Fig. 1. Isolation of TnsB immunity bypass mutants (A) A mini-Tn7lac (mTn7lac) element, consisting of a promoterless lacZY gene cassette flanked by the Tn7 left (Tn7L) and right (Tn7R) sequences required for its mobilization (triangles), resides in a transcriptionally silent location on a plasmid such that, in the absence of transposition, cells containing the plasmid have a Lac^– phenotype. However, when the mTn7lac element inserts into attTn7, it is transcribed by the glmUS promoter upstream of attTn7 (Bainton et al., 1993), generating a cell with a Lac⁺ phenotype, i.e. formation of Lac⁺ papillae on a MacConkey lactose indicator plate. Thus, in a strain containing a non-mobilizable mTn7 element 8 kb from attTn7 (attTn7 + mTn7_8kb) (DeBoy and Craig, 1996), the frequency of mTn7lac insertion into attTn7 is very low, and this strain produces very few Lac⁺ papillae when grown on a MacConkey lactose indicator plate (second column). TnsB mutants that bypass immunity will promote higher-frequency transposition to attTn7 + mTn7_8kb, which increases the number of Lac⁺ papillae per colony (third column). (B) TnsB mutations that affect target immunity and transposition overlap. The TnsB immunity bypass mutations (top) were localized to the C-terminus of TnsB (middle). Alanine scanning mutagenesis was shown previously to block TnsB–TnsC interaction and transposition (bottom) (Skelding et al., 2002).