Biosynthesis of Membrane Lipids
Chapter
37
JOHN E. CRONAN, JR., and CHARLES O. ROCK
The study of the membrane lipids of Escherichia coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) has contributed greatly to our understanding of the synthesis and functions of membrane lipids. In addition to the myriad other advantages of studying these organisms, their lipid compositions are among the simplest found in biology. These bacteria contain three major phospholipids: phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) (also called diphosphatidylglycerol). PE, the major phospholipid of E. coli and S. typhimurium (and of gram-negative bacteria in general), constitutes about 75% of the total phospholipid. The relative amounts of PG and CL depend on the growth phase of the cultures; PG is most abundant in log-phase cells, whereas CL accumulates in stationary-phase cells. The phospholipids, neutral lipids, and lipoproteins of E. coli have similar fatty acid compositions, consisting of the saturated fatty acids palmitic (hexadecanoic) acid and myristic (tetradecanoic) acid and the monounsaturated fatty acids palmitoleic (cis-9-hexadecenoic) acid and cis-vaccenic (cis-11-octadecenoic) acid. Traces of lauric (dodecanoic), stearic (octadecanoic), and cis-7-tetradecenoic acids are also present. Recently, it was reported that stationary-phase cultures of E. coli contained linoleic (cis-9-cis-12-octadecdienoic) acid (219), but (in agreement with the older literature) we were unable to detect this fatty acid in the same E. coli strain (48). Most workers in the field have used E. coli, and more compositional data are available for this bacterium. However, in cases in which S. typhimurium has been examined, the two organisms seem identical; thus, information obtained with one organism can be assumed to be directly applicable to the other. The analytical data on the lipid compositions of these organisms were essentially complete over 15 years ago, and thus we shall defer to previous reviews for the original references (39, 44, 47, 180).
Lipid synthesis in E. coli and S. typhimurium is conveniently divided into two parts: (i) synthesis of the fatty acids which are responsible for the characteristic hydrophobicity of lipids and (ii) attachment of the completed fatty acids to sn-glycerol-3-phosphate (G3P) followed by the addition and modification of the polar head groups to yield the major cellular phospholipids. The synthesis of lipid A is addressed in chapter 69. The fatty acid synthetic pathway is required for the synthesis of the protein- bound coenzymes biotin and lipoic acid (chapter 46), and in other gram-negative bacteria, fatty acid synthesis is required for the synthesis of the acylated homoserine lactones involved in density-dependent signalling (76). Also, host-specific nodulation by the rhizobia requires synthesis of various acylated polysaccharides by a specialized fatty acid synthetic pathway (194), and the syntheses of polyketide antibiotics (120) and poly β-hydroxybutyrate (123) proceed by modified fatty acid pathways. Lipid metabolism in E. coli provides an important paradigm for plants, in which lipid biosynthesis occurs predominantly in the bacterium-derived chloroplast. More recently, the expanding field of plant lipid biosynthesis has engendered a significant counterflow of information, and the expression of several plant genes in E. coli has given some valuable insight into bacterial lipid synthesis. Finally, fatty acid synthesis has recently become a target for new antimicrobial agents (8, 189, 276).
All of the phospholipids and lipid-containing molecules of E. coli and S. typhimurium are membrane components; no cytoplasmic, extracellular, or periplasmic structures are known. The phospholipids are distributed between the inner (cytoplasmic) membrane and the outer membrane. The outer membrane contains roughly one-third of the total phospholipid and is thought to consist largely or exclusively of PE. In contrast, the phospholipids of the inner membrane contain roughly equal amounts of PE and of the two acidic lipids PG and CL. Phospholipid fatty acid compositions of the two membranes are similar, although the inner membrane is enriched in unsaturated fatty acids. This enrichment is attributed to the increased levels of PG in the cytoplasmic membrane, which contains a greater fraction of unsaturated fatty acid than do the other phospholipids.
The enzymes of lipid biosynthesis are distributed between the cytosol and the inner membrane. The fatty acid synthetic enzymes and G3P synthase are cytosolic, whereas the enzymes of phospholipid synthesis are bound to the inner membrane. There is one exception to these generalizations. First, the acyl carrier protein (ACP) was reported to be localized to the inner face of the cytoplasmic membrane on the basis of autoradiography of intact cells. However, a more refined colloidal gold-antibody technique demonstrates that ACP is distributed throughout the cytoplasm of E. coli. Second, the phospholipid biosynthetic enzyme, PS synthase, is found associated with the ribosomes rather than with the inner membranes of disrupted cells. Subsequent work has shown this association to be an artifact of cell disruption. In the presence of the lipid substrate of the enzyme, CDP-diacylglycerol, the enzyme is released from the ribosomes and binds to the inner membrane.
The fatty acid synthase system of E. coli is the archetype of the type II or dissociated fatty acid synthase systems. The individual reactions are carried out by separate proteins readily purified independently of the other enzymes of the pathway and are encoded by unique genes. The mechanism of fatty acid synthesis is conserved in prokaryotes and eukaryotes (the archaea synthesize isoprenoid-derived lipids) and proceeds in two stages, initiation and cyclic elongation. There are often multiple proteins that carry out the same basic chemical reaction, and because of differences in substrate specificity, each plays a unique role in the physiological regulation of the spectrum of products produced by the pathway. One of the principal challenges of current research in this area is determining the number and functions of these isozymes. The known enzymes of fatty acid biosynthesis and their genes are listed in Table 1. Intermediates of the pathway are diverted to introduce the double bond of the unsaturated fatty acid species and to provide the 3-hydroxy fatty acids of lipid A and the unknown intermediates utilized in the biotin and lipoic acid synthetic pathways. The first committed step is the conversion of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA by acetyl-CoA carboxylase (Fig. 1). There are three potential mechanisms for the initiation of fatty acid biosynthesis in E. coli (Fig. 2). First, β-ketoacyl-ACP synthase III catalyzes the condensation of acetyl-CoA with malonyl-ACP to yield acetoacetyl-ACP. Second, the acetate moiety is first transferred from acetyl-CoA to acetyl-ACP by either acetyl-CoA:ACP transacylase or synthase III. The acetyl-ACP is then condensed with malonyl-ACP by synthase I (or by synthase II). The third pathway involves the decarboxylation of malonyl-ACP by synthase I to form acetyl-ACP followed by subsequent condensation with malonyl-ACP.
Table 1Mutants in E. coli lipid biosynthesisa |
The elongation reactions of fatty acid biosynthesis are outlined in Fig. 3. The first step is the condensation of malonyl-ACP with a growing acyl chain by β-ketoacyl-ACP synthase. The resulting β-ketoester is reduced by an NADPH-dependent β-ketoacyl-ACP reductase followed by removal of a water molecule by β-hydroxyacyl-ACP dehydrase. The final reduction is catalyzed by an NADH-dependent enoyl-ACP reductase to form acyl-ACP, which in turn serves as a substrate for another round of elongation. Each of these chemical reactions can be carried out by multiple discrete enzymes. For example, there are at least three β-ketoacyl-ACP synthases and at least two β-hydroxyacyl-ACP dehydrases. Owing to their differing substrate specificities, each isozyme makes a unique contribution to the regulation of the distribution of products from the pathway (see below). There may be multiple β-ketoacyl-ACP reductases and enoyl-ACP reductases; however, definitive genetic and biochemical evidence for their existence is not yet available.
A specific dehydrase enzyme, β-hydroxydecanoyl-ACP dehydrase (the fabA gene product), first described by Bloch (17), catalyzes a key reaction at the point that unsaturated fatty acid biosynthesis diverges from saturated fatty acid synthesis (Fig. 4). This dehydrase catalyzes the dehydration reaction shown in Fig. 3 but is also capable of isomerizing trans-2-decanoyl-ACP to cis-3-decenoyl-ACP and is essential to the synthesis of unsaturated fatty acids. However, this protein is not the only gene product required for unsaturated fatty acid synthesis in E. coli. Analyses of β-ketoacyl-ACP synthase I (fabB) mutants show that the fabB gene product is also required to produce unsaturated fatty acids.
ACP.
A unique feature of this pathway is that all of the intermediates are covalently bound to ACP, a small and highly soluble protein (218). The fatty acid carboxyl group is in thioester linkage to the thiol of the 4'-phosphopantetheine prosthetic group. ACP is one of the most abundant proteins in E. coli, constituting 0.25% of the total soluble protein (∼6 × 104 molecules per cell) (241). The secondary structure predicted from the amino acid sequence (241) has been largely confirmed by high-resolution nuclear magnetic resonance (NMR) spectroscopy (112, 150, 151). ACP (8.860 kDa) is a rod-shaped protein composed of a preponderance of acidic residues largely grouped into three α helices, with recent evidence for a short additional helix as originally predicted. However, modeling of the NMR data indicates that ACP exists in two conformations that differ in the conformation of the loop regions and the orientation of the helices (150, 151). The acyl intermediates of fatty acid biosynthesis are bound to the protein through thioester linkage attached to the terminal sulfhydryl of the 4'-phosphopantetheine prosthetic group (218). The prosthetic group sulfhydryl is the only thiol group of E. coli ACP and is attached to the protein via a phosphodiester linkage to Ser-36 located in a β turn situated between the second and third α-helical segments (150, 151). The fatty acyl chain of an acyl-ACP intermediate extends up along the second helix (150, 151, 241). The protein pocket accommodates a six-carbon acyl group (40, 241) approximately, and occupation of this site by a hydrocarbon moiety stabilizes the protein structure (241). In contrast, when a charged thioester such as malonate is bound to the prosthetic group within the pocket, the acyl-ACP is more susceptible to hydrodynamic expansion (241), increasing the exposure of the reactive thioester bond.
The prosthetic group of ACP undergoes metabolic turnover, and the apoprotein is inactive in fatty acid synthesis. ACP synthase (67) transfers 4'-phosphopantetheine from CoA to apo-ACP, whereas ACP phosphodiesterase (281) cleaves the prosthetic group from ACP (Fig. 5; see also chapter 44). These enzymes are not well studied, although ACP phosphodiesterase was recently purified to homogeneity (74) and a mutant (acpS) deficient in ACP synthase is available. The CoA pool is eightfold larger than the ACP pool in normally growing cells (132), and virtually all of the ACP is maintained in the active, holo form in vivo (133). Thus, the supply of prosthetic group does not limit fatty acid biosynthesis, and the operation of the prosthetic group turnover cycle may be involved in governing the intracellular CoA concentration (chapter 44). During logarithmic growth, a significant pool of unacylated ACP can be found in vivo. Inhibition of acyl transfer, however, causes accumulation of acyl-ACPs, and the supply of unacylated ACP is no longer detectable (137, 245). Factors that restrain either chain elongation or fatty acid transfer to the membrane bilayer may cause the supply of ACP protein to be limiting, but the size of the ACP pool must be severely depleted before an effect on fatty acid and phospholipid synthesis can be detected (133, 216). The cellular concentration of ACP protein is regulated, and overproduction of ACP encoded by an inducible plasmid vector is lethal to E. coli (138, 233). Most of the protein expressed in the inducible systems is apo-ACP, and the toxicity can be explained by the finding that apo-ACP is a potent inhibitor of G3P acyltransferase. ACP plays other roles in cell physiology. ACP is required for the synthesis of membrane-derived oligosaccharides (chapter 70) and is associated with MukB, a protein required for correct chromosome partitioning in E. coli (196). Acyl-ACP has also been reported to be an acyl donor in protein acylation (129), as determined from in vitro results in a crude system.
Acetyl-CoA Carboxylase.
Acetyl-CoA carboxylase catalyzes the first committed step of fatty acid (hence lipid) synthesis (3). The overall reaction is composed of two distinct half reactions (3, 91), the ATP-dependent carboxylation of biotin with bicarbonate to form carboxybiotin followed by transfer of the carboxy group from carboxybiotin to acetyl-CoA to form malonyl-CoA (Fig. 1). Biotin is covalently coupled to a 16.7-kDa protein called biotin carboxyl carrier protein (BCCP) (3). The biotin must be coupled to BCCP for acetyl-CoA carboxylase to function, and the coupling reaction is catalyzed by a specific enzyme, biotin-apoprotein ligase (see below).
The two acetyl-CoA carboxylase half reactions are catalyzed by two different protein subcomplexes. Carboxylation of biotin is catalyzed by biotin carboxylase, a homodimeric enzyme composed of 55-kDa subunits that is copurified in a complex with BCCP (itself a homodimer) (3, 91). The enzyme transferring the carboxy group from the biotin moiety of BCCP to acetyl-CoA is the carboxyltransferase component, a heterotetramer composed of two copies of two dissimilar subunits, called α and β (91, 168). In cell extracts, the overall acetyl-CoA carboxylase reaction (acetyl-CoA to malonyl-CoA) is lost and only the separate BCCP-biotin carboxylase and carboxytransferase half reactions are detected (3, 91). The overall acetyl-CoA carboxylase reaction can be reconstituted in vitro with high concentrations of the purified components (91) or by simultaneous overexpression of all four subunits (M. Davis and J. E. Cronan, Jr., unpublished observations). We assume that the enzyme present in vivo is composed of one copy of each subcomplex, with a molecular mass of 280 kDa. This assumption is based on results obtained with the pseudomonal acetyl-CoA carboxylases, which are composed of subunits similar to those in E. coli (15, 71, 102). This enzyme is isolated in the presence of high salt as a 280-kDa complex containing equal numbers of the four subunits, whereas at low salt concentrations, the complex breaks down into two subcomplexes analogous to those in E. coli.
The genes encoding all four acetyl-CoA carboxylase subunits have been cloned, their sequences have been determined, and their protein products have been overexpressed and purified to homogeneity (158, 168, 169, 289). Temperature-sensitive mutants with lesions in accB and accD are available (169, 171). The two carboxyltransferase subunits are encoded by the accA and accD genes (168, 171), and the functional carboxyltransferase subcomplex is composed of two copies of each subunit (91, 168). Sequence similarities suggest that the acetyl-CoA binding site lies within the AccA subunit, but confirmation awaits structural studies. BCCP (AccB) and biotin carboxylase (AccC) are encoded in a small operon (169), and recent progress in determining the structures of both proteins has been made. The active form of the AccB protein requires covalent attachment of biotin to lysine, the reaction catalyzed by biotin-apoprotein ligase (chapter 46). The N-terminal half of AccB is not required for protein biotination and appears responsible for dimerization and/or perhaps association with biotin carboxylase (27, 169). Mutational analysis of BCCP and related proteins (41, 169) shows that essentially the entire C-terminal half of AccB is required for protein modification by biotin ligase and that the biotinated lysine is located in the center of this minimal sequence. Moreover, the C-terminal half of BCCP is protease resistant (72), consistent with a stable folded domain structure. These findings indicate that biotin ligase recognizes a structural domain rather than a linear sequence of amino acid residues. Biotination is a posttranslational event because C-terminal protein extensions can inhibit biotination (41), a result incompatible with cotranslational modification.
The structures of the biotinated and nonbiotinated forms of the C-terminal segment of BCCP were determined by twodimensional NMR analysis (20, 27). The two forms are essentially identical and are composed of two four-stranded antiparallel β sheets, with the biotin located at the tip of a β-hairpin loop protruding from the globular domain. The coenzyme plus the entire amino acid domain swings between two active sites on the proline/alanine-rich hinges. BCCP residues close to the biotin attachment site are also required for catalysis. A motif conserved in biotinated proteins consists of several methionine residues adjacent to the biotinated lysine residue, and it has been assumed that these residues play a role in recognition by biotin-apoprotein ligase. However, substitution of leucine for these methionine residues in the biotinated subunit of Propionibacterium shermanii transcarboxylase yields proteins that are efficiently biotinated but defective in the carboxyltransferase half reaction (256).
E. coli biotin carboxylase is an intensively studied enzyme because of its unusual ability to accept free biotin in place of the BCCP-bound form (3, 154, 217). This property, although very inefficient and of no physiological significance, greatly simplifies analysis of intermediates and allows the use of biotin analogs. Despite these advantages and a recent structure obtained by X-ray crystallography (289), the mechanism of biotin carboxylation remains elusive. Carboxylation is thought to involve the reaction of ATP and CO2 to form carboxyphosphate, an intermediate with an estimated half-life of 70 ms, but it is uncertain whether it reacts directly with biotin (154).
Initiation of Fatty Acid Biosynthesis.
Malonyl-CoA is utilized for fatty acid biosynthesis only following its conversion to malonyl-ACP by malonyl-CoA:ACP transacylase, the product of the fabD gene (93). FabD is a monomeric protein that accepts the malonyl moiety from malonyl-CoA to form a stable malonyl-serine enzyme intermediate (218). Nucleophilic attack on this ester by the sulfhydryl of ACP yields malonyl-ACP, the major building block of fatty acids. The sequence of FabD is known (181, 285), and diffraction quality crystals of the protein have been obtained (253). The original fabD89 temperature-sensitive allele is an amber mutation (286). The temperature sensitivity results from the amino acid inserted by a suppressor tRNA present in the original strain.
In contrast to the reactions that produce malonyl-ACP, the reactions whereby the methyl carbon atom and the immediately adjacent carbon atom (the last two carbons of the fatty acid chain by chemical nomenclature) are incorporated into fatty acid is unclear (Fig. 2). Isotopic labeling studies demonstrate that these "primer" carbons are derived from acetate (293). The acetate source is acetyl-CoA produced mainly by the decarboxylation of pyruvate (chapter 18), although there are other pathways for acetyl-CoA synthesis (chapter 18). Acetyl-CoA is a substrate for β-ketoacyl-ACP synthase III and is incorporated directly into the first four-carbon fatty acid (135, 273). Acetyl-CoA is also converted into acetyl-ACP by a transacylase activity (4, 177, 294), and the resulting acetyl-ACP can serve as the primer when alternative condensing enzymes such as β-ketoacyl-ACP synthase I catalyze the initial condensation. For many years, the acetyl-CoA:ACP transacylase activity in E. coli was considered to be a discrete protein(4, 177, 294). However, the acetyl-CoA:ACP transacylase reaction is catalyzed by synthase III (131, 135, 273), and the acetyl transacylase activity measured in cell lysates may represent a partial reaction of this condensing enzyme. Malonyl-ACP is usually thought to be utilized only in the elongation steps in fatty acid biosynthesis. However, both β-ketoacyl-ACP synthases I and II are capable of initiating fatty acid synthesis in the absence of an added acetyl-ACP primer through a side reaction, malonyl-ACP decarboxylation to produce acetyl-ACP. This reaction is readily demonstrated in vitro (5, 100a), but its role in initiation in vivo awaits experimental verification. The question of whether one or several routes are used to initiate fatty acid synthesis remains open. Although synthase III is well studied, null mutants are not available. An acetyl-CoA:ACP transacylase has been purified (177), but there are no mutants available and it is not clear whether this enzyme actually was synthase III. Finally, the malonyl-ACP decarboxylase activities of β-ketoacyl-ACP synthases I and II involve the same active sites as the synthase reaction (5, 50, 100a), and thus genetic elimination of this source of acetyl-ACP is problematical. Recent work places acetyl-ACP as a by-product of fatty acid synthesis rather than an active participant in chain initiation (100a).
The β-Ketoacyl-ACP Synthase Reaction.
Three E. coli enzymes are known to catalyze the β-ketoacyl-ACP synthase reaction. These enzymes are referred to as synthases I, II, and III, the products of the fabB, fabF, and fabH genes. The latter two genes are located within the cluster of fatty acid synthetic genes (233), whereas fabB lies alone at a distant site. Recently, Siggaard-Andersen and coworkers (261) reported a putative fourth synthase activity in E. coli and assigned an open reading frame to this activity. However, this report was based on a series of indirect inferences and is in error (179). The gene analyzed was the fabF gene as previously noted (180, 233), and the enzyme activity measured in column fractions may be synthase III mixed with synthase I and/or synthase II.
Synthase I is composed of two identical subunits (82) and has both malonyl-ACP and fatty acyl-ACP binding sites (50, 51). In the condensation reaction, the acyl group is covalently linked to the active-site cysteine (Cys-163) enzyme (50, 145). The acyl enzyme undergoes condensation with malonyl-ACP to form β-ketoacyl-ACP, CO2, and free enzyme. The active site of synthase I has sequence similarity with synthases and mono- and polyfunctional fatty acid synthases that catalyze similar condensation reactions (145).
Further investigation revealed the presence of an additional synthase activity in E. coli, β-ketoacyl-ACP synthase II (52). Like synthase I, synthase II has a dimeric structure and is inhibited by cerulenin (279), although synthase II is less sensitive to cerulenin than is synthase I. Both synthases are capable of participating in saturated and unsaturated fatty acid synthesis. The enzymes have been shown, in vitro, to function similarly to all long-chain acyl-ACPs except palmitoleoyl-ACP; palmitoleoyl-ACP is an excellent substrate for synthase II but not for synthase I (52, 82). Strains lacking synthase I, however, require unsaturated fatty acids for growth; therefore, in vivo, synthase I must catalyze a key reaction in unsaturated fatty acid synthesis that synthase II cannot. This reaction is probably the elongation of cis-3-decenoyl-ACP, although this has not been shown experimentally. This step is also thought to be the rate-limiting step in unsaturated fatty acid synthesis (31).
The fabB gene has been cloned (56, 145) and its sequence has been determined (145). The deduced amino acid sequence encodes a protein of 42.6 kDa, which is consistent with the estimated monomeric molecular weight of purified synthase I (82, 145). Overproduction of synthase I has two effects. First, overproduction of the enzyme compensates for its poor ability to elongate palmitoleate, and an increased amount of cis-vaccenic acid is found in phospholipid (56). Second, excess cellular synthase I renders E. coli resistant to the antibiotic thiolactomycin (see below). In the presence of the antibiotic, excess synthase I appears to allow the cell to bypass the two other initiation pathways, acetyl transacylase and synthase III (Fig. 2), by catalyzing the decarboxylation of malonyl-ACP to form the initiation primer, acetyl-ACP. Given the foregoing observations, synthase I may be the only synthase absolutely required for growth (274).
Synthase II was first detected as a component that was resolved from synthase I by hydroxyapatite chromatography (52). The homogeneous protein differs from synthase I by peptide mapping and antigenicity (82). The gene encoding synthase II was identified by its role in the temperature regulation of fatty acid composition (see below). Mutants lacking temperature control (called Cvc) lack synthase II (83). The Cvc phenotype and the lack of synthase II are due to mutations in the same gene, fabF, which is the structural gene for synthase II. Reversion of a fabF mutation results in restoration of synthase II activity, cis-vaccenic acid synthesis, and temperature regulation (83). Thus, synthase II plays an essential role in the thermal regulation of fatty acid composition of E. coli.
Strains harboring a temperature-sensitive mutation in the fabB gene and an additional mutation in the fabF gene fail to synthesize long-chain fatty acids at the nonpermissive temperature (83). Even supplementation with oleate, an unsaturated fatty acid that allows growth of fabB unsaturated fatty acid auxotrophs, fails to permit growth of a fabB(Ts) fabF double mutant at the nonpermissive temperature owing to the inability of the strain to synthesize saturated fatty acids. Synthases I and II are therefore the only E. coli synthase activities active in the synthesis of long-chain fatty acids.
The investigation of synthase II is hampered by the inability to clone the intact fabF gene (182). The gene encoding ACP, acpP, is adjacent to the fabF locus (233), and since overproduction of ACP is toxic to the cell, the isolation of the fabF gene from clone banks was probably precluded. However, directed cloning of fabF has also failed, although the sequence of the gene has been determined by cloning chromosomal fragments and PCR products (179). The deduced FabF amino acid sequence gives a protein of 43 kDa, a value in close agreement with that of the purified enzyme (83). The FabF sequence can be aligned with FabB over the entire length of the two proteins and shares 38% identical residues with FabB, including a similar active-site sequence.
Synthase III is a monomeric protein of 33.5 kDa first detected as a condensation activity resistant to cerulenin both in vivo and in vitro (135). Although cerulenin blocks the synthesis of long-chain fatty acids, short-chain (C4 to C8) acids linked to ACP accumulate both in vivo and in cell extracts. The fabH gene encodes synthase III, and the deduced amino sequence has little similarity to the FabB or FabF sequence except at the active site, although there is good alignment with sequences of other enzymes known to catalyze condensation reactions. From the chain length of the acyl-ACPs produced and the behavior of fabB fabF double mutants mentioned above, it is clear that synthase III does not participate in the terminal condensation steps of fatty acid synthesis. However, the enzyme could produce the fatty acid synthetic intermediates used in lipoic acid (and perhaps biotin) synthesis.
β-Ketoacyl-ACP Reductase.
An open reading frame encoding a protein with strong similarities to several acetoacetyl-CoA reductases and particularly plant β-ketoacyl-ACP reductases (>50% identical residues) is located within the fab gene cluster between the fabD and acpP genes (233). This gene, designated fabG, is cotranscribed with acpP. Mutants with lesions in this gene have not been isolated. There seems to be only a single NADPH-specific β-ketoacyl-ACP reductase in E. coli which functions with all chain lengths (272). Hence, fabG mutants should block fatty acid synthesis following the initial condensation.
β-Hydroxyacyl-ACP Dehydrase.
This enzyme is not to be confused with the β-hydroxydecanoyl-ACP dehydrase specifically required for introduction of the double bond of the unsaturated acids (both enzymes are more properly called dehydratases). Prior biochemical data give a confused picture of this reaction. One group has reported that this step is catalyzed by a single enzyme active with substrates of all chain lengths (16), whereas another laboratory reported the presence of three enzymes specific for short-, medium-, and long-chain-length substrates (190). An E. coli gene (called fabZ) that encodes a dehydrase active on β-hydroxymyristoyl-ACP has recently been isolated (191). Mutants with reduced enzyme activity are suppressors of mutations in lipid A biosynthesis, and the suppression is thought to be due to increased intracellular levels of β-hydroxymyristoyl-ACP (191). The chain length specificity of FabZ is unknown, but the availability of the cloned enzyme will facilitate study of this enzyme. A partial S. typhimurium open reading frame has an essentially identical sequence (191).
Enoyl-ACP Reductase.
Two forms of enoyl-ACP reductase (Fig. 3), one dependent on NADH and the other dependent on NADPH, have been reported (247, 292). The genes encoding the NADH-dependent enoyl-ACP reductases of E. coli (13, 14) and S. typhimurium (276) have been identified. A mutation in the E. coli gene was reported in 1973 (66) as a temperature-sensitive mutant called envM. The identification of the protein encoded by this gene arose from the study of mutants E. coli and S. typhimurium resistant to diazaborines, a class of antimicrobial agents that inhibit lipid synthesis (276). A diazaborine-resistant mutant of E. coli has a lesion in the envM gene, and plasmids expressing the wild-type gene from either E. coli or S. typhimurium overcome the temperature-sensitive growth of the envM mutant (13, 276). In both organisms, the diazaborine resistance mutation (G93S) is the same (13). The homogeneous wild-type protein has NADH-dependent enoyl-ACP reductase activity (14). Binding of diazaborine to the enzyme requires the presence of NADH, and the enzyme from the diazaborine-resistant mutant fails to bind the inhibitor. Given these data, the gene was renamed fabI. The deduced FabI amino acid sequence (the proteins of E. coli and S. typhimurium are 98% identical) is homologous to a diazaborine-resistant plant enoyl-ACP reductase which can replace the fabI gene in E. coli fatty acid synthesis and give diazaborine-resistant growth (144). The FabI amino acid sequence is also similar to that of an NADH-specific enoyl-ACP reductase encoded by a gene (called inhA) from mycobacteria (8). Missense mutations within the inhA gene result in resistance to the antituberculosis drugs isoniazid and ethionamide. Since these drugs inhibit mycolic acid biosynthesis, it seems that the synthesis of these unusual mycobacterial acids requires an enoyl-ACP reductase (8). Indeed, the residue altered to give resistance to isoniazid and ethionamide in mycobacteria is only one residue removed from the analogous residue altered to give diazaborine- resistant mutants in the enterobacteria and both mutations result in decreased affinities for NADH (14, 218a). The crystal structures of the InhA-NADH complexes of both the wild-type and isoniazid-resistant proteins indicate that the decreased NADH affinity of the mutant protein is due to perturbation of the hydrogen-bonding pattern of the cofactor binding site (56a).
The NADPH-dependent enoyl-ACP reductase has been studied in cell extracts (247, 292), but the physiological role of this enzyme is unclear. The phenotype of the fabI mutant and the effects of diazaborine indicate that the activities are not functionally interchangeable. The NADH enzyme is reported to be active with substrates of chain length C2 to C14 (292), and so a role for a second enzyme is not obvious.
Biosynthesis of Unsaturated Fatty Acids.
β-Hydroxydecanoyl-ACP dehydrase (FabA) specifically catalyzes the dehydration of β-hydroxydecanoyl-ACP to a mixture of trans-2-decenoyl-ACP and cis-3-decenoyl-ACP (17) (Fig. 4). The reaction proceeds via the formation of trans-2-decenoyl-ACP as an enzyme-bound intermediate that can disassociate from the enzyme (17) and be reduced by an enoyl-ACP reductase and subsequently converted to saturated fatty acids as in the standard elongation cycle (31). Enzyme-bound trans-2-decenoyl-ACP, however, is isomerized to cis-3-decenoyl-ACP. The double bond is preserved, and the cis-3 intermediate is elongated to the unsaturated fatty acids of E. coli, palmitoleic acid and cis-vaccenic acid. The nucleotide sequence of the fabA gene has been determined (46), and the deduced amino acid sequence has been confirmed by protein chemistry. The mechanism of the enzyme has been thoroughly examined (17, 46, 104) and FabA has been crystallized (255), but the structure is not available. FabA is a homodimer of 18-kDa subunits (46) and is distinct from the elongation cycle β-hydroxyacyl-ACP dehydrase discussed above, although the two enzymes have sequence similarity (191).
The first mutants isolated in fatty acid biosynthesis, called fabA, lacked β-hydroxydecanoyl-ACP dehydrase (262). These mutants are unable to synthesize unsaturated fatty acids but synthesize saturated fatty acids normally. Mutant fabA enzyme forms neither the cis-3- nor trans-2-decenoyl product in vitro (43). This finding, along with the observation that saturated fatty acid synthesis continues in vivo, indicated that another dehydrase is available for saturated fatty acid synthesis. This second enzyme (presumably the fabZ enzyme) is able to catalyze the formation of trans-2-decenoyl-ACP but fails to catalyze the isomerase reaction. In the absence of thermal regulation, the ratio of unsaturated to saturated fatty acids in E. coli is dependent on the levels of β-hydroxydecanoyl-ACP dehydrase and β-ketoacyl-ACP synthase I. It was shown that overproduction of the fabA gene product in vivo did not increase the level of unsaturated fatty acids but significantly increased the amount of saturated fatty acids incorporated into membrane phospholipid (31). This observation indicated that although FabA is required for the synthesis of unsaturated fatty acids, the level of enzyme activity does not limit the rate of unsaturated fatty acid synthesis. Introduction of multiple copies of the fabB gene (encoding synthase I) reversed the effect of dehydrase overproduction, resulting in wild-type fatty acid composition (31). Thus, the step more likely to limit the rate of unsaturated fatty acid synthesis is the elongation of cis-3-decenoyl-ACP catalyzed by synthase I. The relative levels of fabA and fabB gene expression appear to establish a basal ratio of unsaturated to saturated fatty acid synthesis.
The first step in membrane phospholipid formation is the transfer of the acyl chains of the acyl-ACP end products of fatty acid biosynthesis to G3P (Fig. 5). The first enzyme (the plsB gene product) transfers fatty acids to the 1 position of G3P, and the second enzyme (the plsC gene product) esterifies the 2 position of the glycerol backbone. Like most phospholipids in nature, bacterial phospholipids have an asymmetric distribution of fatty acids between the 1 and 2 positions of the glycerolphosphate backbone that is controlled in part by the acyl chain specificity of the two acyltransferases. The G3P acyltransferase system is not considered to be a component of fatty acid biosynthesis per se; however, the activity of the acyltransferase system does affect both the chain length distribution of the fatty acids found in membrane phospholipids and the rate of fatty acid biosynthesis (see below).
The pathway for the synthesis of membrane phospholipids is shown in Fig. 6. Phosphatidic acid is converted to CDP-diacylglycerol, which serves as an intermediate in the biosynthesis of all membrane phospholipids. PE, the most abundant membrane phospholipid, is synthesized by the exchange of serine for CMP catalyzed by PS synthase followed by PS decarboxylase, yielding PE. PG is synthesized by the exchange of G3P for CMP to form PGP, which is subsequently dephosphorylated to generate PG. CL is synthesized by the condensation of two molecules of PG by CL synthase. The known genes in phospholipid synthesis are listed in Table 1.
Biosynthesis of G3P.
G3P is a water-soluble intermediate that forms the scaffold of all phospholipid molecules and is the precursor to the polar head group of PG. G3P can be synthesized by two routes. Growth on glycerol as the sole carbon source induces the enzymes of glycerol degradation (the glp operon). Glycerol kinase (encoded by glpK) is one of the enzymes induced and converts glycerol to G3P. During growth on carbon sources other than glycerol, G3P is made by the NADH-dependent reduction of the glycolytic intermediate, dihydroxyacetone phosphate (152). The enzyme catalyzing this reaction is called the biosynthetic G3P dehydrogenase, or G3P synthase, and is the product of the gpsA gene (42). This enzyme is sensitive to allosteric feedback regulation by G3P (65), and this mechanism is responsible for the maintenance of a stable intracellular G3P concentration, as verified by the isolation of mutants resistant to feedback inhibition (12). These mutants were isolated as extragenic suppressors of the plsB mutants (see below) and contain elevated levels of G3P (12, 65).
The gpsA gene product is probably the sole enzyme that produces G3P for phospholipid synthesis in cells grown on carbon sources other than glycerol. Mutants (gpsA) lacking G3P synthase activity require exogenous G3P or glycerol for growth. In the absence of G3P, phospholipid synthesis is inhibited >95%. The residual 32Pi incorporation into phospholipid in gpsA mutants is attributed to diacyglycerol phosphorylation largely resulting from PG turnover. The levels of G3P do not limit the rate of phospholipid biosynthesis. Not only are the G3P levels in wild-type strains maintained at a concentration of 200 μM by feedback regulation of GpsA, but the experimental manipulation of the G3P levels over a 10-fold range in vivo has no noticeable effect on the phospholipid biosynthetic rate or membrane phospholipid composition (84).
Biosynthesis of 1-Acyl-G3P.
G3P acyltransferase catalyzes the first acylation of the 1 position of G3P (Fig. 5) and represents the transition from soluble intermediates and enzymes to membrane-bound enzymes and products. A considerable amount of effort has been expended on understanding the substrate specificity of the acyltransferase in an effort to explain the positional asymmetry observed in vivo (Fig. 5). The results of much of this work appear contradictory; however, they generally consistently indicate that the acyltransferase system does possess the appropriate specificity to account for the positional distribution of fatty acid observed in vivo (242). The most detailed study of the membrane-bound system with native acyl-ACPs shows that palmitoyl-ACP and cis-vaccenoyl-ACP are good substrates for the acyltransferase, but palmitoleoyl-ACP is not (244). Although the extent of specificity is dependent on the assay conditions and acyl donors used, overall, the in vitro data are consistent with the in vivo experiments which show that the acylation specificity is not absolute but can be altered by manipulation of the supply of acyl donors (242).
A major advance in the investigation of G3P acyltransferase was the isolation of E. coli mutants (plsB) with defective acyltransferase activity (10). These mutants are G3P auxotrophs and exhibit an increased Michaelis constant for G3P in in vitro acyltransferase assays (10, 11). Therefore, plsB mutants are thought to express a mutant form of G3P acyltransferase with decreased affinity for G3P that requires an artificially high intracellular concentration of G3P for activity. Complementation of these mutants facilitated the cloning, purification, and biochemical characterization of the G3P acyltransferase (86, 87, 164, 172, 173, 251). Although the acyltransferase can be extracted from the inner membranes, only a minimal purification of the enzyme was achieved starting with wild-type strains (265). Hybrid plasmids that suppressed the G3P requirement of plsB strains overexpress G3P acyltransferase activity 10-fold. Extraction of the membrane fraction from these strains and subsequent column chromatography yielded a single protein with an apparent molecular mass of 83 kDa (86, 164). The nucleotide sequence of the plsB gene has been determined and predicts a protein of 91.26 kDa (172). The protein with an apparent molecular mass of 83 kDa has been established to be the product of the plsB gene by comparing amino acid sequence information with that predicted from the DNA (87). The single polypeptide catalyzes the formation of 1-acyl-G3P from either acyl-CoA or acyl-ACP acyl donors (86).
The G3P acyltransferase, solubilized from membranes with Triton X-100 and purified in this nonionic detergent, is inactive unless reconstituted with phospholipid (86, 249). Much of the acyltransferase in the membrane fraction of cells engineered to overexpress the PlsB protein is inactive (latent) until the membranes are solubilized and the 83-kDa polypeptide is reconstituted into unilamellar vesicles made from E. coli phospholipid (250). A mixed micelle assay containing detergent micelles and phospholipid demonstrated that the G3P acyltransferase is specifically activated by acidic phospholipids, PG and CL (86, 249). Hydrodynamic experiments indicate that the enzyme reconstituted into mixed micelles is active as a monomer and that the latency of the acyltransferase in strains overproducing PlsB is attributed to oligomerization at high protein concentrations (250). Monomeric G3P acyltransferase also exhibits negative cooperativity with respect to G3P binding (250), a property that may account in part for the finding that dramatic increases in the intracellular G3P concentration do not increase the amount of phospholipid in E. coli (84).
The interpretation of the enzymatic alteration in the plsB mutants is complicated by the finding that the plsB phenotype depends on two unlinked mutations (165). One mutation is in the plsB gene discussed above, and the second is in a gene called plsX. Both mutations are required for a strain to exhibit a requirement of G3P, since strains harboring only the plsB or plsX lesion do not have a growth phenotype (165). The apparent Km defect in G3P acyltransferase is associated with the plsB mutation, not the plsX mutation. The plsX gene is located between rpmF and fabH near 24 min on the E. coli map and is composed of 346 codons predicted to encode a protein of 37.1 kDa (207). The low Km for G3P found in isolated membranes is converted to a 10-fold-higher Km following detergent solubilization and reconstitution of the G3P acyltransferase (250). The biochemical basis for this behavior is unknown, but it is possible that the native PlsB protein exists in a complex with other proteins. One candidate for such a factor is the plsX gene product. It is interesting that Rhodobacter capsulatus contains a plsX homolog that complements the E. coli mutation (25).
Biosynthesis of Phosphatidic Acid.
The next step in phospholipid biosynthesis is catalyzed by 1-acyl-G3P acyltransferase (the plsC gene product), which acylates the product of the PlsB step to form phosphatidic acid (Fig. 5). Phosphatidic acid constitutes only about 0.1% of the total phospholipid in E. coli and turns over rapidly (78), a property consistent with its role as an intermediate in phospholipid synthesis. Temperature-sensitive mutants that accumulate 1-acyl-G3P at the nonpermissive temperature (32) and possess temperature-sensitive 1-acyl-G3P acyltransferase activity in vitro were isolated. The plsC gene is located between parC and sufI near 65 min of the chromosome (33). The sequence of the homologous gene in S. typhimurium has also been determined, and a plant gene that complements the E. coli mutation has been isolated (21). The amino acid sequence of S. typhimurium plsC (also called parF) is 94% identical to the E. coli enzyme except for an additional 20-amino-acid insert in the E. coli sequence near the carboxy terminus (33). This enzyme utilizes either acyl-CoAs or acyl-ACPs as acyl donors and is thought to transfer unsaturated fatty acids selectively to the 2 position. The cloning and overexpression of PlsC allows more detailed biochemical analysis of its substrate specificity.
The presence of phosphatases that specifically dephosphorylate phosphatidic acid and 1-acyl-G3P is known (283), and mutants deficient in these enzymes have been isolated (127). The function of these phosphatases remains an enigma, but their potential involvement in the acylation of G3P is suggested by the observation that certain acyl-ACPs promote the dephosphorylation of G3P, 1-acyl-G3P, and phosphatidic acid (244).
CDP-Diacylglycerol Synthase.
A key finding in the pathway for phospholipid synthesis was the discovery of an activated form of phosphatidic acid, CDP-diacylglycerol (24, 141). This metabolically active intermediate constitutes only 0.05% of the total phospholipid pool (226). In E. coli, conversion of phosphatidic acid to a mixture of CDP-diacylglycerol and dCDP-diacylglycerol is catalyzed by a single enzyme called CDP-diacylglycerol synthase (Fig. 6). Mutants severely deficient in this enzyme have lesions at a single genetic locus (cds), but despite retaining only 5% of the normal levels of CDP-diacylglycerol synthase, the strains grow normally under standard laboratory conditions (78, 79). However, some of these mutants accumulate substantial amounts of phosphatidic acid (up to 5% of the total phospholipid), which may account for their increased sensitivity to erythromycin and elevated pH. Shifting the growth medium from pH 7.0 to 8.5 triggers massive accumulation (up to 25% of the total phospholipid) of phosphatidic acid and the inhibition of phospholipid synthesis. These data suggest that CDP-diacylglycerol synthase is present in large excess of the minimal amount of enzyme required to sustain phospholipid synthesis. An unlinked mutation that suppresses the phenotype of strains carrying a cds mutant allele has also been isolated (80), although the nature of this mutation and the specificity of its suppression are unknown. The CDP-diacylglycerol synthase structural gene (cdsA) has been cloned, and its sequence has been determined (128).
CDP-diacylglycerol reacts with either G3P or serine to form PGP or PS, respectively. The presence of both ribo and deoxyribo forms of the liponucleotide could play a role in determining the relative rates of the synthesis of these two phospholipids if the respective synthases exhibit selectivity toward dCDP versus CDP-diacylglycerol, which exist in a ratio of 0.88 in vivo (79). However, both ribo- and deoxyriboliponucleotides are substrates for PS synthase in vitro (163, 227), and a change in the ratio of liponucleotides in vivo to 3.1 has no effect on the relative rates of PE and PG synthesis (79). Thus, the significance, if any, of the two forms of liponucleotide remains to be determined.
Biosynthesis of PE.
The first step in the synthesis of PE is the condensation of CDP-diacylglycerol with serine to form PS catalyzed by PS synthase (141) (Fig. 6). PS synthase does not appear in the inner membrane fraction during standard cellular localization procedures, as do the other enzymes of phospholipid synthesis. The enzyme instead is found attached to ribosomes (227). This association is an artifact of cell disruption; the ribosomes act as a ion-exchange trap for the PS synthase and following addition of CDP-diacylglycerol, PS synthase is dissociated from the ribosome and subsequently associates with phospholipid vesicles containing CDP-diacylglycerol (163, 176). The structural gene for PS synthase (pss) has been identified, mapped, and cloned (221, 228). PS synthase has been purified to homogeneity (163), a procedure that is greatly simplified by the overproduction of the enzyme (228). The PS synthase reaction proceeds via a ping-pong mechanism (163, 222).
PS is minor membrane constituent of E. coli since it is rapidly converted to PE by PS decarboxylase (Fig. 6). This inner membrane enzyme has been purified to homogeneity and found to have a subunit molecular mass of 36 kDa (64). PS decarboxylase has a pyruvate prosthetic group that participates in the decarboxylation reaction by forming a Schiff base with PS (248). The two nonidentical subunits of the mature enzyme are formed by cleavage of a proenzyme resulting in the conversion of Ser-254 to an amino-terminal pyruvate residue (166). Protein processing is interrupted by mutational alteration of Ser-254 to either Cys-254 or Thr-254, but although the mutant proteins have only minimal activity, they are able to complement psd(Ts) mutants (167). These data are consistent with the mechanism proposed for the formation of pyruvate prothetic groups in several pyruvate-dependent decarboxylases (236).
Mutants (psd) with a temperature-sensitive decarboxylase accumulate PS at the nonpermissive temperature (94, 95). Despite the reduced levels of PE and the concomitant increase in PS levels, the mutants continue to grow for several hours after the shift to the nonpermissive temperature. The psd gene has been cloned, and strains harboring such clones overproduce the enzyme 30- to 50-fold (278). Under these conditions, only half of the enzyme remains associated with the inner membrane and there is no effect on membrane phospholipid composition.
The sequence of the psd gene has also been determined and has been inactivated by insertional mutagenesis (54). The inability to synthesize PE is lethal. Surprisingly, the lethality is phenotypically suppressed by the addition of divalent cations to the growth medium, although there are perturbations in the function of permeases (18), electron transport (185), and motility and chemotaxis (257). PE is capable of forming the hexagonal (nonbilayer) HII lipid phase, and the divalent cations interact with CL to replace PE in the formation of an HII phase (148, 237, 238). The requirement for CL is consistent with the inability to introduce a null cls allele into either pss or psd::kan strains (54, 202). Thus, PE is essential for the polymorphic regulation of lipid structure. The physiological processes dependent on the formation of local regions of nonbilayer structure remain to be elucidated, but the process of cell division, the formation of contacts between inner and outer membranes, and the translocation of molecules across the membrane are viable candidates.
There is also a salvage pathway for the synthesis of PE from 2-acylglycerophosphoethanolamine (2-acyl-GPE). 2-Acyl-GPE arises from the action of phospholipase A1, as a by-product of protein acylation, or can be taken up from the medium (117, 121, 239). The resulting 2-acyl-GPE is recycled into PE by 2-acyl-GPE acyltransferase/acyl-ACP synthetase (the product of the aas gene). The acyltransferase was first recognized as an inner membrane protein called acyl-ACP synthetase (235). Acyl-ACP synthetase catalyzes the ligation of ACP to fatty acids in the presence of ATP, Mg2+, and high salt concentrations. The acyltransferase contains ACP as a bound subunit that acts as the intermediate acyl acceptor in the acyltransferase reaction, and the high salt concentrations are required to dissociate the acyl-ACP intermediate from the enzyme, thereby uncovering the synthetase activity (35, 240, 246). Although the acyl-ACP synthetase reaction has proven extremely valuable in the preparation of acyl-ACPs that are substrates for other enzymes (243), 2-acyl-GPE acyltransferase is thought to be the only reaction catalyzed by Aas in vivo, consistent with the finding that exogenous fatty acids are not converted to acyl-ACP derivatives that can be utilized by the enzymes of fatty acid biosynthesis. The isolation of aas mutants supports the biochemical studies (122). The aas mutants are defective in both acyl-ACP synthetase and 2-acyl-GPE acyltransferase activities. However, they do not accumulate 2-acyl-GPE in vivo unless they are also defective in the pldB gene, which encodes a lysophospholipase that represents a second pathway for 2-acyl-GPE metabolism. The aas gene was cloned, and strains overexpressing aas gene overproduce both synthetase and acyltransferase enzyme activities (130). The aas gene is the structural gene for the acyltransferase, and in contrast to an earlier report (35), the aas gene product is a protein with a molecular mass of 81 kDa. Analysis of the predicted amino acid sequence reveals extensive homology to other synthetases that employ acyl-adenylates as intermediates (130).
Biosynthesis of PG.
The first step in the synthesis of PG is the condensation of CDP-diacylglycerol with glycerolphosphate to form PGP (Fig. 6). The reaction is analogous to the synthesis of PS, and CMP is the released product (26). The protein has been purified to homogeneity by affinity chromatography on CDP-diacylglycerol-Sepharose (108). The reaction kinetics are consistent with a sequential bi-bi mechanism, and unlike PS synthase, PGP synthase does not catalyze the hydrolysis of CDP-diacylglycerol (108, 222). An additional substrate for the enzyme is the G3P analog 3,4-dihydroxybutyl-1-phosphonate, but the PGP analog synthesized from this substrate cannot be hydrolyzed by PGP phosphatase (260). When cells are grown in the presence of this analog, the abnormal lipid accumulates and growth ceases. This analog has other metabolic fates; therefore, it is not clear that its effects on cell growth can be exclusively attributed to the accumulation of the PGP analog (277). However, more recent work indicates that the conversion of 3,4-dihydroxybutyl-1-phosphonate to its PGP analog accounts for the increased sensitivity of E. coli to this analog in media containing elevated amounts of divalent cations (146).
Mutants defective in PGP synthesis have been isolated by the colony autoradiography approach (199, 220). These mutants (pgsA) contain less than 5% of normal PGP synthase activity in vitro; however, there is no growth phenotype associated with these mutants. A second round of mutagenesis was performed on the pgsA mutants, and temperature-sensitive strains that were severely impaired in the ability to synthesize PG owing to a mutation at a second locus, pgsB, were isolated (197, 199). A surprising finding in the pgsA pgsB double mutants was the accumulation of two novel glycolipid precursors of lipid A (200, 270, 271). The pgsB allele was subsequently identified as an enzyme in lipid A biosynthesis and has been renamed lpxB (see chapter 69 for details). The pgsA gene is the structural gene for PGP synthase; pgsA has been cloned (85), and its sequence along with those of three mutant pgsA alleles has been determined (280).
The insertional inactivation of PGP synthase is lethal (99). There are many important cellular functions that are affected by reduced PG and/or CL content of the membrane. PG is required for protein translocation across the membrane, and SecA is the critical component affected (57, 161, 162, 174), although SecA-independent translocation is also impaired (160). Acidic phospholipids are also required for channel activity of bacterial colicins (284) and the interaction of antibiotics with the membrane (58).
The second step in the synthesis of PG is the dephosphorylation of PGP (Fig. 6). Using a colony autoradiography approach, Icho and Raetz (127) isolated two independent genes, pgpA and pgpB, that encoded PGP phosphatases, as determined by an in vitro assay. Both of these loci have been cloned, and their sequences have been determined (125, 126). On the basis of the fact that the the pgpA-encoded phosphatase specifically hydrolyzed PGP, whereas the PgpB phosphatase also hydrolyzed phosphatidic acid, PgpA was thought to be the enzyme involved in PG biosynthesis. However, Funk and coworkers (75) disrupted both of these genes in a single strain, and although the respective phosphatase activities are reduced, PG synthesis is not impaired. Thus, neither of these phosphatases is required for PG synthesis, and the existence of at least one other phosphatase capable of operating in the PG biosynthetic pathway remains to be discovered.
Biosynthesis of CL.
CL is produced by the condensation of two PG molecules (Fig. 6). Initially, CL was thought to be synthesized by the reaction of CDP-diacylglycerol with PG, the mechanism that exists in mammalian mitochondria. However, results of a series of physiological experiments indicated that unlike synthesis of other phospholipids, CL synthesis occurs under conditions of ATP depletion and led to the identification of the enzyme catalyzing this reaction (111, 275). The physiological role of CL synthase was corroborated by the isolation of a mutant (cls) deficient in both the synthesis of CL and transesterification activity (215). These mutants were initially thought not to have a growth phenotype, but subsequently, growth was shown to be impaired in mutants (pss) deficient in PE synthesis (258). This latter property was used to clone the cls gene (209). E. coli is able to survive the disruption of the cls gene, although the cells grow at a slower rate and to a lower density than the corresponding wild-type cells, indicating that CL may confer a growth or survival function (109). CL accumulation and CL synthase activity increase as the cells enter the stationary phase of growth (101, 109, 275), and CL is the most stable membrane phospholipid during prolonged incubation in stationary phase (109). The cls null mutants lose viability in stationary phase, supporting the idea that CL is important for long-term survival under nongrowing conditions (109). The conclusion that CL is nonessential is complicated by the finding of residual CL in the cls null mutants (202). The origin of the CL may be due to the activity of PS synthase. The cls null allele cannot be transferred to pss mutants, suggesting that a low level of CL may be essential for viability. Amplification of CL synthase, in turn, leads to the overproduction of CL and to a decreased membrane potential and loss of viability (110). Therefore, E. coli tolerates rather large changes in the overall CL content, but the elimination or overproduction of CL leads to significant physiological imbalance. E. coli accumulates phosphatidylmannitol and diphosphatidylmannitol when grown on 0.6 M mannitol, and a number of other sugar alcohols can be incorporated into phospholipid (259). The formation of these unusual analogs is increased when a pss (PS synthase) mutant is shifted to the nonpermissive temperature. The formation of these lipids appears to be catalyzed by CL synthase, since cls mutants fail to accumulate phosphatidylmannitols and a strain carrying a cloned cls gene overproduces these phospholipids. A pathway involving the reversal of the CL synthase reaction coupled with a lack of substrate specificity of CL synthase is proposed to account for these findings (259). Interestingly, cells that contain high levels phosphatidylmannitol and diphosphatidylmannitol grow almost normally, although the ability of the cells to respond to stress has not been evaluated.
The synthesis of cyclopropane fatty acids (CFAs) is a postsynthetic modification since the substrate unsaturated fatty acids are already esterified into membrane-localized phospholipid molecules. The reaction is a methylenation of the double bonds, the methylene donor being S-adenosylmethionine (SAM). Much is known about CFA synthesis, but two interesting questions remain. First, how do the soluble CFA synthase and soluble substrate, SAM, gain access to the phospholipids of the inner and outer membranes? Second, why are these acids made by a large variety of bacteria? Mutants that completely lack CFA synthase activity (owing to null mutations in the cfa gene) exist, but they grow and survive normally under virtually all conditions (89). The only exception to this finding is that cfa mutant strains are more sensitive to freeze-thaw treatment than are isogenic Cfa+ strains (90).
CFA synthase has been purified to homogeneity, and the nucleotide sequence of the cfa gene encodes a 44-kDa protein (291). The only similarities of the deduced amino acid sequence of CFA synthase to sequences of other known proteins are to a putative homolog found in Pseudomonas putida (J. R. Sokatch, unpublished observations) and to a short sequence conserved in other SAM-utilizing enzymes believed to be the SAM binding site (291).
The bulk of CFA synthesis occurs as cultures enter the stationary phase of growth corresponding to a sharp increase in CFA synthase activity owing to increased cfa transcription (290). CFA synthase levels are low in stationary-phase cultures, suggesting that the unstable enzyme is destroyed by proteolysis. Analyses of CFA gene transcription indicate the presence of two promoters of approximately equal strengths (290). The more distal promoter functions throughout the growth cycle, whereas the proximal promoter is active only as cultures enter stationary phase. The proximal promoter requires a special sigma factor (σ S) encoded by the rpoS gene (chapter 93). Indeed, the CFA content of rpoS strains is low, and transcription from the proximal promoter is absent in these strains (290).
In log-phase cultures, only the distal promoter is active, resulting in low CFA synthase activity. As cultures enter stationary phase, RpoS (σ S) is synthesized (chapter 93), activates the proximal promoter, and increases cfa transcription and CFA synthase. Moreover, as growth slows, phospholipid synthesis diminishes, and thus CFA synthase activity no longer encounters an expanding substrate pool. The increased CFA synthase activity efficiently converts the membrane phospholipids accumulated during log phase to their CFA derivatives. The instability of CFA synthase in stationary-phase cells results in little carryover of CFA synthetic capacity when exponential growth resumes. The rationale for such a system is unclear, since high-level production of CFA by log-phase cultures fails to inhibit growth (89).
The supply of ACP does limit fatty acid biosynthesis, since there is a significant pool of unacylated holo-ACP during logarithmic growth (133). In addition to unacylated ACP, there are significant pools of acetyl-ACP and malonyl-ACP (245), but acyl-ACPs with chain lengths of four carbons or more are not major components (245). The discovery that acetyl-CoA:ACP transacylase activity is associated with the β-ketoacyl-ACP synthase III specific for the first condensation reaction in the pathway suggests that regulation of the synthase might be coordinated with regulation of acetyl-CoA carboxylase. The coordinate regulation would act to control the elongation of fatty acids in order to keep pace with the production of primer. The balance between these two enzymes also affects the chain length of fatty acids (see below). The precise role of the initiation steps in regulating fatty acid formation is not obvious, and current research is focused on defining the relationship between synthase III activity and the rate of fatty acid initiation. Recent work on the mechanisms coupling fatty acid synthesis to phospholipid synthesis indicates that an early step(s) in fatty acid synthesis is regulated by long-chain acyl-ACP levels (see below).
Palmitate, palmitoleate, and cis-vaccenate constitute the bulk of the fatty acids found in E. coli membranes. The β-ketoacyl-ACP synthases play a major role in controlling the chain length and production of these fatty acids. In vitro substrate specificity experiments indicate that E. coli membrane phospholipids contain negligible levels of chain lengths >18 carbons because the precursor acyl-ACPs are poor substrates for elongation by the synthases (83, 88). Inactivation of synthase I (FabB) blocks unsaturated fatty acid synthesis and therefore produces a deficiency in cis-vaccenate (83, 279). On the other hand, overexpression of synthase I leads to the overproduction of cis-vaccenate (56). Thus, the elevated activity of synthase I allows it to elongate acyl-ACPs that are poor substrates for this enzyme. Mutants lacking synthase II are unable to synthesize cis-vaccenate and are therefore deficient in C18 fatty acids in the membrane. As noted above, clones that overexpress synthase II have not yet been isolated. Mutants severely impaired in synthase III activity are enriched in C18 fatty acids, whereas the overexpression of synthase III causes a decrease in the average fatty acid chain length and the appearance of significant amounts of myristic acid in the phospholipids (273). This effect is attributed to an increased rate of fatty acid initiation which leads to a deficiency in malonyl-CoA (and/or malonyl-CoA) for the terminal elongation reactions. Overproduction of either malonyl-CoA:ACP transacylase (FabD) (181, 285) or the NADH-dependent enoyl-ACP reductase (FabI) (144) also slightly increases the average chain lengths of the phospholipid fatty acids.
The activity of the glycerolphosphate acyltransferase system is the other important component involved in regulating acyl chain length. When phospholipid synthesis is slowed or arrested at the G3P acyltransferase step (by glycerol starvation of either plsB or gpsA mutants), the fatty acids that accumulate have abnormally long chain lengths (e.g., 20 and 22 carbons) (36, 49). Conversely, overproduction of the acyltransferase results in a somewhat decreased average chain length represented mainly by an increase in myristic acid (36). Thus, competition among the rate of elongation by the synthases, the supply of malonyl-ACP, and the utilization of acyl-ACPs by the acyltransferase is the most significant determinant of fatty acid chain length.
At physiological temperatures, normal cell function requires a membrane bilayer in a largely fluid state and thermal regulation of membrane fluidity in common with all organisms. As growth temperatures are lowered, the membrane undergoes a reversible change from a fluid (disordered) to a nonfluid (ordered) state (43, 45). In E. coli, the temperature at the point this transition occurs depends on the fatty acid composition of the membrane phospholipids. Marr and Ingraham (182) first noted that E. coli adjusts its fatty acid composition in response to a lower growth temperature by increasing the amount of cis-vaccenic acid and decreasing the amount of palmitic acid incorporated into membrane phospholipid. Lower growth temperatures result in an increase in the number of diunsaturated phospholipids in the membrane. At 37°C, palmitic acid occupies position 1 of the phospholipid backbone, whereas palmitoleic acid is found only at position 2 (6, 43). As the growth temperature is lowered, cis-vaccenic acid competes with palmitic acid for position 1 of the newly synthesized phospholipids. This mechanism is thought to allow an organism to regulate the membrane fluidity to optimize function at various growth temperatures.
The elucidation of the mechanism of thermal regulation in E.coli involved a number of independent observations. First, the finding that Cvc strains lacked both synthase I and thermal regulation suggested that the elongation of palmitoleoyl-ACP played a role in thermal regulation. Another key observation was that the increased rate of cis-vaccenic acid synthesis characteristic of thermal regulation is evident within 30 s after a temperature downshift (81). This finding indicated that neither mRNA nor protein synthesis is required for fatty acid composition adjustment; therefore, thermal regulation is controlled by a protein present at all temperatures but active only at low temperatures. Palmitoleoyl-ACP is an excellent substrate for synthase II (52, 82), especially at lower temperatures (82). The demonstration that the Cvc phenotype and the lack of synthase II are due to mutations in the same gene, fabF, firmly established the essential role of synthase II in the thermal regulation of fatty acid composition of E. coli.
Although it was known that fabF mutants lacked temperature control, it remained unclear whether the presence of cis-vaccenate per se conferred thermal regulation or whether the synthesis of cis-vaccenate by synthase II was required for the response. The overproduction of synthase I produces an appreciable increase in the cis-vaccenic acid content of membrane phospholipids (56). Introduction of this plasmid into a fabF mutant does increase the cis-vaccenic acid content of cells but is independent of growth temperature (56). Therefore, synthase II is the sole enzyme responsible for thermal modulation of the fatty acid composition.
There is also evidence for a second, uncharacterized site of temperature-dependent control that functions in the incorporation of exogenously supplied fatty acids (38, 263). When supplemented with saturated and unsaturated fatty acids at lower growth temperatures, E. coli preferentially incorporates the unsaturated fatty acids into phospholipid. As the growth temperature is increased, saturated fatty acid incorporation increases. Differential effects on transport, β oxidation, or synthesis of endogenous acids do not explain these results (38). It is clear from the fabF data, however, that this mechanism is not involved in temperature regulation of lipid compositions that derive their fatty acids by endogenous synthesis. In the presence of exogenous fatty acids, endogenous unsaturated fatty acid synthesis is decreased (via FadR regulation), and thus the FabF pathway cannot operate.
The Vtr mutation causes cells to overproduce cis-vaccenic acid at all temperatures (55, 279). The Vtr mutation is closely linked to the fabF gene (279). Efforts to detect a kinetic defect in synthase II extracted from aVtr mutant have been unsuccessful (279), and recent work shows that both the fabF and acpP genes of a Vtr strain have wild-type coding sequences (179) . It therefore seems that the Vtr mutation may affect the expression of these or other genes of the fab cluster. It is hoped that further investigation of the genetic nature of the Vtr mutation will provide further insight into temperature regulation.
The known genes are scattered about the genome with only two clusters, the minimal accBC operon (168) and the fab cluster, containing the fabH, fabD, fabG, acpP, and fabF genes (207, 233). This latter clustering of genes may have functional significance, since several genes are cotranscribed (233; Y. Zhang, S. Subrahmanyam, and J. E. Cronan, Jr., unpublished observations). However, most genes also appear to have a unique promoter, and the detailed transcription pattern is not yet clear. The gene upsteam of fabH is plsX, a gene playing an undefined role in the G3P acyltransferase reaction. However, the gene (rpmF) upstream of plsX encodes ribosomal protein L32, whereas the gene downstream of fabF is pabC, a gene of p-aminobenzoate synthesis, and thus the extent of the fab gene cluster is delimited. The accB and accC genes are cotranscribed from a promoter located unusually far upstream of the accB gene (169, 170). Although RNA polymerase initiating transcription at this promoter traverses both a region of DNA reported to attain a static curve (bent DNA) and a small open reading frame just upstream of accB, deletion of either or both of these features has no effect on accBC transcription (170). The major accA promoter lies within the coding sequence of the polC (dnaE) gene, although transcription through polC and perhaps other upstream genes also reads through the accA sequence (170). The accD gene is transcribed from a promoter located within the upstream dedA gene (170). Transcription of all four acc genes is under growth rate control, the rate of transcription decreasing with decreased growth rate (170). However, the situation is complex in that the accBC operon seems regulated by a mechanism that differs from the regulation of the accA and accD genes (170). Introduction of many copies of the accBC operon results in only modest (<3-fold) increases in accBC transcription and protein products, whereas similar experiments with the accA and accD genes give the expected overproduction of transcription and translation products. Moreover, replacement of the normal accBC promoter with a heterologous lac promoter gives the expected overproduction of gene products (169). We hypothesize that accBC transcription is regulated by a positive activator in limiting supply.
The fabB, fabI, fabZ, and fabA genes are unlinked to other lipid synthetic genes, and the first three genes are transcribed from unique promoters as monocistonic mRNAs (145; C. O. Rock, unpublished observations; Subrahmanyam and Cronan, unpublished observations). In contrast, the fabA gene has two promoters, with the stronger promoter regulated by transcriptional activation that requires FadR protein (105).
FadR protein was discovered as a repressor regulating the fatty acid degradation (fad) regulon of E. coli, which includes genes of β oxidation and fatty acid transport (chapter 21). In cells growing in the absence of fatty acids, FadR binds to operator sites upstream of the fad gene coding sequences and represses transcription of these genes. Exogenous fatty acids enter the cell and are converted to acyl-CoA thioesters which bind to FadR. When complexed to acyl-CoA, FadR disassociates from the operators, resulting in transcription of the fad regulon genes (chapter 21) (59, 60). This view of FadR function in E. coli is now expanded by the finding that FadR acts as a positive activator in the transcription of a fatty acid synthetic gene, fabA.
The first indication of the dual role of FadR in fatty acid metabolism was the finding that introduction of a fadR mutation into a conditional (temperature-sensitive) fabA mutant strain converted the strain to a nonconditional phenotype (205). A second indication of a role for FadR in fatty acid synthesis was the increased sensitivity of fadR strains to a specific inhibitor of the FabA enzyme (31). Subsequent analyses showed that fadR strains had decreased unsaturated fatty acid contents relative to isogenic wild-type strains, indicating that functional FadR was necessary for either fabA gene expression and/or enzyme function (31, 205). Henry and Cronan (105, 106, 107) then demonstrated that FadR is a positive transcriptional activator of fabA expression and that fabA gene expression is decreased 12-fold in a fadR null mutant.
In fadR null mutants, the fabA gene is transcribed from two weak promoters of about equal strength, whereas in wild-type strains, a 20-fold increase in transcription from the proximal promoter is seen (106). FadR binds to a 17-bp nucleotide sequence located in the –40 region of this promoter (59, 60, 107). This binding site is located at the position most often used by transcriptional activators of σ 70 promoters. The nucleotide sequence is similar to those found within the promoters of two fad genes, fadBA and fadL, at which FadR acts as a repressor (59). Other examples of activator proteins that also act as repressors are known (34). The distinction between these two roles usually depends on the location of the protein binding site relative to the transcriptional start. Most σ 70-dependent promoters have their activator sites positioned such that the bound protein overlaps position –40, whereas DNA binding proteins act as repressors when positioned within a larger downstream region of the promoter, generally between –30 and +10 (34). This simple scheme explains FadR action. The binding site for fabA activation is centered at –40, whereas the fadBA and fadL binding sites (where FadR represses transcription) are centered at +9 and –17, respectively. Thus, by analogy with other systems of similar properties (chapter 79), we expect that FadR binding to the fabA DNA aids RNA polymerase binding or action via protein-protein interactions. Likewise, FadR binding to the fad regulon operators would hinder the binding or action of RNA polymerase (chapter 79). Given this latter role it seems surprising that FadR fails to repress expression of its own gene (107). Recent in vitro experiments by DiRusso and coworkers (59, 60) demonstrate that purified FadR activates the proximal fabA promoter and represses the fadBA and fadL promoters.
Transcriptional activation of fabA gene expression is inhibited by fatty acids in vivo owing to decreased activity of the proximal promoter (107). Fatty acyl-CoAs inhibit the binding of FadR to the –40 region of the proximal promoter, and the acyl chain lengths of the acyl-CoAs effective in FadR release from the DNA accurately reflect those of the fatty acids effective in decreasing fabA expression in vivo (107). A similar pattern is seen for the induction of the β-oxidation genes (60). Thus, FadR monitors the intracellular concentration of long-chain acyl-CoA molecules and coordinately regulates fatty acid synthesis and β oxidation in response to these compounds. FadR binds acyl-CoA, and mutants defective in acyl-CoA binding are not removed from the operator sites by acyl-CoA (232).
The FadR amino acid sequence (61, 98) predicts a helix-turn-helix motif common to DNA-binding proteins, and FadR belongs to a new family of bacterial regulatory proteins (98). The FadR binding site of fadBA has perfect or nearly perfect dyad symmetry (59), suggesting that FadR is homodimeric in structure, although dimerization of FadR in solution has not been detected. Gel filtration experiments indicate that FadR is a monomer; however, analysis of negative trans-dominant mutations suggests that the protein binds in multimeric form (59). This inability to reconcile the biochemical and genetic data is not unprecedented. LexA and BirA, the repressors of SOS-inducible and biotin biosynthetic genes, respectively, both bind as monomers to their respective half-operator sites (1, 149). Subsequent cooperative binding of the second monomer produces dimerization on the DNA. We could envision such a dimerization mechanism for FadR. The unraveling of the FadR regulation is the first example of a regulatory protein that positively regulates the biosynthesis of a molecule and negatively regulates the catabolism of the same family of molecules (106, 107).
A major environment of E. coli, the colon, can be a rich source of unsaturated fatty acids. These fatty acids are used as phospholipid precursors as well as energy and carbon sources. In such an environment, endogenous synthesis of unsaturated fatty acids would be unnecessary and inefficient. The presence of the fatty acyl-CoAs therefore results in decreased fabA expression and hence decreased endogenous synthesis of unsaturated fatty acids. If instead only saturated fatty acids are available, repression of fabA transcription is less efficient and the presence of the second FadR-independent promoter maintains a basal level of dehydrase. This allows synthesis, although decreased, of the unsaturated acids needed for functional phospholipids. When exogenous fatty acids are not available, FadR binds to the DNA, increasing fabA transcription. There are no concrete examples of other FadR-regulated fatty acid biosynthetic genes in E. coli. Transcription of the fabB (106), fabI (Subrahmanyam and Cronan, unpublished observations), fabZ (Rock, unpublished observations), and acpP (Zhang et al., unpublished observations) genes are normal in fadR null mutants.
Studies with model membranes suggest that regulation of the relative levels of PE, PG, and CL in the membrane should be a parameter important to membrane function. Within a given strain of E. coli, the PE/PG/CL ratio is unaffected by manipulating the parameters of cell growth (except the conversion of PG to CL occurring in stationary phase), consistent with the presence of homeostatic mechanisms that regulate membrane composition. However, we know little about the mechanisms that underlie these physiological observations. One idea is that the relative content is controlled by the level of enzyme expression. Support for this idea comes from the observation that CL synthase levels increase as cells enter stationary phase concomitant with an increase in the rate of CL synthesis (101, 109, 275). Furthermore, two regulatory mutations that result in the overexpression of PS synthase (pssR) (266) and diacylglycerol kinase (dgkR) have been isolated (225), suggesting the existence of trans-acting factors that control the expression of these key enzymes. However, the substantial overexpression of PS synthase (210), PGP synthase (211), or CL synthase (209) does not lead to a dramatic change in the membrane phospholipid composition, strongly arguing against a role for control over protein levels in the regulatory scheme. A second idea is that the individual enzymes are independently regulated by feedback inhibition that is sensitive to small changes in membrane phospholipid composition. This model was tested in in vivo experiments in which PG was continuously degraded by the transfer of glycerolphosphate to extracellular arbutin (4-hydroxyphenyl-O-β-d-glucoside) by phosphoglycerol transferase I (mdoB), an enzyme involved in the synthesis of membrane-derived oligosaccharides (136). Arbutin treatment did not significantly alter the membrane phospholipid composition, although there was a sevenfold increase in the rate of PG synthesis without any increase in the cellular content of PG synthase, consistent with the idea that PS synthase and PGP synthase are independently regulated by phospholipid composition. This concept is supported by the finding that purified CL synthase is strongly feedback inhibited by CL and that this inhibition is partially relieved by PE (231). The protein product of the Bacillus subtilis pss gene is similar to the yeast PS synthase but has no homology to its E. coli counterpart (212). Expression of the B. subtilis pss gene in E. coli leads to an increase in PE content to 93% of the total phospholipid, indicating that the regulation of PE content in E. coli is an intrinsic property of the PS synthase. Considerably more research on the biochemical mechanism and feedback regulation of PS and PGP synthases is needed to clarify the mechanistic details underlying the regulation of polar head group composition.
In growing cultures of E. coli, fatty acid synthesis is tightly coupled to phospholipid synthesis; the intracellular pools of fatty acid synthetic intermediates are small (245), indicating that fatty acid synthesis is coordinately regulated with or by phospholipid synthesis. The earliest study (188) reported that free fatty acids (FFAs) did not accumulate following the cessation of phospholipids synthesis by the removal of glycerol from either plsB or gpsA mutants. However, the strain used was capable of FFA degradation. Strains (fadE) blocked in β oxidation were subsequently shown to incorporate [14C]acetate into FFA after glycerol starvation at the same rate as when glycerol is supplied (49). However, later work showed that these labeling experiments were complicated by an unexpected shrinkage of the endogenous acetate (acetyl-CoA) pool in glycerol-starved cells (206). Thus, the specific activities of the cellular acetate pools utilized in fatty acid synthesis differed between the cultures starved for glycerol and the unstarved control cultures. Therefore, equivalent rates of [14C]acetate incorporation did not translate into equivalent rates of lipid synthesis.
One plausible explanation for the shrinkage of the acetyl-CoA pool upon glycerol starvation is that FadR, the repressor of the β-oxidation regulon, also downregulates the expression of the glyoxylate operon (chapters 14 and 18). The FFAs are likely converted to acyl-CoAs which would then neutralize FadR, resulting in increased production of the glyoxylate cycle enzymes. Increased glyoxylate cycle enzyme levels would then result in shrinkage of the acetate pool. For these reasons, the choice of a precursor to measure lipid synthesis during glycerol starvation is problematic. Malonate fails to enter E. coli, and the only direct precursor other than acetate, tritiated water, is diluted by the water of the culture medium such that pulse-labeling is essentially impossible. An alternative approach is to measure the level of fatty acyl-acyl carrier protein (acyl-ACP) molecules by labeling the protein moiety. These experiments show the accumulation of acyl-ACPs following glycerol starvation (245), indicating that fatty acid synthesis did continue in the absence of phospholipid synthesis, although FFA produced by hydrolysis of acyl-ACPs could not be determined. An E. coli strain in which endogenous synthesis of acetate was blocked and the only fate of acetate was as a specific precursor of lipid synthesis was constructed (137). Use of this strain demonstrated that fatty acid synthesis following glycerol starvation proceeds at only 10 to 20% of the rate observed during phospholipid synthesis (137). Acyl-ACPs accumulate under these conditions and are thought to feedback inhibit the fatty acid synthetic pathway, accounting for the observed coupling (see below).
The main evidence for feedback inhibition by acyl-ACPs is that overexpression of either of the E. coli thioesterases allows continued fatty acid synthesis following glycerol starvation (137). Moreover, thioesterase overexpression also eliminated the accumulation of acyl-ACP species and the synthesis of fatty acids of abnormal length (137), indicating that the inhibition of fatty acid synthesis may be caused by the accumulation of acyl-ACP species. Restoration of fatty acid synthesis by thioesterase overproduction could result from either loss of the acyl-ACP per se or the increase in ACP concentration resulting from the cleavage of the acyl group. The latter explanation appears untenable, since the ACP pools of the starved cells are not significantly depleted and overproduction of ACP fails to relieve inhibition of fatty acid synthesis (137).
The most straightforward model is that long-chain acyl-ACP species accumulate and inhibit a key fatty acid synthetic enzyme(s). Long-chain acyl-ACPs seem more likely than short-chain acyl-ACP to be the inhibitory species, since only long-chain species accumulate and thioesterase I, which efficiently relieves inhibition, is unable to cleave short-chain (<C8) acyl thioesters(137). The accumulation of long fatty acyl-ACPs provides a signal that fatty acid synthesis is "ahead" of the utilization of acyl-ACP in phospholipid synthesis and provides a homeostatic mechanism to slow fatty acid synthesis (137).
A very similar (if not identical) feedback inhibition mechanism may account for the dependence of fatty acid synthesis on cellular growth. Normally, E. coli ceases lipid synthesis upon entering into stationary phase (the residual synthesis can be attributed to the small fraction of growing cells present in stationary-phase cultures) (37, 214). However, when thioesterase activity is expressed in the cytosol, FFA synthesis continues. This effect was first seen upon expression in E. coli of a thioesterase from the California bay tree, resulting in the accumulation of a massive amount of lauric acid in the culture medium (287). Expression of another plant thioesterase in E. coli gives a mixture of saturated and unsaturated long-chain fatty acids, consistent with the different in vitro substrate specificity of the thioesterase (63). E. coli thioesterase I gives a mixture of fatty acids when localized in the cytosol (see above), and in this case, labeling with radioactive acetate together with analysis of the radioactive products showed that the excreted fatty acids are produced by cleavage of acyl-ACPs (30). A second possible candidate for the regulatory molecule is acyl-CoA. In this scenario, FFA generated by an endogenous thioesterase would be converted to acyl-CoA by the acyl-CoA synthetase (FadD) and downregulate fatty acid synthesis. However, fadD mutant strains producing the aforementioned thioesterases gave the same results as strains blocked elsewhere in β oxidation or in wild-type strains (30, 63, 287), thus ruling out a role for acyl-CoA.
The identity of the fatty acid synthetic enzyme(s) that is inhibited by the putative inhibitory acyl-ACP is not clear but is under investigation. It seems likely that identification of the inhibited enzyme will require an in vitro system that accurately reflects in vivo metabolism and the isolation of mutants refractory to inhibition.
In wild-type strains of E. coli and S. typhimurium, the inhibition of protein synthesis by starvation for a required amino acid results in a strong inhibition of stable RNA synthesis. The inhibition of RNA synthesis correlates with the accumulation of the novel nucleotide guanosine 5'-diphosphate-3'-diphosphate (ppGpp) following the starvation of wild-type (rel +), but not relA mutant strains (chapter 92). The product of the relA gene (ppGpp synthase I) is a ribosomal protein that produces ppGpp in response to uncharged tRNA. The interaction of ppGpp with RNA polymerase mediates the inhibitory effects of ppGpp on stable RNA synthesis (chapter 92). Several laboratories report that phospholipid synthesis is decreased after the starvation of rel + but not relA strains, and a considerable body of evidence points to ppGpp as the effector of lipid synthesis in vivo. A regulatory role for the G3P acyltransferase was suggested by Merlie and Pizer (183), who reported that ppGpp inhibited the acyltransferase in vitro. However, this conclusion seemed inconsistent with in vivo experiments that indicated a direct effect of the relA gene on fatty acid biosynthesis (204, 267). Additional support for the regulation of fatty acid synthesis as the primary step came from the finding that the acyltransferase was inhibited by ppGpp when acyl-CoA was the acyl donor but was relatively unaffected when acyl-ACP was the acyl donor (178, 234). A passive role for the acyltransferase in the stringent response, however, was not consistent with the demonstration of relA-mediated control of phospholipid synthesis in strains requiring exogenous fatty acids for growth (203). This rather confusing picture has been clarified by Heath et al. (100), who suggest a chain of events that account for the inhibition of both fatty acid and phospholipid synthesis during the stringent response. They report that the induction of ppGpp synthesis was associated with the accumulation of long-chain acyl-ACPs, indicating the inhibition of the acyltransferase in vivo. Overexpression of the acyltransferase prevents the accumulation of acyl-ACP and attenuates the inhibition of both fatty acid and phospholipid synthesis. These findings place the acyltransferase as the proximal target and indicate that fatty acid biosynthesis is in turn downregulated via feedback inhibition by acyl-ACP as described above. Additional work is required to understand the biochemical mechanism responsible for the inhibition of the acyltransferase by ppGpp.
The inhibition of lipid biosynthesis also triggers the stringent response. Using mutants defective in either fatty acid or phospholipid synthesis, Seyfzadeh et al. (254) showed that the inhibition of fatty acid synthesis stimulated the accumulation of ppGpp and that it was dependent on the activity of the spoT gene product (ppGpp synthase II). The mechanistic details of how this regulatory system operates are unknown, and it will be important to determine whether intermediates, such as acyl-ACP, are the intracellular metabolites that mediate the regulation of SpoT activity.
Early observations on phospholipid metabolism showed that the polar head group of PG was lost in a pulse-chase experiment, whereas that of PE was quite stable (140). At first it was thought that the PG was being degraded; however, the discovery that E. coli contains CL made it clear that some of the PG "turnover" is due to the conversion of PG to CL catalyzed by CL synthase. However, CL synthesis does not account for all the loss of 32P-labeled PG observed in pulse-chase experiments, nor does it explain why the nonacylated glycerol of PG is labeled (and chased) more rapidly than the acylated glycerol moiety (7). A nonlipid phosphate-containing compound derived from the head group of PG was sought, and a family of molecules called membrane-derived oligosaccharides (MDOs) was discovered (see chapter 70). These molecules are composed of sn-glycerol-1-phosphate (derived from PG), glucose, and (usually) succinate and ethanolamine moieties, have molecular weights of 4,000 to 5,000, and are found in the osmotically sensitive periplasmic compartment. The synthesis of the MDO compounds is regulated by the osmotic pressure of the growth medium (decreased osmotic pressure gives an increased rate of MDO synthesis) (147); thus, MDO seem to be involved in osmotic regulation.
The discovery of the MDO compounds provided a function for the well-studied, but enigmatic, enzyme diacylglycerol kinase. In the synthesis of MDO, the sn-glycerol-1-phosphate polar group of PG is transferred to the oligosaccharide, with 1,2-diacylglycerol as the other product (Fig. 7). The ethanolamine moieties are derived from PE (186). Diacylglycerol kinase will phosphorylate the diacylglycerol to phosphatidic acid, which can reenter the phospholipid biosynthetic pathway to complete the diacylglycerol cycle. In the overall reaction, only the sn-glycerol-1-phosphate portion of the PG molecule is consumed; the lipid portion of the molecule is recycled back into phospholipid. It is clear that MDO synthesis is responsible for most of the metabolic instability of the polar group of PG, since this turnover is greatly decreased if MDO synthesis is blocked at the level of oligosaccharide synthesis (252).
A key enzyme in this turnover cycle is diacylglycerol kinase (encoded by dgk). This enzyme has been purified (175), its inner membrane topology has been determined (264), and mutants lacking Dgk activity have been isolated (229). The lack of a phenotype in dgk mutants illustrates that the enzyme is not essential for de novo phospholipid biosynthesis. The rate of diacylglycerol accumulation in strains lacking diacylglycerol kinase (Dgk) depends on both the presence of the oligosaccharide acceptor and the osmotic pressure of the growth medium (229, 230), clearly indicating the role of this enzyme in PG turnover.
The only established metabolic cycle for the turnover of membrane phospholipid acyl moieties is the 2-acyl-GPE cycle. This cycle involves the removal of 1-position acyl moieties from PE and their replacement by 2-acyl-GPE acyltransferase (239). The rate of acyl group turnover is low, amounting to only 3 to 5% of the PE pool per generation. Thus, detection of acyl group turnover in pulse-chase metabolic labeling experiments is technically challenging owing to the large amount of PE in the cell (ca. 107 molecules per cell). Although this low rate of turnover seems insignificant, it may play an essential metabolic role when considered in light of the synthesis of membrane components that are 3 to 4 orders of magnitude less abundant. A second approach to detecting phospholipid fatty acid turnover is to look for lysophospholipid substrates and enzymes that reacylate endogenous lysophospholipids (239). In these experiments, 2-acyl-GPE and 1-acylglycerol-3-phosphate were the only lysophospholipids detected in E. coli membranes. These data corroborate the existence of the three acyltransferases described above but do not reveal the presence of other lysophospholipid intermediates that may indicate the operation of additional acyl group turnover cycles. A complete picture of the destination of the 1-position acyl moieties is not available, but the amino-linked fatty acid on the amino terminus of the major outer membrane lipoprotein is one fate (134). Although the transfer of 1-position fatty acids in PE to lipoprotein can be directly demonstrated in vivo (134), the transacylases responsible for lipoprotein acylation do not have an absolute specificity for PE, since the lipoprotein is acylated and correctly processed in pss mutants that lack PE (92). In this case, the fatty acids are derived from PG. Membrane phospholipids are also the source of fatty acid attached to membrane proteins of Mycoplasma capricolum (53). Protein acylation is a driving force for fatty acid turnover in membrane phospholipids, but a role for phospholipases cannot be ruled out (see below).
E. coli has a membrane-bound CDP-diacylglycerol phosphodiesterase that cleaves CDP-diacylglycerol to CMP and phosphatidic acid (223, 224). The hydrolase also catalyzes the transfer (cytidylylation) of CMP or dCMP from the liponucleotide to phosphate or any of a variety of phosphomonoesters (22). Bulawa and Raetz (23) isolated mutants (cdh) that lack this enzyme, and these strains (including some due to Tn10 insertion) lack a phenotype other than a fivefold elevation of intracellular CDP-diacylglycerol levels. The overproduction of the hydrolase does not affect the liponucleotide pools or phospholipid synthesis in general (23). The function of Cdh protein in lipid biosynthesis remains an enigma, and it is possible that it is involved in a physiological process distinct from phospholipid synthesis.
E. coli contains two well-characterized enzymes that cleave the thioester bond of acyl-CoA molecules, yielding CoA and fatty acid. Both enzymes are much less active on palmitoyl-ACP than acyl-CoA owing to the sequestration of the thioester bond by the ACP moiety (268). Thioesterase I is a protein of 20.5 kDa, encoded by the tesA gene, that cleaves acyl-CoAs of >12 carbon atoms and is unable to cleave 3-hydroxyacyl-CoA thioesters (9, 28). The deduced amino acid sequence of TesA has active-site residues arranged similarly to those found in several mammalian thioesterases (28). The active site is also closely related to those of serine proteases, consistent with covalent labeling and inhibition of TesA by serine esterase inhibitors (9, 28). A comparison of the tesA nucleotide sequence and that determined from the purified protein demonstrates that 26 amino acids are removed from the N terminus of the primary translation product, indicating that TesA is a periplasmic enzyme and that thioesterase I is quantitatively released from E. coli cells by osmotic shock treatment (28). Recently, the tesA gene has been modified by deletion of the leader sequence such that the enzyme is trapped in the cytosol rather than exported to the periplasm (30).
The substrate specificity of TesA measured in vitro predicts the accumulation of FFAs with chain lengths of ≥12 carbons, with 16- and 18-carbon fatty acyl thioesters being the preferred substrates (9). Therefore, the large accumulation of C8 and C10 FFAs observed in vivo upon trapping TesA lacking a leader sequence in the cytosol was unexpected (30). However, lower expression levels of cytosolic thioesterase I activity results in the liberation of FFAs consisting mainly of the 16- and 18-carbon acids expected from the in vitro specificity. It therefore seems that the high level of cytosolic thioesterase I activity produced by expression of tesA allows the efficient hydrolysis of normally unfavored substrates, short-chain acyl-ACPs. Although thioesterase I preferentially cleaves long-chain acyl thioesters, the enzyme also efficiently cleaves dissimilar activated oxygen esters (29, 124). Hydrolysis of these molecules (synthetic substrates used in the assay of chymotrypsin) led to the conclusion that TesA was a protease ("protease I"), although the purified protein does not hydrolyze peptide bonds. A clue to this unusual specificity is that only the activated esters of nonpolar amino acids are hydrolyzed, and thus hydrophobicity and a readily hydrolyzed ester (or thioester) bond are the determinants of substrate activity (29, 124). A similar result is seen with the LuxD thioesterase of luminescent bacteria (73). A precedent for this type of specificity is mammalian acyl-CoA-binding protein which binds long chain (>14 carbons) acyl-CoA with high affinity (159). The NMR structure of the complex of the protein with palmitoyl-CoA shows that only long-chain acyl-CoA molecules fill the hydrophobic binding site and allow the cooperative interactions required for tight binding (159).
In contrast, thioesterase II is a cytosolic tetrameric protein composed of 32-kDa subunits encoded by the tesB gene (19, 193). Thioesterase II cleaves acyl-CoAs of more than six carbon atoms and β-hydroxyacyl-CoAs but is unable to cleave acyl-panthetheine thioesters (19, 193). TesB lacks the active-site serine motif found in other thioesterases and shows no sequence similarity to other known proteins (193). Iodoacetamide inhibits thioesterase II, and the modified residue is a histidine residue, thus implicating this base in cleavage of the thioester bond (193). Thioesterase II has been crystallized (269), and detailed structural information should be forthcoming
The physiological functions of thioesterases I and II are unknown, and the presence of these enzymes remains an enigma. These enzymes were assigned a role in phospholipid synthesis on the basis of kinetic isotope effects (192), but these data can be explained by differential isotope effects on the acyltransferases of phospholipid and lipid A synthesis rather than the proposed acyl-ACP hydrolysis. The chromosomal copies of both tesA and tesB have been disrupted to give null mutants (28, 193). Neither the tesA nor the tesB null mutation affects cell growth (the tesA and tesB mutations were isolated by reverse genetics and a brute force screen, respectively). A tesAB double-null mutant strain grows normally (29, 30), indicating that neither protein is essential. However, it remains possible that the functions of both enzymes can be compensated for by another enzyme. Indeed, the tesB null mutant still retains about 10% of the wild-type activity, indicating the existence of a third thioesterase in E. coli (193, 195).
The contribution of phospholipases to membrane phospholipid turnover is undefined. The best-studied phospholipase is phospholipase A1, which is located in the outer membrane. The protein is very stable and has been purified to homogeneity. Mutants (pldA) lacking phospholipase A1 activity have been isolated; the pldA gene has been cloned, and its sequence has been determined (2, 113, 114, 198, 208). Interestingly, cells that lack PldA lack a phenotype, and cells that overexpress the PldA possess increased phospholipase activity in vitro but do not exhibit an obvious phenotype (113). The effect of the presence or absence of this enzyme on 1-position turnover in PE has not been but should be examined to determine the contribution of this enzyme to the turnover rate. However, PldA appears to be present in a latent form in vivo (114). The reason for the inactivity of the enzyme in intact cells is unknown, but the activity can be revealed when the cell envelope formation is perturbed. For example, mutants (envC) defective in an inner membrane protein (153) form chains and accumulate 2-acyl-GPE, because of the activation of PldA (184, 185). Activation of PldA by membrane perturbants is important to the ability of polymorphonuclear leukocytes to kill E. coli (69). Although it is clear that the presence of PldA in the outer membrane determines the sensitivity of E. coli to polymyxin B (68) or cytotoxicity when ingested by leukocytes (295), the importance of this latent enzyme to bacterial physiology remains a mystery.
E. coli also has a lysophospholipase activity that plays a role in preventing the accumulation of lysophospholipids in vivo. The gene for this enzyme (pldB) has been cloned, and its sequence has been determined (115). The pldB gene is found as an operon with pldA (156, 157), and the PldB protein, an inner membrane component, has been purified and characterized (142). PldB hydrolyzes 2-acyl-GPE more readily that the 1-acyl isomer and does not attack diacylphospholipids. PldB also transfers the fatty acid from the 2 position of 2-acyl-GPE to the polar head group of PG, forming acylphosphatidylglycerol (142, 201). These two activities of PldB work in concert to ensure that membrane-damaging lysophospholipids do not accumulate in vivo. In aas mutants, 2-acyl-GPE does not accumulate even though the activity of 2-acyl-GPE acyltransferase cannot be detected (122). Instead, a new phospholipid identified as acylphosphatidylglycerol appears, indicating that the PldB pathway for 2-acyl-GPE metabolism is significant in the absence of the acyltransferase/synthetase activity. Mutants that either lack or overexpress PldB activity do not have a discernible phenotype (155). The accumulation of 2-acyl-GPE in aas pldB double mutants confirms the role of PldB in acylphosphatidylglycerol synthesis and 2-acyl-GPE degradation. However, 2-acyl-GPE does not accumulate to high levels in these double mutants, pointing to yet another pathway for the removal of 2-acyl-GPE. A membrane-associated transacylase activity that converts two 2-acyl-GPEs to PE and GPE has been characterized (116, 118, 119), and there is a second lysophospholipase that cleaves 1-acyl isomers localized in the soluble fraction (62). The role of these two enzymes in membrane phospholipid turnover awaits the isolation of mutants and clones that can be used to manipulate the intracellular levels of these enzymes. The fact that acylphosphatidylglycerol accumulates in aas mutants suggests that reacylation of 2-acyl-GPE is the predominant pathway for the metabolism of 2-acyl-GPE.
The β-hydroxydecanoyl-ACP dehydrase (the fabA gene product) is specifically and irreversibly inhibited by the acetylenic substrate analog 3-decenoyl-N-acetylcysteamine (3-decenoyl-NAC) and its allenic counterpart (70, 103). The inhibitor forms a covalent adduct with histidine, resulting in the loss of all of the partial reactions of the enzyme (46, 104). 3-Decenoyl-NAC concentrations of between 10 and 50 μM completely inhibit unsaturated fatty acid synthesis and bacterial growth, but growth inhibition is relieved by addition of unsaturated fatty acids to the medium (31, 143). Saturated fatty acid synthesis continues normally in the presence of 3-decenoyl-NAC and supplies necessary precursors for lipopolysaccharide production.
Cerulenin, (2R)(3S)-2,3-epoxy-4-oxo-7,10-dodecadienolyamide, is a fungal product that is an irreversible inhibitor of β-ketoacyl-ACP synthases I and II (50, 282) and is extremely effective in blocking growth of a large spectrum of bacteria (213). Cerulenin covalently modifies the active site of the synthases and inhibition correlates with the binding of 1 mol of cerulenin per mol of enzyme (145). Incubation of the synthases with acyl-ACP protects the enzymes from cerulenin inactivation, supporting the concept that cerulenin interacts with the synthases at the fatty acyl binding site on the synthases. Synthase III does not utilize long-chain acyl-ACP substrates and is not inhibited by cerulenin (135). Although cerulenin is a versatile biochemical tool, it is not a suitable antibiotic for clinical use because it is also a potent inhibitor of the multifunctional mammalian fatty acid synthase (213).
Thiolactomycin, (4S)(2E,5E)-2-4-6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide, inhibited type II but not type I fatty acid synthases (189). An analysis of the individual reactions of fatty acid synthesis shows that the β-ketoacyl-ACP synthase and acetyl transacylase activities were the only inhibited activities (96, 97). Malonyl-ACP protects the synthases from thiolactomycin inhibition, indicating that this antibiotic targets a different site on the condensing enzyme from that targeted by cerulenin. Although thiolactomycin inhibits all three condensing enzymes in vivo and in vitro, overproduction of synthase I imparts thiolactomycin resistance; however, overexpression of synthase III did not (274). Since synthase II is not essential, these data suggest that synthase I is the relevant thiolactomycin target in vivo. Although one class of thiolactomycin-resistant mutants exhibits an altered synthase III activity (131), the thiolactomycin resistance phenotype due to a lesion at min 57.5 of the E. coli chromosome results from the activation of the emrAB multidrug resistance pump (77).
Diazoborines, a group of antibacterial heterocyclic compounds containing boron as the third heteroatom, inhibit fatty acid synthesis in E. coli by interfering with the activity of enoyl-ACP reductase (FabI) (14). Inhibition by diazaborines requires the presence of NAD or NADH, but it is not known whether the complex blocks enoyl-ACP binding (14). The discovery that the FabI analog in Mycobacterium tuberculosis (InhA) is the target for isoniazid and ethionamide (8), drugs used to treat tuberculosis, illustrates the potential importance of enoyl-ACP reductase as an antibiotic target.
Although there are new genes involved in the reactions of fatty acid biosynthesis that remain to be discovered, we now understand in considerable detail the biochemistry of the individual steps in fatty acid and phospholipid biosynthesis. Examining the regulation of these steps and their integration with the other major branches of cellular metabolism will be a major focus for future research. Clues to how the rate of fatty acid synthesis is regulated and integrated with cell growth presented in this chapter suggest that the older views of regulation need to be supplanted by more complex scenarios that include multiple enzymes in both the fatty acid and phospholipid biosynthetic pathways. However, these findings only scratch the surface, and the isolation of new mutants that globally affect fatty acid, phospholipid, and macromolecular synthesis will reveal important interrelationships among these processes. The problem of the regulation of phospholipid head group composition is likely to be a property of the individual enzymes in the pathway, and detailed biochemical studies on the regulation of these activities by phospholipids are likely to uncover important allosteric mechanisms that contribute to the control of membrane phospholipid composition. Finally, the study of lipid metabolism in E. coli and S. typhimurium will continue to be directly applicable to advancing the understanding of lipid metabolism in other bacteria, plants, and mammals.
Our work is supported by National Institutes of Health grants AI15650 (J.E.C.) and GM34496 (C.O.R.), Cancer Center support grant CA 21765 (C.O.R.), and the American Lebanese Syrian Associated Charities.
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