New shuttle vectors for gene cloning and expression in multidrug-resistant Acinetobacter species

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.