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N isotopic discrimination was used to determine the ratio of influx to efflux in experiments in which NO3 reduction was decreased (and also net NO-3 uptake, since under steady-state conditions, net NO-3 uptake is equal to the rate of reduction) by tungstate. Exponential growth rate constants for the cyanobacterium Synechococcus sp. R2 grown in media containing 0.18 μM-molybdate decreased linearly (0.040 to 0.013 h-1) with increasing tungstate concentration (0 to 0.2 mM). Values of the overall, observed N isotope effect (βobs, the ratio of the rate of reaction of 14N- to 15N-bearing molecules, normalized to adjust for unequal concentrations) varied inversely and were linear with exponential growth rate. At high tungstate concentration, when the exponential growth rate approached zero (as a result of negligible NO-3 reduction), βobs approached 1.0197. In the absence of tungstate, βobs was equal to 1.0037. From this value, we calculated the ratio of efflux to influx to be about 0.19. That is, in the absence of tungstate, more than 80% of the NO-3 entering the cell underwent reduction with less than 20% released to the medium from the cell. This approach can be used to distinguish changes in influx from changes in metabolic steps in response to factors which affect net uptake rate.

Separating the efflux and influx components of net nitrate uptake by Synechococcus R2 under steady-state conditions