Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina

Abstract

Background 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 more integrating vectors, using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Results Analysis of the genome of A. marina DSM45569 indicated the presence of attB -like sequences for TG1 and R4 integrases. The TG1 and R4 attB s were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from Escherichia coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. Conclusions The homologous TG1 and R4 attB sites within the genus Amycolatopsis have been identified. The results indicate that vectors based on TG1 and R4 integrases could be widely applicable in this genus.

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

“…Plasmids pHG4 [23], pHG5, pHG6 and pHG7 are all integrating vectors encoding the int/attP loci from TG1, SV1, φBT1 and φC31, respectively (). These four plasmids also encode compatible antibiotic resistance loci; apramycin resistance ( aac(3)IV , pHG4), kanamycin resistance ( aphII , pHG5), erythromycin resistance ( ermE , pHG6) and hygromycin resistance ( hygB , pHG7).…”

“…Plasmids pHG4 [ 23 ], pHG5, pHG6 and pHG7 are all integrating vectors encoding the int/attP loci from TG1, SV1, φBT1 and φC31, respectively ( Fig. 1 ).…”

Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

“…Despite the importance of the genus Amycolatopsis , the development of adapted genetic tools has lagged behind that for other actinobacterial genera such as Streptomyces (8, 9). For some time the introduction of DNA into Amycolatopsis has been a limiting factor, until this problem could be circumvented by the development of a method of direct transformation of Amycolatopsis mycelia (10) or by using intergeneric conjugation from Escherichia coli to introduce single-stranded DNA into the Amycolatopsis host (11, 12).…”

“…Despite the importance of the genus Amycolatopsis, the development of adapted genetic tools has lagged behind that for other actinobacterial genera such as Streptomyces (8,9).…”