Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina

Hong Gao,Buvani Murugesan,W Marshall Stark,Janina Hoßbach,Margaret C M Smith,Stephanie K Evans

doi:10.1186/s12896-019-0521-y

Hong Gao, Buvani Murugesan + Show 4 more

Open Access

https://doi.org/10.1186/s12896-019-0521-y

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Abstract

BackgroundFew 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.ResultsAnalysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs 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.ConclusionsThe 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.

Highlights

Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms
The sequences of attB sites recognised by a variety of integrases were used in BLAST searches of the genome sequence of A. marina DSM45569
Both R4 and TG1 attB sites were highly conserved relative to the attB sites originally identified from Streptomyces parvulus [32] (84% for R4 integrase) and Streptomyces avermitilis [30] (62% for TG1 integrase)

Summary

Introduction

Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. Streptomyces bacteria are widely exploited for their abundant bioactive natural products [1]. After decades of exploitation, the rate of discovery of new Streptomyces-derived bioactive products has declined, and interest has grown in other potential non-Streptomycete sources, such as the rare Actinomycetes [2, 3]. Amongst rare Actinomycetes, the Amycolatopsis genus is of particular interest for its production of critically important antibiotics such as vancomycin [4] and rifamycin [5], as well as a diverse range of active natural products [6,7,8]. Similar to Streptomyces, the genome of each Amycolatopsis contains averagely over 20 secondary metabolic gene clusters [9]. The mining of these metabolic clusters offers excellent potential for novel antibiotic discovery

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