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

Nitrogenase activity of suspensions of the unicellular cyanobacterium Gloeothece sp. PCC 6909 plotted against the concentration of dissolved 0, (do,) resulted in a bell-shaped curve, both in the light and in the dark, with optima of 25 or 80 ~M -O , depending on the age of the culture, At the optimum d o 2 , nitrogenase activity [typically 4 to 6 nmol C2H, (mg protein)-' min-l] 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)-l, 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 d o 2 was increased. However, a high level of illumination (30 klx) stimulated or inhibited nitrogenase activity, depending on the external do,, presumably as a consequence of changes in the intracellular O2 concentration; in the presence of DCMU, activity increased twofold, independent of do,.In the dark, the dependence of the rate of respiration on O2 concentration suggested the presence of three 0,-uptake systems with apparent K, values of 1 PM, 5 p~ and 25 p~. The ADP/ATP ratio under anaerobic conditions was 0.47 and showed a marked decrease as d o 2 was increased to 25 VM. 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, of 5 p~. The dependence of the specific activity of nitrogenase on d o 2 indicated that both the high affinity ( K , 1 WM) and low affinity ( K , 25 p~) O2-uptake systems contributed ATP or reductant for N,-fixation. KCN (2.5 mM) completely inhibited nitrogenase activity in the dark and at moderate levels of illumination and d o 2 . 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.

SUMMARYThe diurnal patterns of cyanobacterial acetylene reduction (nitrogen fixation) and seasonal variations in these patterns were investigated in situ on a salt marsh and in a limestone cave. In the field, the non-heterocystous species fixed Ng mainly at night, while heterocystous species fixed Ng mainly during the day. These basic patterns could he infiuenced hy environmental conditions such as O^ availability and temperature. No acetylene reduction was observed in December, January or February.

Oscillutoriu spp. UCSBS and UCSB25 are both capable of aerobic N2 fixation. The optimum temperature for C2H2 reduction was 22 "C for Oscillutoriu sp. UCSBS and 35 "C for Osciffutoriu sp. UCSB25, whilst the optimum temperature for growth on N2 was 25 "C and 30 "C, respectively. In Oscillutoriu sp. UCSB25, but not in UCSBS, inhibition of N2 fixation may limit diazotrophic growth at temperatures above 35°C. When grown under alternating 12 h light and 12 h darkness, both isolates reduced C2H2 predominantly in the dark and both were capable of N2 fixation and photoheterotrophic growth in the presence of 20 PM-DCMU to inhibit photosystem I1 activity. Under these conditions, the best exogenous carbon source for Osciffuforiu sp. UCSBS was glucose, whilst that for Oscillutoriu sp. UCSB25 was fructose. In Osciflutoriu sp. UCSB8, exogenous glucose was catabolized mainly through the oxidative pentose phosphate pathway. Although cultures grown photoheterotrophically showed higher specific activities of nitrogenase than photoautotrophic cultures, they grew more slowly. Furthermore, cultures grown photoheterotrophically under alternating light and darkness reduced C2H2 both in the light and in the dark, but the highest rates of C2H2 reduction were observed in the dark. This cyclic pattern of N2 fixation was independent of photosystem I1 activity.

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