Genetic evidence of a major role for glucose-6-phosphate dehydrogenase in nitrogen fixation and dark growth of the cyanobacterium Nostoc sp. strain ATCC 29133.

Heterocysts, sites of nitrogen fixation in certain filamentous cyanobacteria, are limited to a heterotrophic metabolism, rather than the photoautotrophic metabolism characteristic of cyanobacterial vegetative cells. The metabolic route of carbon catabolism in the supply of reductant to nitrogenase and for respiratory electron transport in heterocysts is unresolved. The gene (zwf) encoding glucose-6-phosphate dehydrogenase (G6PD), the initial enzyme of the oxidative pentose phosphate pathway, was inactivated in the heterocyst-forming, facultatively heterotrophic cyanobacterium, Nostoc sp. strain ATCC 29133. The zwf mutant strain had less than 5% of the wild-type apparent G6PD activity, while retaining wild-type rates of photoautotrophic growth with NH4+ and of dark O2 uptake, but it failed to grow either under N2-fixing conditions or in the dark with organic carbon sources. A wild-type copy of zwf in trans in the zwf mutant strain restored only 25% of the G6PD specific activity, but the defective N2 fixation and dark growth phenotypes were nearly completely complemented. Transcript analysis established that zwf is in an operon also containing genes encoding two other enzymes of the oxidative pentose phosphate cycle, fructose-1,6-bisphosphatase and transaldolase, as well as a previously undescribed gene (designated opcA) that is cotranscribed with zwf. Inactivation of opcA yielded a growth phenotype identical to that of the zwf mutant, including a 98% decrease, relative to the wild type, in apparent G6PD specific activity. The growth phenotype and lesion of G6PD activity in the opcA mutant were complemented in trans with a wild-type copy of opcA. In addition, placement in trans of a multicopy plasmid containing the wild-type copies of both zwf and opcA in the zwf mutant resulted in an approximately 20-fold stimulation of G6PD activity, relative to the wild type, complete restoration of nitrogenase activity, and a slight stimulation of N2-dependent photoautotrophic growth and fructose-supported dark growth. These results unequivocally establish that G6PD, and most likely the oxidative pentose phosphate pathway, represents the essential catabolic route for providing reductant for nitrogen fixation and respiration in differentiated heterocysts and for dark growth of vegetative cells. Moreover, the opcA gene product is involved by an as yet unknown mechanism in G6PD synthesis or catalytic activity.

Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp. BH72.

We report the discovery of novel subcellular structures related to bacterial nitrogen fixation in the strictly respiratory diazotrophic bacterium Azoarcus sp. BH72, which was isolated as an endophyte from Kallar grass. Nitrogenase is derepressed under microaerobic conditions at O2 concentrations in the micromolar range. With increasing O2 deprivation, bacteria can develop into a hyperinduced state, which is characterized by high specific rates of respiration and efficient nitrogen fixation at approximately 30 nM O2. Ultrastructural analysis of cells in the course of hyperinduction revealed that complex intracytoplasmic membrane systems are formed, which consist of stacks of membranes and which are absent under standard nitrogen-fixing conditions. The iron protein of nitrogenase was highly enriched on these membranes, as evidenced by immunohistochemical studies. Membrane deficiency in NifH/K- mutants, a deletion mutant in the nifK gene and the character of NH+4-grown cells suggested, in concert with the membrane localization of nitrogenase, that these structures are specialized membranes related to nitrogen 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 detectable in membrane fractions. Mutational analysis, and determination of the N-terminal amino acid sequence indicate that the nifH gene product is covalently modified by a mechanism probably different from adenosine diphosphoribosylation. Development of diazosomes in nitrogen-fixing cells can be induced in pure cultures and in co-culture with a fungus isolated from the rhizosphere of Kallar grass.