All organisms produce fatty acids via a repeated cycle Suipacha 531 of reactions involving the condensation, reduction, de-2000-Rosario hydration, and reduction of carbon-carbon bonds (Camp-Argentina bell and Cronan, 2001; Rock and Cronan, 1996). In mam-2 Department of Biology mals and other higher eukaryotes, these reactions are Massachusetts Institute of Technology catalyzed on a type I synthase (FAS I), a large multifunc-Building 68, Room 530 tional protein in which the growing fatty acid chain is Cambridge, Massachusetts 02139 covalently attached to the protein (Campbell and Cronan, 2001; Rock and Cronan, 1996). In contrast, bacteria, plant chloroplasts, and Plasmodium falciparum contain Summary a type II system (FAS II) in which each reaction is catalyzed by a discrete protein and reaction intermediates Bacterial cells exert exquisite control over the biosynare carried through the cytosol as a thioester of the thesis of their membrane lipids, but the mechanisms small acyl carrier protein (ACP) (Rock and Jackowski, are obscure. We describe the identification and purifi-2002; Campbell and Cronan, 2001). The chain elongation cation from Bacillus subtilis of a transcription factor, step in fatty acid biosynthesis consists of the condensa-FapR, that controls the expression of many genes intion of acyl groups, which are derived from acyl-ACP or volved in fatty acid and phospholipid metabolism (the acyl-coenzyme A (acyl-CoA), with malonyl-ACP by the fap regulon). Expression of this fap regulon is influ-␤-ketoacyl-ACP synthases (often referred as condensenced by antibiotics that specifically inhibit the fatty ing enzymes) (Cronan and Rock, 1996). These enzymes acid biosynthetic pathway. We show that FapR negaare divided in two groups. The first (FabH) class of contively regulates fap expression and that the effects of densing enzymes is responsible for the initiation of fatty antibiotics on fap expression are mediated by FapR. acid elongation and utilizes acyl-CoA primers (Rock and We further show that decreasing the cellular levels Jackowski, 2002). Escherichia coli produces straight of malonyl-CoA, an essential molecule for fatty acid chain and unsaturated fatty acids, and E. coli FabH elongation, inhibits expression of the fap regulon and selectively uses acetyl-CoA to initiate the pathway (Rock that this effect is FapR dependent. Our results indicate and Jackowski, 2002). In contrast, Bacillus subtilis prothat control of FapR by the cellular pools of malonylduces mainly branched chain fatty acids and contains CoA provides a mechanism for sensing the status of two FabH isozymes (named FabHA and FabHB) that fatty acid biosynthesis and to adjust the expression differ from the E. coli enzyme in that they are selective of the fap regulon accordingly. for branched chain acyl-CoAs (Choi et al., 2000). The second (FabF-FabB) class of condensing enzymes is responsible for the subsequent rounds of fatty acid elon-Introduction gation in the pathway (Campbell and Cronan, 2001). These enzymes condense malonyl-ACP with acyl-A...

Malonyl‐CoA is an essential intermediate in fatty acid synthesis in all living cells. Here we demonstrate a new role for this molecule as a global regulator of lipid homeostasis in Gram‐positive bacteria. Using in vitro transcription and binding studies, we demonstrate that malonyl‐CoA is a direct and specific inducer of Bacillus subtilis FapR, a conserved transcriptional repressor that regulates the expression of several genes involved in bacterial fatty acid and phospholipid synthesis. The crystal structure of the effector‐binding domain of FapR reveals a homodimeric protein with a thioesterase‐like ‘hot‐dog’ fold. 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 helix‐turn‐helix motifs, impairing their productive association for DNA binding. Structure‐based mutations that disrupt the FapR–malonyl‐CoA interaction prevent DNA‐binding regulation and result in a lethal phenotype in B. subtilis, suggesting this homeostatic signaling pathway as a promising target for novel chemotherapeutic agents against Gram‐positive pathogens.

plsX (acyl-acyl carrier protein [ACP]:phosphate acyltransferase), plsY (yneS) (acyl-phosphate:glycerol-phosphate acyltransferase), and plsC (yhdO) (acyl-ACP:1-acylglycerol-phosphate acyltransferase) function in phosphatidic acid formation, the precursor to membrane phospholipids. The physiological functions of these genes was inferred from their in vitro biochemical activities, and this study investigated their roles in gram-positive phospholipid metabolism through the analysis of conditional knockout strains in the Bacillus subtilis model system. The depletion of PlsX led to the cessation of both fatty acid synthesis and phospholipid synthesis. The inactivation of PlsY also blocked phospholipid synthesis, but fatty acid formation continued due to the appearance of acylphosphate intermediates and fatty acids arising from their hydrolysis. Phospholipid synthesis ceased following PlsC depletion, but fatty acid synthesis continued at a high rate, leading to the accumulation of fatty acids arising from the dephosphorylation of 1-acylglycerol-3-P followed by the deacylation of monoacylglycerol. Analysis of glycerol 3-P acylation in B. subtilis membranes showed that PlsY was an acylphosphate-specific acyltransferase, whereas PlsC used only acyl-ACP as an acyl donor. PlsX was found in the soluble fraction of disrupted cells but was associated with the cell membrane in intact organisms. These data establish that PlsX is a key enzyme that coordinates the production of fatty acids and membrane phospholipids in B. subtilis.Phosphatidic acid (PtdOH) is synthesized by the successive acylation of glycerol-P followed by the acylation of 1-acylglycerol-P, and there are two enzyme systems that carry out the first reaction in bacteria. In the Escherichia coli model system, either acyl-acyl carrier protein (ACP) or acyl-coenzyme A (CoA) thioesters are utilized by the membrane