Proton Electrochemical Gradients in Washed Cells of Nitrosomonas europaea and Nitrobacter agilis

Abstract

The components of the proton motive force (Δp), namely, membrane potential (Δψ) and transmembrane pH gradient (ΔpH), were determined in the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis . In these bacteria both Δψ and ΔpH were dependent on external pH. Thus at pH 8.0, Nitrosomonas europaea and Nitrobacter agilis had Δψ values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [ 3 H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids, 14 C-benzoic and 14 C-acetyl salicylic, and the weak base [ 14 C]methylamine. Nitrosomonas europaea accumulated 14 C-benzoic acid and 14 C-acetyl salicylic acid when the external pH was below 7.0 and [ 14 C]methylamine at alkaline pH. Similarly, Nitrobacter agilis accumulated the two weak acids below an external pH of about 7.5 and [ 14 C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a ΔpH (inside alkaline). Thus, above pH 7.0 for Nitrosomonas europaea and pH 7.5 for Nitrobacter agilis , Δψ only contributed to Δp. In Nitrosomonas europaea the total Δp remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In Nitrobacter agilis , Δp decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in Nitrosomonas europaea varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In Nitrobacter agilis , however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In Nitrosomonas europaea , Δp and its components (Δψ and ΔpH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Δp. Such an experiment was not possible with Nitrobacter agilis because of low cell yields. The effects of protonophores and ATPase inhibitors on ΔpH and Δψ in the two nitrifying bacteria are considered.