Coupling of Cellular Processes and Their Coordinated Oscillations under Continuous Light in Cyanothece sp. ATCC 51142, a Diazotrophic Unicellular Cyanobacterium

Abstract

Unicellular diazotrophic cyanobacteria such as Cyanothece sp. ATCC 51142 (henceforth Cyanothece), temporally separate the oxygen sensitive nitrogen fixation from oxygen evolving photosynthesis not only under diurnal cycles (LD) but also in continuous light (LL). However, recent reports demonstrate that the oscillations in LL occur with a shorter cycle time of ~11 h. We find that indeed, majority of the genes oscillate in LL with this cycle time. Genes that are upregulated at a particular time of day under diurnal cycle also get upregulated at an equivalent metabolic phase under LL suggesting tight coupling of various cellular events with each other and with the cell's metabolic status. A number of metabolic processes get upregulated in a coordinated fashion during the respiratory phase under LL including glycogen degradation, glycolysis, oxidative pentose phosphate pathway, and tricarboxylic acid cycle. These precede nitrogen fixation apparently to ensure sufficient energy and anoxic environment needed for the nitrogenase enzyme. Photosynthetic phase sees upregulation of photosystem II, carbonate transport, carbon concentrating mechanism, RuBisCO, glycogen synthesis and light harvesting antenna pigment biosynthesis. In Synechococcus elongates PCC 7942, a non-nitrogen fixing cyanobacteria, expression of a relatively smaller fraction of genes oscillates under LL condition with the major periodicity being 24 h. In contrast, the entire cellular machinery of Cyanothece orchestrates coordinated oscillation in anticipation of the ensuing metabolic phase in both LD and LL. These results may have important implications in understanding the timing of various cellular events and in engineering cyanobacteria for biofuel production.

Fig 1. Sinusoidal model fitting to cyclic genes and estimation of model parameters.

A: Results shown for nifK gene (cce_0561) as a representative under continuous light with the observed expression profile (●), linear term of the model (grey straight line, a + b*t from Eq (1)), data points after subtracting the linear term (○) and sinusoidal fit (grey sinusoidal curve). B: Histogram showing periodicity of oscillation for n = 2152 genes, deemed to be cyclic by sinusoidal model with a cut-off of 25 percent residual percent energy as a measure of goodness of fit.

Fig 2. Scatter plot to demonstrate 11 h as the predominant periodicity among the cyclic genes.

A scatter plot for 2152 genes that show satisfactory goodness of fit in the sinusoidal model with a randomly selected time point of t_121.92h as x-axis and its respective t+5.5h (t_127.13h) or t+11h (t_132.42h) as the y-axix. A: Scatter plot showing a negative correlation between the gene expression at t and t+ 5.5 h. B: Scatter plot showing a positive correlation between the gene expression at t and t+11 h.

Fig 3. Heat map and hierarchical clustering of cyclic genes.

Heat map was generated by performing hierarchical clustering for 1202 genes, which show 11h periodicity in oscillations and satisfy both Sinusoidal and Fourier Transform based models. Genes that peak under photosynthesis or respiration based metabolism are marked notionally as dawn peaking and dusk peaking, respectively. Normalized gene expression value, log₂ (fold change) is plotted as per the scale bar shown below the x-axis.

Fig 4. Day peaking oscillatory genes of Cyanothece 51142.

Genes were selected based on 1.5-fold change cutoff and clustered using the gene expression data under continuous light (LL) (present study) and light/dark (LD) condition [19]. Panels (a) and (b) show the essential genes that are involved in the organization of Phycobilisomes and Phycobiliproteins; panels (c) and (d) show the genes that participate in biogenesis, repair and assembly of Photosystem II. Panels (e) and (f) refer to the genes that are involved in functioning of Photosystem I, peaking during the mid-day or mid of the photosynthetic phase. Panel (g)-(h) represent genes that participate in the electron transport chain and panels (i)-(k) represent the genes that play an important role in carbon fixation, glycogen synthesis and central carbon metabolism. Panels (l)-(n) show the genes that are involved in cell division, ribosome recycling, RNA polymerase sigma factor, where all up-regulate at dawn or during the onset or middle of the photosynthetic phase to support cell growth and maintenance. Grey and White filled and continuously white filled boxes in the X-axis, indicate the culture growth conditions of LD and LL, respectively.

Fig 5. Night peaking oscillatory genes of Cyanothece 51142.

Panels (a)-(c) show the genes that are involved in NADH dehydrogenase, NADH quinone reductase and terminal oxidase of oxidative phosophorylation pathway, peaking at dusk or prior to the onset of respiration phase. Panels (d)-(g) represent the genes that participate in the nitrogen fixation mechanism, such as ferrous iron transport, translational initiation, nitrogen fixing activity and nitrogen regulatory protein. Panels (h)-(j) show the genes that take part in RNA polymerase sigma factors, cyanophycin and phycobilisomes degradation. Panels (k)-(n) represent the genes that are involved in biogenesis of ribosomes and RNA metabolism, such as ribosomal proteins, poly(A) polymerase and RNA helicase. See legend to Fig 4 for other details.

Fig 6. Comparison of oscillatory behavior of cyclic genes in nitrogen fixing (Cyanothece 51142) and non-nitrogen fixing organism (Synechococcus 8942).

Cyclic genes were selected for Cyanothece 51142 and their homologs in non-nitrogen fixing Synechococcus 7942 (data from [32]). Panels (a) and (b) show the genes that are involved in day metabolism, such as carbon concentrating mechanism (CCM) and ATP synthase complex, peaking in the dawn or at the onset of photosynthetic phase. Panels (c)-(f) show the genes that peak during night metabolism, such as glycogen degradation, central carbon metabolism, hydrogenase and housekeeping gene, all peaking at dusk or at the onset of respiration phase or at subjective night. See legend to Fig 4 for details.

Fig 7. Oscillation in circadian clock genes.

Selection of clock genes in Cyanothece 51142 was performed by considering the homologues of Synechococcus 7942. A: Panel (a-f) shows the oscillation of clock genes in Cyanothece51142 under continuous light (LL), involving in input pathway (ldpA), output pathway (cpmA, labA), master clock regulator (rpaA) and core structural clock mechanism (kaiB3 and kaiB4). B: Panel (a) and (b) shows the comparison of clock genes involved in core structural clock mechanism (kaiC1 and kaiB1) and input pathway of clock (cikA), showing 24h periodicity for Synechococcus 7942 and approximately 11 h periodicity for Cyanothece 51142 under continuous light. White filled boxes in the X-axis, indicate the culture growth under LL condition.

Fig 8. Peaking behavior of Cyanothece 51142 genes under diurnal cycles (LD) and continuous light (LL).

Clock dial shows the cellular events that peak at a particular metabolic phase under LL or at a particular time of the day under LD. Cellular events or broad cellular functions are marked on the dial if majority of genes associated with it peak at the same time or at the same phase, under LD and LL, respectively.