Abstract
Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N2) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N2 fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N2 fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N2 fixation and photosynthesis commonly observed. However, N2 fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell.
Highlights
Dinitrogen (N2) fixation and photosynthesis are two crucial metabolic processes in diazotrophic cyanobacteria
Cyanobacterial photosynthesis leads to the production of O2, which inactivates the key enzyme for N2 fixation, nitrogenase [1,2]
N2 Fixation and Photosynthesis at the Population Level N2 fixation rates in C. watsonii determined via the acetylene reduction assay (ARA) followed the diel pattern known for this organism [24,25] during the first 24 h of the experimental phase encompassing a 12:12 h dark:light cycle with N2 fixation occurring during the dark phase (Figure 1A)
Summary
Dinitrogen (N2) fixation and photosynthesis are two crucial metabolic processes in diazotrophic cyanobacteria. Several studies suggest that photosynthesis and nitrogen fixation could occur concurrently, at least on the population level, during transitions from dark to light [12] or in studies where the light conditions are experimentally shifted relative to the clock [13] Both studies did not directly measure N2 fixation and photosynthesis at the single-cell level leaving the question whether individual cells do perform both processes. N2 fixation of single cells has already been analyzed in previous studies including field studies [21]; for example, Teredinibacter turnerae was shown to exhibit population heterogeneity in N2 fixation [22] whereas vegetative cells in Anabaena oscillarioides filaments had a rather uniform distribution of recently fixed nitrogen from the heterocysts [23] The former is a heterotrophic proteobacterium which is diazotrophic but not photosynthetic, and the latter is a heterocystous cyanobacterium which fixes N2 and is photosynthetic but has the capability of spatially separating N2 fixation and photosynthesis. This allowed us to assess the level of phenotypic heterogeneity during regular light-dark cycles, and under conditions where populations that are ‘scheduled’ to perform O2sensitive N2 fixation are exposed to light, and potentially are subject to a physiological dilemma posed by the opportunity to simultaneously carry out two incompatible processes
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