Contrary to the general view point that respiratory control does not exist in Escherichia coli, we have been able to demonstrate this phenomenon in this micro-organism. E. coli cells growing aerobically on an oxidizable carbon source respire at a high rate, which cannot be increased by the addition of uncouplers of oxidative phosphorylation. Upon removal of the carbon source, the rate of respiration decreases progressively to reach, after 10 min, less than 1/20 of its previous value. Under these conditions the addition of an uncoupler increases the residual respiration by a factor of 3 to 9. This increase is due to the release of respiratory control. Release of respiratory control cannot be observed upon addition of ADP which does not penetrate across the membrane. Respiratory control can also be shown in the presence of non-limiting respiratory substrate provided that it is not a carbon source. This was shown by measuring the rate of oxidation of ascorbate and phenazine methosulfate.NADH oxidase activity of membrane vesicles obtained by high pressure disintegration was increased by a factor of about 1.5 upon the addition of uncouplers and also upon addition of ADP plus phosphate. In contrast, membrane vesicles depleted from BF1 ATPase (a soluble bacterial coupling factor presenting ATPase activity) did not exhibit respiratory control. It could be restored either by rebinding of BF1 ATPase or by addition of dicyclohexylcarbodiimide, an ATPase inhibitor.The limited respiratory rate of mitochondria during so called 'state 4' which can be increased by uncouplers or by ADP plus phosphate is attributed to respiratory control [l].The lack of respiratory stimulation by the same agents on respiring Escherichia coli led several investigators [2--41 to state that there was no respiratory control in E. coli. It appears that growing E. coli cells respire at maximal rate, presumably because ATP utilising synthetic processes continuously liberate ADP plus phosphate. The increase in respiration of starved cells observed upon addition of an actively transported lactose analogue, methyl p-Dthiogalactoside was interpreted also as a release of respiratory control by the utilization of metabolic energy [5]. In this paper, we describe conditions which allow the demonstration of respiratory control in intact E. coli without added carbon source by its release upon addition of uncouplers. That this is not due to limiting oxidizable substrate can be shown by adding excess ascorbate and phenazine methosulfate.Ahhreviations. CCCP, carbonylcyanide-p-trichloromethoxyphenylhydrazone; BF1 ATPase: soluble bacterial coupling factor F1 presenting ATPase activity.Addition of ADP, a non-penetrating solute, does not cause the same respiratory increase. We also describe a less pronounced increase of respiration, with NADH as the reducing substrate in a cell-free membrane preparation which responds to the addition of uncouplers as well as of ADP plus Pi.
MATERIALS AND METHODSGrowth of Bacteria E. coli K12 strain 3300 constitutive for the lactose oper...