CONDITIONALLY REPLICATING MYCOBACTERIOPHAGES - A SYSTEM FOR TRANSPOSON DELIVERY TO MYCOBACTERIUM-TUBERCULOSIS

Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a vari ety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculos is has been limited in part because of the inefficiency of the deliver y systems, This report describes the development of conditionally repl icating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast-and slow-growi ng mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a mini-Tn10(kan) or Tn5367 inserted i nto a nonessential region of the phage genome, Thermosensitive mutatio ns 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 i n highly efficient transposon delivery to the entire population of myc obacterial cells, Transposition of mini-Tn10(kan) occurred in a site-s pecific fashion in M, smegmatis whereas Tn5367 transposed apparently r andomly in M, phlei, Bacille Calmette-Guerin (BCG), and M, tuberculosi s, Sequence analysis of the M. tuberculosis and BCG chromosomal region s adjacent to Tn5367 insertions, in combination with M, tuberculosis g enomic sequence and physical map data, indicates that the transpositio ns have occurred randomly in diverse genes in every quadrant of the ge nome, Using this system, it has been readily possible to generate libr aries containing thousands of independent mutants of M, phlei, BCG, an d M, tuberculosis.