Abstract
The Mycobacterium-Escherichia coli 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 Mycobacterium yongonense. 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 Mycobacterium smegmatis (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 EGFP 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 in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.
Highlights
Mycobacterium bovis BCG (BCG) is a live attenuated vaccine that is used to treat tuberculosis and is currently the most frequently administered vaccine worldwide [1,2,3,4]
The mycobacterial cultures were grown at 37°C in 7H9 broth or on 7H10 agar plates supplemented with the appropriate antibiotics
Based on the genome sequences, a restriction map for the pMyong2 linear plasmid, containing nine restriction enzymes with a single recognition site, was constructed using the MapDraw program (DNAstar) (S1 Fig). This plasmid sequence contains 68-bp terminal inverted repeats and covalently closed hairpin loops at both termini, suggesting that this plasmid belongs to a class of genetic elements called invertrons [40], which is similar to the linear plasmids of other Actinomycetales
Summary
Mycobacterium bovis BCG (BCG) is a live attenuated vaccine that is used to treat tuberculosis and is currently the most frequently administered vaccine worldwide [1,2,3,4]. BCG represents the most stable and safe live vaccine developed; the vaccine demonstrates excellent adjuvant properties, induces long-lasting immunity, and has low production costs [5,6,7]. BCG has many properties that make these bacteria attractive live vectors for the development of PLOS ONE | DOI:10.1371/journal.pone.0122897. A Novel Mycobacterium-E. coli Shuttle Vector, pMyong study design, data collection and analysis, decision to publish, or preparation of the manuscript BCG has many properties that make these bacteria attractive live vectors for the development of PLOS ONE | DOI:10.1371/journal.pone.0122897 March 30, 2015
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