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Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination

Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK ¹
Molecular Biology Unit, South African Institute for Medical Research, PO Box 1038, Johannesburg 2000, South Africa ²
Immunopathology Unit, Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, UK³
Department of Haematology and Molecular Medicine, University of the Witwatersrand, Johannesburg, South Africa⁴

Author for correspondence: Neil G. Stoker. Tel: +44 20 7927 2425. Fax: +44 20 7637 4314. e-mail: neil.stoker{at}lshtm.ac.uk

The ability to generate mutants of Mycobacterium tuberculosis will be important if we are to understand the biology of this major pathogen. However, allelic replacement methods have only recently achieved success. We have developed a reproducible method for generating defined mutants of M. tuberculosis using homologous recombination. The transforming DNA was used following pre-treatment either with UV light or alkali denaturation in order to stimulate homologous recombination and abolish illegitimate recombination. Suicide vectors carrying one of nine amino acid biosynthesis genes were electroporated into M. tuberculosis, and homologous recombinants were obtained in all nine genes; eight resulted from single-crossover events (SCOs) and one from a double-crossover event (DCO) (in the metB gene). The remaining colonies were spontaneous hygromycin-resistant mutants; no products of illegitimate recombination were observed. To more efficiently distinguish spontaneous mutants, the lacZ gene was cloned into five vectors (two containing genes not previously tested), and the transformations were repeated. SCO mutants were identified by screening for blue colonies on indicator plates. White transformants were tested for auxotrophy and trpD, hisD and proC auxotrophic mutants were obtained. Only blue SCOs were obtained for argF and glnE. Thus, using this methodology we have obtained homologous recombination in 11 genes, and DCOs in 4 genes, showing that it is possible to generate targeted mutants in a reproducible way.

Keywords: auxotrophs, gene replacement, lacZ , Casamino acids, pre-treated DNA

Abbreviations: DCO, double crossover; HR, homologous recombination; IR, illegitimate recombination; hyg^R, hygromycin resistant; SCO, single crossover

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