Figure 2

RepB sites chosen for mutagenesis. Panel A represents the output of the PredBur program used to select sites for mutagenesis. Sites were chosen by the PredBur average hydrophobicity algorithm (black) or the PredBur hydrophobic moment algorithm (red). The PredBur score indicates the probability of burial of the residue. Higher PredBur values suggest a higher likelihood of the given residue being buried and thus a higher likelihood that mutation of that residue will result in a Ts plasmid. Panel B shows the 10 sites that were selected for mutagenesis (triangles) as well as annotation of putative catalytic domains (green ovals) [27] and a putative DNA binding region (red arrow) [28]. Panel C shows the results of the initial screen for temperature sensitivity for mutations in the pMU770 backbone. A black outline indicates the number of mutants generated at each site (3 or 4). Blue bars indicate the number of mutants able to confer thiamphenicol resistance at 48°C and orange bars indicate the number of mutants able to confer thiamphenicol resistance at 58°C. The difference between the blue bars and the orange bars indicates the number of temperature-sensitive mutants at each site.