AcrIIA22 nicks supercoiled plasmids in vivo and in vitro.
(A) Gel electrophoresis of plasmids purified from overnight E. coli cultures expressing either acrIIA22, or a null mutant with an early stop codon, or neither. Compared to the null mutant, more plasmid runs in a slowly migrating, OC conformation rather than SC plasmid with the wild-type acrIIA22 allele, suggesting that acrIIA22 may impact plasmid topology. %SC indicates the percentage of DNA in the SC form for each sample. (B) N-terminally His6-tagged AcrIIA22 nicks supercoiled plasmids in vitro. (C) C-terminally twin-strep-tagged AcrIIA22 nicks supercoiled plasmids in vitro with higher specific activity than shown in panel B (compare protein concentrations). Original, uncropped versions of images depicted in figure may be found in the Supporting information file, S1 Raw Images. OC, open-circle; SC, supercoiled.