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

Section of Microbiology, Cornell University¹, and Agricultural Research Service, USDA², Ithaca, NY 14853, USA

Author for correspondence: J. B. Russell. Tel: +1 607 255 4508. Fax: +1 607 255 3904. e-mail: jbr8{at}cornell.edu

Because the DCCD (dicyclohexylcarbodiimide)-sensitive, F-ATPase-mediated, futile ATP hydrolysis of non-growing Streptococcus bovis JB1 cells was not affected by sodium or potassium, ATP hydrolysis appeared to be dependent only upon the rate of proton flux across the cell membrane. However, available estimates of bacterial proton conductance were too low to account for the rate of ATP turnover observed in S. bovis. When de-energized cells were subjected to large pH gradients (2·75 units, or -170 mV), internal pH declined at a rate of 0·15 pH units s^-1. Based on an estimated cellular buffering capacity of 200 nmol H⁺ (mg protein)^-1 per pH unit, H⁺ flux across the cell membrane (at -170 mV) was 108 mmol (g protein)^-1 h^-1. When potassium-loaded cells were treated with valinomycin in low-potassium buffers, initial K⁺ efflux generated membrane potentials in close agreement with values predicted 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) mmol K⁺ (g protein)^-1 h^-1 at -170 mV, but the potassium-sensitive fluorescent 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, the 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 protonmotive forces, and the assumption that H⁺ influx rate would be ohmic. Rates of H⁺ influx into S. bovis cells [as high as 9x10^-11 mol H⁺ (cm membrane)^-2 s^-1] were similar to rates reported for respiring mitochondria, but were at least 20-fold greater than any rate previously reported in lactic acid bacteria.

Keywords: protonmotive force, proton conductance, energy spilling, Streptococcus bovis

Abbreviations: 9-AA, 9-aminoacridine; BCECF, 2,7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; BCECF-AM, acetoxymethyl ester derivative of BCECF; DCCD, dicyclohexylcarbodiimide; TCS, tetrachlorosalicylanilide; TPP, tetraphenylphosphonium