Regulation of fatty acid metabolism in bacteria

Authors


*E-mail yfujita@bt.fubt.fukuyama-u.ac.jp; Tel. (+81) 84 936 2111; Fax (+81) 84 936 2023.

Summary

In Escherichia coli, the main player in transcription regulation of fatty acid metabolism is the FadR protein, which is involved in negative regulation of fatty acid degradation and in positive and negative regulation of the cellular processes related to it, as well as in positive regulation of the biosynthesis of unsaturated fatty acids in a concerted manner with negative regulation of FabR. On the other hand, Bacillus subtilis possesses two global transcriptional regulators, FadR (YsiA) and FapR. B. subtilis FadR represses fatty acid degradation, whereas FapR represses almost all the processes in the biosynthesis of saturated fatty acids and phospholipids. Furthermore, Streptococcus pneumoniae FabT represses the genes of fatty acid biosynthesis that are clustered in its genome. Long-chain acyl-CoAs appear to be metabolic signals for fatty acid degradation by bacteria in general, and antagonize the FadR protein from either E. coli or B. subtilis. However, malonyl-CoA is a metabolic signal for fatty acid and phospholipid biosynthesis by Gram-positive low-GC bacteria, and it antagonizes FapR. These would be the primary aspects for understanding the elaborate and complex regulation of fatty acid metabolism in bacteria to maintain membrane lipid homeostasis.

Introductory remarks

Fatty acids are essential components of membranes and are important sources of metabolic energy in all organisms. Thus, fatty acid degradation and biosynthesis pathways must be switched on and off according to the availability of fatty acids to maintain membrane lipid homeostasis. The regulation of these pathways has been mainly studied in a model prokaryote, Escherichia coli (Cronan and Rock, 1996; Cronan and Subrahmanyam, 1998; DiRusso and Nyström, 1998; Zhang et al., 2002). In the last 5 years, global regulation of fatty acid metabolic pathways has been demonstrated in another model prokaryote, Bacillus subtilis (Schujman et al., 2003; 2006; Matsuoka et al., 2007) as well as in Streptococcus pneumoniae (Lu and Rock, 2006).

The Gram-negative E. coli is an enteric γ-proteobacterium that mostly possesses straight-chain fatty acids. The global transcriptional regulator FadREc, a member of the GntR family (Fujita et al., 1986; Haydon and Guest, 1991), is involved mainly in fatty acid degradation and the cellular processes related to it, and is antagonized by long-chain acyl-CoAs (Cronan and Rock, 1996; Cronan and Subrahmanyam, 1998; DiRusso and Nyström, 1998). This protein also regulates the synthesis of unsaturated fatty acids (Henry and Cronan, 1991; Campbell and Cronan, 2001a) in a concerted manner with FabR, a TetR family member (Zhang et al., 2002). On the other hand, the Gram-positive B. subtilis is a soil bacterium of low-GC content that possesses both straight-chain and branched-chain fatty acids (Kaneda, 1977). Two transcriptional global regulators [FadRBs (YsiA), a TetR family member (Matsuoka et al., 2007), and FapR, a DeoR family member (Schujman et al., 2003; 2006)] are involved in fatty acid degradation and biosynthesis respectively. FadRBs is antagonized by long-chain acyl-CoAs (Matsuoka et al., 2007), whereas FapR is antagonized by malonyl-CoA, a building block for fatty acid biosynthesis (Schujman et al., 2003; 2006). Moreover, the Gram-positive S. pneumoniae is a human pathogen that mostly possesses straight-chain fatty acids. The FabT protein, a MarR family member, is involved in fatty acid biosynthesis of this pathogen (Lu and Rock, 2006). A metabolic signal for the regulation of FabT is unknown.

Fatty acid metabolism in E. coli and its regulation through the FadREc protein

Fatty acid degradation in E. coli

Escherichia coli can use fatty acids with various chain lengths as sole carbon and energy sources. After uptake, fatty acids can either be degraded via the β-oxidation pathway or used as precursors for membrane phospholipid biosynthesis. The degradation pathway is catalysed by the enzymes encoded by the fad regulon, which are responsible for the transport and activation of long-chain fatty acids, and their β-oxidative cleavage into acetyl-CoAs (Fig. 1). Long-chain fatty acids are transported across the cell membrane via a transport/acyl-activation mechanism involving an outer membrane protein, FadL (Black, 1991), and an inner membrane-associated acyl-CoA synthase, FadD (Black et al., 1992). Although eukaryotic systems possess multiple acyl-CoA synthases with different fatty acid chain-length specificities, E. coli has a single acyl-CoA synthase, FadD, of broad substrate specificity (Overath et al., 1969; Kameda and Nunn, 1981). The first step in the β-oxidation cycle involves the conversion of acyl-CoA to enoyl-CoA via FadE, which appears to be the only acyl-CoA dehydrogenase in E. coli (Campbell and Cronan, 2002). The remaining steps of hydration, oxidation, and thiolytic cleavage in fatty acid degradation are performed by a tetrameric complex consisting of two copies each of FadB and FadA (Pramanik et al., 1979). The β-oxidation pathway acts in a cyclic manner, each cycle resulting in shortening of the input acyl-CoA by two carbon atoms to give acetyl-CoA.

Figure 1.

Fatty acid metabolism in E. coli and its regulation through the FadR protein. Fatty acids are degraded as follows. Long-chain fatty acids are transported across the cell membrane via a transport/acyl-activation mechanism involving an outer membrane protein, FadL, and inner membrane-associated FadD [acyl-CoA synthase (fatty acid-CoA ligase)]. The first step in the β-oxidation pathway involves the conversion of acyl-CoA to enoyl-CoA through acyl-CoA dehydrogenase (FadE). Then, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (FadB) converts enoyl-CoA to 3-ketoacyl-CoA via 3-hydroxylacyl-CoA through hydration and oxidation. The last step is catalysed by 3-ketoacyl-CoA thiolase (acetyl-CoA C-acyltransferase) (FadA) that cleaves 3-ketoacyl-CoA, resulting in shortening of the input acyl-CoA by two carbon atoms to give acetyl-CoA. For degradation of unsaturated fatty acids, 2,4-dienoyl-CoA reductase (FadH) transforms 2,4-dienolyl-CoA derived from unsaturated fatty acids into enoyl-CoA. On the other hand, the first step of bacterial fatty acid synthesis pathway (FASII) is the formation of malonyl-CoA from acetyl-CoA catalysed by acetyl-CoA carboxylase (AccABCD). The malonyl moiety is transferred to ACP by malonyl-CoA:ACP transacylase (FabD). The initial condensation reactions are catalysed by 3-ketoacyl-ACP synthase III (FabH), which condenses acetyl-CoA with malonyl-ACP to form acetoacetyl-ACP. The next reaction in the elongation cycle involves the NADPH-dependent 3-ketoacyl-ACP reductase (FabG). The resulting 3-hydroxyacyl-ACP is dehydrated by 3-hydroxyacyl-ACP dehydratase (FabZ). The final step in the cycle is catalysed by enoyl-ACP reductase (FabI). The resulting acyl-ACP can be further elongated by 3-ketoacyl-ACP synthases I (FabB) and/or II (FabF). To synthesize unsaturated fatty acids, FabA also catalyses the dehydration of 3-hydroxydecanoyl-ACP and isomerizes the trans-2-enoyl bond of the ACP-bound substrate to the cis-3 isomer. The FabB enzyme also condenses cis-3-decenoyl-ACP with malonyl-ACP to produce cis-5-ene-3-ketododecenoyl-ACP, which is able to undergo all the subsequent reactions catalysed the FabG, FabZ and FabI. The FadR protein represses the expression of the fadL, fadD, fadE, fadB, fadA and fadH genes as well as that of the uspA, yfcX and yfcY genes, the products of which are green-boxed except UspA. The uspA gene encodes universal stress protein, whereas yfcX and yfcY are involved in anaerobic fatty acid utilization. FadR triggers the expression of the fabA and fabB genes as well as the iclR gene, the products of which are red-boxed except IclR. FadR is antagonized by long-chain acyl-CoAs (over 14 carbon atoms). The iclR gene encodes a negative regulator of glyoxylate shunt. Furthermore, FabR only represses the fabA and fabB genes (blue-boxed). The signal(s) modulating the FabR binding activity (X) is unknown. In addition, acylated derivatives of ACP (acyl-ACPs) regulate fatty acid biosynthesis by feedback inhibition of AccABCD, FabH and FabI (in light blue).

The FadREc protein

Transcriptional control of the fatty acid degradation genes occurs via the FadREc regulatory protein. When long-chain fatty acids of more than 14 carbons are present in the growth medium, they are converted to acyl-CoAs, which bind to FadREc, causing release of the regulatory protein from the operator and thus derepression of transcription of the fad genes (Fig. 1) (Henry and Cronan, 1991; 1992; DiRusso et al., 1992). FadREc also negatively regulates transcription from the uspA promoter. UspA is unique in its almost universal responsiveness to diverse stresses, and this regulation of cellular uspA expression is primarily exerted at the transcription level (Nyström and Neidhardt, 1994). In addition, FadR triggers expression of the iclR gene, which negatively regulates the aceB-aceA-aceK operon encoding the glyoxylate shunt enzymes (Maloy and Nunn, 1981; Gui et al., 1996). Furthermore, FadREc negatively regulates the expression of yhcX-yhcY, the products of which are homologues of FadB and FadA, and are involved in anaerobic growth on fatty acids (Campbell et al., 2003). Thus, FadREc functions as a repressor of many genes and operons mainly involved in fatty acid degradation, and as a regulator of the cellular processes associated with it (Farewell et al., 1996; DiRusso and Nyström, 1998). Moreover, many fad genes are positively regulated by the cyclic-AMP receptor protein/cyclic-AMP system (Pauli and Overath, 1974; Clark and Cronan, 1996).

According to the completed bacterial genomes, FadREc is found only in the γ-proteobacteria that colonize vertebrates (or in one case plants), and it functions as a dimer (Raman et al., 1997). Four FadREc crystal structures have been reported; one is of the unliganded protein (van Aalten et al., 2000), one is an FadR–myristoyl-CoA complex (van Aalten et al., 2001), and the other two are complexes of FadREc with the fadB-fadA operator DNA (van Aalten et al., 2001; Xu et al., 2001). The three-dimensional structures of the complexes of FadREc–operator DNA and FadREc–myristoyl-CoA are shown in Fig. 2A and B respectively. The N-terminal DNA-binding domain has the winged helix structure often found in prokaryotic regulator proteins (Haydon and Guest, 1991; Brennan, 1993), whereas the C-terminal half of FadREc contains an acyl-CoA-binding domain (Raman and DiRusso, 1995; van Aalten et al., 2001). The adenosine 3′-phosphate and bisphosphate moieties of acyl-CoA embedded in FadREc are exposed to the solvent. Furthermore, the atoms of acyl-CoA embedded in FadREc interact exclusively with amino acids of one subunit of the dimerized protein (van Aalten et al., 2000; Xu et al., 2001) (Fig. 2B).

Figure 2.

Structural mechanisms of induction of the FadREc and FadRBs proteins.
A. Overlay of FadREc bound to DNA (in deep blue). The structure of FadREc bound to the fadB-A operator is deposited under Protein Data Base (PDB) identification code (1H9T) (van Aalten et al., 2001). FadREc is a homodimer that is divided into an N-terminal DNA-binding domain (in blue) and a C-terminal acyl-CoA binding site; 10 α-helices (1–8, 10 and 11) of one subunit are indicated.
B. Overlay of FadREc with myristoyl-CoA. The structure of one subunit of an FadREc homodimer complexed with a ligand of myristoyl-CoA (PDB: 1H9G) (van Aalten et al., 2001), where atoms of myristoyl-CoA are shown and 10 helices (1–4, 5–6 and 8–11) are indicated: helices 5 and 6 in (A) are combined to helix 5–6, and helix 9 emerges.
C. Overlay of FadRBs with lauroyl-CoA (PDB: 1VI0) (Badger et al., 2005). FadRBs is a homodimer that is divided into an N-terminal DNA-binding domain (in blue) and a C-terminal acyl-CoA binding site; 8 α-helices (1–8) of one subunit are indicated. The atoms of one lauroyl-CoA molecule per dimer, which are indicated, interact with amino acids derived from both subunits.

Fatty acid biosynthesis in E. coli

As shown in Fig. 1, acetyl-CoA carboxylase catalyses the first committed step in fatty acid biosynthesis, the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate. In contrast to eukaryotic forms, the E. coli acetyl-CoA carboxylase is composed of four distinct proteins (AccABCD) (Cronan and Waldrop, 2002). All organisms produce fatty acids via a repeated cycle of reactions involving the condensation, reduction, dehydration and reduction of carbon—carbon bonds (Rock and Cronan, 1996; Campbell and Cronan, 2001b) (Fig. 1). In mammals and other higher eukaryotes, these reactions are catalysed by a large multifunctional protein, type I synthase (FAS I) (Rock and Cronan, 1996; Campbell and Cronan, 2001b). In contrast, bacteria contain a type II synthase (FAS II) for which each reaction is catalysed by a discrete protein and reaction intermediates are carried in the cytosol as thioesters of the small acyl carrier protein (ACP) (Campbell and Cronan, 2001b; Rock and Jackowski, 2002). The chain elongation step in fatty acid biosynthesis consists of the condensation of acyl groups, which are derived from acyl-CoA or acyl-ACP, with malonyl-ACP by the 3-ketoacyl-ACP synthases. These enzymes are divided into two groups. The first class of 3-ketoacyl-ACP synthase III (FabH) is responsible for the initiation of fatty acid elongation and utilizes acyl-CoA primers. The second class of enzymes (FabF and FabB) is responsible for the subsequent rounds of fatty acid. Each cycle of reduction, dehydration and reduction of carbon-carbon bonds to produce acyl-ACP is catalysed by three enzymes [NADPH-dependent 3-ketoacyl-ACP reductase (FabG), 3-hydroxyacyl-ACP dehydratase (FabZ) and NAD(P)H-dependent enoyl-ACP reductase (FabI)] respectively. Additional cycles are initiated by FabF and FabB. This cycle of fatty acid biosynthesis is regulated by feedback inhibition of AccABCD (Heath and Rock, 1996a; Davis and Cronan, 2001) and FabH and FabI (Heath and Rock, 1995; 1996a,b) with the end-products of acyl-ACPs.

Only two unique biochemical reactions catalysed by FabA and FabB are specifically required to produce unsaturated fatty acids in the overall course of fatty acid biosynthesis in E. coli (Fig. 1) (Lu et al., 2004; Schujman and de Mendoza, 2005). Out of the two 3-hydroxyacyl-ACP dehydratases (FabA and FabZ), FabZ is involved in the dehydration of 3-hydroxyacyl-ACPs of all chain lengths (Mohan et al., 1994). FabA activity is restricted to 10 carbon substrates, and not only catalyses the dehydration of 3-hydroxydecanoyl-ACP but also isomerizes the trans-2-enoyl bond of the ACP-bound substrate to the cis-3 isomer (Bloch, 1971). This isomerization places the nascent acyl chain in the unsaturated fatty acid synthetic pathway. 3-Ketoacyl-ACP synthase I (FabB) is likely required for elongation of the cis-3-decenoyl-ACP produced by FabA and is known to be the primary factor determining the cellular unsaturated fatty acid content (Clark et al., 1983).

The FadREc protein, a global regulator of fatty acid degradation, is a transcriptional activator that binds to the fabA promoter region (Henry and Cronan, 1991; 1992). Furthermore, FadREc is a positive regulator of the fabB gene (Campbell and Cronan, 2001a). Thus, FadREc acts as a repressor of β-oxidation genes and an activator of the two genes required for unsaturated fatty acid synthesis (Fig. 1) (Cronan and Subrahmanyam, 1998). However, the reason why a regulatory factor for fatty acid degradation is involved in the regulation of unsaturated fatty acid biosynthesis remains obscure.

The regulation of unsaturated fatty acid biosynthesis is complex. Another transcriptional regulatory site is bioinformatically localized immediately downstream of the FadREc binding site in the promoter regions of the fabA and fabB genes, to which FabR specifically binds (Fig. 1) (McCue et al., 2001). The FabR protein functions as a repressor of fabB expression as well as of fabA one, which, in turn, modulates the physical properties of the membrane by altering the level of unsaturated fatty acid production (Zhang et al., 2002). Regulation of the unsaturated fatty acid content by an FabR repressor requires the presence of an FadREc activator. So, FabR likely regulates the two genes through repression and antagonizes the action of the FadREc activator. However, it remains unclear which signal(s) modulates its binding activity. This pathway of unsaturated fatty acid synthesis is not widely distributed in bacteria. Genome analysis indicates that only the α- and γ-proteobacteria have the proteins of this pathway (Campbell and Cronan, 2001a,b).

Fatty acid metabolism and its regulation in B. subtilis and S. pneumoniae

Fatty acid degradation in B. subtilis and its repression through the FadRBs protein

Bacillus subtilis cannot grow well on acetate as the sole carbon source, due to its lack of the glyoxylate shunt enzymes (Kunst et al., 1997; Kanehisa et al., 2002). Growing B. subtilis cannot degrade straight-chain fatty acids added to the culture medium (Kaneda, 1971). These results suggest that fatty acid degradation through β-oxidation is insignificant. However, B. subtilis possesses a considerable number of genes that are involved in the β-oxidation of fatty acids, such as lcfA, lcfB (yhfL), fadB (ysiB), fadN (yusL), fadA (yusK) and fadE (yusJ) (Bryan et al., 1996; Kunst et al., 1997; Kanehisa et al., 2002; Koburger et al., 2005; Matsuoka et al., 2007). Long-chain fatty acid-CoA ligases (LcfA and LcfB) are soluble enzymes, as judged with the SOSUI method (Yanagihara et al., 1989), in contrast to E. coli FadD, which is an inner membrane protein. This conservation implies that the β-oxidation of fatty acids has an indispensable function under certain physiological conditions. In fact, the fadN-A-E operon encoding the β-oxidation enzymes is induced at the onset of sporulation. This induction requires the YvbA protein involved in cannibalism by sporulating cells (González-Pastor et al., 2003). Furthermore, the fadR (ysiA)-fadB-etfB-etfA operon encoding a β-oxidation enzyme and an electron transfer flavoprotein is necessary for calcium carbonate biomineralization (Barabesi et al., 2007).

DNA microarray analysis of an fadR disruptant and subsequent transcription analysis revealed that the FadRBs protein represses five operons (15 genes), i.e. lcfA-fadR-fadB-etfB-etfA, fadH-fadG (ykuF-ykuG), lcfB, fadM (yusM)-fadN-A-E and fadF (ywjF)-acdA-rpoE (Fig. 3A). Among these genes, lcfA, fadB, etfA, etfB, lcfB, fadN, fadA, fadE, fadH and acdA are involved in fatty acid β-oxidation in B. subtilis (Fig. 3A) (Matsuoka et al., 2007). FadRBs (YsiA) boxes are present in the promoter regions upstream of fadR, fadH, fadN, lcfB and fadF. In a similar manner to FadREc, the binding of FadRBs to these boxes is specifically inhibited by long-chain acyl-CoAs including branched chains with 14–20 carbon atoms. Furthermore, the knock-out of acyl-CoA dehydrogenation through fadE, etfA or etfB disruption results in FadRBs inactivation, due to the accumulation of long-chain acyl-CoAs in the cells. In addition, the disruption of fadE, fadN, fadA, etfA, etfB or fadG affects fatty acid utilization significantly, although even the wild-type cells grow only poorly on it.

Figure 3.

Fatty acid metabolism in B. subtilis and its regulation through the FadR and FapR proteins.
A. Fatty acid degradation and its repression through FadR. The fadR regulon comprises five operons: lcfA-fadR (ysiA)-fadB (ysiB)-eftB-eftA, lcfB (yhfL), fadM (yusM)-fadN (yusL)-fadA (yusK)-fadE (yusJ), fadH (ykuF)-fadG (ykuG) and fadF (ywjF)-acdA-rpoE. LcfA and LcfB are long-chain fatty acid-CoA ligases; FadE and AcdA, acyl-CoA dehydrogenases; FadB and FadN, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase complexes; FadA, 3-ketoacyl-CoA thiolase; FadH, 2,4-dienoyl-CoA reductase; and EtfA and EtfB, electron transfer flavoprotein α- and β-subunits. The rpoE encodes δ-subunit of RNA polymerase, whereas fadF codes for a 4Fe-4S ferredoxin protein possibly associated with acyl-CoA hydrogenation. The fadM and fadG genes code for a unknown protein possibly involved in fatty acid degradation and a wall protein possibly involved in fatty acid transport respectively. The FadR protein represses the expression of the above 15 genes. Out of 15, the 10 gene products green-boxed are involved in fatty acid degradation. The FadR protein is antagonized by long-chain acyl-CoAs (over 14 carbon atoms).
B. Fatty acid biosynthesis in B. subtilis and its repression through the FapR protein. The fapR regulon comprises presumed six operons (10 genes); fabHA-fabF, fapR-plsX-fabD-fabG, fabI, fabHB, yhfC and plsC (yhdO). AccABCD are acetyl-CoA carboxylase subunits; FabD, malonyl-CoA:ACP transacylase; FabHA and FabHB, β-ketoacyl-ACP synthases (initial condensation with acetyl-CoA and branched acyl-CoAs); FabF, β-ketoacyl-ACP synthase (chain elongation); FabG, NADPH-dependent β-ketoacyl-ACP reductase; YwpB and YcsD, β-hydroxyacyl-ACP dehydratases; FabI, enoyl-ACP reductase; PlsX, fatty acid/phospholipid synthesis protein; and PlsC, 1-acylglycerol-3-phosphate O-acyltransferase. PlsX and PlsY produce lyso-phosphatidic acid, which is converted to phospholipids by PlsC (Schujman and de Mendoza, 2006). The yhfC gene encodes a transmembrane protein probably involved in fatty acid synthesis. The FapR protein represses the expression of fabHA, fabHB, fabD, fabF, fabG, fabI, plsX, plsC and yhfC, the products of which are blue-boxed except YhfC. FapR is antagonized by malonyl-CoA.

Many E. coli fad genes are under catabolite repression, and are positively regulated by the cyclic-AMP receptor protein/cyclic-AMP system, as described above. In B. subtilis and close relatives, global regulation of carbon catabolite control occurs on the binding of the complex of CcpA (catabolite control protein A) and P-Ser-HPr (seryl-phosphorylated form of a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system) to the catabolite responsive elements (cre) of the target operons, the constituent genes of which are roughly estimated to number three hundred (Fujita et al., 1995; 2007; Deutscher et al., 2002). Among the members of the FadRBs regulon, the lcfA-fadR-B-etfB-A, lcfB and fadN-A-E operons all carry cres within the lcfA, lcfB and fadN genes respectively (H. Matsuoka and Y. Fujita, unpubl. data). This coincides with the findings from genome-wide mRNA profiling that the lcfA-fadR-B-etfB-A and fadN-A-E operons are under catabolite repression or induced upon glucose starvation (Blencke et al., 2003; Koburger et al., 2005).

The FadRBs protein is highly conserved in many Gram-positive bacteria including Bacillus, Clostridium, Streptomyces and other related genera (Matsuoka et al., 2007). Moreover, its orthologues are unexpectedly present in more diverse genera, such as Metanosarcina (Archaea), and Bordetella, Burkholderia and Chromobacteria (β-proteobacteria). It is notable that FadRBs is not conserved in the Listeria and Staphylococcus genera, which possess FapR homologues, as described below. This diverse but unique distribution of FadRBs orthologues implies that horizontal gene transfer(s) might have occurred during their molecular evolution.

The FadRBs protein is a homodimeric protein, and the N-terminal region of FadRBs carries a helix–turn–helix (HTH) motif (Matsuoka et al., 2007). The crystallographic structure of FadRBs was recently determined in the framework of a bacterial structural genomics project (Badger et al., 2005), which is shown in Fig. 2C. The crystallized FadRBs protein contains one molecule of lauroyl-CoA, which is incorporated during FadRBs synthesis in E. coli, leading to the identification of the amino acids forming hydrogen bonds with the H and O atoms of lauroyl-CoA. However, lauroyl-CoA hardly interferes with the formation of the FadRBs and FadRBs box complex, so it is unlikely to cause the conformational change. The amino acids of FadRBs that interact with lauroyl-CoA are unexpectedly located in the C-terminal regions of both subunits, that is, the atoms of lauroyl-CoA interact with amino acids derived from both subunits (Fig. 2C). In contrast, the atoms of acyl-CoA embedded in FadREc interact exclusively with those of one subunit of the dimerized protein (van Aalten et al., 2000; Xu et al., 2001). As in the case of FadREc, the adenosine 3′-phosphate and bisphosphate moieties of acyl-CoA embedded in FadRBs are exposed to the solvent.

Fatty acid biosynthesis in B. subtilis and its repression through the FapR protein

Figure 3B shows a schema of fatty acid and phospholipid biosynthesis in B. subtilis, the reactions of which are catalysed by distinct enzymes. In contrast to that E. coli contains straight-chain fatty acids, B. subtilis possesses straight- and branched-chain fatty acids. This microorganism contains two FabH isozymes (named FabHA and FabHB) that differ from the E. coli enzyme in that they can initiate the straight- and branched-chain fatty acid synthesis cycle by condensing acetyl-CoA, isobutyryl-CoA, isovaleryl-CoA or α-methylbutyryl-CoA with malonyl-ACP (Choi et al., 2000). Although E. coli produces the two types of condensing enzymes for chain elongation (FabB and FabF), as described above, the FabF protein is the sole condensing enzyme responsible for subsequent elongation in B. subtilis (Schujman et al., 2001; de Mendoza et al., 2002).

A major advance in our understanding of the transcriptional control of bacterial fatty acid and phospholipid biosynthesis occurred following the identification of FapR, a global transcriptional repressor that controls the expression of the fap regulon involved in the biosynthesis of fatty acids and phospholipids in B. subtilis. The fap regulon members are fabHA-fabF, fapR-plsX-fabD-fabG, fabI, fabHB, yhfC and plsC (plsX and plsC are involved in phospholipid biosynthesis) (Schujman et al., 2003) (Fig. 3B). There are cis-sequences in the promoter regions upstream of fabHA, fapR, fabI, fabHB, yhfC and plsC, to which FapR binds. This specific binding of FapR is exclusively inhibited by malonyl-CoA, an essential intermediate in fatty acid synthesis in all living cells, the cellular pools of which provide a mechanism for sensing the status of fatty acid biosynthesis and for adjusting the expression of the fap regulon accordingly (Schujman et al., 2003; 2006). Thus, malonyl-CoA plays a novel role as a signalling molecule regulating the activity of FapR to control membrane lipid homeostasis, indicating that the cellular pool of malonyl-CoA is rigorously regulated, through elaborate regulation of the activity of acetyl-CoA carboxylase (AccABCD) by unidentified regulation systems not involving FapR. The regulation system might be feedback inhibition of acetyl-CoA carboxylase with acyl-ACPs, the end-products of fatty acid biosynthesis. This feedback inhibition is well established in E. coli, as described above.

The FapR protein is highly conserved in many Gram-positive organisms. The gene is present in all species of the Bacillus, Listeria and Staphylococcus genera, and also in Clostridium and other related genera. It is noteworthy that several organisms containing FapR are human pathogens, including Bacillus anthracis, Bacillus cereus and Listeria monocytogenes. The fapR gene in these bacteria is associated with the plsX gene, as in B. subtilis (Schujman et al., 2003). All FapR primary structures have an HTH motif in their N-terminal region. As described above, malonyl-CoA is a direct and specific inducer of FapR. The crystal structure of the effector-binding domain of FapR indicates a homodimeric protein with a thioesterase-like ‘hot dog’ fold (Schujman et al., 2006). Binding of malonyl-CoA promotes a disorder-to-order transition, which transforms an open ligand-binding groove into a long tunnel occupied by the effector molecule in the complex. This ligand-induced modification propagates to the HTH motifs, impairing their productive association for DNA binding.

Besides the FabAB pathway in E. coli described above, the Des pathway in B. subtilis regulates the synthesis of the cold shock-induced membrane-bound enzyme Δ5-fatty acid desaturase (Δ5-Des) (Aguilar et al., 2001). The Des pathway is positively regulated by the two-component regulatory system of DesK–DesR; DesK is a sensor histidine kinase that responds to a decrease in membrane lipid fluidity and DesR is the cognate response regulator (Cybulski et al., 2002). Only the phosphorylated form of the DesR protein is able to bind to a regulatory region immediately upstream of the promoter of the Δ5-Des gene (Cybulski et al., 2004). The mechanism of post-biosynthetic modification of the biophysical properties of membrane phospholipids is thought to allow B. subtilis to adapt to an abrupt decrease in membrane fluidity after cold shock (Aguilar et al., 2001; Lu et al., 2004).

Fatty acid biosynthesis in S. pneumoniae and its regulation through the FabT protein

The human pathogen S. pneumoniae produces the same spectrum of saturated and unsaturated fatty acids as E. coli. The fatty acid degradation in this pathogen is not well investigated. However, the pathway of fatty acid synthesis in S. pneumoniae is well studied as illustrated in Fig. 4, the reactions of which are catalysed by distinct enzymes. The FabM protein is an enoyl-ACP isomerase involved in unsaturated fatty acid synthesis (Marrakchi et al., 2002), whereas a flavoprotein of FabK is enoyl-ACP reductase which is distinct from E. coli FabI (Marrakchi et al., 2003). The FabF protein is only a single FabF type of condensing enzyme. All the genes required for fatty acid biosynthesis are located in a single cluster in the S. pneumoniae genome in the order of fabM, fabT, fabH, acpP, fabK, fadD, fabG, fabF, accB, fabZ, accC, accD and accA; acpP codes for ACP. Similar biosynthetic gene clusters are found in other groups of Gram-positive bacteria, such as Enterococcus, Clostridium and Lactococcus. The second gene in the cluster (fabT) encodes a global transcription regulator belonging to the MarR family, which represses all the 12 genes except fabM (Lu and Rock, 2006). The FabT regulon comprises two operons of fabT-H-acpP and fabK-D-G-F-accB-fabZ-accC-D-A. The cis-sequences for FabT are identified in the promoter regions of fabT and fabK (Lu and Rock, 2006). However, a ligand for the regulation of FabT is not uncovered. The branch point in unsaturated fatty acid synthesis in S. pneumoniae is located one step downstream from the branch point in E. coli (Figs 1 and 4). The FabK protein is a major determinant of the physical properties of the S. pneumoniae membrane, which diverts intermediates to saturated fatty acid formation (Lu and Rock, 2006).

Figure 4.

Fatty acid biosynthesis in S. pneumoniae and its repression through the FabT protein. The genes involved in fatty acid biosynthesis cluster at a single location within the S. pneumoniae genome, in the order of fabM, fabT, fabH, acpP, fabK, fabD, fabG, fabF, accB, fabZ, accC, accD and accA. AccABCD are acetyl-CoA carboxylase subunits; FabD, malonyl-CoA:ACP transacylase; FabH, β-ketoacyl-ACP synthase (initial condensation with acetyl-CoA); FabF, β-ketoacyl-ACP synthase (chain elongation); FabG, NADPH-dependent β-ketoacyl-ACP reductase; FabZ, β-hydroxyacyl-ACP dehydratases; FabK, enoyl-ACP reductase; and FabM, enoyl-ACP isomerase. The acpP gene encodes ACP. The FabT regulon comprises two operons (fabT-H-acpP and fabK-D-G-F-accB-fabZ-accC-D-A). The FabT protein represses these 12 genes, the products of which are blue-boxed except ACP. The fabM gene, whose expression is not repressed by FabT, is involved in unsaturated fatty acid synthesis. The signal(s) modudulating the FabT binding activity (X) is unknown.

Concluding remarks

The pathways of fatty acid degradation and biosynthesis and the enzymes involved in them are well conserved. Bacteria must exert elaborate and highly sophisticated regulation over them during growth and stationary phases under various nutrient conditions. It is rational to consider that the primary regulation of the fatty acid metabolic pathways is conducted at the transcriptional level, as for other biological processes.

In E. coli, the main player in the regulation of fatty acid metabolism is FadREc, which is involved in negative regulation of fatty acid degradation and in negative and positive regulation of the cellular processes related to it, as well as in positive regulation of the biosynthesis of unsaturated fatty acids involving fabA and fabB through a concerted action with negative regulation of FabR whose signal to modulate its binding activity is unknown. It is notable that transcriptional regulation of the genes involved in fatty acid biosynthesis has not been reported except for the two genes in E. coli. It is well established that acyl-ACPs can regulate fatty acid biosynthesis by feedback inhibition of AccABCD, FabH and FabI. Nevertheless, it remains to be investigated whether or not only such post-transcriptional regulation is sufficient to co-ordinately regulate the genes of fatty acid biosynthesis. On the other hand, B. subtilis possesses two global transcriptional regulators, FadRBs and FapR. FadRBs represses the genes whose products catalyse all the steps in fatty acid degradation, while FapR regulates all of the genes except ywpB, ycsD, plsY and accA-B-C-D, which are involved in saturated fatty acid and phospholipid synthesis. In addition, the Des pathway for the biosynthesis of unsaturated fatty acids is known in B. subtilis. Furthermore, S. pneumoniae possesses a global regulator of FabT for fatty acid biosynthesis. This pathogen possesses a unique pathway to unsaturated fatty acid which is distinct from those in E. coli and B. subtilis. The pathway of unsaturated fatty acid biosynthesis appears to be most diversified among fatty acid metabolisms in bacteria. It is noteworthy that the two Gram-positive bacteria, B. subtilis and S. pneumoniae, possess the global transcription regulation system for fatty acid biosynthesis.

Escherichia coli is an enteric bacterium that is able to grow on fatty acid as the sole carbon source, whereas B. subtilis is a soil bacterium that does not grow well on fatty acid. Moreover, the molecular evolution of FadREc and FadRBs, belonging to the GntR and TetR families, respectively, appears to be very divergent. However, long-chain acyl-CoAs are common metabolic signals for fatty acid degradation by E. coli and B. subtilis, and antagonize both the FadREc and FadRBs proteins, implying that they might be general metabolic signals for fatty acid degradation by bacteria. This would be one of the examples, showing that general metabolic signals are likely more conserved than the regulatory proteins themselves to interact with them. It is amazing that the amino acids of FadRBs interacting with long-chain acyl-CoAs are located in the C-terminal regions of both subunits, in contrast to the atoms of acyl-CoA embedded in FadREc that interact exclusively with those of one subunit of the dimerized protein. This fact suggests that the domains of the two FadR proteins interacting with long-chain acyl-CoA developed independently during their molecular evolution. On the other hand, malonly-CoA is a metabolic regulatory signal for fatty acid and phospholipid biosynthesis in Gram-positive low-GC bacteria, and it antagonizes FapR. This compound might be a more general metabolic signal for the biosynthesis of fatty acid in bacteria. However, the binding of malonyl-CoA to the FabT repressor has not been directly demonstrated (Lu and Rock, 2006). The cellular pool of malonyl-CoA would be rigorously regulated, through elaborate regulation of the activity of acetyl-CoA carboxylase by unidentified regulation systems. This regulation system might be feedback inhibition of fatty acid biosynthesis by acyl-ACPs, which is well established in E. coli.

The regulation of fatty acid metabolism described in this review is obviously the first step for understanding the highly sophisticated and finely tuned overall regulation of fatty acid metabolism in bacteria that maintain membrane lipid homeostasis in the growth and stationary phases as well as in various physical and nutrient states.

Acknowledgements

This work was supported by grants-in-aid for Scientific Research on Priority Areas, Scientific Research (B), and the High Tech Research Project for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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