RESUMEN
The mupirocin trans-AT polyketide synthase pathway, provides a model system for manipulation of antibiotic biosynthesis. Its final phase involves removal of the tertiary hydroxyl group from pseudomonic acid B, PA-B, producing the fully active PA-A in a complex series of steps. To further clarify requirements for this conversion, we fed extracts containing PA-B to mutants of the producer strain singly deficient in each mup gene. This additionally identified mupM and mupN as required plus the sequence but not enzymic activity of mupL and ruled out need for other mup genes. A plasmid expressing mupLMNOPVCFU + macpE together with a derivative of the producer P. fluorescens strain NCIMB10586 lacking the mup cluster allowed conversion of PA-B to PA-A. MupN converts apo-mAcpE to holo-form while MupM is a mupirocin-resistant isoleucyl tRNA synthase, preventing self-poisoning. Surprisingly, the expression plasmid failed to allow the closely related P. fluorescens strain SBW25 to convert PA-B to PA-A.
Asunto(s)
Antibacterianos/metabolismo , Mupirocina/biosíntesis , Pseudomonas fluorescens/metabolismo , Antibacterianos/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Mupirocina/química , Mutagénesis , Plásmidos/genética , Plásmidos/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Policétidos/química , Policétidos/metabolismo , Pseudomonas fluorescens/genéticaRESUMEN
RA3 is a low-copy-number, broad-host-range (BHR) conjugative plasmid of the IncU incompatibility group isolated originally from Aeromonas spp. A 4.9-kb fragment of RA3 is sufficient to stabilize an otherwise unstable replicon in Escherichia coli. This fragment specifies the korA-incC-korB-orf11 operon coding for an active partition system related to the central control operon of IncP-1 plasmids and found also in BHR environmental plasmids recently classified as the PromA group. All four genes in the cassette are necessary for segregation. IncC and KorB of RA3 belong to the ParA and ParB families of partitioning proteins, respectively. In contrast with IncP-1 plasmids, neither KorB nor IncC are involved in transcriptional autoregulation. Instead, KorA exerts transcriptional control of the operon by binding to a palindromic sequence that overlaps the putative -35 promoter motif of the cassette. The Orf11 protein is not required for regulation, but its absence decreases the stabilization potential of the segregation module. A region discontiguous from the cassette harbors a set of unrelated repeat motifs distributed over â¼300 bp. Dissection of this region identified the centromere sequence that is vital for partitioning. The â¼300-bp fragment also encompasses the origin of conjugative transfer, oriT, and the promoter that drives transcription of the conjugative transfer operon. A similar set of cis-acting motifs are evident in the PromA group of environmental plasmids, highlighting a common evolutionary origin of segregation and conjugative transfer modules in these plasmids and members of the IncU group.
