Cyclopropane ring formation in membrane lipids of bacteria.

It has been known for several decades that cyclopropane fatty acids (CFAs) occur in the phospholipids of many species of bacteria. CFAs are formed by the addition of a methylene group, derived from the methyl group of S-adenosylmethionine, across the carbon-carbon double bond of unsaturated fatty acids (UFAs). The C1 transfer does not involve free fatty acids or intermediates of phospholipid biosynthesis but, rather, mature phospholipid molecules already incorporated into membrane bilayers. Furthermore, CFAs are typically produced at the onset of the stationary phase in bacterial cultures. CFA formation can thus be considered a conditional, postsynthetic modification of bacterial membrane lipid bilayers. This modification is noteworthy in several respects. It is catalyzed by a soluble enzyme, although one of the substrates, the UFA double bond, is normally sequestered deep within the hydrophobic interior of the phospholipid bilayer. The enzyme, CFA synthase, discriminates between phospholipid vesicles containing only saturated fatty acids and those containing UFAs; it exhibits no affinity for vesicles of the former composition. These and other properties imply that topologically novel protein-lipid interactions occur in the biosynthesis of CFAs. The timing and extent of the UFA-to-CFA conversion in batch cultures and the widespread distribution of CFA synthesis among bacteria would seem to suggest an important physiological role for this phenomenon, yet its rationale remains unclear despite experimental tests of a variety of hypotheses. Manipulation of the CFA synthase of Escherichia coli by genetic methods has nevertheless provided valuable insight into the physiology of CFA formation. It has identified the CFA synthase gene as one of several rpoS-regulated genes of E. coli and has provided for the construction of strains in which proposed cellular functions of CFAs can be properly evaluated. Cloning and manipulation of the CFA synthase structural gene have also enabled this novel but extremely unstable enzyme to be purified and analyzed in molecular terms and have led to the identification of mechanistically related enzymes in clinically important bacterial pathogens.     

Production of conjugated fatty acids: A review of recent advances

Conjugated fatty acids (CFAs) have received a deal of attention due to the increasing understanding of their beneficial physiological effects, especially the anti-cancer effects and metabolism-regulation activities. However, the production of CFAs is generally difficult. Several challenges are the low CFAs content in natural sources, the difficulty to chemically synthesize target CFA isomers in high purity, and the sensitive characteristics of CFAs. In this article, the current technologies to produce CFAs, including physical, chemical, and biotechnical approaches were summarized, with a focus on the conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLNAs) which are the most common investigated CFAs. CFAs usually demonstrate stronger physiological effects than other non-conjugated fatty acids; however, they are more sensitive to heat and oxidation. Consequently, the quality control throughout the entire production process of CFAs is significant. Special attention was given to the micro- or nano-encapsulation which presented as an emerging technique to improve the bioavailability and storage stability of CFAs. The current applications of CFAs and the potential research directions were also discussed.     

Selection and properties of Escherichia coli mutants defective in the synthesis of cyclopropane fatty acids.

Mutants of Escherichia coli K-12 defective in the synthesis of cyclopropane fatty acids (CFA) have been selected and isolated by a L-[methyl-3H]methionine suicide procedure. Two mutants were isolated. Stationary-phase cultures of both mutants contain less than 0.7% of the CFA content found in the parental strain. The CFA deficiency is attributed to a deficiency of CFA synthetase activity. Extracts of both mutants contain less than 10% of the CFA synthetase activity found in extracts of the parental strain. Experiments in which parental and mutant extracts were mixed indicate that the lack of activity in the mutant strains is not due to an inhibitor of CFA synthetase present in the mutant extracts. We have not yet detected a physiological phenotype for these mutants. These strains grow normally at various temperatures in a variety of media. We have tested survival (colony-forming ability) in response to (i) prolonged incubation in stationary phase, (ii) exposure to drying, and (iii) exposure to detergents, heavy metals, low pH, high salt concentration, and a variety of other environmental conditions. The survival of both mutants is identical to that of the parental strain under all conditions tested. The compositions (excepting the CFA deficiency) and metabolic turnover rates of the phospholipids of both mutant strains are indistinguishable from those of the wild-type strain. The transport of several amino acids also seems normal in these mutants.     

Fatty acid biosynthesis is involved in solvent tolerance in Pseudomonas putida DOT-T1E

The unusual tolerance of Pseudomonas putida DOT-T1E to toluene is based on the extrusion of this solvent by constitutive and inducible efflux pumps and rigidification of its membranes via phospholipid alterations. Pseudomonas putida DOT-T1E-109 is a solvent-sensitive mutant. Mutant cells were less efficient in solvent extrusion than the wild-type cells, as shown by the limited efflux of 14C-1,2,4-trichlorobenzene from the cell membranes, despite the fact that the efflux pumps are overexpressed as a result of increased expression of the ttgDEF and ttgGHI efflux pump operons. This limitation could be the result of alterations in the outer membrane because the mutant cells released more beta-lactamase to the external medium than the wild-type cells. The mutant P. putida DOT-T1E-109 showed negligible synthesis of fatty acids in the presence of sublethal concentrations of toluene as revealed by analysis of 13CH3-13COOH incorporation into fatty acids. In contrast, the mutant strain in the absence of solvents, and the wild-type strain, both in the presence and in the absence of toluene, incorporated 13CH3-13COOH at a high rate into de novo synthesized lipids. The mutation in P. putida DOT-T1E-109 increases sensitivity to the solvent because of a limited efflux of the solvent from the cell membranes with the concomitant inhibition of fatty acid biosynthesis.     

Cellular fatty acids as chemical markers for differentiation of Yersinia pestis and Yersinia pseudotuberculosis

Aims:  Gas chromatography (GC) was utilized to investigate the cellular fatty acids (CFAs) composition of 141 Yersinia pestis isolates from different plague foci of China, and 20 Yersinia pseudotuberculosis strains as well.
Methods and Results:  The whole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation and extraction followed with analysis using a standardized Microbial Identification System (MIS). Y. pestis and Y. pseudotuberculosis strains are quite similar in major CFA profiles, which include 16:0, 17:0 cyclo, 3-OH-14:0, 16:1ω7c and 18:1ω7c, accounting for more than 80% of the total CFAs.
Conclusions:  Yersinia pestis could be easily differentiated from Y. pseudotuberculosis by plotting the ratios of some CFA pairs, i.e.,14:0/18:0 vs 18:1ω7c/18:0, 3-OH-14:0/18:0 vs 18:1ω7c/18:0, 16:1ω7c/18:0 vs 18:1ω7c/18:0, 12:0/18:0 vs 18:1ω7c/18:0 and 12:0 ALDE/18:0 vs 16:1ω7c/18:0 fatty acids.
Significance and Impact of the Study:  In the present study, the normalized Sherlock MIS and Sherlock standard libraries were used to analyse the fatty acid composition of different strains of Y. pestis and Y. pseudotuberculosis . Meanwhile, ratios of certain CFA components were found to serve as chemical markers for differentiating the two closely related bacteria that are difficult to be differentiated by simply comparing CFA profiles based on other researches.     

Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers.

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied. PHAs isolated from P. putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition. In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate. The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance. The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis. Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature. These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis. Analysis of a P. putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.     

Fatty acids synthesized by oral treponemes in chemically defined media

OMIZ-W68, a chemically defined medium that contains no long-chain fatty acids and yet supports in vitro proliferation of a wide range of fastidious oral anaerobes, is described. The type strains of Treponema denticola, Treponema lecithinolyticum, Treponema maltophilum, Treponema pectinovorum, Treponema socranskii, and an as yet unpublished canine Treponema species could be propagated indefinitely in this medium with sugar supplements for the saccharolytic species. Analysis of the cellular fatty acids (CFA) of these treponemes by gas chromatography demonstrated the synthesis of C14, C15, C16, and C17 fatty acids (linear-, iso-, and anteiso-forms) in various proportions, but neither hydroxy- nor unsaturated fatty acids. However, between 0% and 40% of the eluted material could not be identified. The proportions of CFAs differed not only between species but also between the eight strains of Treponema denticola investigated. Replacing OMIZ-W68 by a derivative minimal essential medium (OMIZ-M/TDCDK) developed for Treponema denticola had little effect on the CFA profiles. In contrast, the CFA profiles of treponemes grown in OMIZ-W68 showed at best minor similarity to the strains from the Moore library of the Virginia Polytechnic Institute, which had been grown in media containing serum, peptones, and yeast extract.     

Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers.

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied. PHAs isolated from P. putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition. In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate. The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance. The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis. Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature. These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis. Analysis of a P. putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.     

Involvement of cyclopropane fatty acids in the response of Pseudomonas putida KT2440 to freeze-drying

Pseudomonas putida KT2440, a saprophytic soil bacterium that colonizes the plant root, is a suitable microorganism for the removal of pollutants and a stable host for foreign genes used in biotransformation processes. Because of its potential use in agriculture and industry, we investigated the conditions for the optimal preservation of the strain and its derivatives for long-term storage. The highest survival rates were achieved with cells that had reached the stationary phase and which had been subjected to freeze-drying in the presence of disaccharides (trehalose, maltose, and lactose) as lyoprotectants. Using fluorescence polarization techniques, we show that cell membranes of KT2440 were more rigid in the stationary phase than in the exponential phase of growth. This is consistent with the fact that cells grown in the stationary phase exhibited a higher proportion of C17:cyclopropane as a fatty acid than cells in the exponential phase. Mutants for the cfaB gene, which encodes the main C17:cyclopropane synthase, and for the cfaA gene, which encodes a minor C17:cyclopropane synthase, were constructed. These mutants were more sensitive to freeze-drying than wild-type cells, particularly the mutant with a knockout in the cfaB gene that produced less than 2% of the amount of C17:cyclopropane produced by the parental strain.     

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.     

The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources

We recently identified the phaG(Pp) gene encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase in Pseudomonas putida, which directly links the fatty acid de novo biosynthesis and polyhydroxyalkanoate (PHA) biosynthesis. An open reading frame (ORF) of which the deduced amino acid sequence shared about 57% identity with PhaG from P. putida was identified in the P. aeruginosa genome sequence. Its coding region (herein called phaG(Pa)) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The resulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phaG(Pp)-negative mutant P. putida PhaG(N)-21. The PHA was composed of 5 mol% 3-hydroxydodecanoate, 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate and 5 mol% 3-hydroxyhexanoate. Furthermore, an isogenic phaG(Pa) knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaG(Pa) mutant did not show any difference in growth rate, but PHA accumulation from gluconate was decreased to about 40% of wild-type level, whereas from fatty acids wild-type level PHA accumulation was obtained. These data suggested that PhaG from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity and strongly enhances the metabolic flux from fatty acid de novo synthesis towards PHA(MCL) synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known.     

A Pseudomonas putida cardiolipin synthesis mutant exhibits increased sensitivity to drugs related to transport functionality

Biological membranes have evolved different mechanisms to modify their composition in response to chemical stimuli in a process called 'homeoviscous adaptation'. Among these mechanisms, modifications in the ratio of saturated/unsaturated fatty acids and in cis/trans fatty acid isomers, cyclopropanation and changes in the phospholipids head group composition have been observed. To further understand the role of phospholipid head groups in solvent stress adaptation, we knocked out the cls (cardiolipin synthase) gene in Pseudomonas putida DOT-T1E. As expected, cls mutant membranes contained less cardiolipin than those of the wild-type strain. Although no significant growth rate defect was observed in the cls mutant compared with the wild-type strain, mutant cells were significantly smaller than the wild-type cells. The cls mutant was more sensitive to toluene shocks and to several antibiotics than the parental strain, suggesting either that the RND efflux pumps involved in the extrusion of these drugs were not working efficiently or that membrane permeability was altered in the mutant. Membranes of the cls mutant strain seemed to be more rigid than those of the parental strain, as observed by measurements of fluorescence polarization using the DPH probe, which intercalates into the membranes. Ethidium bromide is pumped out in Pseudomonas putida by at least one RND efflux pump involved in antibiotic and solvent resistance, and the higher rate of accumulation of ethidium bromide inside mutant cells indicated that functioning of the efflux pumps was compromised as a consequence of the alteration in phospholipid head group composition.     

A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes

P450 monooxygenases from microorganisms, similar to those of eukaryotic mitochondria, display a rather narrow substrate specificity. For native P450 BM-3, no other substrates than fatty acids or an indolyl-fatty acid derivative have been reported (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem. J. 327, 537-544). We thus were quite surprised to observe, in the course of our investigations on the rational evolution of this enzyme towards mutants, capable of hydroxylating shorter-chain fatty acids, that a triple mutant P450 BM-3 (Phe87Val, Leu188-Gln, Ala74Gly, BM-3 mutant) could efficiently hydroxylate indole, leading to the formation of indigo and indirubin (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Indole is not oxidized by the wild-type enzyme; it lacks the carboxylate group by which the proper fatty acid substrates are supposed to be bound at the active site of the native enzyme, via hydrogen bonds to the charged amino acid residues Arg47 and Tyr51. Our attempts to predict the putative binding mode of indole to P450 BM-3 or the triple mutant by molecular dynamics simulations did not provide any useful clue. Encouraged by the unexpected activity of the triple mutant towards indole, we investigated in a preliminary, but systematic manner several alkanes, alicyclic, aromatic, and heterocyclic compounds, all of which are unaffected by the native enzyme, for their potential as substrates. We here report that this triple mutant indeed is capable to hydroxylate a respectable range of other substrates, all of which bear little or no resemblance to the fatty acid substrates of the native enzyme.     

A specific region in the N terminus of a replication initiation protein of plasmid RK2 is required for recruitment of Pseudomonas aeruginosa DnaB helicase to the plasmid origin

Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44Delta2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37-55 (TrfA-44Delta3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71-88 (TrfA-44Delta4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.