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.     

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.     

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     

Molecular cloning and sequencing of the phenol hydroxylase gene from Pseudomonas putida BH

A genomic library of the phenol-degrading bacterium Pseudomonas putida BH was constructed in the broad host range cosmid pVK100 and introduced into Escherichia coli HB101. One of the recombinant cosmids recovered from catechol- and/or 2-hydroxymuconic semialdehyde-accumulating clones, pS10–45, had a 19.6-kb insert fragment which allowed P. putida KT2440 to grow on phenol as a sole carbon and energy source. Subcloning and expression studies indicated that the phenol hydroxylase gene cluster (pheA) is located on a 6.1-kb SacI fragment. The results of DNA sequencing of the SacI fragment revealed that the pheA gene cluster encodes a multicomponent phenol hydroxylase.     

Cis/trans isomerisation of unsaturated fatty acids in a cardiolipin synthase knock-out mutant of Pseudomonas putida P8

The gene encoding cardiolipin synthase ( cls) from the phenol-degrading bacterium Pseudomonas putida P8, which rapidly adapts its membrane lipids to the presence of organic solvents by cis/trans isomerisation of unsaturated fatty acids, was isolated and completely sequenced. The functionality of the predicted gene product was proven by constructing a knock-out mutant that was significantly reduced in its growth rate both at elevated temperatures and in the presence of membrane-active solvents. Though the mutant showed a clear phenotype it was still able to synthesise trace amounts of cardiolipin. As an increase in cardiolipin (diphosphatidylglycerol) content is known to function as a long term membrane adaptation mechanism in pseudomonads, we tested whether the mutant compensates for the lack of the Cls by increased cis/trans isomerisation of unsaturated fatty acids. Increase in cis/trans isomerisation of unsaturated fatty acids was observed for the mutant at zero and low concentrations of 4-chlorophenol; however, cis/trans isomerisation is not able to fully compensate for the lack of cardiolipin production. Possibly, other long-term adaptation mechanisms are instrumental in compensating for the missing cardiolipin synthesis. As the cis/trans isomerase is activated similarly in the mutant and the wildtype, cis/trans isomerisation and cardiolipin production do not display mutual dependency.     

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.     

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.     

P450cam gene cloning and expression in Pseudomonas putida and Escherichia coli

The gene camC, which encodes the cytochrome P450 monoxygenase protein, was cloned into the shuttle vector pKT240 and recovered as the recombinant pKG201 with a 2.3 kb insert from the CAM plasmid in the PstI site. The gene product is expressed constitutively in P. putida and in E. coli whereas the inverted insert clone lacks expression, indicating absence of an insert promoter.     

Molecular cloning and sequencing of a pectinesterase gene from Pseudomonas solanacearum

Two pectinesterase-positive Escherichia coli clones, differing in expression levels, were isolated from a genomic library of Pseudomonas solanacearum. Both clones contained a common DNA fragment which included the pectinesterase-encoding region. The different expression levels found with the two clones could be ascribed to different positioning of the pectinesterase gene with respect to a vector promoter. Restriction analysis, subcloning, and further exonuclease deletion mapping revealed that the genetic information for pectinesterase was located within a 1.3 kb fragment. A protein of 41 to 42 kDa was expressed from this fragment. Nucleotide sequence analysis of the respective region disclosed an open reading frame of 1188 bp. The deduced polypeptide had a calculated molecular mass of 41,004 Da, which is consistent with the determined size of the pectinesterase protein. The predicted amino acid sequence showed significant homology to pectinesterases from Erwinia chrysanthemi and tomato. In cultures of E. coli clones up to 30% of total pectinesterase activity was transported into the medium. However, no significant pectinesterase activity could be detected in the periplasm.     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.     

Production of methanol from aromatic acids by Pseudomonas putida.

When grown at the expense of 3,4,5-trimethoxybenzoic acid, a strain of Pseudomonas putida oxidized this compound and also 3,5-dimethoxy-4-hydroxybenzoic (syringic) and 3,4-dihydroxy-5-methoxybenzoic (3-O-methylgallic) acids; but other hydroxy- or methoxy-benzoic acids were oxidized slowly or not at all. Radioactivity appeared exclusively in carbon dioxide when cells were incubated with [4-methoxyl-14C]trimethoxybenzoic acid, but was found mainly in methanol when[methoxyl-14C]3-O-methylgallic acid was metabolized. The identity of methanol was proved by analyzing the product from [methoxyl-13C]3-O-methylgallic acid by nuclear magnetic resonance spectroscopy and by isolating the labeled 3,5-dinitrobenzoic acid methyl ester, which was examined by mass spectrometry. These results, together with measurements of oxygen consumed in demethylations catalyzed by cell extracts, showed that two methoxyl groups of 3,4,5-trimethoxybenzoate and one of syringate were oxidized to give carbon dioxide and 3-O-methylgallate. This was then metabolized to pyruvate; the other product was presumed to be the 4-methyl ester of oxalacetic acid, for which cell extracts contained an inducible, specific esterase. P. putida did not metabolize the methanol released from this compound by hydrolysis. Support for the proposed reaction sequence was obtained by isolating mutants which, although able to convert 3,4,5-trimethoxybenzoic acid into 3-O-methylgallic acid, were unable to use either compound for growth.     

Molecular cloning of the Pseudomonas putida glyoxalase I gene in Escherichia coli

The glyoxalase I gene of Pseudomonas putida was cloned onto a vector plasmid pBR 322 as a 7.5 kilobase Sau 3AI fragment of chromosomal DNA and the hybrid plasmid was designated pGI 318. The gene responsible for the glyoxalase I activity in pGI 318 was recloned in pBR 322 as a 2.2 kilobase Hin dIII fragment and was designated pGI 423. The P. putida glyoxalase I gene on pGI 318 and pGI 423 was highly expressed in E. coli cells and the glyoxalase I activity level was increased more than 150 fold in the pGI 423 bearing strain compared with that of E. coli cells without pGI 423. The E. coli transformants harboring pGI 318 or pGI 423 could grow normally in the presence of methylglyoxal, although the E. coli cells without plasmid were inhibited to grow and showed the extremely elongated cell shape.     

Tryptophan 7-halogenase from Pseudomonas aureofaciens ACN strain: gene cloning and sequencing and the enzyme expression

The gene of tryptophan 7-halogenase was isolated from the Pseudomonas aureofaciens ACN strain producing pyrrolnitrin, a chlorocontaining antibiotic, and sequenced. A high homology degree (over 95%) was established for the genes and the corresponding halogenases from P. aureofaciens ACN and P. fluorescens BL915. The tryptophan 7-halogenase gene was amplified by PCR, and the corresponding enzyme was expressed in Escherichia coli cells using the pBSII SK+ vector.     

Physiological function of the Pseudomonas putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids.

Pseudomonas putida PpG6 is able to utilize purified n-alkanes of six to ten carbon atoms for growth. It can also grow on the primary terminal oxidation products of these alkanes and on 1-dodecanol but not on the corresponding 2-ketones or 1,6-hexanediol, adipic acid, or pimelic acid. Revertible point mutants can be isolated which have simultaneously lost the ability to grow on all five n-alkane growth substrates but which can still grow on octanol or nonanol. An acetate-negative mutant defective in isocitrate lysase activity is unable to grow on even-numbered alkanes and fatty acids. Analysis of double mutants defective in acetate and propionate or in acetate and glutarate metabolism shows that alkane carbon is assimilated only via acetyl-coenzyme A and propionyl-coenzyme A. These results support the following conclusions: (i) The n-alkane growth specificity of P. putida PpG6 is due to the substrate specificity of whole-cell alkane hydroxylation; (ii) there is a single alkane hydroxylase enzyme complex; (iii) the physiological role of this complex is to initiate the monoterminal oxidation of alkane chains; and (iv) straight-chain fatty acids from butyric through nonanoic are degraded exclusively by beta-oxidation from the carboxyl end of the molecule.     

Evidence for cis-trans isomerization of a double bond in the fatty acids of the psychrophilic bacterium Vibrio sp. strain ABE-1.

Vibrio sp. strain ABE-1 was grown in a medium that contained as its stable isotope tracer either [2,2-2H2]cis-9-hexadecenoic or [2,2-2H2]trans-9-hexadecenoic acid. Gas chromatographic-mass spectrometric analysis of the cis-9-hexadecenoic and trans-9-hexadecenoic acid fractions from the cells revealed the formation of an intracellularly isomerized 2,2-2H2-fatty acid which differed from the tracer only in the geometrical configuration of the double bond. This observation shows that cis-trans isomerization without a shift in double-bond position between these two geometric hexadecenoic acid isomers can occur in the cells.     

The parAB gene products of Pseudomonas putida exhibit partition activity in both P. putida and Escherichia coli

The bacteria for which there is evidence that proteins of the ParAB family act in chromosome segregation also undergo developmental transitions that involve the ParAB homologues, raising the question of whether the partition activity is equivalent to that of plasmid partition systems. We have investigated the role in partition of the parAB locus of a free-living bacterium, Pseudomonas putida, not known to pass through developmental phases. A parAB deletion mutant, compared with wild type, showed slightly higher frequencies of anucleate cells in exponentially growing cultures but much higher frequencies in deceleration phase. This increase was growth medium dependent. Oversupply of ParA and ParB proteins also raised anucleate cell levels, specifically in the deceleration phase, in wild-type and mutant strains and regardless of medium, as well as generating abnormal cell morphologies. Absence or oversupply of ParAB function had either slight or considerable effects on growth rate, depending on temperature and medium. The need for the Par proteins in chromosome partition thus appears to be subject to the cell's physiological state. Three sequences similar to cis-acting stabilization sites of Bacillus subtilis are present in the P. putida oriC-parAB region. One was inserted into an unstable mini-F and shown to stabilize it in E. coli in a ParAB-dependent manner.