Plasmid supercoiling decreases during the dark phase in cyanobacteria: a clarification of the interpretation of chloroquine-agarose gels.

Abstract

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with the circadian transcription program and (Woelfle et al. [2007], Vijayan et al. [2009], PNAS). Specifically, Woelfle et al. have reported that DNA supercoiling of an endogenous plasmid became progressively higher during prolonged dark phases in Synechococcus elongatus PCC 7942. This is counterintuitive, since higher levels of negative DNA supercoiling are commonly associated with exponential growth and high metabolic flux. Vijayan et al. then have reverted the interpretation of plasmid mobility on agarose gels supplemented with chloroquine diphosphate (CQ), but not further discussed the differences. Here, we set out to clarify this open issue in cyanobacterial DNA supercoiling dynamics. We first re-capitulate Kellers band counting method (1975, PNAS) using CQ instead of ethidium bromide as the intercalating agent. A 500x-1000x higher CQ concentration is required in the DNA relaxation reaction (topoisomerase I) than in the agarose gel buffer to quench all negative supercoiling of pUC19 extracted from Escherichia coli. This is likely due to the dependence of both, the DNA binding affinity of CQ and the induced DNA unwinding angle, on the ionic strength of the buffer. Lower levels of CQ were required to fully relax in vivo pUC19 supercoiling than were used by Woelfle et al. Next, we analyzed the in vivo supercoiling of endogenous plasmids of Synechocystis sp. PCC 6803, at two different CQ concentrations. These experiments indicate that negative supercoiling of plasmids does not increase but decreases in the dark phase, and progressively decreases further in prolonged darkness.