Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis

Abstract

The authors have developed a simple and highly efficient system for generating allelic exchanges in both fast-and slow-growing mycobacteria. In this procedure a gene of interest, disrupted by a selectable marker, is cloned into a conditionally replicating (temperature-sensitive) shuttle phasmid to generate a specialized transducing mycobacteriophage. The temperaturesensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 SC but prevent replication at the nonpermissive temperature of 37 SC. Transduction at a non-permissive temperature results in highly efficient delivery of the recombination substrate to virtually all cells in the recipient population. The deletion mutations in the targeted genes are marked with antibiotic-resistance genes that are flanked by γδ-res (resolvase recognition target) sites. The transductants which have undergone a homologous recombination event can be conveniently selected on antibiotic-containing media. To demonstrate the utility of this genetic system seven different targeted gene disruptions were generated in three substrains of Mycobacterium bovis BCG, three strains of Mycobacterium tuberculosis, and Mycobacterium smegmatis. Mutants in the lysA, nadBC, panC, panCD, leuCD, Rv3291c and Rv0867c genes or operons were isolated as antibiotic-resistant (and in some cases auxotrophic) transductants. Using a plasmid encoding the γδ-resolvase (tnpR), the resistance genes could be removed, generating unmarked deletion mutations. It is concluded from the high frequency of allelic exchange events observed in this study that specialized transduction is a very efficient technique for genetic manipulation of mycobacteria and is a method of choice for constructing isogenic strains of M. tuberculosis, BCG or M. smegmatis which differ by defined mutations.

MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

“…Various Mtb mutant strains were constructed using the temperature-sensitive mycobacteriophage phAE87, as described previously 69 . Briefly, to generate the mutant strains, 800 bp of upstream and downstream regions flanking the gene of interest were amplified by PCR and cloned into pYUB854 flanking the desired antibioticresistance gene.…”

Transcription factors Rv0081 and Rv3334 connect the early and the enduring hypoxic response of Mycobacterium tuberculosis

“…To better understand the roles of Rv0081 and Rv3334 in hypoxic response of M. tb , we constructed two deletion mutants, Δ Rv0081 and Δ Rv3334 , of M. tb H37Rv using a transducing phage system [38]. The entire open reading frame (ORF) of each gene was replaced by a hygromycin resistance gene via homologous recombination using the fragments immediately up- and down-stream of the coding sequence (Figure 1(a)).…”

“…M. tb Δ Rv0081 and Δ Rv3334 were constructed from M. tb H37Rv using the TM4 phage-mediated transduction system [38] and described below. Mycobacteria were routinely grown in Middle brook 7H9 broth containing 0.2% glycerol, 10% OADC (oleic acid, bovine serum albumin, dextrose, and catalase; Difco) and 0.05% Tween 80 or 7H11 agar supplemented with 0.2% glycerol and 10% OADC at 37°C under aerobic conditions.…”

“…To generate Δ Rv0081 and Δ Rv3334 mutant strains, the ORF of Rv0081 or Rv3334 of M. tb H37Rv was replaced with a hygromycin-resistance cassette using TM4 phage-mediated specialized transduction [38]. The primer pairs used to PCR amplify the left and right fragments of Rv0081 to generate the allelic exchange substrate were LF-0081 (5ʹ-GGCACTAGTCTGATT GTGAATTCGCGTCCG-3ʹ), LR-0081 (5ʹ-GTGAAGCTTCGCGTCTCCTGAGAGCTTC-3ʹ), RF-0081 (5ʹ-GGTTCTAGACGTAACGCCATGGGT-3ʹ) and RR-0081 (5ʹ-ATAGGTACC GACGTCGACGGGTA-3ʹ); and for Rv3334 were: LF-3334 (5ʹ-CGCACTAGTGCCGCACTG ATAAATCGG-3ʹ), LR-3334 (5ʹ-TACAAGCTTGCGACTCCCCTTGACCTT C-3ʹ), RF-3334 (5ʹ-CGCTCTAGAAGGTCGCTGGGTACGC-3ʹ) and RR-3334 (5ʹ-TACGGTACCTGCGCA CCAAACTGTC-3ʹ).…”

“…The resulting phLR/pKO0081 and phLR/pKO3334 phasmid DNA from the transductants was electroporated into M. smegmatis mc 2 -155 and plated for mycobacteriophage plaques at the permissive temperature of 30°C. A high-titre phage lysate, prepared from a temperature-sensitive phage plaque, was used to infect M. tb H37Rv at the non-permissive temperature of 37°C as described previously [38]. Hygromycin-resistant colonies were selected at 6-weeks post-transduction and confirmed by PCR analysis.…”