Production of unmarked mutations in mycobacteria using site-specific recombination.

Abstract:

:Gene disruption experiments play an important role in the functional characterization of genes in mycobacteria and rely mostly on the use of one or two antibiotic resistance markers. We have developed a system for mycobacteria which features both the advantages of the use of antibiotic resistance markers for gene disruption experiments and the ability to efficiently rescue the marker leaving an unmarked mutation on the chromosome. This new genetic tool relies on the transposon gammadelta site-specific recombination system. A res-OmegaKm-res cassette was used to generate an insertional mutation by allelic exchange both in Mycobacterium smegmatis and Mycobacterium bovis BCG. Upon expression in the mutated strains of tnpR, the transposon gammadelta resolvase gene, res-OmegaKm-res, was excised efficiently leaving behind a single res sequence at the mutated locus. A plasmid was engineered allowing expression of tnpR from an easily curable mycobacterial vector. This system will be useful for simple construction of unmarked mutations or repeated use of the same antibiotic marker to generate multiple mutants.

journal_name

FEMS Microbiol Lett

authors

Malaga W,Perez E,Guilhot C

doi

10.1016/S0378-1097(03)00003-X

keywords:

subject

Has Abstract

pub_date

2003-02-28 00:00:00

pages

261-8

issue

2

eissn

0378-1097

issn

1574-6968

pii

S037810970300003X

journal_volume

219

pub_type

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