FIGURE 3.

Unigenic evolution analysis of LtrA variants that bind DIVa. E. coli DH5α containing a pBDG-LtrA library with random PCR-induced mutations in the LtrA ORF was induced with L-arabinose and screened by using a colony-based fluorescence assay to identify functional LtrA variants that bind DIVa to down-regulate GFP expression (GFP⁻ phenotype). Plasmids isolated from putative GFP⁻ colonies were retransformed into DH5α for FACS assay to confirm the GFP⁻ phenotype (defined as <15% of the L-arabinose-induced GFP fluorescence increase in a parallel FACS assays with the control plasmid pBDG), and then sequenced to identify mutations. The plot shows mutability (M) values at the center of a 25-amino acid sliding window based on the sequences of 107 functional LtrA variants that showed a GFP⁻ phenotype and contained 442 missense and 140 silent mutations (summarized in Supplemental Fig. S2). Negative M-values indicate hypomutable regions, with a minimum value of −1 indicating no missense mutations in the 25-amino acid residue window, and positive M-values indicate hypermutability normalized to a maximum value of +1. P-values are shown for hypomutable regions A and B, and the statistically significant hypermutable region (amino acid residues 306–374). Although two other peaks have higher M-values suggesting hypermutability, they are not statistically significant based on the numbers of missense and silent mutations observed in these regions compared with those expected (P > 0.17; see Materials and Methods). A schematic of LtrA showing different regions aligned with the mutability plot is below. RT-1 to RT-7 are conserved sequence blocks characteristic of all RTs, and RT-0, RT-2a, RT-3a, RT-4a, RT-7a, and ti are RT or thumb domain insertions found in group II intron RTs (Blocker et al. 2005).