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 mini-Tn10(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 degrees C but not at 37 degrees C (TM4) or 38.5 degrees C (D29). Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells. Transposition of mini-Tn10(kan) occurred in a site-specific fashion in M. smegmatis whereas Tn5367 transposed apparently randomly in M. phlei, Bacille Calmette-Guérin (BCG), and M. tuberculosis. Sequence analysis of the M. tuberculosis and BCG chromosomal regions adjacent to Tn5367 insertions, in combination with M. tuberculosis genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome. Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of M. phlei, BCG, and M. tuberculosis.

Figure 1

Transposon delivery in mycobacteria using conditionally replicating shuttle phasmids. Shuttle phasmids are mycobacteriophage molecules into which an E. coli cosmid has been inserted in a nonessential region. These molecules can thus replicate in E. coli as cosmids and replicate in mycobacteria as phages. The cosmid is flanked by restriction sites that are not found in the shuttle phasmid, thus allowing for simple excis-ion of the cosmid. A cosmid containing a transposon is readily cloned into the phage backbone by cosmid cloning in E. coli. Cosmids are isolated from E. coli and transfected into M. smegmatis cells by electroporation. At permissive temperatures (30°C), shuttle phasmids undergo growth as a lytic mycobacteriophage to high titers. Infection of various mycobacteria with the mycobacteriophages containing the transposons results in the delivery to every cell in a population of mycobacterial cells. The temperature-sensitive mutations in the mycobacteriophages prevent phage propagation, which allows for transpositions into the mycobacterial chromosome to occur. The broad host ranges of D29 and TM4 mycobacteriophages allow for delivery of transposons to a variety of different mycobacterial species, including BCG and M. tuberculosis.

Figure 2

Southern blot analysis of transposon mutants. (A) kan^r M. smegmatis mc² 155 mutagenized with mini-Tn10(kan) using phAE78 as a delivery phage. Chromosomal DNAs were isolated from 11 independent mutants, digested with PstI (which does not cleave in the transposon), and hybridized with a DNA fragment containing the gene that confers kan^r. (B) kan^r M. phlei cells mutagenized with Tn5367 using phAE77 as a delivery phage. Chromosomal DNA digested with PstI and ApaLI and hybridized with the aph gene. Both restriction sites are located within the transposon, so a common band is generated as well as the random fragment generated from the insertion of Tn5367 into the M. phlei chromosome. Lane 1 contains pYUB553 digested with PstI and ApaLI. (C and D) Southern blot analysis of IS1096 in fast-growing mycobacteria. Ethidium bromide-stained agarose gel of chromosomal DNAs of various M. smegmatis strains (lanes 2–10); M. phlei (lane 11) and M. vaccae (lane 12) were digested with ApaLI (C) and hybridized with tnpA of IS1096 (D).

Figure 3

Distribution of Tn5367 insertions on the genome map of M. tuberculosis. The location of the Tn5367 insertions in mutants generated from M. tuberculosis Erdman [mc² 3000s] or BCG [mc² 1500s] were deduced from the determination of the sequence adjacent to the insertion and comparison to the M. tuberculosis genomic database and the M. tuberculosis physical map (28).