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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 0.1 M solution of NazS20, in the buffer were passed through the column to remove oxygen before the extract was introduced. Chlorophyll-containing particles were prepared essentially as described for particles from Chlorobium thiosulfatophilum (Evans & Buchanan, 1965). Enzyme reactions were carried out in 7.0 ml. glass vials sealed with Suba Seal rubber caps under an atmosphere of argon at 30' in a shaking water bath, and stopped by the injection of 0.1 ml. of 60 % perchloric acid. Nitrogenase activity was assayed by measuring the reduction of acetylene to ethylene (Dilworth, 1966). Ethylene formation was estimated by gas chromatography using a 6 ft x 4 in. column of Poropak R in a Perkin Elmer FII or Varian Aerograph 1400 gas chromatograph with a flame ionization detector. Ferredoxin was prepared essentially as described previously (Evans, Hall, Bothe & Whatley, 1968). Preparations used in these experi7

Nitrogen Fixation by the Green Photosynthetic Bacterium Chloropseudomonas ethylicum