Formation of trans Fatty Acids Is Not Involved in Growth-Linked Membrane Adaptation of Pseudomonas putida

Fatty acid compositions in growing and resting cells of severalstrains of Pseudomonas putida (P8, NCTC 10936, and KT 2440)were studied, with a focus on alterations of the saturationdegree, cis-trans isomerization, and cyclopropane formation.The fatty acid compositions of the strains were very similarunder comparable growth conditions, but surprisingly, and contraryto earlier reports, trans fatty acids were not found in eitherexponentially growing cells or stationary-phase cells. Duringthe transition from growth to the starvation state, cyclopropanefatty acids were preferentially formed, an increase in the saturationdegree of fatty acids was observed, and larger amounts of hydroxyfatty acids were detected. A lowered saturation degree and concomitanthigher membrane fluidity seemed to be optimal for substrateuptake and growth. The incubation of cells under nongrowth conditionsrapidly led to the formation of trans fatty acids. We show thatharvesting and sample preparation for analysis could provokethe enzyme-catalyzed formation of trans fatty acids. Freeze-thawingof resting cells and increased temperatures accelerated theformation of trans fatty acids. We demonstrate that cis-transisomerization only occurred in cells that were subjected toan abrupt disturbance without having the possibility of adaptingto the changed conditions by the de novo synthesis of fattyacids. The cis-trans isomerization reaction was in competitionwith the cis-to-cyclopropane fatty acid conversion. The potentialfor the formation of trans fatty acids depended on the cyclopropanecontent that was already present.

INTRODUCTION

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Introduction

The bacterial cytoplasmic membrane is an important target and/orreceptor for stress factors. As a response to permanent physicaland chemical changes in the cell environment, several protectivemechanisms and metabolic adaptation reactions have evolved (10,52, 56). At the level of membrane lipids and fatty acids, theseprocesses are often referred to as homeoviscous adaptation (57).Cells control the fluidity of their membranes by altering thelipid composition to compensate for changes in fluidity inducedby certain environmental factors, such as temperature or thepresence of toxic, membrane-active compounds. In most cases,however, microorganisms are not able to compensate for externallyinduced fluidity changes with 100% efficacy (7, 37, 38). Theycan tolerate and maybe even need a wider range of differentlipid compositions to establish homeostasis, especially duringgrowth. Lipids can coexist in physically separated microdomainswith more or less fluid- or gel-phase behavior, and membranefunctions are locally influenced by many factors other thanfluidity (20, 43). These are the reasons for attempts to enlargethe term homeoviscous adaptation (to maintain membrane fluidity)by use of the term homeophasic adaptation (to adjust membranefluidity).

At the level of lipid membrane composition, the predominantresponse of many bacteria to environmental perturbations isthe alteration of lipid acyl chain structures by changing theratio of saturated to unsaturated fatty acids during growth(8, 15, 19, 26, 42, 53). Several other mechanisms have beenfound in some bacteria to adjust fluidity, such as shorteningor growth-dependent elongation of fatty acid chain lengths (6,8, 14, 53), growth-dependent changes in the ratio of terminallybranched iso and anteiso fatty acids (28, 30), growth-dependentmethylation of cis unsaturated fatty acids to methyl branched(31) or cyclopropane saturated ones (3, 29, 33, 42), and isomerizationof cis to trans double bonds (11, 16, 18, 22, 25, 40, 47).

Pseudomonas putida is a ubiquitous gram-negative bacterium anda potent pollutant degrader. Strains of this species are ofecophysiological interest because most of them are able to useat least the following three adaptation mechanisms at the levelof lipids to respond to physical and chemical stresses: (i)changes in the overall degree of saturation of fatty acids (22,35, 37), (ii) the formation of cyclopropane fatty acids (35),and (iii) cis-trans isomerization with a double bond configuration(11, 22, 35, 36, 37, 46, 59). The kind and degree of the responsedetermine the stability and resilience of the cells and decidetheir fate. Commonly, the different adaptation mechanisms ofP. putida have been investigated independently. The purposeof this study was the integral examination of these three adaptationresponses of P. putida and their dependence on different growthphases. To gain insights into strain-dependent differences andto verify the measured effects, we chose three well-known strainsof this species for use in this study.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and growth conditions.
Three strains of P. putida were used for this study. P. putidaP8 was kindly supplied by H. J. Heipieper (UFZ Centre for EnvironmentalResearch Leipzig-Halle, Leipzig, Germany), and P. putida NCTC10936 and KT 2440 were obtained from the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (Braunschweig, Germany).All three strains were maintained on Trypticase soy agar slantsat 4°C and cultivated at 30°C. Shaking flasks containing150 ml of growth medium were inoculated with cells from an overnightpreculture (10% [vol/vol]). Standard mineral medium, designedfor the development of 2 g of biomass/liter, was composed ofthe following reagents (in milligrams per liter): NH₄Cl, 761;KH₂PO₄, 681; K₂HPO₄, 871; CaCl · 6H₂O, 5.5; MgSO₄ ·7H₂O, 71.2; ZnSO₄ · 7H₂O, 0.4; MnSO₄ · 4H₂O, 0.8;CuSO₄ · 5H₂O, 0.8; Na₂MoO₄ · 2H₂O, 0.3; and FeSO₄· 7H₂O, 5.0. Bacteria were grown in batches on sodiumsuccinate (2 g/liter) as the sole carbon and energy source.For the study of growth-phase-dependent changes in the fattyacid composition, samples were harvested between the early andlate exponential growth phases (3 to 8 h), during a transientphase of deprivation (24 h), and during stationary phase (48h), as monitored by measuring the optical densities of the culturesat 700 nm (data not shown).

Sample preparation.
During different growth phases, the cells were rapidly harvestedwithout temperature changes by centrifugation at 4,000 x g for5 min, washed in imidazole buffer (2 mM, pH 7), and recollected.This procedure was adhered to as far as possible with respectto the short centrifugation time and the constancy of temperature.From the data presented here, it seems that a brief spin at4°C would be acceptable because it would minimize preparationartifacts. The pellets from different batches were processedin parallel in three different ways: (i) pellets were directlysaponified to liberate the fatty acids without any changes intheir physiological status quo, (ii) the pellets were incubatedfor 1 to 3 h at 30°C and analyzed immediately after incubation,and (iii) the biomass was put in a deep freezer at –20°C,stored for several days at this temperature, thawed for 1 or15 min at 30°C, and then analyzed either immediately orafter 1, 3, or 5 h of incubation in a shaking water bath at30°C. To explore the effect of freezing and thawing, wemodified the third procedure further. In some experiments, thecells were frozen twice, and in other experiments, the thawingtemperature during the 2-h incubation period was set to 10,20, 30, or 40°C prior to fatty acid analysis. Typically,two or more independent batches were pooled to collect samplesfor the analysis. Each experiment was performed at least twice,sometimes after a variation of the cultivation times to confirmobserved trends of earlier results.

Fatty acid determination.
Fatty acids were determined as methyl esters from whole-cellhydrolysates according to the procedure of the SHERLOCK microbialidentification system (MIDI Inc., Newark, Del.). Samples ofabout 5 to 10 mg of cell dry mass were subjected to derivatizationin Teflon screw-cap test tubes. For the preparation of esters,cells were saponified at 100°C for 30 min with 1 ml of reagentI (45 g of sodium hydroxide, 150 ml of methanol, 150 ml of water),methylated at 80°C for 10 min with reagent II (325 ml of6 M hydrochloric acid, 275 ml of methanol), extracted with 1.25ml of reagent III (200 ml of n-hexane, 200 ml of methyl tert-butylether), and base washed with 3 ml of reagent IV (10.8 g of sodiumhydroxide, 40 g of sodium chloride, 900 ml of water). Methylationfollowing saponification was preferred for total fatty acidanalysis because transesterification procedures are suspectedto cause underestimations of cyclopropane acids and are lesseffective at releasing cellular hydroxyl fatty acids (45). Theseparation and determination of the fatty acid methyl esterswere performed with a 6890 GC gas chromatography system (Agilent,Palo Alto, Calif.) equipped with a flame ionization detector(GC-FID). An automatic sample inlet was used (split injection,split ratio of 100:1, 250°C), and the separation was carriedout in an Ultra2 nonpolar capillary column (length, 25 m; insidediameter, 0.20 mm; film thickness, 0.33 µm; Agilent),with hydrogen as the carrier gas (9 lb of constant pressure/in²).The temperature program was as follows: 170 to 260°C at5 K min^–1, 260 to 310°C at 40 K min^–1, and 310°Cfor 1.5 min. Data acquisition and data analysis were controlledby ChemStation software (version 10.01; Agilent) and the Sherlocksoftware package (version 4.5; MIDI). Fatty acid assignmentswere checked by comparisons of the retention times with thoseof known standards measured on columns with different polaritiesand were analyzed further after common derivatization procedures,including hydrogenation and silylation. The position of thedouble bond was determined by derivatization with pyrrolidinefollowed by an analysis of electron impact fragmentation byGC-mass spectrometry (GC-MS) (1). The cis-trans geometry ofthe double bonds was confirmed by comparisons of chromatographicdata (retention times) and mass spectrometric data (double bondpositions). The fatty acid composition was calculated from fractionsof individual peaks of the total peak area. Replicate determinationsindicated reproducible fatty acid profiles with relative errorsof 2 to 5%, calculated as standard errors of the means (SEM).

The following fatty acid nomenclature was applied: the numberpreceding the colon indicates the total number of carbon atoms,the number following the colon indicates the number of doublebonds, and the suffix designates the position of the doublebonds (from the methyl end of the molecule, cis or trans indicatestheir configuration) or the position of hydroxyl groups (fromthe carboxyl end of the molecule). It was helpful for the sakeof discussion to combine single fatty acids into groups withcommon structural characteristics. Such groups of interest arestraight-chain saturated fatty acids (n:0), straight-chain unsaturatedfatty acids with a cis (n:1 cis) or trans (n:1 trans) configuration,cyclopropane fatty acids (n:0 cyclo), and ratios of these groups.The degree of saturation, S, was defined as the ratio of straight-chainsaturated fatty acids to the total amount of fatty acids. Theaverage carbon chain length of fatty acids, ACCL, was expressedas ${Sigma}$ (FA x C)/100, where FA is the percentage of each fatty acidand C is the number of carbon atoms in the straight chain.

RESULTS AND DISCUSSION

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Results and Discussion

Fatty acid compositions of individual strains are very similar under comparable growth conditions.
The fatty acid compositions of P. putida P8, NCTC 10936, andKT 2440 grown in batch cultures at 30°C on succinate asthe sole source of carbon and energy were determined. Table1 shows data obtained with exponentially grown cells (harvestedafter 6 h of cultivation) and cells from stationary phase (48h). When the organisms were harvested during the same growthphase, their basic fatty acid profiles were qualitatively andquantitatively very similar and comprised saturated, cis (mono)unsaturated,hydroxyl, and cyclopropane fatty acids, mainly in the rangeof 10- to 18-carbon-atom chain lengths. Differences were foundbetween the growth phases. At 6 and 48 h, saturated straight-chainfatty acids were in the ranges of 33 to 34% and 40 to 43%, respectively,whereas fatty acids originating from cis unsaturated ones (sumof cis, cyclo, and trans fatty acids) were in the ranges of47 to 53% and 46 to 50%, respectively. The observed fatty acidpatterns are typical for many gram-negative bacteria (34). Palmiticacid (16:0), palmitoleic acid (16:1 ${omega}$ 7c), and vaccenic acid (18:1 ${omega}$ 7c)were always the dominant fatty acid components of the cells.The occurrence of vaccenic acid indicated the presence of theanaerobic pathway of fatty acid biosynthesis and the absenceof a fatty acid synthesis-independent desaturase system foundin organisms possessing the aerobic pathway (e.g., Acinetobactercalcoaceticus [19]). Indeed, P. putida is known to be incapableof changing the saturation degree of existing fatty acids andrather relies on de novo biosynthesis of fatty acids (11).

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TABLE 1. Total fatty acid compositions (wt/wt) of P. putida strains grown on 0.2% succinate at 30°C

No trans fatty acids are formed in exponentially growing cells or stationary-phase cells.
P. putida is known for possessing trans unsaturated fatty acidsin its outer membrane. Surprisingly, and in contrast to earlierreports with the same strain, P. putida P8 (11, 35), and relatedstrains of P. putida (46, 51), trans unsaturated fatty acidswere not found in this study, regardless of the time of sampling.As shown in Table 1, trans fatty acids were not formed duringexponential growth or stationary phase by any of the strains,provided that the cells were analyzed immediately after sampling.This was all the more surprising since trans acids were alsoabsent from cells of P. putida P8, which were grown in the presenceof the inhibitor phenol at a concentration leading to 50% growthinhibition on succinate (data not shown). Only in long-termstarved cells were small amounts of trans fatty acids detectable,as seen in an 11-day sample of P. putida P8 (2% 16:1 ${omega}$ 7t). Thisphenomenon, already observed in cells of Vibrio cholerae thatwere starved for 30 days (25), will not be discussed here furtherbecause these trans fatty acids appeared in cells with a veryundefined physiological state.

To exclude the possibility that the composition of the growthmedium prevented the formation of trans fatty acids during growth,we alternatively cultivated strain P8 in a complex nutrientmedium. The results were the same as those for minimal medium(Table 2). Moreover, growth experiments with P. putida P8 wererepeated for media described by others (22, 24) that containedsuccinate as the sole carbon and energy source. In the past,such media had been chosen to investigate the impact of organicsolvents on cis-trans isomerization. We performed our experimentsunder exactly the same growth conditions used by these laboratories,including the centrifugation, lipid extraction, and transesterificationsteps. In contrast to previous findings, we never found transfatty acids in cells which had not been incubated with the toxicantif they were analyzed immediately (Table 2).

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TABLE 2. Percentage of trans fatty acids in P. putida P8 grown on complex medium or minimal medium

Cyclopropane fatty acids are predominantly formed during the transition from the exponential to the stationary growth phase.
Cyclopropane fatty acids are typical for many gram-negativebacteria using the anaerobic pathway of fatty acid biosynthesis.Usually, only minor amounts of cyclopropane fatty acids areobserved during exponential growth, and increasing amounts aredetected during the transition to a starvation state, when growthceases (3, 29, 33, 42). We were able to confirm these findings.As shown in Table 1 and in contrast to our findings for transfatty acids, cyclopropane fatty acids were always present, althoughonly in small amounts during the exponential growth phase. Instationary-phase cells, the amount of cyclopropane acids wasup to 12 times higher than that during exponential growth. Thecyclo/cis ratio was lower in P. putida NCTC 10936 (0.05 and0.57 for 6 and 48 h, respectively) than in both P. putida KT2440 (0.10 and 1.40 for 6 and 48 h, respectively) and P. putidaP8 (0.11 and 1.79 for 6 and 48 h, respectively). Note that theincrease in n:0 cyclo occurred at the expense of n:1 cis. Itis known that the energy- and S-adenosyl-L-methionine-dependentcyclopropane synthase acts on phospholipid-esterified cis unsaturatedfatty acids (32, 61). Although the molecular mechanisms controllingthe formation of cyclopropane fatty acids as a function of thegrowth phase have been well established (5, 13, 58), the roleof these fatty acids is not fully understood yet. Obviously,cyclopropane fatty acids stabilize lipids against turnover anddegradation, so the energetic investment might be worthwhilebecause it minimizes membrane alterations that may lead to life-threateningchanges in membrane fluidity during starvation (17, 33). Thereare reports that the presence of cyclopropane fatty acids seemsto improve survival rates of acid-stressed cells (4), and itwas established that especially large amounts of cyclopropaneacids accumulated when starvation took place at high temperatures(9). Moreover, in comparison to cis unsaturated fatty acids,the presence of cyclopropane fatty acids should make the membranesomewhat more rigid because of their higher lipid melting points.This effect is small but significant (39) and may also contributeto stabilization of the lipid bilayer during the subsequenttransition to starvation without much affecting the cells' abilityto resume growth. The formation of cyclopropane fatty acidsat the expense of cis unsaturated fatty acids toward the endof growth is favored and appears to represent an adaptationto starvation conditions.

Fatty acid chain length and hydroxy fatty acid content are only marginally affected by the growth phase.
As shown in Table 1, the calculated ACCLs were very similarfor all three strains. ACCLs ranged from 15.1 to 15.5 and 15.5to 15.7 for 6 and 48 h of growth, respectively. The increasingchain lengths indicate a somewhat higher rigidity of membranestowards the end of exponential growth. However, the increasewas only marginal compared with values found for Micrococcuscryophilus (53) and Pseudomonas oleovorans (6) and does notjustify discussion in terms of adaptive strategies in P. putida.The proportion of hydroxy fatty acids was slightly decreasedtowards the beginning of stationary phase. The sum of hydroxyfatty acids ranged between 13 and 18% and 6 and 12% after 6and 48 h of growth, respectively. The purpose of this decreaseis unclear.

The saturation degree increases during transition from exponential to stationary growth phase.
Growth-linked changes of the saturation degree (S) are the mostfrequently observed membrane adaptation to altering environmentalconditions. The calculated saturation degrees were similar forall three strains provided that growth occurred under the samecultivation conditions (Table 1). For 6 and 48 h of growth,S ranged from 47 to 52% and 50 to 54%, respectively. This increasewith prolonged cultivation and depletion of the growth substrateconfirms earlier data obtained with P. putida P8 (35) and resemblesdata obtained with chemostat cultures of Escherichia coli atdifferent dilution rates (2, 55). Increased contents of saturatedfatty acids enhancing the rigidity of the cytoplasmic membraneare a well-known mechanism of adaptation to strong membrane-fluidizingfactors such as a rise in temperature, growth on or in the presenceof lipophilic organic solvents, etc. (26, 54). As shown here,an increase in saturation can also be the result of a transitionto the starvation state. The variability of S along the growthcurve was closely linked to the changing substrate supply, indicatingthat adaptive responses adjust rather than maintain membranefluidity. Less saturation leading to higher membrane fluidityseemed to be optimal for growth. This may be explained by therole of membrane fluidity in the activity of membrane proteinsinvolved in substrate uptake (8).

Reincubation of cells under nongrowth conditions leads to sudden formation of trans fatty acids.
The extent to which the capacity of an organism to adapt isrealized depends on its physiological state. We were interestedin determining the possible changes in fatty acid compositionin cells of P. putida under nongrowth conditions. To gain insightsinto the behavior of resting cells, we investigated variationsin the fatty acid patterns of the strains after different treatments.The washed biomass was incubated in buffer (without growth substrates)for 1 to 3 h at 30°C before fatty acid analysis. Figure1 shows the development of selected groups of fatty acids. Variouscombinations of cultivation times and incubation times as restingcells were studied. Whereas P. putida P8 and KT 2440 (8-h values)showed only marginal changes upon incubation as resting cells,a small percentage of palmitelaidic acid (16:1 ${omega}$ 7t) appeared inresting cells of P. putida NCTC 10936 (grown for 8 h). The longerthe incubation in buffer solution was, the more trans fattyacids formed at the expense of cis acids were found, whereasno further formation of cyclopropane acids was observed (Fig.1). A similar behavior, but delayed and occurring to a somewhatlesser extent, was found for cells that had been harvested fromstationary phase. This was consistent with a higher contentof cyclopropane acids in stationary-phase cells, as it may beexplained by the smaller amounts of cis acids available forcis-trans isomerization and the fact that the cells were alreadysufficiently adapted (via cis-cyclo conversion) at the timeof incubation in buffer.

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FIG. 1. Fatty acid compositions (wt/wt) of P. putida strains (P8 [A], NCTC 10936 [B], and KT 2440 [C]) grown on 0.2% succinate at 30°C. Groups of straight-chain saturated, cyclopropane, cis, and trans fatty acids are shown. Samples were taken after 8 h (exponential growth phase) and 48 h (stationary growth phase) and processed in different ways. One sample (control, 0 h) was analyzed immediately, and the other samples were incubated for 1 or 3 h in a nutrient-free buffer at 30°C before analysis. Representative results are shown. At least two independent experiments were performed (SEM, <5%).

For verification of the analytical data, two original gas chromatogramsof fatty acid methyl esters from exponentially growing cellsof P. putida NCTC 10936 are shown as examples (Fig. 2). Palmitelaidicacid was only detectable in the sample representing restingcells (Fig. 2B). To prove that the peak at 10.22 min was reallythe trans compound and not an artifact or a cis fatty acid withanother double bond position, we confirmed our findings by GC-MS,using a column with more polarity. For this experiment, thesample giving the chromatogram shown in Fig. 2B was analyzedin a polar column (DB 23 instead of HP Ultra2). Figure 3 showsthe total ion chromatogram of the mass spectra. The elutionorder of fatty acid methyl esters changed as expected, and thepeak interpreted as 16:1 ${omega}$ 7t was found again and was baselineseparated from 16:1 ${omega}$ 7c. We verified the identities of both peaksby derivatization with pyrrolidine (1). Figure 3 shows massspectra typical for 16:1 pyrrolidide compounds. They are identical,thus proving the coexistence of two chromatographically separated16:1 compounds with double bond ${omega}$ 7, as seen from the unique 12-atomic-mass-unitdifference between m/z 196 and 208. They thus undoubtedly representthe cis and trans isomers. Moreover, in the past, the unambiguousoccurrence of these trans fatty acids (16:1 ${omega}$ 7t and, in minoramounts, 18:1 ${omega}$ 7t) was proven in strain P8 by infrared spectrometry(22).

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FIG. 2. GC-FID separation of fatty acid methyl esters of P. putida NCTC 10936 in a nonpolar column (HP Ultra2). Cells were grown on 0.2% succinate for 8 h. (A) The sample was analyzed immediately; (B) the sample was resuspended in imidazole buffer at 30°C for 3 h before analysis. Peaks representing 16:1 and 16:0 are enlarged.

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FIG. 3. GC-MS-total ion chromatogram (TIC) of fatty acid methyl esters of P. putida NCTC 10936 in a polar column (DB-23). The sample used was the same as that described for Fig. 2B. Mass spectra of the peaks representing 16:1 ${omega}$ 7c and 16:1 ${omega}$ 7t after derivatization of the fatty acid methyl esters to their corresponding pyrrolidide compounds are shown above the total ion chromatogram.

The surprising results that no trans fatty acids were formedduring exponential and stationary growth but suddenly occurredduring incubation in buffer may serve as a plausible explanationfor the large amounts of trans acids previously found in V.cholerae (17) and in a marine bacterial isolate (41). Both organismshave in common the ability to form trans fatty acids, but possiblythe trans acid contents measured were not characteristic forshort-term-starved cells. For these starvation studies, theorganisms were harvested and reincubated with an energy- andnutrient-free medium. Therefore, possibly the reincubation andnot the short-term starvation was the cause for the large observedamounts of trans fatty acids.

Freeze-thawing of resting cells and increasing temperatures accelerate the formation of trans fatty acids.
Figure 4 shows that the formation of trans fatty acids was promotedby a freeze-thaw procedure prior to fatty acid analysis. A combinationof freeze-thawing and prolonged incubation of resting cellsgave rise to the largest quantities of trans fatty acids. Thisobservation is important because in analysis protocols frommany laboratories freezing is a preferred technique for storingsamples. Based on our results, this may be a source of erroneousdata with regard to trans fatty acids. The occurrence of a smallamount of trans fatty acids in some 0-h samples (Fig. 4) appearsto be caused by the treatment prior to incubation (concentrationof the biomass by centrifugation, freezing, and thawing withouttemperature control). To understand the influence of freeze-thawingon the formation of trans acids in more detail, we studied theeffects of the temperature changes during these processes. Figure5 shows two results obtained with P. putida KT2440. Sample freezingand thawing, even when repeated, did not lead to the formationof n:1 trans fatty acids provided that there was no increasein the temperature beyond the original growth temperature. Whensamples were incubated for 2 h after thawing at different temperatures,cis-trans isomerization was seen at the higher temperaturestested (30 and 40°C). At incubation temperatures that werelower than the cultivation temperature, no trans acids appeared(20 or 10°C). The strong temperature influence on the transfatty acid formation is in accordance with earlier results (35).To verify our findings of the strong impact of sample treatment,we repeated some experiments that were done by other authors(22, 24). While no trans fatty acids could be found in samplescultivated according to the authors' specifications and analyzedimmediately, the formation of trans fatty acids may be provokedby changing the sample treatment (Table 2).

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FIG. 4. Fatty acid compositions (wt/wt) of P. putida strains (P8 [A], NCTC 10936 [B], and KT 2440 [C]) grown on 0.2% succinate at 30°C. Groups of straight-chain saturated, cyclopropane, cis, and trans fatty acids are shown. Samples were harvested after 8 h (exponential growth phase) or 48 h (stationary growth phase), washed rapidly, and then frozen (–20°C). After thawing (30°C, 15 min), the samples were analyzed immediately (control, 0 h) or after an incubation time of 1 or 5 h in a nutrient-free buffer at 30°C. Representative results are shown. At least two independent experiments were performed (SEM, <5%).

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FIG. 5. Development of straight-chain saturated, cyclopropane, cis, and trans fatty acids of P. putida KT 2440 grown for 4 h on 0.2% succinate at 30°C. Samples were harvested, washed rapidly, and analyzed immediately after the following alternate treatments: 1, control sample without further treatment; 2, one freeze (–20°C)-thaw (30°C, 1 min) cycle; 3, two freeze-thaw cycles without a longer dwell time; 4 to 7, a freeze (–20°C)-thaw (10°C) cycle followed by a 2-h incubation at 10, 20, 30, and 40°C, respectively. At least two independent experiments were performed (SEM, <5%).

Conclusions.
Our results show for the first time that under conditions ofundisturbed growth, no trans fatty acids are found in lipidsof P. putida, regardless of the growth phase. However, upona change in the environment of cells affecting membrane fluiditythat was too abrupt to be compensated for by altered fatty acidsynthesis, trans fatty acids were formed rapidly (21, 36, 59).The highest rate of trans fatty acid formation was found forcells that were harvested from the exponential growth phaseand subsequently incubated for several hours in buffer at anelevated temperature. As expected, the contents of cyclopropaneand saturated fatty acids remained largely constant during thistreatment. The sudden appearance of trans fatty acids suggeststhat the cytoplasmic membrane was more fluid at the time ofdisturbance than was appropriate for functioning under the newconditions. Therefore, the formation of trans unsaturated fattyacids can be considered an adaptation response to increase themembrane rigidity, i.e., an emergency reaction rather than away to fine-tune (48) the membrane fluidity during growth andmultiplication (23).

The formation of trans fatty acids is catalyzed by a nonreversiblecis-trans isomerase without a shift in the double bond positionand without a requirement of a cofactor or energy (12, 44, 50).The enzyme has been purified (49, 50), and the encoding genewas cloned and sequenced (24, 27). It has been verified thatthe isomerization reaction is possible without growth (22, 36).The fact that trans fatty acids are formed rapidly in cellsincubated in an energy- and nutrient-free medium suggests thatthe cis-trans isomerase is already present (22, 47). If thatis true, then the constitutively expressed enzyme exists ina nonactive state or is spatially separated to act on the phospholipidmolecules of the membrane during the exponential and stationarygrowth phases. There is evidence that changes in the membranefluidity are the primary signals eliciting the isomerizationreaction (26, 46). However, if a high membrane fluidity is aprecondition for the cis-trans conversion, it cannot be theonly one. In fact, exponentially growing cells are characterizedby lower degrees of saturation but do not contain trans acids.

The extent to which trans fatty acids can be formed obviouslydepends on the amounts of cyclopropane fatty acids already formedfrom the common precursor pool of cis unsaturated fatty acids.Only the remaining cis fatty acids can be used for the formationof trans acids. The relatively stable cyclopropane acids areunavailable for conversion into trans acids. Therefore, in contrastto a former hypothesis (17) and confirmative of later reports(11), the trans/cis ratio is a less robust indicator for lownutrient levels in ecosystems than the cyclo/cis ratio (60).

Theoretically, the content of trans fatty acids can serve asa stress indicator in ecosystems because cis-trans isomerizationis a way for some bacteria to respond to a temperature riseor an exposure to toxic substances (59). However, the practicaluse of trans fatty acids as a quantitative biomarker of specificenvironmental stress is questionable because of their generallylow quantities, their occurrence in only a small proportionof all bacteria, the competing cyclopropane fatty acid synthesis,and the fact that they are formed only under nongrowth conditionsand upon shock impacts.

ACKNOWLEDGMENTS

We thank Hermann J. Heipieper for a critical discussion duringthe preparation of the manuscript and Angelika Wichmann forher technical assistance.

FOOTNOTES

* Corresponding author. Mailing address: UFZ Centre for Environmental Research Leipzig-Halle, Department of Environmental Microbiology, P.O. Box 500136, 04301 Leipzig, Germany. Phone: 49 341 235 2363. Fax: 49 341 235 2247. E-mail: claus.haertig{at}ufz.de.

REFERENCES

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