RESUMO
Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916-sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na(+)/H(+) antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway.
Assuntos
Proteínas de Bactérias/genética , Cloranfenicol/biossíntese , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas/genética , Streptomyces/genética , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Análise em Microsséries , Anotação de Sequência Molecular , Família Multigênica , Mutação , Análise de Sequência de DNA , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Streptomyces/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
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 proteins (ACPs) are required for the transfer of acyl intermediates during fatty acid and polyketide syntheses. In Sinorhizobium meliloti 1021 there are five known ACPs: AcpP, NodF, AcpXL, the ACP domain in RkpA and SMb20651. The genome sequence of S. meliloti 1021 also reveals the ORF SMc01553, annotated as a putative ACP. smc01553 is part of a 6.6 kb DNA region that is duplicated in the chromosome and in the pSymb plasmid, the result of a recent duplication event. SMc01553 overexpressed in Escherichia coli was labelled in vivo with [(3)H]beta-alanine, a biosynthetic building block of the 4'-phosphopantetheine prosthetic group of ACPs. The purified SMc01553 was modified with 4'-phosphopantetheine in the presence of S. meliloti holo-ACP synthase, and this modification resulted in a major conformational change of the protein structure, since the holo-form runs faster in native PAGE than the apo-form. SMc01553 could not be loaded with a malonyl group by malonyl-CoA-ACP transacylase from S. meliloti. Using RT-PCR we could show the presence of mRNA for SMc01553 and of the duplicated ORF SMb22007 in cultures of S. meliloti. However, a mutant in which the two duplicated regions were deleted did not show any different phenotype with respect to the wild-type in the free-living or symbiotic lifestyle.
Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Sinorhizobium meliloti/genética , Proteína de Transporte de Acila/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Panteteína/análogos & derivados , Estrutura Secundária de Proteína , RNA Bacteriano/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinorhizobium meliloti/metabolismoRESUMO
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
Acyl carrier proteins (ACPs) are small acidic proteins that carry growing acyl chains during fatty acid or polyketide synthesis. In rhizobia, there are four different and well-characterized ACPs: AcpP, NodF, AcpXL and RkpF. The genome sequence of Sinorhizobium meliloti 1021 reveals two additional ORFs that possibly encode additional ACPs. One of these, smb20651, is located on the plasmid pSymB as part of an operon. The genes of the operon encode a putative asparagine synthetase (AsnB), the predicted ACP (SMb20651), a putative long-chain fatty acyl-CoA ligase (SMb20650) and a putative ammonium-dependent NAD+ synthetase (NadE1). When SMb20651 was overexpressed in Escherichia coli, [3H]beta-alanine, a biosynthetic building block of 4'-phosphopantetheine, was incorporated into the protein in vivo. The purified SMb20651 was modified with 4'-phosphopantetheine in the presence of S. meliloti holo-ACP synthase (AcpS). Also, holo-SMb20651 was modified in vitro with a malonyl group by malonyl CoA-ACP transacylase. In E. coli, coexpression of SMb20651 together with other proteins such as AcpS and SMb20650 led to the formation of additional forms of SMb20651. In this bacterium, acylation of SMb20651 with C12 : 0 or C18 : 0 fatty acids was detected, demonstrating that this protein is involved in fatty acid biosynthesis or transfer. Expression of SMb20651 was detected in S. meliloti as holo-SMb20651 and acyl-SMb20651.
Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Sinorhizobium meliloti/metabolismo , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/imunologia , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/genética , Ligases/metabolismo , Medicago sativa/microbiologia , Mutagênese Sítio-Dirigida , Óperon , Panteteína/análogos & derivados , Panteteína/metabolismo , Coelhos , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/crescimento & desenvolvimentoRESUMO
Acyl carrier protein (ACP) is a central cofactor for de novo fatty acid synthesis, acyl chain modification and chain-length termination during lipid biosynthesis in living organisms. Although the structural and functional organization of the ACPs in bacteria and plant are highly conserved, the individual ACP is engaged in the generation of sets of signature fatty acids required for specific purpose in bacterial cells and plant tissues. Realizing the fact that the bacterial ACP being originated early in molecular evolution is characteristically different from the plant's counterpart, we explored the property of an ACP from Azospirillum brasilense (Ab), a plant-associative aerobic bacterium, to find its role in changing the fatty acid profile in heterologous systems. Functional expression of Ab-ACP in Escherichia coli, an enteric bacterium, and Brassica juncea, an oil-seed crop plant, altered the fatty acid composition having predominantly 18-carbon acyl pool, reflecting the intrinsic nature of the ACP from A. brasilense which usually has C18:1 rich membrane lipid. In transgenic Brassica the prime increment was found for C18:3 in leaves; and C18:1 and C8:2 in seeds. Interestingly, the seed oil quality of the transgenic Brassica potentially improved for edible purposes, particularly with respect to the enhancement in the ratio of monounsaturated (C18:1)/saturated fatty acids, increment in the ratio of linoleic (C18:2)/linolenic (C18:3) and reduction of erucic acid (C22:1).
Assuntos
Proteína de Transporte de Acila/genética , Azospirillum brasilense/genética , Brassica napus/genética , Escherichia coli/genética , Ácidos Graxos/metabolismo , Proteína de Transporte de Acila/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Brassica napus/microbiologia , Clonagem Molecular , Isopropiltiogalactosídeo/farmacologia , Cinética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Acyl carrier protein (ACP) plays a crucial role in bacterial fatty acid synthesis. Cloning genes encoding ACPs from Gram-negative bacteria in Escherichia coli is difficult due to adverse effects of the cloned gene on host cell viability, and we were unsuccessful in cloning the full length ACP gene (acpP) from Azospirillum brasilense using conventional methods. Therefore, ACP from A. brasilense was purified to homogeneity and a part of the acpP gene was cloned using the polymerase chain reaction (PCR) technique with two primers, one designed from the N-terminal amino acid sequence of the purified ACP and the other from the highly conserved amino acid sequence of bacterial ACPs. The nucleotide sequence of the gene was obtained by cloning and sequencing inverse PCR products containing the acpP region generated by two oppositely oriented internal primers designed from the partial acpP gene sequence using restriction-enzyme-digested, self-circularized chromosomal DNA fragments as templates. Characterization of the purified ACP and analysis of the derived amino acid sequence of the acpP gene of A. brasilense revealed that: (a) the mature ACP, composed of 78 amino acids, is a highly expressed protein (about 2.0-3.0 x 10(4) molecules per cell), (b) compared to E. coli ACP, it has a more compact structure and contains significantly more hydrophobic amino acid residues and (c) the potential mRNA sequence of the ACP gene has some structural features typical of a stable mRNA.