The fadL gene product of Escherichia coli is an outer membrane protein required for uptake of long-chain fatty acids and involved in sensitivity to bacteriophage T2.

The fadL+ gene of Escherichia coli encodes an outer membrane protein (FadL) essential for the uptake of long-chain fatty acids (C12 to C18). The present study shows that in addition to being required for uptake of and growth on the long-chain fatty acid oleate (C18:1), FadL acts as a receptor of bacteriophage T2. Bacteriophage T2-resistant (T2r) strains lacked FadL and were unable to take up and grow on long-chain fatty acids. Upon transformation with the fadL+ clone pN103, T2r strains became sensitive to bacteriophage T2 (T2s), became able to take up long-chain fatty acids at wild-type levels, and contained FadL in the outer membrane.     

The amino-terminal region of the long-chain fatty acid transport protein FadL contains an externally exposed domain required for bacteriophage T2 binding

The fatty acid transport protein FadL from Escherichia coli is predicted to be rich in beta-structure and span the outer membrane multiple times to form a long-chain fatty acid specific channel. Proteolysis of FadL within whole cells, total membranes, and isolated outer membranes identified two trypsin-sensitive sites, both predicted to be in externally exposed loops of FadL. Amino acid sequence analysis of the proteolytic fragments determined that the first followed R93 and yielded a peptide beginning with 94S-L-K-A-D-N-I-A-P-T-A104 while the second followed R384 and yielded a peptide beginning with 385S-I-S-I-P-D-Q-D-R-F-W395. Proteolysis using trypsin eliminated the bacteriophage T2 binding activity associated with FadL, suggesting the T2 binding domain within FadL requires elements within one of these extracellular loops. A peptide corresponding to the amino-terminal region of FadL (FadL28-160) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the amino-proximal extracellular loop of the protein confers T2 binding activity. Using an artificial neural network (NN) topology prediction method in combination with Gibbs motif sampling, a predicted topology of FadL within the outer membrane was developed. According to this model, FadL spans the outer membrane 20 times as antiparallel beta-strands. The 20 antiparallel beta-strands are presumed to form a beta-barrel specific for long-chain fatty acids. On the basis of our previous studies evaluating the function of FadL using site-specific mutagenesis of the fadL gene, proteolysis of FadL within outer membranes, and studies using the FadL28-160 peptide, the predicted extracellular regions between beta-strands 1 and 2 and beta-strands 3 and 4 are expected to contribute to a domain of the protein required for long-chain fatty acid and bacteriophage T2 binding. The first trypsin-sensitive site (R93) lies between predicted beta-strands 3 and 4 while the second (R384) is between beta-strands 17 and 18. The trypsin-resistant region of FadL is predicted to contain 13 antiparallel beta-strands and contribute to the long-chain fatty acid specific channel.     

Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli

We report the purification and localization of the fadL gene product (FLP), an essential component of the long-chain fatty acid transport machinery in Escherichia coli. FLP was extracted from total membranes by differential extraction with the nonionic detergents Tween 20 and Triton X-100. This protein was further purified from a Tween 20-insoluble-Triton X-100-soluble extract by salt fractionation, gel filtration chromatography, and hydrophobic interaction chromatography. This regime results in a 95-fold purification of FLP from total membranes. The purified protein preparation was homogeneous based on silver staining and gave the characteristic behavior established for the fadL gene product in the presence of sodium dodecyl sulfate at different temperatures prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr of 33,000 when heated at 25 degrees C and Mr of 43,000 when heated at 100 degrees C) and on two-dimensional polyacrylamide gels (pI of 4.6 and a Mr of 33,000). Purified FLP was rich in hydrophobic residues accounting for approximately 45% of the total amino acid composition. To localize FLP, antisera were raised against the purified protein and were used to probe differentially fractionated membranes by Western immunoblotting. This procedure demonstrated the presence of this protein only in the outer membrane fraction of fadL+ strains. We confirmed the outer membrane localization of FLP by measuring long-chain fatty acid transport in fadL+ and fadL strains treated with EDTA to alter outer membrane permeability and in spheroplasts generated from fadL+ and fadL strains. Both EDTA-treated cells and spheroplasts transported long-chain fatty acids at essentially the same rate regardless of whether they contained a wild-type or mutant fadL gene. These data imply that FLP is a protein in the outer membrane which is specifically involved in long-chain fatty acid transport.     

Microbial production of medium-chain-length 3-hydroxyalkanoic acids by recombinant Pseudomonas putida KT2442 harboring genes fadL, fadD and phaZ

Monomers of microbial polyhydroxyalkanoates, mainly 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO), were produced by overexpressing polyhydroxyalkanoates depolymerase gene phaZ, together with putative long-chain fatty acid transport protein fadL of Pseudomonas putida KT2442 and acyl-CoA synthetase (fadD) of Escherichia coli MG1655 in P. putida KT2442. FadL(Pp), which is responsible for free fatty acid transportation from the extracellular environment to the cytoplasm, and FadD(Ec), which activates fatty acid to acyl-CoA, jointly reinforce the fatty acid beta-oxidation pathway. Pseudomonas putida KT2442 (pYZPst01) harboring polyhydroxyalkanoates depolymerase gene phaZ of Pseudomonas stutzeri 1317 produced 1.37 g L(-1) extracellular 3HHx and 3HO in shake flask studies after 48 h in the presence of sodium octanoate as a sole carbon source, while P. putida KT2442 (pYZPst06) harboring phaZ(Pst), fadD(Ec) and fadL(Pp) achieved 2.32 g L(-1) extracellular 3HHx and 3HO monomer production under the same conditions. In a 48-h fed-batch fermentation process conducted in a 6-L fermentor with 3 L sodium octanoate mineral medium, 5.8 g L(-1) extracellular 3HHx and 3HO were obtained in the fermentation broth. This is the first time that medium-chain-length 3-hydroxyalkanoic acids (mcl-3HA) were produced using fadL(Pp) and fadD(Ec) genes combined with the polyhydroxyalkanoates depolymerase gene phaZ.     

Long-chain fatty acid transport in Escherichia coli. Cloning, mapping, and expression of the fadL gene

Transport of long-chain fatty acids across the inner membrane of Escherichia coli K-12 requires a functional fadL gene (Maloy, S. R., Ginsburgh, C. L., Simons, R. W., and Nunn, W. D. (1981) J. Biol. Chem. 256, 3735-3742). Mutants defective in the fadL gene lack a 33,000-dalton inner membrane protein as evaluated using two-dimensional pI/sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (Ginsburgh, C. L., Black, P. N., and Nunn, W. D. (1984) J. Biol. Chem. 259, 8437-8443). In an effort to determine whether the fadL gene is the structural gene for this 33,000-dalton protein, we have cloned, mapped, and analyzed the expression of the fadL gene. The fadL gene has been localized on a 2.8-kilobase EcoRV fragment of E. coli genomic DNA. Plasmids containing this gene (i) complement all fadL mutants, (ii) increase the long-chain fatty acid transport activity of fadL strains harboring them by 2- to 3-fold, and (iii) direct the synthesis of a membrane protein which has the same molecular weight and isoelectric point as that described by Ginsburgh et al. This is a heat-modifiable protein which has an apparent molecular weight of 43,000 daltons when solubilized at 100 degrees C in the presence of SDS and 33,000 daltons when solubilized at 50 degrees C in the presence of SDS.     

New locus (ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source.

The nature of resistance to phage T2 in Escherichia coli K-12 was investigated by analyzing a known phage T2-resistant mutant and by isolating new T2-resistant mutants. It was found that mutational alterations at two loci, ompF (encoding the outer membrane protein OmpF) and ttr (T-two resistance), are needed to give full resistance to phage T2. A ttr::Tn10 mutation was isolated and was mapped between aroC and dsdA, where the fadL gene (required for long-chain fatty acid transport) is located. The receptor affected by ttr was the major receptor used by phage T2 and was located in the outer membrane. Phage T2 was thus able to use two outer membrane proteins as receptors. All strains having a ttr::Tn10 allele and most of the independently isolated phage T2-resistant mutants were unable to grow on oleate as the sole carbon and energy source, i.e., they had the phenotype of fadL mutants. The gene fadL is known to encode an inner membrane protein. The most likely explanation is that fadL and ttr are in an operon and that ttr encodes an outer membrane protein which functions in translocating long-chain fatty acids across the outer membrane and also as a receptor for phage T2.     

Transport of long-chain fatty acids in Escherichia coli. Evidence for role of fadL gene product as long-chain fatty acid receptor

Transport of long-chain fatty acids (LCFA) across the cytoplasmic membrane of Escherichia coli requires functional fadL and fadD genes. The fadD gene codes for an acyl-CoA synthetase (fatty acid: CoA ligase (AMP forming] which has broad chain length specificity and is loosely bound to the cytoplasmic membrane. The fadL gene codes for a 43,000-dalton cytoplasmic membrane protein which, acting by an unknown mechanism, is needed specifically for LCFA transport. As a first step to define the role of the fadL gene product, studies were performed to determine if it functions as a LCFA receptor. The LCFA-binding activity was quantitated in intact cells in the absence of LCFA transport by comparing the binding of LCFA in fadD fadL and fadD fadL+ strains. These studies revealed that (i) fadD fadL+ strains bind 6-fold more LCFA than fadD fadL strains; (ii) fadD fadL strains harboring a plasmid containing the fadL gene bind 16-fold more LCFA than fadD fadL strains harboring only the plasmid vector; and (iii) the fadL-specific LCFA-binding activity is regulated by the fadR gene and catabolite repression. Studies with fadL strains harboring fadL plasmids containing in vitro constructed deletions indicate that mutations which alter the physical properties of the 43,000-dalton fadL gene product also affect fadL gene product-specific LCFA-binding activity. Overall, these studies suggest that one role of the fadL gene product in the LCFA transport process is to sequester LCFA at sites in the cell membrane for transport.     

Long-chain fatty acid transport in bacteria andyeast. Paradigms for defining the mechanism underlying this protein-mediated process

Protein-mediated transport of exogenous long-chain fatty acids across the membrane has been defined in a number of different systems. Central to understanding the mechanism underlying this process is the development of the appropriate experimental systems which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both [1] exhibit saturable long-chain fatty acid transport at low ligand concentration; [2] have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus; and [3] can be easily manipulated using the tools of molecular genetics. In E. coli, this process requires the outer membrane-bound fatty acid transport protein FadL and the inner membrane associated fatty acyl CoA synthetase (FACS). FadL appears to represent a substrate specific channel for long-chain fatty acids while FACS activates these compounds to CoA thioesters thereby rendering this process unidirectional. This process requires both ATP generated from either substrate-level or oxidative phosphorylation and the proton electrochemical gradient across the inner membrane. In S. cerevisiae, the process of long-chain fatty acid transport requires at least the membrane-bound protein Fat1p. Exogenously supplied fatty acids are activated by the fatty acyl CoA synthetases Faa1p and Faa4p but unlike the case in E. coli, there is not a tight linkage between transport and activation. Studies evaluating the growth parameters in the presence of long-chain fatty acids and long-chain fatty acid transport profiles of a fat1 strain support the hypothesis that Fat1p is required for optimal levels of long-chain fatty acid transport.     

Rapid selection of mutants of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> for carbohydrate and fatty acid metabolism by fourier transform infrared spectroscopy and gas chromatography combined with multivariate analysis

Transgene-tagged mutants of Chlamydomonas reinhardtii were generated by random insertional mutagenesis for screening of mutants of carbohydrate and fatty acid metabolism. Approximately 2,500 insertion mutants tagged with the aph7″ gene were produced from one mutagenesis in three weeks. To establish a rapid screening system for numerous insertional lines, whole cell extracts of 100 insertional lines were subjected to Fourier transform infrared spectroscopy (FT-IR) and gas chromatography (GC) analysis combined with multivariate analysis. Mutant lines 28, 67, and 90 showed dramatic differences in the carbohydrate (1,000∼1,200 cm⁻¹) and amide (1,500∼1,700 cm⁻¹) regions of the FT-IR spectrum compared to wild type strain CC-124. Separate GC analysis also showed that 16:0 iso, palmitic acid (16:0), and oleic acid (18:1) were the major fatty acids in the wild type strain. In mutant 80, the relative content ratio of 16:0 iso in total fatty acids was significantly lower than in wild type, whereas the ratios of palmitic acid and oleic acid to 16:0 iso were higher. In mutant 95, the ratio of 16:0 iso to total fatty acids was increased, whereas ratios of palmitic acid and oleic acid to 16:0 iso were decreased. In particular, mutant 57 showed remarkably different fatty acid patterns with novel peaks of long-chain fatty acids having more than 20 carbon atoms. The results of this study show that FT-IR and GC combined with multivariate analysis enable rapid selection of mutants of carbohydrate and fatty acid metabolism in C. reinhardtii.     

Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification.

The processes that govern the regulated transport of long-chain fatty acids across the plasma membrane are quite distinct compared to counterparts involved in the transport of hydrophilic solutes such as sugars and amino acids. These differences stem from the unique physical and chemical properties of long-chain fatty acids. To date, several distinct classes of proteins have been shown to participate in the transport of exogenous long-chain fatty acids across the membrane. More recent work is consistent with the hypothesis that in addition to the role played by proteins in this process, there is a diffusional component which must also be considered. Central to the development of this hypothesis are the appropriate experimental systems, which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both (i) exhibit saturable long-chain fatty acid transport at low ligand concentrations, (ii) have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus, and (iii) can be easily manipulated using the tools of molecular genetics. In both systems, central players in the process of fatty acid transport are fatty acid transport proteins (FadL or Fat1p) and fatty acyl coenzyme A (CoA) synthetase (FACS; fatty acid CoA ligase [AMP forming] [EC 6.2.1.3]). FACS appears to function in concert with FadL (bacteria) or Fat1p (yeast) in the conversion of the free fatty acid to CoA thioesters concomitant with transport, thereby rendering this process unidirectional. This process of trapping transported fatty acids represents one fundamental mechanism operational in the transport of exogenous fatty acids.     

Fatty acid transport in Saccharomyces cerevisiae. Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p

The fatty acid transport protein Fat1p functions as a component of the long-chain fatty acid transport apparatus in the yeast Saccharomyces cerevisiae. Fat1p has significant homologies to the mammalian fatty acid transport proteins (FATP) and the very long-chain acyl-CoA synthetases (VLACS). In order to further understand the functional roles intrinsic to Fat1p (fatty acid transport and VLACS activities), a series of 16 alleles carrying site-directed mutations within FAT1 were constructed and analyzed. Sites chosen for the construction of amino acid substitutions were based on conservation between Fat1p and the mammalian FATP orthologues and included the ATP/AMP and FATP/VLACS signature motifs. Centromeric and 2 mu plasmids encoding mutant forms of Fat1p were transformed into a yeast strain containing a deletion in FAT1 (fat1Delta). For selected subsets of FAT1 mutant alleles, we observed differences between the wild type and mutants in 1) growth rates when fatty acid synthase was inhibited with 45 microm cerulenin in the presence of 100 microm oleate (C(18:1)), 2) levels of fatty acid import monitored using the accumulation of the fluorescent fatty acid 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-S-indacene-3-dodecanoic acid and [(3)H]oleate, 3) levels of lignoceryl (C(24:0)) CoA synthetase activities, and 4) fatty acid profiles monitored using gas chromatography/mass spectrometry. In most cases, there was a correlation between growth on fatty acid/cerulenin plates, the levels of fatty acid accumulation, very long-chain fatty acyl-CoA synthetase activities, and the fatty acid profiles in the different FAT1 mutants. For several notable exceptions, the fatty acid transport and very long-chain fatty acyl-CoA synthetase activities were distinguishable. The characterization of these novel mutants provides a platform to more completely understand the role of Fat1p in the linkage between fatty acid import and activation to CoA thioesters.     

Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli

Two distinct pathways for the incorporation of exogenous fatty acids into phospholipids were identified in Escherichia coli. The predominant route originates with the activation of fatty acids by acyl-CoA synthetase followed by the distribution of the acyl moieties into all phospholipid classes via the sn-glycerol-3-phosphate acyltransferase reaction. This pathway was blocked in mutants (fadD) lacking acyl-CoA synthetase activity. In fadD strains, exogenous fatty acids were introduced exclusively into the 1-position of phosphatidylethanolamine. This secondary route is related to 1-position fatty acid turnover in phosphatidylethanolamine and proceeds via the acyl-acyl carrier protein synthetase/2-acylglycerophosphoethanolamine acyltransferase system. The turnover pathway exhibited a preference for saturated fatty acids, whereas the acyl-CoA synthetase-dependent pathway was less discriminating. Both pathways were inhibited in mutants (fadL) lacking the fatty acid permease, demonstrating that the fadL gene product translocates exogenous fatty acids to an intracellular pool accessible to both synthetases. These data demonstrate that acyl-CoA synthetase is not required for fatty acid transport in E. coli and that the metabolism of exogenous fatty acids is segregated from the metabolism of acyl-acyl carrier proteins derived from fatty acid biosynthesis.     

FadD is required for utilization of endogenous fatty acids released from membrane lipids

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth.     

Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism

The Arabidopsis acyl-CoA oxidase (ACX) family comprises isozymes with distinct fatty acid chain-length specificities that together catalyse the first step of peroxisomal fatty acid beta-oxidation. We have isolated and characterized T-DNA insertion mutants in the medium to long-chain (ACX1) and long-chain (ACX2) acyl-CoA oxidases, and show that the corresponding endogenous activities are decreased in the mutants. Lipid catabolism during germination and early post-germinative growth was unaltered in the acx1-1 mutant, but slightly delayed in the acx2-1 mutant, with 3-day-old acx2-1 seedlings accumulating long-chain acyl-CoAs. In acx1-1 and acx2-1, seedling growth and establishment in the absence of an exogenous supply of sucrose was unaffected. Seedlings of the double mutant acx1-1 acx2-1 were unable to catabolize seed storage lipid, and accumulated long-chain acyl-CoAs. The acx1-1 acx2-1 seedlings were also unable to establish photosynthetic competency in the absence of an exogenous carbon supply, a phenotype that is shared with a number of other Arabidopsis mutants disrupted in storage lipid breakdown. Germination frequency of the double mutant was significantly reduced compared with wild-type seeds. This was unaffected by the addition of exogenous sucrose, but was improved by dormancy-breaking treatments such as cold stratification and after-ripening. We show that the acx1-1, acx2-1 and acx1-2 acx2-1 double mutants and the ketoacyl-CoA thiolase-2 (kat2) mutant exhibit a sucrose-independent germination phenotype comparable with that reported for comatose (cts-2), a mutant in a peroxisomal ABC transporter which exhibits enhanced dormancy. This demonstrates an additional role beyond that of carbon provision for the beta-oxidation pathway during germination or in dormant seeds.     

Selection of fatty acid auxotrophs from the oleaginous yeast Cryptococcus curvatus and production of cocoa butter equivalents in batch culture

Summary A search was carried out for mutants, defective in the conversion of stearic acid to oleic acid in effort to improve the quality of lipids produced by Cryptococcus curvatus ATCC, 20509. Mutants were selected as unsaturated fatty acid (Ufa) auxotrophs. After treatment of parent organism with Ethyl methanesulfonate (EMS), 11 oleate-requiring auxotrophs were isolated. Only 3 of them were real unsaturated fatty acid (Ufa) mutants, while the other 8 were designated as fatty acid synthetase (Fas) mutants. The amount of saturated fatty acid (SFA) was about 65.2 % in the lipids extracted from an Ufa mutant named UfaM3 and it was significantly higher than that of the wild-type (WT) (46.6 %) and similar to that of cocoa butter (60.4 %).     

Control of triglyceride synthesis by the intracellular level of long-chain acyl coenzyme A for lipid synthesis

Candida lipolytica mutants defective in acyl coenzyme A synthetase I synthesized triglyceride to a markedly less extent than did the wild-type yeast, when grown on oleic acid. The synthesis of triglyceride was controlled by the level of long-chain acyl coenzyme A available for lipid synthesis, whereas the synthesis of phospholipids was hardly affected.