Involvement of the N- and C-terminal domains of Mycobacterium tuberculosis KatG in the protection of mutant Escherichia coli against DNA-damaging agents

1 Department of Medical Microbiology, University of Cape Town and Groote Schuur Hospital, Medical School, Observatory, 7925 Cape Town, South Africa 2 Faculté de Médecine Necker-Enfants malades, Laboratoire de Microbiologie, 156, rue de Vaugirard, 75730 Paris Cedex 15, France 3 Department of Microbiology, University of Cape Town, Rondebosch, 7800 Cape Town, South Africa Author for correspondence: Lafras M. Steyn. Tel: + 27 21 4066389. Fax: + 27 21 4488153. e-mail: Isteyn@medmicro.uct.ac.za ABSTRACT The Mycobacterium tuberculosis KatG enzyme, like most hydroperoxidase I (HPI)-type catalases, consists of two related domains, each with strong similarity to the yeast cytochrome c peroxidase. The catalase-peroxidase activity is associated with the amino-terminal domain but currently no definite function has been assigned to the carboxy-terminal domain, although it may play a role in substrate binding. This paper reports another possible function of the KatG protein involving protection of the host cell against DNA-damaging agents. The M. tuberculosis katG gene, the 5' domain and the 3' domain were cloned separately, in-frame with the maltose-binding protein, into the vector pMAL-c2. These constructs were introduced into four DNA-repair mutants of Escherichia coli , DK1 ( recA ), AB1884 ( uvrC ), AB1885 ( uvrB ) and AB1886 ( uvrA ), which were then tested for their ability to survive treatment with UV light (254 nm), hydrogen peroxide (1·6 mg ml -1 ) and mitomycin C (6 µg ml -1 ). All three constructs conferred resistance to UV upon the recA E. coli cells, whereas resistance to mitomycin C was found in all repair mutants tested. Protection against hydrogen peroxide damage was less pronounced and predominantly found in the recA host. These results indicated that the M. tuberculosis katG gene can enhance DNA repair in E. coli , and that the 5' and 3' domains can function separately. UV sensitivity tests on Mycobacterium intracellulare and M. tuberculosis strains mutant in katG revealed that the katG gene product does not play an additive role in the survival of mycobacterial cells after exposure to short-wavelength UV irradiation, in repair-competent cells. Keywords: Mycobacterium tuberculosis , KatG, catalase, DNA repair The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is L14268 . Abbreviations : ADC, albumin dextrose catalase; EMS, ethyl methane-sulfonate; HPI, hydroperoxidase I; INH, isoniazid; MBP, maltose-binding protein.