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Molecular Genetics and Immunobiology of Mycobacteria |
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK1
Department of Medical Microbiology, Barts and the London, Queen Mary’s School of Medicine and Dentistry, 32 Newark Street, London E1 2AA, UK2
Author for correspondence: Tanya Parish. Tel: +44 20 7377 0444. Fax: +44 20 7247 3428. e-mail: t.parish{at}qmul.ac.uk
Attempts to construct Mycobacterium tuberculosis strains with a defect in the common aromatic amino acid biosynthesis pathway were made. In other bacteria the genes of this pathway (aro) can be disrupted in the presence of suitable media supplements. The genomic organization of the aro genes in M. tuberculosis reveals that there is one operon (aroCKBQ) and three isolated aro genes (aroE, aroG and aroA). The aroK gene was chosen as a target for disruption; this encodes shikimate kinase, which catalyses the fifth step in chorismate biosynthesis. Attempts to replace the wild-type aroK gene with a disrupted allele (aroK
::hyg) by a two-step homologous recombination procedure were unsuccessful in a wild-type strain. When a second functional copy of aroK was integrated into the chromosome, it was possible to isolate a strain carrying the disrupted gene. Excision of the L5-integrated copy of aroK by the L5 excisionase could be not be achieved in the strain carrying the disrupted copy, but was possible in a strain carrying a wild-type copy. These results demonstrate that the chorismate pathway is essential for the viability of M. tuberculosis.
Keywords: chorismate biosynthesis, auxotrophs, gene replacement, bacteriophage L5, excisionase
Abbreviations: BCG, bacille Calmette–Guérin; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate
a Present address: Department of Pathology and Infectious Diseases, Royal Veterinary College, Royal College Street, London NW1 0TU, UK.
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