Temporal Patterns and Intra- and Inter-Cellular Variability in Carbon and Nitrogen Assimilation by the Unicellular Cyanobacterium Cyanothece sp. ATCC 51142

Abstract

Unicellular nitrogen fixing cyanobacteria (UCYN) are abundant members of phytoplankton communities in a wide range of marine environments, including those with rapidly changing nitrogen (N) concentrations. We hypothesized that differences in N availability (N2 vs. combined N) would cause UCYN to shift strategies of intracellular N and C allocation. We used transmission electron microscopy and nanoscale secondary ion mass spectrometry imaging to track assimilation and intracellular allocation of 13C-labeled CO2 and 15N-labeled N2 or NO3 at different periods across a diel cycle in Cyanothece sp. ATCC 51142. We present new ideas on interpreting these imaging data, including the influences of pre-incubation cellular C and N contents and turnover rates of inclusion bodies. Within cultures growing diazotrophically, distinct subpopulations were detected that fixed N2 at night or in the morning. Additional significant within-population heterogeneity was likely caused by differences in the relative amounts of N assimilated into cyanophycin from sources external and internal to the cells. Whether growing on N2 or NO3, cells prioritized cyanophycin synthesis when N assimilation rates were highest. N assimilation in cells growing on NO3 switched from cyanophycin synthesis to protein synthesis, suggesting that once a cyanophycin quota is met, it is bypassed in favor of protein synthesis. Growth on NO3 also revealed that at night, there is a very low level of CO2 assimilation into polysaccharides simultaneous with their catabolism for protein synthesis. This study revealed multiple, detailed mechanisms underlying C and N management in Cyanothece that facilitate its success in dynamic aquatic environments.

Electron & Biomass Dynamics of Cyanothece Under Interacting Nitrogen & Carbon Limitations

“…As a consequence, instantaneous growth rates progressively revert to very low values (Supplementary Figure 3B). The decline in photosynthetic rates following the draw-down of DIC was confirmed by trends in 13 C incorporation shown in a companion paper (Polerecky et al, 2021), with a decrease in C specific assimilation rates from 1.7 ± 0.35 d −1 (morning) to 0.39 ± d −1 (afternoon) in N 2 -fixing cultures and from 0.84 ± 0.15 d −1 (morning) to 0.42 ± 0.07 d −1 (afternoon) in NO 3 − cultures (Polerecky et al, 2021). When CO 2 concentration in the inflow air is increased, the DIC limitation is relaxed and O 2 concentration increases during the light phase (Supplementary Figure 4).…”

“…This induction, however, does not depend upon DIC limitation because a similar pattern occurred even in bioreactors bubbled with CO 2 to relieve the diel onset of DIC limitation (Supplementary Figure 1). Taken together, the data show a constitutive induction of alternate electron flows in late subjective morning, possibly after cellular requirements for CH 2 O accumulation have been met or saturated during the initial period of rapid net oxygen evolution (Polerecky et al, 2021).…”

“…The incorporation of labelled C and N presented in a companion paper clearly indicates that N 2 -fixation occurs primarily in the first half of the dark period, and decreases by two orders of magnitude during the second half of the dark phase (1.34 ± 0.79 d −1 in early night vs. 0.012 ± 0.047 d −1 in late night; Polerecky et al, 2021). This result is in good agreement with the OD 720 dynamics, confirming that the active respiration of carbohydrates to fuel N 2 -fixation also stops by the mid-dark phase.…”

“…In this mode, direct reductive NO 3 − assimilation competes with inorganic carbon assimilation for photosynthetic reductant, which slows the drawdown of the DIC pool in our turbidostat experiments. The subsequent dark phase is then a period of maintenance respiration and synthesis of cell components like Chl a, and proteins (Polerecky et al, 2021). In marked contrast, Cyanothece grown in obligate diazotrophy does not take up nitrogen in the light and therefore can funnel more photosynthetic reductant to CO 2 assimilation, thereby accumulating a larger stock of refractile carbohydrate, a necessity to subsequently support the e − :ATP requirements of N 2 -fixation during the succeeding dark period.…”

“…During the light period, electrons provided by photosynthetic water-splitting in the N 2 -fixing culture cells are allocated for CO 2 fixation, while none are used directly for N acquisition. In contrast, in cells growing on NO 3 − as a nitrogen source diurnal patterns in NO 3 − assimilation follow those in C assimilation, with highest rates in both processes measured in the morning (Polerecky et al, 2021). NO 3 − reduction requires 8 + 2 electrons per NO 3 − ion assimilated to glutamate (see Introduction), originating ultimately from the photosynthetic electron transport.…”