FUNCTIONAL-ANALYSIS OF PAL5000, A PLASMID FROM MYCOBACTERIUM-FORTUITUM - CONSTRUCTION OF A MINI-MYCOBACTERIUM ESCHERICHIA-COLI SHUTTLE VECTOR

Functional domains of pAL5000 were determined by gene disruption and deletion analysis. Of the five plasmid open reading frames (ORFs), ORF1 to ORF5, and a putative origin of replication previously identified (J. Rauzier, J. Moniz-Pereira, and B. Gicquel-Sanzey, Gene 71:315-321), two of the ORFs (ORF3 and ORF4) were deemed dispensable for plasmid replication. A 'mini' mycobacterium-Escherichia coli shuttle plasmid applicable for general recombinant DNA studies in mycobacteria was constructed by using the gene for Kan(r) (Tn903) as a selective marker. Heterologous expression of the gene for Kan(r) was confirmed by Western blotting (immunoblotting) analysis.