Cyanobacteria have played a profound role in shaping the biosphere, most notably through the Great Oxygenation Event (GOE) with the advent of photosynthesis. Cyanobacteria also contribute to global primary production through biological nitrogen fixation (BNF) using nitrogenase, an oxygen-labile enzyme complex that evolutionarily predates the GOE. Current literature reports nitrogenase activity in unicellular cyanobacteria is protected from oxygen through a diurnal separation of photosynthesis and BNF. However, historic conditions of continuous-light and warm temperature at polar latitudes during the Triassic and Cretaceous may have created a selective advantage amongst unicellular cyanobacteria for non-temporal mechanisms of maintaining nitrogenase activity in the presence of oxygen. Here we report constitutive nitrogenase activity concurrent with a net-gain of oxygen through photosynthesis in a continuous-light adapted culture of the unicellular cyanobacteria, Cyanothece sp. ATCC 51142. Nitrogenase activity in the adapted culture exhibited dependence on light and increased resilience to artificially raised oxygen-tension compared to traditional culture. We predict cyanobacteria closely related to Cyanothece sp. ATCC 51142 also possess this physiology and found an accessory predicted proteome with functional relevance. This work provides a model of light-driven, oxygen-tolerant, constitutive nitrogenase activity and suggests this physiology may be conserved in closely related unicellular diazotrophic cyanobacteria with implications for primary production in polar ecosystems and potential biotechnological application in sustainable agriculture production.

Some cyanobacteria carry out both O2-producing photosynthesis and O2-sensitive nitrogen fixation, making them unique contributrors to global carbon and nitrogen cycles and potential contributors industrial and agricultural processes either spatially, in filamentous N2-fixing cyanobacteria such as Anabaena spp. become heterocysts that are present singly at semiregular intervals along the filaments. Heterocysts are morphologically and biochemically specialized for N2-fixation. By sequestering nitrogenase within heterocysts, Anabaena spp. can carry out, simultaneously, oxygenic photosynthesis and the O2-labile assimilation of N2. Heterocysts have three mechanisms to protect nitrogenase. They form an additional two-layers of cell wall with a layer of glycolipids and an outer protective layer of specific polysaccharides to block the environmental O2, stop O2 production by shutting down PSII, and increase respiration to consume O2. Unlike filamentous cyanobacteria, Cyanothece ATCC 51142 (hereafter cyanothece), a unicellular cyanobacterium, rhythmically separates photosynthes and nitrogen fixation. However, cyanothece still has to deal with encironmental oxygen. Little is known about how cyanothece protects nitrogenase from inactivation by environmental O2. In this study, cyanothece cells were grown in nitrogen fixing and non-nitrogen fixing conditions and screened with fluorescein conjugated lectins, allowing a comparative fluorescent microscopy study of polysaccharides containing N-acetylgalactosamine and N-acetylglucosamine, but a relative lack of polysaccaride diversity. These findings suggest that the two cell types (N2-fixing and non-N2-fixing) have different cell wall poolysaccharides, which warrants further investigation regarding the cell wall’s role in nitrogen fixation.