title: Enhanced gene replacement in mycobacteria.
abstract: Allelic replacement will be a vital tool for understanding gene function in mycobacteria. Disruption of the chromosomal hisD gene of Mycobacterium smegmatis by standard gene replacement methods was surprisingly difficult, with most products being caused by illegitimate recombination (IR) events. A recombination assay was therefore developed and used to optimize conditions for homologous recombination (HR) in M. smegmatis. Treatment of competent cells with UV, hydrogen peroxide or mitomycin C did not improve the frequency of HR; however, treatment of the DNA with alkali or UV enhanced recombination frequency, while boiling did not. Applying these observations to allele replacement, UV and alkali treatment of transforming DNA increased HR events with pyrF and hisD, while the level of IR was unchanged. The introduction of ss phagemid DNA improved the level of HR and abolished IR. In Mycobacterium intracellulare the use of alkali-denatured DNA increased the numbers of recombinants obtained with an inactivated 19Ag gene, while in Mycobacterium tuberculosis, inactivation of a putative haemolysin gene, tlyA, was achieved using both UV-irradiated DNA and ss phagemid DNA. Significantly, IR, which has been reported to be a problem in this species, was not observed. Thus, four genes in three species were successfully knocked-out using non-replicating DNA pretreated with alkali, UV or in an ss form. The use of these methods to enhance HR will greatly facilitate experiments to inactivate other genes in these important species.
authors.affiliation: Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
authors.forename: Jason
authors.initials: J
authors.lastname: Hinds
authors.affiliation: Department of Pediatrics, University of British Columbia and B. C. Children's Hospital, British Columbia, Canada.
authors.forename: Eshwar
authors.initials: E
authors.lastname: Mahenthiralingam
authors.affiliation: Glaxo Wellcome Research and Development, Stevenage, Herts, UK.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Kempsell
authors.affiliation: Glaxo Wellcome Research and Development, Stevenage, Herts, UK.
authors.forename: Ken
authors.initials: K
authors.lastname: Duncan
authors.affiliation: Department of Pediatrics, University of British Columbia and B. C. Children's Hospital, British Columbia, Canada.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Stokes
authors.affiliation: Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
authors.affiliation: Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
authors.forename: Neil G
authors.initials: NG
authors.lastname: Stoker
mesh_terms: D000429:Alcohol Oxidoreductases; D001426:Bacterial Proteins / Q000235:genetics*; D018274:Electroporation; D005798:Genes, Bacterial; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009161:Mycobacterium / Q000235:genetics*; D015269:Mycobacterium avium Complex / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D001426:Bacterial Proteins; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase