INDUCTION OF COMPLEX INTRACYTOPLASMIC MEMBRANES RELATED TO NITROGEN-FIXATION IN AZOARCUS SP BH72

We report the discovery of novel subcellular structures related to bac terial nitrogen fixation in the strictly respiratory diazotrophic bact erium Azoarcus sp, BH72, which was isolated as an endophyte from Kalla r grass, Nitrogenase is derepressed under microaerobic conditions at O -2 concentrations in the micromolar range. With increasing O-2 depriva tion, bacteria can develop into a hyperinduced state, which is charact erized by high specific rates of respiration and efficient nitrogen fi xation at approximately 30 nM O-2 Ultrastructural analysis of cells in the course of hyperinduction revealed that complex intracytoplasmic m embrane systems are formed, which consist of stacks of membranes and w hich are absent under standard nitrogen-fixing conditions, The iron pr otein of nitrogenase was highly enriched on these membranes, as eviden ced by immunohistochemical studies, Membrane deficiency in NifH/K- mut ants, a deletion mutant in the nifK gene and the character of NH4+-gro wn cells suggested, in concert with the membrane localization of nitro genase, that these structures are specialized membranes related to nit rogen fixation. We propose the term 'diazosomes' for them, Development of intracytoplasmic membranes coincides with the appearance of a high -molecular-mass form of the iron protein of nitrogenase, which was det ectable in membrane fractions, Mutational analysis, and determination of the N-terminal amino acid sequence indicate that the nifH gene prod uct is covalently modified by a mechanism probably different from aden osine diphosphoribosylation. Development of diazosomes in nitrogen-fix ing cells can be induced in pure cultures and in co-culture with a fun gus isolated from the rhizosphere of Kallar grass.