Direct observation with an electrode of uncoupler-sensitive assimilatory nitrate uptake by Rhodopseudomonas capsulata

A major deficiency in our understanding of bacterial transport is the question of how nitrate enters bacteria, to act either as an electron acceptor or as a source of nitrogen. One of the reasons for the dearth of information about nitrate transport is probably an experimental one; transport is generally studied using radioisotopes, but the radioisotope of nitrogen, 13N, has a half-life of only 10 min. Despite this unhelpful property 13N has been successfully used in order to study nitrate uptake into Pseudomonasjluorescens [I]. The bacteria were grown aerobically with nitrate as the source of nitrogen and therefore, as there was evidently repression of any transport systems associated with nitrate respiration in this denitrifying organism, the first measurements of assimilatory nitrate uptake in bacteria were made [ 11. Here, we report that the assimilatory uptake of nitrate by Rhodopseudomonas capsulata, which does not denitrify, can be readily and continuously followed using a nitrate-specific electrode. Use of this method has shown that nitrate uptake is rapidly inhibited upon collapsing the proton electrochemical gradient across the plasma membrane either by addition of a protonophore uncoupler, or by transferring the cells from an illuminated to a darkened state. These data indicate that nitrate uptake is directly dependent on the proton electrochemical gradient rather than on the availability of intra-cellular AlT. It is also shown that nitrate uptake is rapidly but reversibly inhibited by ammonium ions, but that the ammonium analogue