RESUMO
A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.
Assuntos
Bacillus subtilis/metabolismo , Ácidos Graxos/biossíntese , Fosfolipídeos/biossíntese , Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Ácidos Graxos/metabolismo , Lipogênese , Fosfolipídeos/metabolismo , RegulonRESUMO
UNLABELLED: Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant protein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichia coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl-ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a different phenotype in B. subtilis. The structural, biochemical and physiological data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin. DATABASE: Crystallographic data (including atomic coordinates and structure factors) have been deposited in the Protein Data Bank under accession codes 4LS5, 4LS6, 4LS7 and 4LS8.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cerulenina/farmacologia , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/genética , Inibidores da Síntese de Ácidos Graxos/farmacologia , Genes Bacterianos , Humanos , Modelos Moleculares , Micotoxinas/farmacologia , Mutação Puntual , Estrutura Quaternária de Proteína , Eletricidade EstáticaRESUMO
Spore formation in Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of cell-cell communication. One pathway, which couples the proteolytic activation of the mother cell transcription factor σ(E) to the action of a forespore synthesized signal molecule, SpoIIR, has remained enigmatic. Signalling by SpoIIR requires the protein to be exported to the intermembrane space between forespore and mother cell, where it will interact with and activate the integral membrane protease SpoIIGA. Here we show that SpoIIR signal activity as well as the cleavage of its N-terminal extension is strictly dependent on the prespore fatty acid biosynthetic machinery. We also report that a conserved threonine residue (T27) in SpoIIR is required for processing, suggesting that signalling of SpoIIR is dependent on fatty acid synthesis probably because of acylation of T27. In addition, SpoIIR localization in the forespore septal membrane depends on the presence of SpoIIGA. The orchestration of σ(E) activation in the intercellular space by an acylated signal protein provides a new paradigm to ensure local transmission of a weak signal across the bilayer to control cell-cell communication during development.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Comunicação Celular , Fator sigma/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo , Acilação , Bacillus subtilis/fisiologia , Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Peptídeo Hidrolases/metabolismo , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Esporos Bacterianos/fisiologiaRESUMO
The FapR protein of Bacillus subtilis has been shown to play an important role in membrane lipid homeostasis. FapR acts as a repressor of many genes involved in fatty acid and phospholipid metabolism (the fap regulon). FapR binding to DNA is antagonized by malonyl-CoA, and thus FapR acts as a sensor of the status of fatty acid biosynthesis. However, malonyl-CoA is utilized for fatty acid synthesis only following its conversion to malonyl-ACP, which plays a central role in the initiation and elongation cycles carried out by the type II fatty acid synthase. Using in vitro transcription studies and isothermal titration calorimetry, we show here that malonyl-ACP binds FapR, disrupting the repressor-operator complex with an affinity similar to that of its precursor malonyl-CoA. NMR experiments reveal that there is no protein-protein recognition between ACP and FapR. These findings are consistent with the crystal structure of malonyl-ACP, which shows that the malonyl-phosphopantetheine moiety protrudes away from the protein core and thus can act as an effector ligand. Therefore, FapR regulates the expression of the fap regulon in response to the composition of the malonyl-phosphopantetheine pool. This mechanism ensures that fatty acid biosynthesis in B. subtilis is finely regulated at the transcriptional level by sensing the concentrations of the two first intermediates (malonyl-CoA and malonyl-ACP) in order to balance the production of membrane phospholipids.
Assuntos
Proteína de Transporte de Acila/química , Ácidos Graxos/biossíntese , Proteína de Transporte de Acila/genética , Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Malonil Coenzima A/química , Modelos Moleculares , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transcrição GênicaRESUMO
Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid biosynthesis. The molecular mechanisms of regulation of the acpP structural gene, as well as the function of its gene product, are poorly characterized in Bacillus subtilis and other Gram-positive organisms. Here, we report that transcription of acpP takes place from two different promoters: PfapR and PacpP. Expression of acpP from PfapR is coordinated with a cluster of genes involved in lipid synthesis (the fapR operon); the operon consists of fapR-plsX-fabD-fabG-acpP. PacpP is located immediately upstream of the acpP coding sequence, and is necessary and sufficient for normal fatty acid synthesis. We also report that acpP is essential for growth and differentiation, and that ACP localizes in the mother-cell compartment of the sporangium during spore formation. These results provide the first detailed characterization of the expression of the ACP-encoding gene in a Gram-positive bacterium, and highlight the importance of this protein in B. subtilis physiology.
Assuntos
Proteína de Transporte de Acila/genética , Bacillus subtilis/genética , Proteína de Transporte de Acila/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Família Multigênica , Óperon , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Bacteria stringently regulate the synthesis of their membrane phospholipids, but the responsible regulatory mechanisms are incompletely understood. Bacillus subtilis FabF, the target of the mycotoxin cerulenin, catalyses the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. Here we show that B. subtilis strains containing the fabF1 allele, which codes for the cerulenin-insensitive protein FabF[I108F], overexpressed several genes involved in fatty acid and phospholipid biosynthesis (the fap regulon) and had significantly elevated levels of malonyl-CoA. These results pinpointed FabF[I108F] as responsible for the increased malonyl-CoA production, which in turn acts as an inducer of the fap regulon by impairing the binding of the FapR repressor to its DNA targets. Synthesis of acyl-ACPs by a cell-free fatty acid system prepared from fabF1 cells showed the accumulation of short- and medium-chain acyl-ACPs. These results indicate that the acyl-ACP chain length acceptance of FabF[I108F] is biased towards shorter acyl-ACPs. We also provide evidence that upregulation of FabF[I108F] is essential for survival and for resistance to cerulenin of fabF1 cells. These findings indicate that malonyl-CoA is a key molecule to monitor lipid metabolism functioning and trigger appropriate genetic and biochemical adjustments to relieve dysfunctions of this essential metabolic pathway.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Bacillus subtilis/enzimologia , Regulação Bacteriana da Expressão Gênica , Metabolismo dos Lipídeos/genética , Malonil Coenzima A/genética , Proteínas Repressoras/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/efeitos dos fármacos , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Bacillus subtilis/genética , Cerulenina/farmacologia , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Malonil Coenzima A/metabolismo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Regulon , Proteínas Repressoras/genéticaRESUMO
Bacterial cells stringently regulate the synthesis of their membrane phospholipids but the responsible mechanisms are incompletely understood. Recent biochemical, genetic and structural analyses have greatly expanded the knowledge of lipid metabolism in Gram-positive bacteria, revealing that these organisms use novel mechanisms to regulate this essential pathway. A remarkable progress was the identification of a new pathway for the initiation of phospholipid biosynthesis that uncovered a mechanism that coordinates fatty acid and phospholipid biosynthesis. Recent advances in structure determination of a global transcription factor have led to significant insights of the underlying complexities and functional elegance of membrane lipid homeostasis in Gram-positive bacteria.
Assuntos
Ácido Graxo Sintase Tipo II/metabolismo , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Positivas/enzimologia , Lipídeos de Membrana/biossíntese , Ácido Graxo Sintase Tipo II/genética , Bactérias Gram-Positivas/genética , Lipídeos de Membrana/química , Modelos Moleculares , Fosfolipídeos/biossíntese , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Lipídeos/biossíntese , Malonil Coenzima A/química , Dobramento de Proteína , Proteínas Repressoras/biossíntese , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Dimerização , Malonil Coenzima A/genética , Malonil Coenzima A/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/genéticaRESUMO
The synthesis of L-cysteine, the major mechanism by which sulfur is incorporated into organic compounds in microorganisms, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis the cysH operon, encoding several proteins involved in cysteine biosynthesis, is induced by sulfur starvation and tightly repressed by cysteine. We show that a null mutation in the cysK gene encoding an O-acetylserine-(thiol)lyase, the enzyme that catalyzes the final step in cysteine biosynthesis, results in constitutive expression of the cysH operon. Using DNA microarrays we found that, in addition to cysH, almost all of the genes required for sulfate assimilation are constitutively expressed in cysK mutants. These results indicate that CysK, besides its enzymatic role in cysteine biosynthesis, is a global negative regulator of genes involved in sulfur metabolism.
Assuntos
Bacillus subtilis/enzimologia , Cisteína Sintase/metabolismo , Regulação Bacteriana da Expressão Gênica , Enxofre/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/metabolismo , Cisteína/biossíntese , Cisteína Sintase/genética , Escherichia coli , Fusão Gênica , Genes Reporter , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
Fatty acid synthesis is coordinately regulated with phospholipid, macromolecular synthesis and growth as part of the response to changes in the environment. Many of these processes are rapid responses of the integrated biochemical network and do not involve changes in gene expression. An important recent development is the identification and characterization of transcription factors that modify pathway activity by either altering the expression levels of a few important genes or controlling a global adjustment in the expression of the entire pathway. For most of these transcription factors the signaling molecules controlling their activities are still poorly defined.
Assuntos
Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Lipídeos de Membrana/biossíntese , Transcrição Gênica , Bactérias/metabolismoRESUMO
Bacterial cells exert exquisite control over the biosynthesis of their membrane lipids, but the mechanisms are obscure. We describe the identification and purification from Bacillus subtilis of a transcription factor, FapR, that controls the expression of many genes involved in fatty acid and phospholipid metabolism (the fap regulon). Expression of this fap regulon is influenced by antibiotics that specifically inhibit the fatty acid biosynthetic pathway. We show that FapR negatively regulates fap expression and that the effects of antibiotics on fap expression are mediated by FapR. We further show that decreasing the cellular levels of malonyl-CoA, an essential molecule for fatty acid elongation, inhibits expression of the fap regulon and that this effect is FapR dependent. Our results indicate that control of FapR by the cellular pools of malonyl-CoA provides a mechanism for sensing the status of fatty acid biosynthesis and to adjust the expression of the fap regulon accordingly.