<i>55</i>.<i>2</i> expression affects the control of DNA topology and plasmid copy number in <i>E</i>. <i>coli</i>.

<p>(A) Plasmid topoisomers analysis. Left panel: plasmid DNA was extracted from exponentially growing DH5α harboring pYM58 (<i>55</i>.<i>2</i>_IS) or pDB2114 (<i>55</i>.<i>2</i>) plasmids. Plasmid topoisomers were resolved on TBE agarose gels containing the indicated amount of CLQ. The position of migration of relaxed and/or nicked circular DNA is indicated (R). Right panel: Densitometry analysis of the topoisomer distribution on 1.5 μg ml^-1 CLQ gel of four independent samples of pYM58 or pDB2114 plasmid DNA (gel images are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124309#pone.0124309.s004" target="_blank">S4A Fig</a>). Plotted is the average (lines) and standard deviation (shaded area) of relative plasmid density as a function of negative supercoiling. (B) 2D electrophoretic separation of plasmid topoisomers. Left panel: schematic representation of a 2D gel. The migration positions of negatively supercoiled (–), positively supercoiled (+), and form II topoisomers (N) are indicated. Right panel: gel images show the 2D topoisomer distribution of plasmid DNA samples prepared as in A. Chloroquine concentration was 1.5 μg ml^-1 and 25 μg ml^-1 in the first and second dimension, respectively. (C) Plasmid copy number analysis. Linearized plasmid DNA samples from the experiment shown in A were quantified and normalized to the amount (A_600nm) of bacteria used to extract the plasmids. The data represents means and standard error of four independent cultures. (D) Plasmid DNA was extracted from overnight cultures of DHB3, DB503, and DH5α transformed with pBAD101 (<i>55</i>.<i>2</i> –) or pDB2114-101 (<i>55</i>.<i>2</i> +), and a reporter plasmid (pDB868-2). Linearized plasmids were analyzed by agarose gel electrophoresis.