Nitrogen Fixation by the Green Photosynthetic Bacterium Chloropseudomonas ethylicum

Abstract

Nitrogen fixation by autotrophic green photosynthetic bacteria of the genus Chlorobium was first demonstrated by Lindstrom, Tove & Wilson (1950). We have now shown that the green heterotrophic photosynthetic bacterium Chloropseudomonas ethylicum can also grow with Nz as nitrogen source. The mechanism of nitrogen fixation by green bacteria has not been investigated and extracts with nitrogenase activity have not previously been prepared from this group of bacteria.Chloropseudomonas ethylicum strain N 2, obtained from Dr N. Kondratieva, was grown in a modified Pfennig's medium with ethanol as substrate as described previously (Evans, I 968). For growth under nitrogen-fixing conditions, ammonium chloride was omitted and the medium gassed with 95 % nitrogen, 5 % carbon dioxide. Chloropseudomonas ethylicum has been subcultured continuously for up to 3 months under these conditions. Growth was initiated by employing a 5 % inoculum of a N, grown culture, and after 48 h. growth the organisms were harvested with a continuous flow centrifuge. The paste was stored frozen until required.Frozen paste was suspended I : 2 (w/v) in 0.02 M-tris-HCl (pH 7-3). The buffer was deoxygenated by bubbling with N, before use. The suspension was cooled in ice under argon for 5 min. and exposed to sonic oscillation under an atmosphere of argon or nitrogen using a Dawe Soniprobe on setting 8 for omi in. The broken suspension was centrifuged for 30 min. at 150,000g av. and the clear brown supernatant used as the crude extract without further treatment.In some experiments oxygen-free 0.02 M-tris-HCl pH 7.3 containing 0.25 M-NaCl was used. Ferredoxin was removed by passing these extracts through an anaerobic 2 x 1-5 cm. column of DEAE cellulose equilibrated with the same buffer. Two ml. of an 0

The bioenergetics of electron transport to nitrogenase

“…Oxygen concentrations above 1 /zM inhibit light-driven nitrogenase activity [73,74]. Only for the Chlorobiaceaea could it be demonstrated that illuminated chromatophores generate reducing power for N2-fixation [75]. For the other photosynthetic bacteria the redox potential of the primary electron acceptor is too high [-50 to -150 mV) to reduce ferredoxin directly.…”