Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify integrating vectors, derived using phageint/attPloci, that would efficiently integrate site-specifically in the rare Actinomycete,Amycolatopsis marinaDSM45569. Analysis of the genome of A.marinaDSM45569 indicated the presence ofattB-like sequences for TG1 and R4 integrases. The TG1 and R4attBswere active inin vitrorecombination assays with their cognate purified integrases andattPloci. Integrating vectors containing either the TG1 or R4int/attPloci yielded exconjugants in conjugation assays fromE. colitoA. marinaDSM45569. Site-specific recombination of the plasmids into the host TG1 or R4attBsites was confirmed by sequencing. The presence of homologous TG1 and R4attBsites in other species of this genus indicates that vectors based on TG1 and R4 integrases could be widely applicable.ImportanceRare Actinomycetes have the same potential of natural product discovery as Streptomyces, but the potential has not been fully explored due to the lack of efficient molecular biology tools. In this study, we identified two serine integrases, TG1 and R4, which could be used in the rare Actinomycetes species,Amycolatopsis marina, as tools for genome integration. The high level of conservation between theattBsites for TG1 and R4 in a number of Amycolatopsis species suggested that plasmids with the integration systems from these phages should be widely useful in this genus.