Mass Spectrometric Detection and Analysis of Nitrogen Fixation in Oscillatoria chalybea

Abstract

By means of mass spectroscopic measurements in an artifical gas atmosphere containing the stable nitrogen isotope 15N2 we were able to demonstrate nitrogen fixation capacity in the filamentous cyanobacterium Oscillatoria chalybea. Our technique proved to be wellsuited also for investigations on the light-induced nitrogen fixation in the purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus. Oscillatoria chalybea grown without combined nitrogen showed a substantial 15N2-uptake which could clearly be correlated with nitrogen fixation. Nitrate grown cultures did not show this nitrogen uptake or only to a minimal extent. Addition of ammonium chloride resulted in a rapid deactivation of the nitrogenase system. Similar observations have been made with other so-called switch-off effectors like phenazine methosulfate. The structural integrity of the filaments appeared to be a prerequisite for nitrogen fixation also in this organism, as even mild mechanical homogenization strongly inhibited the N2-uptake signals. Illumination of the assays under conditions where the photooxidition of water is not operational (Bader, K. P. (1994), Biochim. Biophys. Acta 1188, 213 -219) did not affect the nitrogen fixation in Oscillatoria chalybea. Illumination of cultures with concomitant release of oxygen from the water splitting reaction resulted in strong inhibition of 15N2-uptake.