(A) Schematic representation of bont/E with its upstream and downstream genes in C. botulinum NCTC 11219. Locus tags for all genes are shown. (B) Position of the ClosTron insert after the 211th base of the bont/E open reading frame in the NCTC 11219 bontE211a::CT mutant, with ermB positioned antisense to bont/E. Also shown are primers bontE_F/bontE_R, annealing up- and downstream of the ClosTron target site, and primers upbontE_F/downbontE_R, annealing outside the loci used in double homologous recombination to delete bont/E. (C) NCTC 11219 ΔbontE::ermB mutant with bont/E deleted and replaced by ermB, confirmed by PCR using primers upbontE_F/downbontE_R. The positions of the cloning primers bontE_5′F/bontE_5′R and bontE_3′F/bontE_3′R, used for construction of pMTL84151_Δbont, also are indicated.