Figure 1.

A comparison of the nucleotide and amino acid sequence changes in the key in cis yopN mutations used in this study. Shown is nucleotide (lower case font) and amino acid (upper case font; single letter code) sequence encompassing codon positions 277–293 of YopN and the overlapping 1–7 codons of TyeA. Derived from the native sequence (Parent), three different polypeptides can be generated—YopN_native, TyeA _native, and a YopN-TyeA hybrid fusion product resulting from an unconfirmed +1 frameshift mutation after codon 279 (Ferracci et al., 2004; Amer et al., 2013). Shading in light gray indicates the YopN_native amino acid sequence. Amino acids shaded in light blue are YopN sequences that differ from the native protein due to a natural or engineered alteration to the codon sequence. Introduced site-directed nucleotide substitutions are highlighted by an overlying filled-in circle. Open arrowheads above the nucleotide sequence specifically locate positions of nucleotide deletions that result in a +1 frameshift, and filled-in arrowheads identify nucleotide insertions that serve as compensatory −1 frameshifts. No mutation altered the coding sequence of overlapping tyeA as shown by routine retention of the first 6–7 TyeA residues in green (TyeA_native); the start codon of which is highlighted in bold italic font. However, bacteria producing Mutant 2 (YopN_288STOP) and Mutant 3 [YopN_{279(F+1), 287(F−1)}] have a displaced tyeA initiation codon relative to a putative Shine-Dalgarno sequence (“agaggg” in bold purple font) by n + 2 [TyeA_native(n+2)] and n + 1 [TyeA_native(n+1)], respectively. Also note that in these bacteria and in bacteria producing Mutant 4 [YopN_{279(F+1), 287STOP}], tyeA coding sequence assumes a different reading from the native sequence. Native or introduced yopN termination codons are indicated by an asterisk (red shade). Two additional mutations were genetically engineered and are designated Mutant 1 (YopN_{288(scramble)293}) and Mutant 5 (YopN_279STOP).