experiments were

started. Chloramphenicol (Sigma, Deisenhofen, Germany)was added shortly after the suspension of cells from an

ethanolic stock solution to a final concentration of 50 mg/liter. This concentration was sufficient to inhibit the growthof P. putida P8 completely.

Lipid extraction and analysis. Cells of 45-ml suspensions(about 3 x 1010 cells) were sedimented 3 h after addition ofthe phenolic agent and washed with saline. The lipids were

extracted with chloroform-methanol-water as described byBligh and Dyer (4). Fatty acid methyl esters were preparedby a 15-min incubation at 95'C in boron trifluoride-methanolby the method of Morrison and Smith (24).A determination of the fatty acid composition was per-

formed by using gas chromatography (GC) (capillary columnDB 225, 30 m and 180'C, with a flame ionization detector).The instrument used was a GC-14A gas chromatograph(Shimadzu, Kyoto, Japan). The second identification of thetrans unsaturated fatty acids employed a GC-9A gas chro-matograph (Shimadzu) with an apolar capillary column (DB5, 30 m and 180°C, with a flame ionization detector). Thefatty acids were identified with the aid of standards. Therelative amounts of the fatty acids were determined from thepeak areas of the methyl esters by using a Chromatopak

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800 1000 1200 1400

wave number (cm'1)FIG. 1. Infrared absorption spectroscopy of fatty acid methyl esters from total lipids of P. putida P8 separated by silver nitrate TLC.

Spectra were taken from the fraction detected as trans unsaturated fatty acids (b) and as cis-unsaturated fatty acids (d) by comparison withstandards. Methyl esters of the authentic standard represent 16:1 9trans (a) and 16:1 9cis (c).

C-R4A integrator (Shimadzu). Replicate determinations in-dicated that the relative error, calculated as [standard devi-ation/mean] x 100, of the values was 2 to 5%.

Argentation chromatography and infrared absorption spec-trometry. For the verification of trans and cis configurationsof double bonds in the unsaturated fatty acids, thin-layerchromatography (TLC) was carried out on Silica Gel 60plates (Merck, Darmstadt, Germany) impregnated with sil-ver nitrate (7). The plates were developed at -20°C with asolvent system composed of chloroform and ethanol (99:1,vol/vol). After drying, the entire plate was sprayed with0.01% primuline in acetone-water (4:1, vol/vol) and the spotsof fatty acid methyl esters were detected under UV light.The separated methyl esters were extracted from the appro-priate regions of the silica gel by chloroform-methanol (1:1,vol/vol). The configuration of the double bond was deter-mined by infrared absorption spectrometry (Perkin-Elmer1420 infrared spectrophotometer) using samples prepared inpotassium bromide discs (6).

Analysis of de novo lipid synthesis. In order to examine thede novo synthesis of lipids, 120 nCi of sodium [1-14C]acetate(48.9 mCi/mmol; Sigma) was added to 1 ml of growing ornongrowing cells containing equal amounts of cells. In caseof the nongrowing cells, the labeled acetate was added at thesame time that 4-chlorophenol was used to start the conver-sion of the fatty acids. After incubation for 2 h at 30°C, thecells were harvested and lipids were extracted as describedabove. The radioactivity of extracted lipids recovered in thechloroform layer was determined by liquid scintillation spec-trometry. Amounts of lipids synthesized de novo wereexpressed in nanomoles of labeled acetate found in thechloroform layer.Measurement of cellular K+ content. One-milliliter samples

were withdrawn before and after addition of the phenoliccompound. Separation of cells from the supernatant wascarried out by rapid centrifugation through silicone oil asdescribed by Bakker and Mangerich (1). The cell pellet was

disrupted by boiling in 5% trichloroacetic acid, and debriswas removed by centrifugation. The released K+ of thesupernatant was measured by flame photometry in an SP9atomic absorption spectrophotometer (Pye Unicam, Cam-bridge, Great Britain).

RESULTS

Identification of fatty acids in P. putida P8. The lipids of P.putida P8 were extracted, and the methyl esters of the fattyacids were subjected to silver nitrate TLC. We separatedthree fractions, which corresponded to saturated (fastest-running band), cis monounsaturated (slowest-running band),and trans monounsaturated fatty acids, as judged from acomparison of their Rf values with those of standards (datanot shown).To verify the trans and cis configurations, the fatty acid

methyl esters were eluted from the corresponding regions ofthe silica gel and subjected to infrared absorption analysis(Fig. 1). The spectrum of the tentatively trans unsaturatedfatty acid showed a peak at 966 cm-1 which is characteristicof a trans double bond. This peak was missing in theotherwise very similar absorption spectrum of the fractionidentified by TLC as fatty acids with a cis configuration.The common existence of trans unsaturated fatty acids

and the corresponding cis compounds in the lipids of P.putida P8 was documented by GC (Fig. 2). Palmitic acid(16:0), palmitelaidic acid (16:1 9trans), palmitoleic acid (16:19cis), and cis-vaccenic acid (18:1 llcis) were identified asmajor fatty acids of this bacterium. trans-Vaccenic acid (18:1lltrans), a second fatty acid with a trans configuration, andalso myristic acid (14:0), stearic acid (18:0) and the cyclo-propane fatty acids 17cyclo and l9cyclo could be detected intrace amounts of between 0.5 and 2%. Because of the factthat the occurrence of trans unsaturated fatty acids has beenreported only relatively seldom, their identities were con-firmed by using a second GC column and method as de-

966

a

bC

d

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P. PUTIDA CONVERSION OF cis FATTY ACIDS TO trans 1849

- 0

I

-

E1-I0E

U)0

4J0L)S.

-3

4

- 516:1 cis

-717 cyclo

18:018:1 trans

- 9 18: 1 cis

19 cyclo

FIG. 2. Chromatogram of capillary GC (column, DB 225) of fatty acid methyl esters of P. putida P8. Cells grown in minimal medium withsodium succinate as the carbon source were harvested 2 h after the addition of 300 mg of 4-chlorophenol per liter.

scribed in Materials and Methods. GC analysis also showedthat the fraction of the argentation chromatography identi-fied as trans unsaturated fatty acids consisted of 16:1trans(99%) and 18:1trans.

Fatty acid compositions of cells adapted or not adapted tophenol. Figure 3 shows the relative amounts of the majorfatty acids of cells adapted and nonadapted to phenol atdifferent phenol concentrations. The proportion of 16:0 inthe lipids of cells adapted to phenol as the sole growthsubstrate was significantly higher than in nonadapted cells,while the content of 18:1cis was decreased in adapted cells.When various phenol concentrations were added to grow-

ing cells of P. putida P8, the fatty acid composition of thesecells was changed in the following manner. The contents of16:0 and 16:1trans increased and 16:1cis and 18:1cis de-creased in cells in which the phenol concentration permittedcontinuation of a retarded growth.Only after the addition of 1.25 g of phenol per liter to

nonadapted cells was the growth completely blocked; thesecells showed contents of 16:0 and 18:1cis, like the controlcells. However, their content of 16:1trans was markedlyincreased and that of 16:1cis was decreased.The fatty acid composition of the membranes was simi-

larly modified when 4-chlorophenol instead of phenol wasadded to growing cells (data not shown). Cells of P. putidaP8 can apparently change their cis-to-trans unsaturated fattyacid ratio by an unknown mechanism. A modification of themembrane composition by biosynthesis of new lipids cannotbe expected when high concentrations of phenolic sub-stances completely block the propagation of cells.Membrane permeability of cells in the presence of phenol.

Changes in the fatty acid composition induced by phenolmay also affect the permeability of the cell membrane. Thismembrane property was studied by using K+ ions as anindicator. Phenol was added to adapted and nonadaptedgrowing cells and caused a concentration-dependent K+efflux. At lower concentrations of phenol, this efflux wasreversible and the cells could restore their K+ gradientsacross the membrane in 30 to 60 min. At higher and thereforemore toxic concentrations, the K+ efflux was irreversible, as

shown in Fig. 4. Adapted cells which had a higher degree ofsaturation of their membrane lipids (Fig. 3) restored the K+gradient across the membrane up to phenol concentrationsof 750 mg/liter. On the other hand, the nonadapted cells werenot able to do this at phenol concentrations higher than 250mg/liter. Also, the K+ efflux rates of the adapted cells weresmaller compared with those of the nonadapted cells (datanot shown).

Conversion of cis-to-trans fatty acids in nongrowing cells.The following experiments were made by using 4-chlorophe-nol as the toxic substance. Since the change in the cis-to-trans ratio of the fatty acids apparently did not depend on thegrowth of the bacteria, as shown for cells with high phenolconcentrations, the effect was further studied in nongrowingcells which were prepared as described in Materials andMethods. Also, in these cells, 4-chlorophenol induced anincrease in the proportion of the 16:1trans fatty acid. Simul-taneously, the content of the corresponding 16:lcis fatty aciddecreased. The effect correlated with the 4-chlorophenolconcentrations used (Fig. 5). All other fatty acids showed nochange in their contents. The total amount of the two 16:1fatty acids also stayed constant (38 to 39%) at all concentra-tions of 4-chlorophenol. The conversion of the fatty acidswas also observed in the absence of an energy source likesuccinate, but to a slightly reduced extent (data not shown).

In these cells, de novo synthesis of lipids was completelyabsent as was documented by incorporation studies with[14C]acetate. Growing cells were able to incorporate 2.20nmol of acetate per ml of cell suspension into the lipidfraction over the course of 2 h. If nongrowing cells wereincubated with labeled acetate in the absence or in thepresence of 300 mg of chlorophenol per liter, the incorpora-tion was less than 0.001 nmol/ml (0.03% of growing cells).The time course of conversion of the 16:1 unsaturated

fatty acids induced by 4-chlorophenol is shown in Fig. 6.However, the formation of the trans fatty acid did not occurin cells boiled for 5 min previous to the addition of thephenolic substance (data not shown). This rules out a strictlychemical reaction and indicates an enzymatic involvement.

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1850 HEIPIEPER ET AL.

50

40

-

-0(n

4-'

%4--

30

20

10

0

50

40

(-

~0C.)-0

-10

(n

30

20

A

16:0

- control

0,25L 0,5

0,75

1,0

1,25

16:1 trans 16:1 Cis

B

18:1

- control

0,25

1 0,5_ 0,75W 1,03 1,25

10 | | M l l |

16:0 16 1 trans 16 1 c's 18:1

FIG. 3. Fatty acid composition of phenol-adapted (A) and -nonadapted (B) cells of P. putida P8. Phenol (concentrations as shown in grams

per liter) was added to growing cells 3 h before they were harvested for fatty acid analysis.

The conversion of the double bond in the fatty acids isapparently catalyzed by a yet unknown cis-trans isomerase.When the cells were treated with chloramphenicol before

the addition of 4-chlorophenol, the conversion of 16:1cis into16:ltrans fatty acids showed a similar course (Fig. 6).However, the cells already had an enhanced content of16:1trans at the time of 4-chlorophenol addition. Also, inthese experiments, the sum of the amounts of the two 16:1fatty acids stayed constant. The formation of 16:1trans in theabsence of fatty acid and protein biosynthesis indicates a

constitutive enzyme system that directly isomerases theunsaturated fatty acids as constituents of phospholipids.

DISCUSSION

The change in fatty acid composition leading to an in-crease of the degree of saturation of the membrane lipids isa well-known reaction of bacteria against membrane-activesubstances like long-chain alcohols and aromatic compounds(15, 16). Also, phenols which are membrane active in bac-

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P. PUTIDA CONVERSION OF cis FATITY ACIDS TO trans 1851

0 250 500 750 1000

phenol (mg/I)

FIG. 4. Influence of phenol on the K+ content of phenol-adapted(0) and -nonadapted (0) cells of P. putida P8. The K+ content ofcells 1 h after the addition of the phenol which elicited a K+ effluxis shown. After this time, the K+ gradients were restored if possible.

teriostatic concentrations induce an increase in the degree ofsaturation of the membrane lipids of Escherichia coli K-12(17). This membrane modification was found to correlatewith an increase in cell tolerance against the toxic com-

pounds. The reason for this may be that both the membranechanges and the presence of phenols affect the membranefluidity, but in an opposite way, and, as a further conse-

quence, the permeability of membranes (5, 19, 23). Anotherreaction of bacteria against phenol is a change in the lipid-to-protein ratio of the cytoplasmic membrane, which alsoinfluences the membrane viscosity (18, 29).The constant contents of the fatty acids, including the total

amounts of the 16:1 isomers at different concentrations ofthe toxin in nongrowing cells of P. putida P8, can beexplained by the mechanism of procaryotes to change theirmembrane fluidity. Only growing bacteria were thought to beable to influence this membrane property by de novo syn-

1-

0-0

4-

4-chlorophenol (mg/I)FIG. 5. Content of 16:1cis (-) and 16:1trans (O) and the total

16:1 (V) content in nongrowing cells of P. putida P8 at different4-chlorophenol concentrations. Cells were harvested during theexponential growth phase and suspended in phosphate buffer (50mM, pH 7.0) with 4 g of succinate per liter as the energy source.

4-Chlorophenol was added to the cells 2 h before the lipids were

extracted for fatty acid analysis.

20

cn

c

15

10

50 30 60 90 120

time (min)FIG. 6. Time course of formation of 16:ltrans in nongrowing

cells of P. putida P8 after the addition of 300 mg of 4-chlorophenolper liter as indicated by the arrow. Cells were treated with (0) or

without (-) the addition of chloramphenicol, as described in thetext.

thesis of lipids and the incorporation into lipids of fatty acidsin a changed saturated-to-unsaturated ratio (9). One excep-

tion to this restriction is the conversion of cis to transunsaturated fatty acids found in this study for cells of P.putida P8. Also, the formation of cyclopropane fatty acids isknown as a postsynthetic modification process of unsatura-ted fatty acids, but the function of this modification isunclear at present (9, 11) since the fluidity of the membraneis not significantly influenced by this conversion. Theisomerization of the double bond is possibly a specialmechanism of phenol degraders in reaction to high substrateconcentrations under conditions not allowing growth andthereby inhibiting lipid de novo synthesis. However, thecommon occurrence of cis- and trans fatty acid isomers inthe cellular lipids for some other strains of Pseudomonas (8,12, 26), for Vibrio sp. (14, 26), and for methylothrophicbacteria (22) has also been reported. In Vibrio sp., probablya very similar mechanism of conversion of fatty acids ispresent (25). A cis-to-trans isomerization which is indepen-dent of lipid biosynthesis is induced in this psychrophilicstrain after a sharp increase in the ambient temperature.The cis configuration effects a strong increase in mem-

brane fluidity by its bended steric structure. In contrast tothis, the long, extended steric structure of the trans config-uration is able to insert itself into the membrane similarly tosaturated fatty acids, which are also mostly in all-transconformations. Therefore, the conversion of cis-to-transunsaturated fatty acids should reduce the membrane fluidity.Physicochemical investigations of the steric behavior oftrans unsaturated fatty acids in membranes are in agreementwith this argument (21). Moreover, different phospholipidscontaining a trans instead of a cis unsaturated fatty acidexhibited increases in phase transition temperatures of from18 to 31°C, indicating a reduction in the membrane fluiditydue to the changed double-bond configuration (25).The function of the alterations in the membrane structure

which change the fluidity of the lipid bilayer may be toreduce the permeability of the membrane. Adapted cells ofP. putida P8, which had lipids with an enhanced degree ofsaturation, were characterized by maintenance of membranegradients for K+ ions in the presence of high phenol concen-

800

700

600

500

I-

._

E

E-.

c

-

- ...9

. --. --.- ----I .

0% P..% 1- -- I---

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1852 HEIPIEPER ET AL.

trations. Thus, these cells could maintain limited growth atphenol concentrations at which nonadapted cells were to-tally inhibited in growth. Also, the conversion of cis-to-transunsaturated fatty acids resulting in a diminution of themembrane fluidity should contribute to protection of cellsfrom the toxicity of phenolic substrates.The observation that, besides phenol and 4-chlorophenol,

the aromatic compound chloramphenicol also induced cis-trans isomerization can be explained by an unspecific mech-anism of enzyme regulation. The isomerase should be asso-ciated with the cell membrane due to gain access to thephospholipids as the probable substrates. Then one attract-ive possibility of regulation is that the system can directlysense the viscosity of the lipid bilayer. The conformation ofthe membrane protein, the displacement of the positioninside the membrane, and the rate of lateral diffusion can beinfluenced by the viscosity of the lipid environment and cantherefore be involved in the control of activity (29).

In contrast to the probable broad range of activators, thesystem of isomerization apparently has a high specificity forC16 unsaturated fatty acids as substrates. The affinity tounsaturated fatty acids with a C18 chain is rather low,because 18: ltrans was detected only in traces even when theamount of 18:1trans was higher than that of the correspond-ing cis fatty acid.

ACKNOWLEDGMENT

This study was supported by a grant from the Deutsche For-schungsgemeinschaft.

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