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 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. 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.

中文翻译：

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在罕见的放线菌（Amycolatopsis）滨海地区进行遗传研究的整合载体。

背景技术由于难以在这些遗传上难治的生物中应用分子方法，因此很少研究稀有放线菌的天然产物途径。在这项研究中，我们试图使用噬菌体int / attP基因座来鉴定更多整合载体，以有效地在稀有的放线菌（Amycolatopsis marina DSM45569）中进行位点特异性整合。结果对滨海拟南芥DSM45569的基因组分析表明，TG1和R4整合的attB样序列存在。TG1和R4 attB在体外重组测定中具有同源的纯化整合子和attP基因座。包含TG1或R4 int / attP位点的整合载体在大肠杆菌和滨海假单胞菌DSM45569的缀合测定中产生了超缀合剂。通过测序证实了质粒到宿主TG1或R4 attB位点的位点特异性重组。结论已经鉴定出了淀粉样芽胞杆菌属中的同源TG1和R4 attB位点。结果表明，基于TG1和R4整合的载体可广泛应用于该属。