John R. Gallon

The Effects of Methyl Viologen on Gloeocapsa sp. LB795 and Their Relationship to the Inhibition of Acetylene Reduction (Nitrogen Fixation) by Oxygen

Summary: Methyl viologen (10 μM) markedly inhibited acetylene reduction (nitrogen fixation) by old but not young cultures of Gloeocapsa sp. LB795, apparently by causing the alga to produce H2O2. H2O2 inhibited acetylene reduction when added to cultures at concentrations greater than 10 μM. As catalase (EC 1.11.1.6) is not present in Gloeocapsa sp. LB795, H2O2 is probably removed by a non-enzymic reaction with ascorbate and also by an enzyme-catalysed reaction with glutathione. Enzymes catalysing the decomposition of H2O2, were most active in young cells which were therefore better able than old cells to metabolize H2O2 produced in the presence of methyl viologen. The maximum activities of these enzymes coincided with maximum nitrogenase activity during the growth of batch cultures, and may provide a protective mechanism for nitrogenase.

Nitrogen Fixation by Gloeothece sp. PCC 6909: Respiration and Not Photosynthesis Supports Nitrogenase Activity in the Light

SUMMARY: Nitrogenase activity of suspensions of the unicellular cyanobacterium Gloeothece sp. PCC 6909 plotted against the concentration of dissolved O2 (dO2) resulted in a bell-shaped curve, both in the light and in the dark, with optima of 25 or 80 μm-O2 depending on the age of the culture. At the optimum dO2, nitrogenase activity [typically 4 to 6 nmol C2H4 (mg protein)-1 min-1] was similar in the light or in the dark. Alteration of light intensity from zero to 2 klx, or addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), had no effect on nitrogenase activity. At 1 klx the ADP/ATP ratio was 0·2 and showed only a marginal increase as the dO2 was increased. However, a high level of illumination (30 klx) stimulated or inhibited nitrogenase activity, depending on the external dO2, presumably as a consequence of changes in the intracellular O2 concentration; in the presence of DCMU, activity increased twofold, independent of dO2. In the dark, the dependence of the rate of respiration on O2 concentration suggested the presence of three O2-uptake systems with apparent K m values of 1 μm, 5 μm and 25 μm. The ADP/ATP ratio under anaerobic conditions was 0·47 and showed a marked decrease as dO2 was increased to 25 μm. A CN-insensitive respiratory activity, which neither supported nitrogenase activity nor was coupled to ATP synthesis, was associated with the system with the apparent K m of 5 μm. The dependence of the specific activity of nitrogenase on dO2 indicated that both the high affinity (K m 1 μm) and low affinity (K m 25 μm) O2-uptake systems contributed ATP or reductant for N2-fixation. KCN (2·5 mm) completely inhibited nitrogenase activity in the dark and at moderate levels of illumination and dO2. We conclude that respiration is the major source of reductant and ATP for nitrogenase activity both in the dark and in the light, but that photosystem I can contribute ATP at very high levels of illumination.

Metabolic Changes Associated with the Diurnal Pattern of N2 Fixation in Gloeothece

When grown in alternating cycles of light and darkness, non-synchronous cultures of Gloeothece fixed N2 mainly in the dark phase. This diurnal pattern of N2 fixation was independent of the doubling time (69 ± 16 h) of the organism and, since cell division was asynchronous, N2 fixation does not seem to be confined to a specific phase in the cell cycle. Fluctuations in the rate of N2 fixation coincided with similar fluctuations in the rate of nitrogenase synthesis. Diurnal fluctuations also occurred in the utilization of glucan and acid-soluble polyphosphate and in the ADP/ATP ratio. Based on these observations, it is proposed that specific metabolic changes are involved in the regulation of the diurnal pattern of N2 fixation by Gloeothece.

Effects of a Light to Dark Transition on Carbon Reserves, Nitrogen Fixation and ATP Concentrations in Cultures of Gloeocapsa (Gloeothece) sp. 1430/3

Between 30 s and 5 min after transfer from light to darkness, the rate of acetylene reduction by cultures of the unicellular cyanobacterium Gloeocapsa sp. 1430/3 decreased rapidly. During the same period, there was a rapid disappearance of storage glucan. There was also a sharp fall in the intracellular concentration of ATP followed by a slower recovery. It is concluded that, in the dark, the catabolism of storage glucan is the only potential source of ATP and/or reductant and that the low rate of N2 fixation in the dark is not due to limitations in the supply of ATP.

Calcium Ions, Oxygen and Acetylene Reduction (Nitrogen Fixation) in the Unicellular Cyanobacterium Gloeocapsa sp. 1430/3

Cultures of Gloeocapsa sp. 1430/3 rapidly and irreversibly lost their ability to reduce acetylene when incubated with 1 mm-EDTA, either in the light or aerobically in the dark. However, EDTA did not inhibit acetylene reduction by cultures of Gloeocapsa incubated anaerobically in the dark. It is suggested that EDTA depletes the cyanobacterial cells of Ca2+ and thereby destroys a Ca2+-dependent process by which nitrogenase is protected from inactivation by oxygen.

Fluoroacetate Metabolism in Gloeocapsa sp. LB795 and its Relationship to Acetylene Reduction (Nitrogen Fixation)

Summary: Sodium fluoroacetate (1 mM) caused an accumulation of citrate and altered the lipid composition in cells of Gloeocapsa sp. LB795. It also inhibited acetylene reduction (nitrogen fixation) by the alga - markedly under aerobic conditions, but much less so in the absence of oxygen. This inhibition is largely the result of the conversion of fluoroacetate to fluorocitrate which, by inhibiting aconitate hydratase (EC 4.2.1.3), interrupts the synthesis of the 2-oxoglutarate required for the assimilation of NH4 +. The consequent accumulation of NH4 + within the cells of Gloeocapsa sp. inhibits nitrogenase synthesis and, since oxygen rapidly inactivates pre-existing nitrogenase, nitrogen fixation by Gloeocapsa sp. decreases under aerobic conditions.

Synthesis of Nitrogenase in the Cyanobacterium Gloeothece (Gloeocapsa) sp. CCAP 1430/3

SUMMARY: 55FeCI3 labelling and non-denaturing gel electrophoresis were used to study nitrogenase synthesis in Gloeothece sp. CCAP 1430/3. Nitrogenase synthesis was inhibited by addition of NHX but was unaffected by elevated concentrations of O2. Upon transfer of cultures of Gloeothece from light to darkness, there was initially a slight decrease in the rate of synthesis of nitrogenase but after 4-5 h there was an almost complete cessation of synthesis. This delayed effect of darkness on nitrogenase synthesis could not be related to any change in RNA synthesis, in protein synthesis or in the rate of breakdown of storage glucan. In cultures of Gloeothece, mRNA, including nif mRNA, was unstable, having a half-life of about 5 min. The synthesis of nif mRNA did not stop immediately upon transfer of cultures to the dark. If darkness exerts its effect on nitrogenase synthesis by inhibiting the synthesis of nif mRNA, it does so only after a lag of about 4 h.

Nitrogen Fixation in Cultures of the Cyanobacterium Gloeocapsa (Gloeothece) sp. 1430/3 Incubated in the Dark

SUMMARY: Gloeocapsa sp. 1430/3 fixed N2 in the dark, but at a lower rate than in the light. Following the transfer of exponentially growing cultures to the dark, the rate of N2 fixation increased for between 2 and 5 h and then decreased, becoming negligible after 12 h. No substantial increase in activity occurred for about 10 h following re-illumination. It is suggested that the decrease in nitrogenase activity which occurred about 5 h after transfer to the dark was not caused by exhaustion of carbon reserves but by a cessation of nitrogenase synthesis coupled with an irreversible inactivation of the enzyme, probably by O2. Subsequent recovery of activity apparently depended upon resynthesis of nitrogenase.

The Effects of Structural Analogues of Amino Acids on Ammonium Assimilation and Acetylene Reduction (Nitrogen Fixation) in Gloeocapsa (Gloeothece) sp. CCAP 1430/3

SUMMARY: In Gloeocapsa, NH4+ formed by N2 fixation was assimilated by the glutamine synthetase-glutamate synthase pathway. The inhibition of acetylene reduction following addition of NH4+ to N2-fixing cultures was not caused by NH4+ itself but was, most probably, related to increased intracellular levels of glutamine. Analogues of several amino acids inhibited acetylene reduction. Apart from glutamine analogues, which interfered with NH4+ assimilation, these analogues probably acted by inhibiting nitrogenase synthesis. Certain analogues of tryptophan and phenylalanine, which have been reported to overcome NH4+ inhibition of cell differentiation and, possibly, nitrogenase synthesis in heterocystous cyanobacteria growing on combined nitrogen, did not prevent NH4+ from inhibiting acetylene reduction in cultures of Gloeocapsa. In contrast, l-methionine-dl-sulphoximine, which similarly counteracts the effect of NH4+ on heterocyst differentiation, also prevented NH4+ from inhibiting acetylene reduction in Gloeocapsa.