Cloning and expression in Escherichia coli of a recA homologue from Mycobacterium tuberculosis

Nair, S.K.; Steyn, Lafras M.

doi:10.1099/00221287-137-10-2409

Cited by 7 publications

(5 citation statements)

References 27 publications

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“…The construction of the PstI genomic library of M. tuberculosis H37Rv in the suicide vector pEcoR252 was described previously (Nair & Steyn, 1991). The library was screened by selection of transformed E. coli DK 1 cells on plates containing 0.1 O/ O (w/v) ethyl methane sulfonate (EMS).…”

Section: Methodsmentioning

confidence: 99%

“…coli DK1 (recA) transformants on 0.1% (w/v) EMS. Two clones were isolated that were able to survive this treatment: one, encoding a putative recA gene, was described earlier (Nair & Steyn, 1991) and the other, designated pD3, is described here. T h e 1747 bp insert from pD3 was sequenced (GenBank accession number L14268) and found to contain three possible ORFs, one of which was truncated at the 5' end.…”

Section: Isolation and Sequencing Of A Clone That Confers Resistance mentioning

confidence: 99%

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

et al. 1999

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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 DNAdamaging 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 pMALc2. These constructs were introduced into four DNArepair mutants of Escherichia coli, DK1 ( r e d ) , AB1884 (uvrC), A61885 (uvrf3) and AB1886 (uvrA), which were then tested for their ability to survive treatment with UV light (254 nm), hydrogen peroxide (1.6 mg ml-l) and mitomycin C (6 pg ml-l). All three constructs conferred resistance to UV upon the r e d 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 r e d 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. encodes a dual-function catalase-peroxidase enzyme which protects the organism against hydrogen peroxide and thus contributes to its survival in macrophages (Middlebrook & Cohn, 1953;Mitchison et al., 1960; Jackett et al., 1978;Wilson et al., 1995;Heym et al., 1997). M . tuberculosis KatG also plays an important role in susceptibility of the organism to the front-line antituberculosis drug, isoniazid (INH) (Middlebrook, 1954;Winder, 1960;Zhang et al., 1992;Heym et al., 1993;Stoeckle et al., 1993;Altamirano et al., 1994; Rouse & Morris, 1995 Repair of damaged DNA is also expected to be an important factor involved in the survival of mycobacteria in macrophages. DNA repair has been extensively studied in Escherichia coli and found t o be complex, involving a number of specialized proteins and pathways, including the SOS response and genetic recombination (Friedberg et a/., 1995). However, relatively little is known about these systems in mycobacteria. It has been shown that there is an SOS response system active in M . tuberculosis (Durbach et al., 1997; Movahedzadeh et al., 1997a, b ; Papavinasasundaram et ul., 1997). T h e lexA repressor has been implicated in the induction of a recA gene in Mycobacterizrm smegmatis (Durbach et al., 1997). T h e M . tuberczrlosis genome data have recently been analysed with respect to genes involved in DNA repair (Mizrahi & Anderse...

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Section: Methodsmentioning

confidence: 99%

Section: Isolation and Sequencing Of A Clone That Confers Resistance mentioning

confidence: 99%

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

et al. 1999

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“…An unexpected finding was that the coding capacity of the clone was substantially larger than predicted from the size of the mycobacterial recA gene product (117). Although the mycobacterial recA gene product was approximately the same size as and reacted with antibody against the E. coli recA gene product (117,372), the mycobacterial recA gene shared nucleotide sequence homology with the E. coli recA gene only at its 5Ј and 3Ј ends (117). Further work demonstrated that the product of the M. tuberculosis recA gene was an 85-kDa precursor protein that was spliced to yield the mature 38-kDa RecA protein (116).…”

Section: Genetics Of Nontuberculous Mycobacteriamentioning

confidence: 96%

“…The key protein of recombination and DNA repair, the recA gene product, has been cloned by complementation of RecA Ϫ mutants of E. coli (372) or by hybridization with a recA gene probe to genomic fragments (117). The M. tuberculosis recA gene was able to confer ethyl methanesulfonate resistance upon RecA Ϫ strains of E. coli, did not increase UV light (254 nm) resistance, and increased recombination proficiency (117,372). An unexpected finding was that the coding capacity of the clone was substantially larger than predicted from the size of the mycobacterial recA gene product (117).…”

Section: Genetics Of Nontuberculous Mycobacteriamentioning

confidence: 99%

Epidemiology of infection by nontuberculous mycobacteria

1996

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“…The RecA protein was the first recombination enzyme to be identified in M. tuberculosis (Davis et al 1991;Nair & Steyn 1991) and it is also the recombination gene/gene product about which we know most. It is a complex protein that binds adenosine triphosphate (ATP) and DNA, polymerizing in a helical filament along the DNA and actively performing the homology search and DNA strand exchange .…”

Section: Recamentioning

confidence: 99%