Microbiology (2000), 146, 1969–1975 Printed in Great Britain Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement Tanya Parish and Neil G. Stoker Author for correspondence : Tanya Parish. Tel : 44 20 7927 2425. Fax: 44 20 7637 4314. e-mail : Tanya.Parishlshtm.ac.uk Department of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK Progress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of Mycobacterium tuberculosis. Four mutants were constructed : a marked deletion of the plcABC cluster, which encodes three phospholipases C ; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin) ; and a double unmarked mutant tlyAΔ plcABCΔ. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by PacI restriction enzyme sites. The final suicide 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 of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations. Keywords : homologous recombination, haemolysin, phospholipase C, rapid cloning system INTRODUCTION Tuberculosis remains a serious public health problem in many parts of the world and is responsible for approxi- mately 2 million deaths per year (Dye et al., 1999). New drugs and vaccines are required to control the disease. However, progress has until recently been hampered by the lack of suitable tools with which to manipulate the genome of the causative pathogen, Mycobacterium tuberculosis. In particular, it has not been possible to generate defined mutants of M. tuberculosis by hom- ologous recombination routinely despite early successes (Balasubramanian et al., 1996 ; Berthet et al., 1998; Pelicic et al., 1997). By making such mutants, the function of individual genes may be analysed, revealing potential targets for novel chemotherapies. Further- ................................................................................................................................................. Abbreviations : hyg, hygromycin; kan, kanamycin; suc, sucrose; MCS, multiple cloning site. more, now that the completed genome sequence is available (Cole et al., 1998), rationally attenuated strains which are potential vaccine candidates can be con- structed. For these reasons, we have been developing an efficient system for introducing specific mutations in M. tu- berculosis. We have previously reported the construc- tion of five M. tuberculosis strains with defined mutations in genes encoding a haemolysin (tlyA) (Hinds et al., 1999) and enzymes from amino acid biosynthesis pathways (hisD, metB, proC and trpD) (Parish et al., 1999). Our method uses a suicide plasmid vector to deliver the recombination substrate to the recipient cell. The plasmids lack a mycobacterial origin of replication (oriM) and are thus unable to replicate in M. tu- berculosis. Plasmid DNA is pretreated with UV light or alkali to stimulate homologous recombination and abolish illegitimate recombination in the recipient cells. Construction of the suicide delivery vector has often 0002-4043  2000 SGM 1969