Transposon Tn916 mutagenesis in Bacillus anthracis

Abstract

Mutagenesis of Bacillus anthracis by the streptococal tetracycline resistance transposon Tn916 is described. Tn916 was transferred from Streptococcus faecalis DS16C1 to B. anthracis VNR-1 by conjugation in a standard filter mating procedure. Tetracycline-resistant (Tc(r)) transconjugants were obtained at a frequency of 1.6 x 10-8 per donor CFU. When donor and recipient cells were treated with nafcillin before conjugation, the frequency was increased nearly 10-fold. Nafcillin pretreatment of donor and recipient strains was used in all subsequent conjugation experiments. S. faecalis CG110, containing multiple chromosomal insertions of Tn916, transferred the transposon to B. anthracis VNR-1 at a frequency of 9.3 x 10-5. A Tc(r) B. anthracis transconjugant, strain VNR-1-tet-1, transferred Tn916 to B. anthracis UM23-1 and Bacillus subtilis BST1 at frequencies of 2.1 x 10-4 and 5.8 x 10-6, respectively. The transfer of Tn916 occurred only on membrane filters, since no Tc(r) transconjugants were obtained when strains VNR-1-tet-1 and UM23-1 were mixed and incubated in broth culture. The presence of the Tn916-associated tetM gene in Tc(r) B. anthracis and B. subtilis transconjugants was confirmed in hybridization experiments by using a 5-kilobase-pair DNA fragment containing the tetM gene as a probe. Of 3,000 B. anthracis UM23-1 Tc(r) transconjugants tested, 21 were phenylalanine auxotrophs and 2 were auxotrophic for phenylalanine, tyrosine, and tryptophan.