Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement

Progress in the field of mycobacterial research has been hindered by the in ability to readily generate defined mutant strains of the slow-growing inab ility to readily generate defined mutant strains of the slow-growing mycoba cteria to investigate the function of specific genes. An efficient method i s described that has been used to generate several mutants, including the f irst double unmarked deletion strain of Mycobacterium tuberculosis. Four mu tants were constructed: a marked deletion of the plcABC cluster, which enco des three phospholipases C; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin); and a double unmarked mutant tlyA Delta plcABC Del ta. To accomplish this, two series of vectors were designed, the first of w hich, named pNIL, allows manipulation of the target gene sequence at a vari ety of convenient restriction sites. The second series, named pGOAL, contai ns marker cassettes flanked by Pad restriction enzyme sites. The final suic ide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single PacI site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross -over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion o f antibiotic resistance markers, the ability to make multiple defined mutat ions and the possibility of making more subtle defined mutations, such as p oint mutations.