Synthesis of trans unsaturated fatty acids in Pseudomonas putida P8 by direct isomerization of the double bond of lipids

The phospholipids of Pseudomonas putida P8 contain monounsaturated fatty acids in the cis and trans configuration. Cells of this phenol-degrading bacterium change the proportions of these isomers in response to the addition or elimination of a membrane active compound such as 4-chlorophenol. This study undoubtedly reveals that the cis unsaturated fatty acids are directly converted into trans isomers without involvement of de novo synthesis of fatty acids. Oleic acid, which cannot be synthesized by this bacterium, was incorporated as a cis unsaturated fatty acid marker in the membrane lipids of growing cells. The conversion of this fatty acid into the corresponding trans isomer was demonstrated by gas chromatographic-mass spectrometric analysis and use of ¹⁴C-labeled oleic acid. Separation and isolation of the cellular membranes showed that the fatty acid isomerase is located in the cytoplasmic membrane of P. putida P8.Abbreviation 4-CP 4-chlorophenol     

cis/trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type.

From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.     

Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity.

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.     

Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity.

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.     

Mechanism of cis-trans isomerization of unsaturated fatty acids in Pseudomonas putida

We studied the pattern of the cis-trans isomerization of unsaturated fatty acids in cells of Pseudomonas putida S12 grown in a medium supplemented with oleic acid which was deuterated at both of the C atoms of its double bond. Direct evidence that isomerization does not include a transient saturation of the double bond was obtained. In addition, analysis of the amino acid sequences of the seven known Cti proteins identified them as heme-containing proteins of the cytochrome c type.     

cis-trans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8.

Transposon mutants of Pseudomonas putida P8 were generated by applying a mini-Tn5 mutagenesis system. The mutants obtained were checked for their ability to tolerate increased temperatures and elevated phenol concentrations. Approximately 5,800 transposon mutants were used to generate a pool of 600 temperature-sensitive strains; one of these strains was identified as being damaged in its ability to perform cis-trans isomerization of fatty acids. A gene library of P. putida P8 was constructed and screened by using as a probe sequences immediately adjacent to the mini-Tn5 insertion. A DNA fragment that complemented the mutation was isolated and cloned. The corresponding gene, termed cti, is located close to the methionine synthase locus (metH) in P. putida P8. A cti-carrying fragment integrated into a plasmid also conferred the ability for cis-trans isomerization to Escherichia coli; the cti gene was completely sequenced, and the amino acid sequence was deduced.     

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 several strains of Pseudomonas putida (P8, NCTC 10936, and KT 2440) were studied, with a focus on alterations of the saturation degree, cis-trans isomerization, and cyclopropane formation. The fatty acid compositions of the strains were very similar under comparable growth conditions, but surprisingly, and contrary to earlier reports, trans fatty acids were not found in either exponentially growing cells or stationary-phase cells. During the transition from growth to the starvation state, cyclopropane fatty acids were preferentially formed, an increase in the saturation degree of fatty acids was observed, and larger amounts of hydroxy fatty acids were detected. A lowered saturation degree and concomitant higher membrane fluidity seemed to be optimal for substrate uptake and growth. The incubation of cells under nongrowth conditions rapidly led to the formation of trans fatty acids. We show that harvesting and sample preparation for analysis could provoke the enzyme-catalyzed formation of trans fatty acids. Freeze-thawing of resting cells and increased temperatures accelerated the formation of trans fatty acids. We demonstrate that cis-trans isomerization only occurred in cells that were subjected to an abrupt disturbance without having the possibility of adapting to the changed conditions by the de novo synthesis of fatty acids. The cis-trans isomerization reaction was in competition with the cis-to-cyclopropane fatty acid conversion. The potential for the formation of trans fatty acids depended on the cyclopropane content that was already present.     

cis/trans Isomerase of Unsaturated Fatty Acids of Pseudomonas putida P8: Evidence for a Heme Protein of the Cytochrome c Type

From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.     

Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.     

Homology modeling and function of trehalose synthase from Pseudomonas putida P06

Trehalose is a non-reducing disaccharide that has wide applications in the food industry and pharmaceutical manufacturing. Trehalose synthase (TreS) from Pseudomonas putida P06 catalyzes the reversible interconversion of maltose and trehalose and may have applications in the food industry. However, the catalytic mechanism of TreS is not well understood. Here, we investigated the structural characteristics of this enzyme by homology modeling. The highly conserved Asp294 residue was identified to be critical for catalytic activity. In addition, flexible docking studies of the enzyme–substrate system were performed to predict the interactions between TreS and its substrate, maltose. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the biochemical characterization of the relevant mutants generated by site-directed mutagenesis.     

Characterization of a benzoate permease mutant of Pseudomonas putida

A spontaneous mutant of Pseudomonas putida (PRS 2017) has been isolated which is incapable of growth on benzoate, does not induce the enzymes of the catechol branch of the -ketoadipate pathway when grown in the presence of benzoate, cannot accumulate radioactively labeled benzoate, yet grows well with mandelate as sole source of carbon and energy. This strain apparently lacks a benzoate permease, which in the wild type shows a K _mof about 0.1 mM for benzoate, is inducible, and is not under the control of the regulatory system which governs the induction of the enzymes of the catechol branch of the -ketoadapate pathway. The lesion in PRS 2017 is apparently single site and maps near other genes governing benzoate dissimilation.Dedicated to R. Y. Stanier on the occasion of his 60th birthday     

Genetic and biochemical characterization of a mutation (fatA) that allows trans unsaturated fatty acids to replace the essential cis unsaturated fatty acids of Escherichia coli.

Unsaturated fatty acid auxotrophs of Escherichia coli are able to use only unsaturated fatty acids of the cis configuration as the required growth supplement. A mutation in the fatA gene allows such auxotrophs to utilize unsaturated fatty acids with a trans double bond as well as fatty acids having a cis double bond. The fatA gene was mapped to min 69 near argG, and the allele studied (fatA1) was found to be dominant over the wild-type gene. fatA1 mutant strains grew at similar rates when supplemented with elaidate (trans-9-octadecenoate) or oleate (cis-9-octadecenoate). The fat+ strain, however, lysed when supplemented with the trans fatty acid. Physiological characterization of the fatA mutant strain was undertaken. The mutation appeared not to be involved with long-chain fatty acid transport. Introduction of lesions in known fatty acid transport genes abolished trans fatty acid utilization in the fatA mutant strain. Also, growth characteristics of the fat+ and the fatA1 mutant strains on elaidate as the sole carbon source were identical, which indicated comparables rate of fatty acid accumulation. The mutation appeared to be involved with recognition of the trans configuration after uptake into the cell. The levels of trans fatty acid incorporation into the phospholipids of the fat+ and the fatA strains differed considerably, with the mutant incorporating much higher levels. No significant accumulation of elaidate into nonphospholipid cellular components was observed. The fatA mutation did not appear to be involved with the cellular metabolic state, as cyclic AMP had no effect on the ability of the strains to utilize trans fatty acids.     

Cis and trans conformational changes of bacterial fatty acids in comparison with analogs of animal and vegetable origin

The conditions of the formations of trans isomers of fatty acids, depending on the method of processing and storage of the raw material of microbial, plant and animal origin, were investigated. In the composition of lipids, except for the main trans-isomer elaidic acid, nonsignificant amounts of trans -2-hexen-4-ynal, trans-2-formlcyclopro-panecarboxylate, methyl octadeca-9-yn-l1-trans-enoate, trans-2, 2-dimethyl-3-(2-propenyl)-ethyl ester, trans-9-octadecenoic acid, and trans-1,5-heptadiene, and mixed isomers of methyloctadeca-9-yn-11-trans-enoate,-methyl-9-cis, 11-trans-octadecadienoate, l-[trans-4-(2-iodo-ethyl) cyclohexyl]-trans-4-pentylcyclo-hexane and cis-9, and trans 11-octadecenoic acid. The major trans elaidic acid component was detected in natural objects of different origin in quantities not exceeding 0.05–0.11%. The combination of thermal processing with other parameters, especially enzymatic treatment, led to an increased proportion of trans isomers. The content of trans isomers is usually proportional to the time of storage of materials.     

Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida

The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.