Transposition of Tn917 in Bacillus megateriumt

Transposon Tn9l7 from Steptococcus faecalis (12) is presently the most useful transposon for Bacillus subtilis genetic manipulation. It is 5.3 kilobases in length, confers inducible erythromycin resistance, and has terminal inverted repeats very closely related to the Tn3 family of transposons of gram-negative organisms (11). Youngman et al. (16) constructed a Tn917-carrying plasmid, pTV1, that is temperature sensitive for replication and demonstrated that Tn917 could transpose efficiently at 48°C in B. subtilis. They also constructed plasmids capable of making transpositional lacZ or cat-86 fusions or both and of cloning genes carrying Tn917 insertions into a pBR322 derivative (14, 15, 17). These plasmids are presently being used to analyze the temporal expression of developmentally regulated sporulation and germination genes (18) as well as DNA-damage-inducible (din) loci (10). Our laboratory has been developing genetic and cloning techniques for B. megaterium (3, 5, 7, 13). The introduction of a transposon would greatly enhance the genetic versatility of this species. Here we report the successful introduction and transposition of Tn9O7 in B. megaterium. Plasmid pTV1 carrying Tn917 was isolated from B. subtilis PY143 (16) by use of a modified alkaline-sodium dodecyl sulfate rapid plasmid preparation (7) and was transformed into B. megaterium PV204 protoplasts with selection for chloramphenicol resistance (5 ,ug/ml). Strain PV204 is a prototrophic derivative of strain QM B1551 that has been cured of six of seven resident plasmids by growth in sublethal concentrations of ethidium bromide. It retains only the 8.0-kilobase pVY105 plasmid (7). Growth conditions