Increasing the level of topo IV overproduction with pET11-parEC slightly reduces hypernegative supercoiling but does not affect the level of R-loop formation in topA topB null cells.
(A) One-dimensional chloroquine gel electrophoresis (7.5 μg/ml of chloroquine) of pACYC184Δtet5’ extracted from JB137 (ΔtopB topA20::Tn10 gyrB(Ts))/pACYC184Δtet5’ and JB208 (JB137 pET11-parEC)/pACYC184Δtet5’ cells grown at 37°C to an OD600 of 0.4 (lanes 1 and 3), or 30 min after a transfer from 37 to 30°C (lanes 2 and 4) as described in Materials and Methods. Arrows indicate hyper-negatively supercoiled DNA. Below is the densitometry tracing of the topoisomers in each lane. (B) Dot-blot with S9.6 antibodies of genomic DNA from JB137 (ΔtopB topA20::Tn10 gyrB(Ts)) and JB208 (JB137/pET11-parEC) cells grown at 30°C. The amount of genomic DNA spotted on the membrane is indicated. +RNase HI indicates that the genomic DNA was treated with RNase HI.