Ability of mutant β clamp proteins to support viability of E. coli.

aSee Table 1 for a description of the plasmids.bAmino acid substitutions are indicated in superscript (e.g., Q61K represents a lysine substitution of residue Q61).cAmpR CFU/CamR CFU is a direct measure of the fraction of CamR pACM clones bearing the AmpR pAMPdnaN+ plasmid. It was determined by selecting at random colonies that had been passaged for ∼100 generations on LB-Cam plates and patching them onto LB-Amp and LB-Cam plates. Ratios (AmpR CFU/CamR CFU) observed for each plasmid are shown, while the % frequency is shown in parentheses. At least 1 representative clone for each CamR and AmpS strain identified was further characterized to verify the presence of the chromosomal dnaN–1FS allele using diagnostic PCR and XhoI restriction, as well as nucleotide sequence of the plasmid-encoded dnaN allele.dViability refers to the ability of the CamR transforming plasmid to support growth of E. coli in the absence of pAMPdnaN+. Symbols are as follows: –, plasmid is unable to support viability of E. coli, meaning 100% of the CFUs are resistant to both Amp and Cam after ∼100 generations of growth under selection for CamR; +, plasmid is able to support viability of E. coli.ePlasmid pACMβ5A expresses the β148–152 mutant, which contains alanines in place of residues H148-R152 [10]. This mutation failed to support E. coli viability when crossed onto the bacterial chromosome [6], and serves as an additional negative control for the plasmid shuffle assay.