ENERGY BUFFERING OF DNA-STRUCTURE FAILS WHEN ESCHERICHIA-COLI RUNS OUT OF SUBSTRATE

To study how changes in the [ATP]/[ADP] ratio affect the level of DNA supercoiling in Escherichia coli, the cellular content of H+-ATPase wa s modulated around the wild-type level, A relatively large drop in the [ATP]/[ADP] ratio from the normal ratio resulted in a small increase in the linking number of our reporter plasmid (corresponding to a smal l decrease in negative supercoiling). However, when cells depleted the ir carbon and energy source, the ensuing drop in energy state was acco mpanied by a strong increase in linking number, This increase was not due to reduced transcription of the DNA in the absence of growth subst rate, since rifampin had virtually no effect on the plasmid linking nu mber. To examine whether DNA supercoiling depends more strongly on the cellular energy state at low [ATP]/[ADP] ratios than at high ratios, we used cells that were already at a low energy state after substrate depletion; after the addition of an uncoupler to these cells, the [ATP ]/[ADP] ratio decreased further, which resulted in a strong increase i n plasmid linking number, Our results suggest that the strong thermody namic control of DNA supercoiling takes over at low [ATP]/[ADP] ratios , whereas at high ratios homeostatic control mechanisms attenuate ther modynamic control.