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

TLDR

Temperature‑sensitive phage mutations allow replication at 30 °C but block it at 37 °C. The study aimed to demonstrate the utility of the system by generating seven targeted gene disruptions across multiple mycobacterial strains. The method uses a temperature‑sensitive shuttle phasmid carrying the target gene with antibiotic markers flanked by γδ‑res sites; transductants are selected on antibiotic media and the markers are excised by γδ‑resolvase to yield unmarked deletions. The system achieved highly efficient allelic exchange, delivering the recombination substrate to nearly all cells and yielding a high frequency of targeted deletions, making specialized transduction a preferred method for constructing isogenic mycobacterial strains.

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 temperature-sensitive mutations in the mycobacteriophage genome permit replication at the permissive temperature of 30 °C but prevent replication at the non-permissive temperature of 37 °C. 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.