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 al kali-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.

Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability

“…The gene, including a 274 bp upstream region, which might contain the native promoter, was amplified by PCR using the specific oligonucleotides 59-ATAAAGCTTCCGGATCCGCAGGACGTCGA-TC-39 and 59-ATTTCTAGAGAGCTAGTGGAACTGGCCCTCTTC-GGTGG-39 and inserted into pMV306, containing an attachment site and an integrase gene derived from mycobacteriophage L5, which allows the integration of the vector into the genome of M. tuberculosis by site-specific recombination . In contrast to the pMV306 complementation vector, the final plasmid preparation of the pYUB854 knockout construct was UV-irradiated prior to electroporation into M. tuberculosis (Hinds et al, 1999).…”

Exoribonuclease R in Pseudomonas syringae Is Essential for Growth at Low Temperature and Plays a Novel Role in the 3′ End Processing of 16 and 5 S Ribosomal RNA

“…Plasmid construct was confirmed by sequencing and PCR analysis. For rnr knockout, the pBSrnr-tet was electroporated into P. syringae following denaturation of the plasmid as described earlier (21), which apparently increases homologous recombination and hence gene disruption frequency. Recombinants were selected by tetracycline resistance, and the rnr knock-out mutation was confirmed by Southern and PCR analysis.…”

Comparative and functional genomics of the Mycobacterium tuberculosis complex a aThis review is based on the 2002 Marjory Stephenson Prize Lecture delivered by the author at the 150th Meeting of the Society for General Microbiology, 9 April 2002.

“…Do specific mycobacterial factors determine the outcome? Answers to these questions will be provided by functional genomics and, in recent years, there have been spectacular advances in gene replacement technology (Bardarov et al, 1997 ;Hinds et al, 1999 ;Parish & Stoker, 2000 ;Pelicic et al, 1997). It is now relatively straightforward to construct knockout mutants although this remains a lengthy process owing to the slow growth of tubercle bacilli.…”