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Applied and Environmental Microbiology, January 2003, p. 517-523, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.517-523.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Gene Replacement in Mycobacteria by Using Incompatible Plasmids

Carey A. Pashley,^1, Tanya Parish,^1, Ruth A. McAdam,² Ken Duncan,² and Neil G. Stoker^1*

Department of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT,¹ GlaxoSmithKline Research and Development, Medicines Research Centre, Stevenage, United Kingdom²

Received 26 April 2002/ Accepted 15 October 2002

A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.

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* Corresponding author. Present address: Department of Pathology and Infectious Diseases, Royal Veterinary College, Royal College St., London NW1 0TU, United Kingdom. Phone: 44 (0) 20 7468 5272. Fax: 44 (0) 20 7468 5306. E-mail: nstoker{at}rvc.ac.uk.

Present address: Department of Medical Microbiology, Barts and the London, Queen Mary's School of Medicine and Dentistry, London E1 2AA, United Kingdom.

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Applied and Environmental Microbiology, January 2003, p. 517-523, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.517-523.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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