The Development of a Novel <i>Mycobacterium-Escherichia coli</i> Shuttle Vector System Using pMyong2, a Linear Plasmid from <i>Mycobacterium yongonense</i> DSM 45126^T

<div><p>The <i>Mycobacterium</i>-<i>Escherichia coli</i> shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from <i>Mycobacterium yongonense</i>. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant <i>Mycobacterium smegmatis</i> (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the <i>EGFP</i> gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the <i>in vivo</i> delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.</p>