The toxicity of <i>55</i>.<i>2</i> in <i>E</i>. <i>coli</i> is suppressed by an increase in the copy number of <i>topA</i>.

<p>(A) Liquid growth assay. Overnight cultures of DB503 cells harboring pBAD101 (ctr) or pDB2114-101 (<i>55</i>.<i>2</i>) plasmids were diluted in fresh LB medium and optical density (A_600nm) was measured at the indicated times. At A_600nm = 0.3, cultures were split in two and one half was induced with 0.2% arabinose (vertical arrow). When A_600nm reached ≈ 1, cultures were diluted 10-fold in prewarmed medium plus or minus arabinose. The graph depicts the data of a representative experiment. (B) Reversibility assay. During a growth curve assay, aliquots of arabinose-induced DB503 cultures, harboring pBAD101 (<i>55</i>.<i>2</i> –) or pDB2114-101 (<i>55</i>.<i>2</i> +) plasmids, were withdrawn at the indicated times, washed in cold media without arabinose, and adjusted to the same A_600nm. Serial 10-fold dilutions were spotted on LB plates without arabinose. (C) DB503 cells transformed with pBAD101 (vector) or pDB2114-101 (<i>55</i>.<i>2</i>) and one of the compatible plasmids, pDB868-2 (vector, 1–2), pDB34-8-4 (<i>topA</i>, 3–4), or pDB34-8 (<i>topA</i>, 5–6) were streaked on LB plates with or without 0.2% arabinose. (D) Plasmid based lethality assay. Overnight cultures of AS1047 (<i>topA</i> +) or AS1050 (<i>topA</i>-) transformed with pBAD33-K (<i>55</i>.<i>2</i> –) or pDB2114-33-K (<i>55</i>.<i>2</i> +) were diluted and outgrown as indicated in the Materials and Methods section. Aliquots were diluted, and plated on M63 plates supplemented with glucose and X-gal. The number of blue and white colonies was scored after 36h at 37°C. Representative photographs are shown in the upper panel (the position of a rare white colony in inset IV is indicated by an arrow). The lower panel depicts percentage of white colonies; average and standard deviation are from three independent experiments.