RESUMEN
Mupirocin, a polyketide-derived antibiotic from Pseudomonas fluorescens NCIMB10586, is a mixture of pseudomonic acids (PA) that target isoleucyl-tRNA synthase. The mup gene cluster encodes both type I polyketide synthases and monofunctional enzymes that should play a role during the conversion of the product of the polyketide synthase into the active antibiotic (tailoring). By in-frame deletion analysis of selected tailoring open-reading frames we show that mupQ, mupS, mupT, and mupW are essential for mupirocin production, whereas mupO, mupU, mupV, and macpE are essential for production of PA-A but not PA-B. Therefore, PA-B is not simply produced by hydroxylation of PA-A but is either a precursor of PA-A or a shunt product. In the mupW mutant, a new metabolite lacking the tetrahydropyran ring is produced, implicating mupW in oxidation of the 16-methyl group.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Familia de Multigenes , Mupirocina/metabolismo , Pseudomonas fluorescens/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Cromatografía Líquida de Alta Presión , Ácidos Grasos/biosíntesis , Ácidos Grasos/genética , Isoleucina-ARNt Ligasa/genética , Isoleucina-ARNt Ligasa/metabolismo , Espectroscopía de Resonancia Magnética , Mupirocina/análogos & derivados , Mutación , Sistemas de Lectura Abierta , Oxidación-Reducción , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Regiones Promotoras Genéticas , Pseudomonas fluorescens/genéticaRESUMEN
The polyketide antibiotic mupirocin (pseudomonic acid) produced by Pseudomonas fluorescens NCIMB 10586 competitively inhibits bacterial isoleucyl-tRNA synthase and is useful in controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus. The 74 kb mupirocin biosynthesis cluster has been sequenced, and putative enzymatic functions of many of the open reading frames (ORFs) have been identified. The mupirocin cluster is a combination of six larger ORFs (mmpA-F), containing several domains resembling the multifunctional proteins of polyketide synthase and fatty acid synthase type I systems, and individual genes (mupA-X and macpA-E), some of which show similarity to type II systems (mupB, mupD, mupG, and mupS). Gene knockout experiments demonstrated the importance of regions in mupirocin production, and complementation of the disrupted gene confirmed that the phenotypes were not due to polar effects. A model for mupirocin biosynthesis is presented based on the sequence and biochemical evidence.
Asunto(s)
Antibacterianos/biosíntesis , Genes Bacterianos , Familia de Multigenes , Mupirocina/biosíntesis , Pseudomonas fluorescens/genética , 3-Hidroxiacil-CoA Deshidrogenasas/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Secuencia de Aminoácidos , Antibacterianos/farmacología , Clonación Molecular , Farmacorresistencia Microbiana , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroliasas/genética , Metiltransferasas/genética , Modelos Químicos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Pseudomonas fluorescens/metabolismoRESUMEN
Mupirocin (pseudomonic acid) is a polyketide antibiotic, targeting isoleucyl-tRNA synthase, and produced by Pseudomonas fluorescens NCIMB 10586. It is used clinically as a topical treatment for staphylococcal infections, particularly in contexts where there is a problem with methicillin-resistant Staphylococcus aureus (MRSA). In studying the mupirocin biosynthetic cluster the authors identified two putative regulatory genes, mupR and mupI, whose predicted amino acid sequences showed significant identity to proteins involved in quorum-sensing-dependent regulatory systems such as LasR/LuxR (transcriptional activators) and LasI/LuxI (synthases for N-acylhomoserine lactones--AHLs--that activate LasR/LuxR). Inactivation by deletion mutations using a suicide vector strategy confirmed the requirement for both genes in mupirocin biosynthesis. Cross-feeding experiments between bacterial strains as well as solvent extraction showed that, as predicted, wild-type P. fluorescens NCIMB 10586 produces a diffusible substance that overcomes the defect of a mupI mutant. Use of biosensor strains showed that the MupI product can activate the Pseudomonas aeruginosa lasRlasI system and that P. aeruginosa produces one or more compounds that can replace the MupI product. Insertion of a xylE reporter gene into mupA, the first ORF of the mupirocin biosynthetic operon, showed that together mupR/mupI control expression of the operon in such a way that the cluster is switched on late in exponential phase and in stationary phase.