摘要

了解细菌发病机制需要足够的遗传工具，以通过诱变和互补测定以及克隆基因的同源和异源表达来评估个体毒力决定簇的作用。我们对鲍曼不动杆菌发病机制的了解迄今一直受到遗传工具稀缺性的限制，因为这些遗传工具缺乏对多数感染负责的多重耐药（MDR）流行株的操纵。在这里，我们报道了新的多功能穿梭质粒的构建，即pVRL1和pVRL2，它们可以在不动杆菌属细菌中高效复制。并在大肠杆菌中。 pVRL1质粒已通过组合：i）来自醋酸钙不动杆菌的隐蔽质粒pWH1277，其提供不动杆菌属的复制起点; ii）ColE1样复制起点; iii）用于抗生素选择的庆大霉素或zeocin抗性盒; iv）含有几个独特限制性位点的多接头。 pVRL1的修饰导致pVRL2质粒的产生，其允许阿拉伯糖诱导的基因转录，在未诱导的条件下具有检测不到的克隆基因的基础表达水平以及对诱导物的高动态范围的响应性。耐药菌株pVRL1和pVRL2均可轻松选择，鲍氏不动杆菌的宿主范围较窄，拷贝数较高，在不动杆菌属中稳定存在，并与流行毒株携带的土着质粒兼容。通过毒素 - 抗毒素系统的存在保证了质粒的维持，从而提供对Acinetobacter sp。中质粒稳定性机制的更多见解。

Understanding bacterial pathogenesis requires adequate genetic tools to assess the role of individual virulence determinants by mutagenesis and complementation assays, as well as for homologous and heterologous expression of cloned genes. Our knowledge of Acinetobacter baumannii pathogenesis has so far been limited by the scarcity of genetic tools to manipulate multi drug resistant (MDR) epidemic strains which are responsible for most of infections. Here, we report the construction of new multi-purpose shuttle plasmids, namely pVRL1 and pVRL2, which can efficiently replicate in Acinetobacter sp. and in Escherichia coli. The pVRL1 plasmid has been constructed by combining: i) the cryptic plasmid pWH1277 from Acinetobacter calcoaceticus, which provides an origin of replication for Acinetobacter sp.; ii) a ColE1-like origin of replication; iii) the gentamicin or zeocin resistance cassette for antibiotic selection; iv) a multilinker containing several unique restriction sites. Modification of pVRL1 led to the generation of the pVRL2 plasmid, which allows arabinose-inducible gene transcription, with undetectable basal expression level of cloned genes under un-induced conditions and high-dynamic range of responsiveness to the inducer. Both pVRL1 and pVRL2 can easily be selected in MDR A. baumannii, have narrow host range and high copy number, are stably maintained in Acinetobacter sp., and appear compatible with indigenous plasmids carried by epidemic strains. Plasmid maintenance is guaranteed by the presence of a toxin-antitoxin system, providing more insights into the mechanism of plasmids stability in Acinetobacter sp.

http://aac.asm.org/content/early/2018/01/09/AAC.02480-17.abstract