Nitrogen fixation by cultures and cell-free extracts of Mycobacterium flavum 301.

Abstract

SUMMARY: Growth, nitrogen fixation and acetylene reduction by Mycobacterium flavum 301 (ncib 10,071) were increased with sodium lactate, pyruvate, gluconate or succinate as compared with ethanol, a recommended substrate. Yeast extract could be replaced with (NH4)2SO4; in continuous culture a source of fixed nitrogen could be omitted altogether. Growth, nitrogen fixation and acetylene reduction all increased at lowered pO2 values. Wholly anaerobic conditions did not support growth. Nitrogen fixation was confirmed isotopically. Cell-free extracts performed the following reductions: N2 to NH3, H+ to H2, C2H2 to C2H4, KCN to CH4, CH3NC to CH4 + C2H4 + C2H6. An ATP-generating system, Mg2+, Na2S2O4, and anaerobic conditions during preparation and assay of extracts were required. 3·5 mole ATP were hydrolysed to release 1 mole H2. Pyruvate, α-ketobutyrate, α-ketoglutarate, succinate, glucose and glucose-6-phosphate did not replace dithionite. ADP, AMP or high concentrations of ATP inhibited reduction. Activity was associated with a particle which sedimented at 145,000 g over 3·1/2 hr. The nitrogenase system of M. flavum thus resembles the particulate system of Azotobacter, rather than the soluble pyruvate-utilizing system of Clostridium pasteurianum.