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A trans unsaturated fatty acid was found as a major constituent in the lipids of Pseudomonas putida P8. The fatty acid was identified as 9-trans-hexadecenoic acid by gas chromatography, argentation thin-layer chromatography, 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 of P. putida, the cells were still able to increase the content of the trans unsaturated fatty acid and simultaneously to decrease the proportion of the corresponding 9-cis-hexadecenoic acid. This conversion of fatty acids was also induced by 4-chlorophenol in nongrowing cells in which the de novo synthesis of lipids had stopped, as shown by incorporation experiments with labeled acetate. The isomerization of the double bond in the presence of chloramphenicol indicates a constitutively operating enzyme system. The cis-to-trans modification of the fatty acids 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 occurrence of trans unsaturated fatty acids as by-products of fatty acid transformations carried out by the obligate anaerobic ruminal microflora has been well known for a long time. In recent years, fatty acids with trans configurations also have been detected in the membrane lipids of various aerobic bacteria. Besides several psychrophilic organisms, bacteria-degrading pollutants, such as Pseudomonas putida, are able to synthesize these compounds de novo. In contrast to the trans fatty acids formed by rumen bacteria, the membrane constituents of aerobic bacteria are synthesized by a direct isomerization of the complementary cis configuration of the double bond without a shift of the position. This system of isomerization is located in the cytoplasmic membrane. The conversion of cis unsaturated fatty acids to trans changes the membrane fluidity in response to environmental stimuli, particularly where growth is inhibited due to the presence of high concentrations of toxic substances. Under these conditions, lipid synthesis also stops so that the cells are not able to modify their membrane fluidity by any other mechanism.

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The physiological significance of trans unsaturated fatty acids, which are constituents of membrane lipids of the phenol-degrading bacterium Pseudomonas putita P8, was studied. The addition of phenol or phenol derivatives to the cells induced the formation of trans unsaturated fatty acids, yielding an overall maximal amount of 41.3°70 of total fatty acids. The inhibition of de-novo lipid synthesis by cerulenin prevented the change in the degree of saturation in the lipids. However, the cells could still respond to phenols with an amplified conversion of cis into trans unsaturated fatty acids, which is apparently a post-synthetic mechanism of isomerization of the double bond. The cis/trans conversion correlated with growth inhibition induced by toxic concentrations of 4-chlorophenol, whereas only growing cells were able to change the degree of saturation. In cells that were protected against phenol by immobilization in calcium alginate, the conversion of cis into trans fatty acids occurred at higher toxin concentrations compared with free ceils. Cells entering the stationary growth phase increased the proportion of saturated to unsaturated fatty acids but maintained a constant trans/cis ratio. P. putida P8 reacted to an increase or decrease in the growth temperature with an appropriate change in the ratio of saturated to unsaturated fatty acids and in ceils inhibited by cerulenin with a change in the trans/cis ratio. This study shows that the physiological role of the cis/trans conversion is probably the regulation of membrane fluidity when the most important mechanism for this, the modification of the degree of saturation, cannot be used by the cells due to inhibition of growth and lipid biosynthesis.

The inhibition of the exponential growth of Escherichia coli K-12 by different phenolic compounds was examined. Cells entrapped in calcium alginate showed a greater tolerance than cells grown in suspension. The extent of inhibition of growth of the immobilized cells depended on the period of growth in the gel matrix. After the addition of bacteriostatic concentrations of phenol or 4-chlorophenol, a dose-dependent efflux of metabolites such as ATP and of K+ ions was elicited. Provided that glucose was supplied as an energy substrate, a reaccumulation of K+ ions at low phenol concentrations was observed. The restoration of the membrane gradient for K+ always preceded the continuation of growth in the presence of the toxic compounds. Compared with free cells, those cells immobilized and grown in alginate suffered a smaller loss of cations after the addition of 4-chlorophenol. The reestablishment of gradients was observed at higher concentrations of the pollutants with entrapped cells than with free cells. Corresponding to the increase in tolerance, the membrane damage was reduced in cells grown in immobilized form for longer times. These data offer a mechanistic explanation of the protection of immobilized microorganisms from phenolic solvents. The data point to the membrane as an important cell component in the toxicity of these pollutants.

Membranes of Escherichia coli cells grown in the presence of phenol were examined after isolation of the cytoplasmic and outer membrane fractions. Both membrane types showed reduced lipid-to-protein ratios compared to cells grown without phenol. Phenol-induced differences in the expression of individual proteins of the inner membrane were established. Different proteins of the outer membrane, probably involved in the uptake of iron, were expressed in smaller quantities after phenol addition. Growth in the presence of phenol increased the respiratory activity of the cytoplasmic membrane, whereas the direct inhibition of O2 consumption by phenol was not affected by the presence of this compound in the growth medium. E. coli cells grown entrapped in calcium alginate showed low lipid-to-protein ratios even without phenol in the growth medium. Immobilization of cells also markedly changed the protein pattern of the outer membrane.