Enhanced gene replacement in mycobacteria

Citation
J. Hinds et al., Enhanced gene replacement in mycobacteria, MICROBIO-UK, 145, 1999, pp. 519-527
Citations number
31
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
MICROBIOLOGY-UK
ISSN journal
1350-0872 → ACNP
Volume
145
Year of publication
1999
Part
3
Pages
519 - 527
Database
ISI
SICI code
1350-0872(199903)145:<519:EGRIM>2.0.ZU;2-3
Abstract
Allelic replacement will be a vital tool for understanding gene function in mycobacteria, Disruption of the chromosomal hisD gene of Mycobacterium sme gmatis by standard gene replacement methods was surprisingly difficult, wit h most products being caused by illegitimate recombination (IR) events. A r ecombination assay was therefore developed and used to optimize conditions for homologous recombination (HR) in M, smegmatis, Treatment of competent c ells with UV, hydrogen peroxide or mitomycin C did not improve the Departme nt of Pediatrics, frequency of HR; however, treatment of the DNA with alkal i or UV enhanced recombination frequency, while boiling did not. Applying t hese observations to allele replacement, UV and alkali treatment of transfo rming DNA increased British Columbia, Canada HR events with pyrF and hisD, while the level of IR was unchanged. The introduction of ss phagemid DNA im proved the level of HR and abolished IR, In Mycobacterium intracellulare th e use of alkali-denatured DNA increased the numbers of recombinants obtaine d with an inactivated 19Ag gene, while in Mycobacterium tuberculosis, inact ivation of a putative haemolysin gene, tlyA, was achieved using both UV-irr adiated 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 thr ee species were successfully knocked-out using non-replicating DNA pretreat ed 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 imp ortant species.