Conditionally replicating mycobacteriophages: A system for transposon delivery to Mycobacterium tuberculosis

Abstract

Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast-and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a miniTn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30°C but not at 37°C (

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.…”

Role of KatG catalase‐peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst

“…The DkatG mutant strain was constructed by single-step homologous recombination using the specialized transducing phage phjsc334 (Bardarov et al, 1997;Glickman et al, 2000). The mutation replaced a 1899 bp internal region of katG (amino acids 15-648) with a hygromycin resistance (hyg) cassette.…”

In vivo transposition of mariner -based elements in enteric bacteria and mycobacteria

“…However, these transposons do not appear functional in many other bacterial species. For example, Tn10, which has been used widely in Escherichia coli, appears to integrate site specifically in Mycobacterium smegmatis (2). In fact, in mycobacteria, the only transposons that have been found to produce insertion mutations in diverse sites are derived from other mycobacteria (3)(4)(5)(6).…”