Marcia A. Murry

Immunochemical evidence that nitrogenase is restricted to the heterocysts in Anabaena cylindrica

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under anaerobic conditions was studied by immunoferritin labelling of the Fe-Mo protein (Component I). Differentiating cultures, incubated under an argon atmosphere, were treated with DCMU 12 h following initiation of induction. DCMU inhibited photosynthetic O2 production, thus insuring strict anaerobic conditions, but had no effect on nitrogenase induction. Fe-Mo protein levels, as determined by rocket immunoelectrophoresis, increased 5-fold within 24h of DCMU treatment. Immunoferritin labelling of aldehyde fixed, ultrathin cryosections of anaerobically induced filaments showed that the Fe-Mo protein was restricted to the heterocyst. Ferritin labelling was shown to be specific by the following criteria: (a) substituting preimmune goat serum for the anti-Fe-Mo protein IgG prevented ferritin labelling; (b) ferritin-conjugated, non-homologous rabbit anti-goat IgG did not bind; (c) incubation of anti-Fe-Mo protein IgG treated sections with rabbit anti-goat IgG prior to the treatment with the ferritin label also prevented labelling. The results provide direct immunochemical evidence that nitrogenase is restricted to the heterocysts even under strictly anaerobic conditions.

Oxidation of diaminobenzidine in the heterocysts ofAnabaena cylindrica

Hemoproteins were localized in the cyanobacteriumAnabaena cylindrica with diaminobenzidine (DAB). Incubation of whole cells in the light with DAB resulted in deposition of oxidized DAB on the lamellae of the vegetative cells and central heterocyst region. This reaction was greatest at pH 7.5, light-dependent, insensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, and abolished by glutaraldehyde fixation. A light-independent oxidation of DAB was also observed with light and electron microscopy in the “honeycomb” region and periphery of heterocysts. This reaction was greatest at pH 7.5, enhanced by H2O2, and active in glutaraldehyde-fixed frozen sections. Inhibitors such as sodium cyanide, sulfide, and hydroxylamine severely reduced DAB oxidation and nitrogenase activity under aerobic but not anaerobic conditions. These results indicate that the heme proteins, localized in heterocysts by light-independent DAB oxidation, are involved in the oxygen-protection mechanism of the O2-labile nitrogenase.

Role of ammona in regulation of nitrogenase synthesis and activity in Anabaena cylindrica

Abstract The regulation of nitrogenase biosynthesis and activity by ammonia was studied in the heterocystous cyanobacterium Anabaena cylindrica. Nitrogenase synthesis was measured by in vivo acetylene reduction assays and in vitro by an activity-independent, immunoelectrophoretic measurement of the Fe-Mo protein (Component I). When ammonia was added to differentiating cultures after a point when heterocyst differentiation became irreversible, FeMo protein synthesis was also insensitive to ammonia. Treating log-phase batch cultures with 100% O2 for 30 min resulted in a loss of 90% of nitrogenase activity and a 50% loss of the FeMo protein. Recovery was inhibited by chloramphenicol but not by ammonia or urea. The addition of ammonia to log-phase cultures resulted in a decrease in specific levels of nitrogenase activity and FeMo protein that occurred at the same rate as algal growth and was independent of O2 tension of the culture media. However, in light-limited linear-phase cultures, ammonia effected a dramatic inhibition of nitrogenase activity. These results indicate that nitrogenase biosynthesis becomes insensitive to repression by ammonia as heterocysts mature and that ammonia or its metabolites act to regulate nitrogen fixation by inhibiting heterocyst differentiation and by inhibiting nitrogenase activity through competition with nitrogenase for reductant and/or ATP, but not by directly regulating nitrogenase biosynthesis in heterocysts.

Hydrogenase activity in the thermophile mastigocladus laminosus

Abstract Hydrogenase activity in the thermophilic cyanobacterium, Mastigocladus laminosus was studied both in vivo and in vivo hydrogen consumption required oxygen but not light, was about ten-fold higher than in mesophilic cyanobacteria, and was relatively insensitive to carbon monoxide. H 2 -supported acetylene reduction in reductant-limited cultures was a light-dependent, but O 2 -independent reaction. In vitro hydrogen evolution was unaffected by carbon monoxide, and this activity could be partially purified using a procedure developed for Anabaena cylindrica .