Protonmotive force regulates the membrane conductance of Streptococcus bovis in a non-ohmic fashion

Because the DCCD (dicyclohexylcarbodiimide)-sensitive. F-ATPase-mediated fu tile ATP hydrolysis of non-growing Streptococcus bovis JB1 cells was not af fected by sodium or potassium, ATP hydrolysis appeared to be dependent Beca use the DCCD (dicyclohexylcarbodiimide)-sensitive. F-ATPase-mediated, only upon the rate of proton flux across the cell membrane. However, available e stimates of bacterial proton conductance were too low to account for the ra te of ATP turnover observed in S, bovis, When de-energized cells were subje cted to large pH gradients (2.75 units, or -170 mV), internal pH declined a t a rate of 0.15 pH units s(-1). Based on an estimated cellular buffering c apacity of 200 nmol H+ (mg protein)(-1) per pH unit, H+ flux across the cel l membrane (at -170 mV) was 108 mmol (g protein)(-1) h(-1). When potassium- loaded cells were treated with valinomycin in low-potassium buffers, initia l K+ efflux generated membrane potentials in close agreement with values pr edicted by the Nernst equation. These artificial membrane potentials drove H+ uptake, and H+ influx was counterbalanced by a further loss of cellular K+. Flame photometry indicated that the rate of K+ loss was 215 (+/-26) mmo l K+ (g protein)(-1) h(-1) at -170 mV, but the potassium-sensitive fluoresc ent compound CD222 indicated that this rate was only 110 (+/-44) mmol K+ (g protein)(-1) h(-1). As pH gradients or membrane potentials were reduced, t he rate of H+ flux declined in a non-ohmic fashion, and all rates were <25 mmol (g protein)(-1) h(-1) at a driving force of -80 mV, Previous estimates of bacterial proton flux were based on low and unphysiological protonmotiv e forces, and the assumption that H+ influx rate would be ohmic, Rates of H + influx into S, bovis cells [as high as 9 x 10(-11) mol H+ (cm membrane)(- 2) s(-1)] were similar to rates reported for respiring mitochondria, but we re at least 20-fold greater than any rate previously reported in lactic aci d bacteria.