Ultradian metabolic rhythm in the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142

Abstract

The unicellular cyanobacterium Cyanothece sp. American Type Culture Collection (ATCC) 51142 is capable of performing oxygenic photosynthesis during the day and microoxic nitrogen fixation at night. These mutually exclusive processes are possible only by temporal separation by circadian clock or another cellular program. We report identification of a temperature-dependent ultradian metabolic rhythm that controls the alternating oxygenic and microoxic processes of Cyanothece sp. ATCC 51142 under continuous high irradiance and in high CO₂ concentration. During the oxygenic photosynthesis phase, nitrate deficiency limited protein synthesis and CO₂ assimilation was directed toward glycogen synthesis. The carbohydrate accumulation reduced overexcitation of the photosynthetic reactions until a respiration burst initiated a transition to microoxic N₂ fixation. In contrast to the circadian clock, this ultradian period is strongly temperature-dependent: 17 h at 27 °C, which continuously decreased to 10 h at 39 °C. The cycle was expressed by an oscillatory modulation of net O₂ evolution, CO₂ uptake, pH, fluorescence emission, glycogen content, cell division, and culture optical density. The corresponding ultradian modulation was also observed in the transcription of nitrogenase-related nifB and nifH genes and in nitrogenase activities. We propose that the control by the newly identified metabolic cycle adds another rhythmic component to the circadian clock that reflects the true metabolic state depending on the actual temperature, irradiance, and CO₂ availability.

Cyanobacteria have helped form the Earth’s biosphere since they first evolved some 3 billion years ago. These microorganisms made our atmosphere oxygenic and their fixation of CO₂ and N₂ has made important contributions to the elemental cycles. In past decades, cyanobacteria of the genus Cyanothece have attracted strong attention for their contributions to the nitrogen cycle (1); as unique models to study the relationship between N₂ fixation, photosynthesis, and respiration (2); and as promising candidates for bioenergy production (3–5). The strain Cyanothece sp. American Type Culture Collection (ATCC) 51142 (hereafter Cyanothece) has particularly robust properties (2, 4). Cyanothece is a unicellular cyanobacterium in which spatial compartmentalization of the mutually exclusive oxygenic photosynthesis and microoxic nitrogen fixation is impossible (6). The strategy used by this organism is to temporally separate the molecular oxygen released by photosynthesis from the nitrogenase that would otherwise be irreversibly O₂-inactivated. The capacity to separate the antagonistic metabolic processes in time is usually attributed to circadian control. The circadian clock in cyanobacteria relies on cyclic (de-)phosphorylation involving complexes of the KaiA, KaiB, and KaiC proteins (7–9). The clock mechanism has been studied in great detail in Synechococcus elongatus PCC 7942 and also in vitro (10). The clock period in this organism has been shown to be temperature-compensated—a feature essential for controlling the daily rhythm, particularly in organisms that do not sustain a stable temperature for their metabolism (11).

In Cyanothece the kai genes exist in multiple copies (12), and the Kai proteins have not been studied in the detail achieved for S. elongatus. The daily modulation of the metabolic activity and gene transcription, namely alternation of photosynthetic and N₂-fixation phases, has been attributed to the control by the circadian clock largely because the observed period was ∼24 h, even in continuous light following a 12-h light/12-h dark entrainment (13). Certain genes, especially those associated with nitrogenase assembly and function, demonstrated a circadian-type of regulation when the light-dark pattern was modified for growth under continuous light or short day–night periods (14).

However, there are no published data on Cyanothece to show that the period of the oscillatory modulation in continuous light is temperature-compensated as expected for circadian control. One can expect an involvement of metabolic processes (see ref. 2) that may result in distinct ultradian rhythms, such as those demonstrated in yeast, which occur with temperature-dependent periods that are significantly shorter than 24 h. The hypothesis suggesting involvement of an ultradian metabolic cycle in Cyanothece is supported by oscillations with ca. 12-h periods that occur in continuous light following an initial 12-h light/12-h dark entrainment (15) as well as in continuous light in a batch culture (16). Components with an approximate 12-h period have also been detected by a Fourier transform analysis in transcript data in continuous light following a 12-h light/12-h dark entrainment (17).

In searching for a potential ultradian metabolic rhythm in Cyanothece we have performed experiments similar to those in yeast in which ultradian oscillations were induced by a starvation period (18). Cyanothece was grown to late exponential or linear phase in regular medium containing nitrate and supplied with saturating CO₂ and light. Strong ultradian oscillations occurred after the cells were moved to a minus-nitrate medium. This newly identified ultradian metabolic rhythm is strongly temperature-dependent. We also show that the circadian cycle is well temperature-compensated. The contrasting temperature dependencies document that the ultradian and circadian cycles are independent. The ultradian rhythm dominates in saturating CO₂ and light, whereas the circadian rhythm prevails when irradiance and/or CO₂ concentration are lowered.

Results

Ultradian and Circadian Rhythms in a Diurnally Entrained Culture.

Cyanothece was grown in flat-panel photobioreactors with highly time-resolved, automated sampling to follow cyclic processes over days and weeks (19). Before the experiment shown in Fig. 1, the culture was entrained in 12-h light/12-h dark cycles in a turbidostat mode in which the culture optical density was kept in a narrow range by a photobioreactor-controlled feedback dilution. The experiment started after the last period of the diurnal entrainment (interval 0–24 h in Fig. 1) by switching off the dilution that allows culture batch growth and by keeping the culture in continuous light for the subsequent 10 d (interval 24–264 h in Fig. 1). In response to the pretreatment, the culture exhibited a complex oscillatory pattern in which both the shorter ultradian and the longer circadian rhythms combined. The periods in which the ultradian rhythm dominated (see the 72–96 h interval in Fig. 1, Lower Left) were followed by periods dominated by the circadian rhythm (see the 216–264 h interval in Fig. 1, Lower Right).

Fig. 1.

Transition from ultradian to circadian rhythms in diurnally entrained batch culture. Cyanothece culture was diurnally entrained in a turbidostat during 28 d with 12-h light/12-h dark, 36 °C, in the ASP2 medium without nitrate. The dark–light entrainment was followed by constant light 136 μmol photons m⁻²⋅s⁻¹ of 627 nm and 10 μmol photons m⁻²⋅s⁻¹ of cool white light with turbidostat switched off. (Upper) The experiment period starting with the last 24-h entrainment period and ending after 10 d of continuous light. The black rectangle at the bottom of the graph between 12 and 24 h shows the last night dark period; the subsequent striped rectangles show subjective (anticipated) night periods. (Lower) Typical ultradian (72–96 h) and circadian (216–264 h) rhythms. The solid line shows the culture growth dynamics as measured by OD₆₈₀. The thick line with open circles shows relative changes in the dissolved O₂ (dO₂). The other measurements are shown only in the lower two panels: The thin full line represents the relative change in the dissolved CO₂, the dashed line represents the relative change of pH, and the long-dashed line shows the relative change of the steady-state pigment fluorescence emission Ft.

The concentration of dissolved oxygen oscillated with a minimum in the first half of subjective night in the circadian phase (216–264 h, see open circles in Fig. 1, Lower Right). The minimum in the concentration of the dissolved oxygen during the ultradian phase (∼48–144 h, open circles in Fig. 1, Lower Left) was not firmly anchored to the subjective day–night cycle, because its period was neither equal to 12 h nor 24 h. The minima of the dissolved oxygen preceded the maxima of the dissolved CO₂ concentration by 1–2 h (thin full line in Fig. 1). The oscillations of the medium pH (short-dashed line in Fig. 1, all Lower panels) were nearly antiparallel to the dissolved CO₂ concentration, as anticipated from the water chemistry of inorganic carbon forms. During the rapid growth (Fig. 1, Lower Left), OD₆₈₀ of the suspension was weakly modulated with a period of the ultradian rhythm so that the maxima of the dissolved oxygen concentration matched the steepest increase of the optical density. This suggests that high photosynthetic activity indicated by the high concentration of dissolved O₂ correlated with an increase in chlorophyll concentration represented by the surrogate OD₆₈₀. The maxima of the stationary pigment fluorescence emission (long-dashed line in Fig. 1, all Lower panels) approximately coincided with the O₂ minima and with the CO₂ maxima, a pattern that was not always reproduced in parallel experiments. This dynamic pattern can be tentatively interpreted as high fluorescence resulting from a reduction of the plastoquinone pool by high rates of respiration and low rates of photosynthesis and vice versa.

Metabolic Rhythms in a Diurnally Nonentrained Culture.

A complex oscillatory pattern similar to the diurnally entrained culture (Fig. 1) was also observed in continuous light, i.e., without the diurnal entrainment, when cyanobacteria growing rapidly in a nitrate-rich medium were transferred to a medium without nitrate (time 0 in Fig. 2). The dynamic pattern during the first 3 d after the media replacement exhibited a dominant ultradian character (period ∼10.5 h). As the culture grew (OD₆₈₀ ∼2.2), the oscillations became dominated by the circadian rhythm (Fig. 2, Lower Right). Note that the residual involvement of the ultradian dynamics remained also in the dense culture, as demonstrated by the two high amplitudes at hours 160.5 and 234 of the experiment. The period separating the two high amplitudes corresponded to seven ultradian periods (7 × 10.5 h = 73.5 h) and to, roughly, three circadian periods (3 × 24 h = 72 h). Thus, at every seventh ultradian and every third circadian period, the respective amplitudes combined rather than canceled each other.

Fig. 2.

Batch culture transition from ultradian to circadian rhythms after an exchange to nitrate-less medium. The culture growing rapidly in nitrate supplemented ASP2 medium was harvested and resuspended in nitrate-less medium (time 0). The transition introduced oscillations in dO₂, in OD₆₈₀ optical density, steady-state fluorescence emission F_t, and in pH. The dissolved CO₂ is represented here by the antiparallel pH dynamics. The symbols are the same as in Fig. 1.

We argue that the transition from the dominant ultradian to circadian rhythm in Fig. 2 was induced by decreasing the mean effective irradiance in the dense culture. This hypothesis is supported by the results in Fig. 3 in which the culture density was stabilized by the turbidostat that was diluting the suspension to the same optical density and, thus, the mean irradiance was fully controlled by the photobioreactor to high irradiance in the initial days and to low irradiance toward the end of the experiment. The data demonstrate that the ultradian rhythm can be converted to a circadian cycle by lowering of the incident irradiance in the photobioreactor. This result raises the question as to what is the decisive factor controlling the transition of the ultradian phenomena to the circadian rhythm: Is it light as the result in Fig. 3 suggests, CO₂ availability, or is it fast growth that requires both high light and high CO₂? Data shown in Figs. S1 and S2 suggest that high light and high CO₂, rather than fast growth, lead to the dominating ultradian rhythm. Another condition necessary for the ultradian rhythm to occur is lack of nitrate in the medium. An addition of nitrate quickly damped the ultradian oscillations to zero (Fig. S3). Also, the ultradian oscillations in Fig. 2 were induced by transferring the cells to a nitrate-free medium.

Fig. 3.

Transition from ultradian to circadian rhythms after decrease of light in a turbidostat. The oscillations of dissolved O₂, CO₂, fluorescence F_t, and pH were dominated by the ultradian rhythm during the first 64 h when exposed to high irradiance (160 μmol photons m⁻²⋅s⁻¹). Switching to low irradiance (50 μmol photons m⁻²⋅s⁻¹) led to a decrease in the ultradian rhythm amplitude and to prevalence of the circadian oscillation. Noteworthy is the saw-like OD₆₈₀ curve that shows that the culture was automatically diluted whenever its density became >0.7. The dilutions were less frequent around minima in the concentration of dissolved O₂, indicating slow growth when photosynthesis is suppressed.

Temperature is another important factor affecting the ultradian metabolic rhythm. The oscillations shown in Figs. 1–3 occurred at 36 °C with an ultradian period of 10.55 ± 0.05 h (mean and SD of five biological replicates). We found that the period of ultradian oscillations was strongly temperature-dependent. It increased to 17 h at 27 °C and decreased to 10 h at 39 °C (Fig. 4). The temperature coefficient (Q₁₀) was 1.55, documenting the ultradian temperature dependence. We also measured the periods of the 24-h rhythms in different temperatures and found Q₁₀ close to 1, typical for the temperature-compensated circadian rhythm. The different response to temperature change shows that the ultradian rhythm is truly an independent phenomenon that is not derived from the circadian clock.

Fig. 4.

Temperature dependence of the ultradian (open circles) and circadian (closed circles) cycles. The data were obtained as averages of at least two independent experiments. At 33 °C and 36 °C, more independent experiments were performed to determine the SD of ±0.05 h. The Q₁₀ rates were calculated to quantify the temperature dependence of a rate proxy of 1 per period.

Data in Fig. 5A demonstrate the ultradian oscillations of dissolved O₂ concentration, pH, and partial synchronization of cell division (bars). The increasing dissolved oxygen and rising pH (i.e., decreasing dissolved CO₂) reflected active photosynthesis leading to an accumulation of biomass carbon between ca. the seventh and 13th hours (closed squares in Fig. 5B). A large part of the organic carbon was stored in the form of semiamylopectin granules (20), frequently referred to as glycogen (Fig. 5B, open squares). The proportion of glycogen carbon to total biomass carbon increased during the photosynthesis phase, likely because of the lack of nitrogen that limited protein synthesis. The imbalance between the surplus of fixed carbon and the lack of nitrate in the medium grew to a point where photosynthesis was down-regulated; the dissolved oxygen and, ca. 2 h later, the pH started decreasing; and the accumulation of organic carbon was suspended. The glycogen content started decreasing and oxygen dropped to a level that allowed nitrogen fixation. Nitrogen fixation dominated between ca. the third and seventh hours and between hours 13.5 and 18 of the experiment—periods indicated by the dashed lines surrounding the respective segments in Fig. 5. The rising nitrogen content of the biomass is indicated by the dashed line and closed circles in Fig. 5C. The genes nifH and nifB necessary for nitrogen fixation are expressed to higher mRNA levels in the early phase of the nitrogen fixation periods.

Fig. 5.

The processes occurring during the ultradian oscillations relative to the photosynthesis and nitrogen fixation phases. (A) Oscillation of the dissolved O₂ (open circles), pH (closed circles), and percentage of dividing cells as counted through a microscope (bars). (B) Total carbon in cells (closed rectangles) increased during the photosynthesis phase, whereas it remained stagnant during the nitrogen fixation phase. Glycogen content (open rectangles) decreased during the nitrogen-fixing phase. (C) Dynamics of the total nitrogen content of photobioreactor biomass together with expression levels of nitrogenase genes. The dashed line shows the increase of N content during the nitrogen-fixation phase and stagnant level during the oxygenic photosynthesis phase. The solid lines show expression levels (NRQ) of the nifB (×) and nifH (+) genes together with measured nitrogenase activity (open triangles).

Discussion

Linking the Ultradian Metabolic Rhythm to Other Cyclic Processes.

This paper has demonstrated that Cyanothece displays ultradian rhythms of alternating photosynthesis and N₂ fixation when grown without nitrate in continuous culture conditions. Whether the cultures were entrained (Fig. 1) or not (Fig. 2), the exponential growth phase showed ultradian rhythms that eventually degenerated into more circadian-like rhythms. This finding ties together many disparate results for Cyanothece over the years and may provide a basis for the interrelationship of the circadian and ultradian types of temporal regulation.

The earlier experiments typically used low light/low CO₂ and showed circadian patterns without an obvious ultradian modulation (3, 21). Here, we demonstrated that the ultradian rhythm occurred when photosynthesis was saturated by high light and high CO₂ concentration. In respect to high carbon availability, it is important to note that an ultradian component was also observed over a circadian pattern when Cyanothece was grown heterotrophically with glycerol (22).

Both the ultradian and circadian cycles exhibit many similar features. During the photosynthesis phase, cells accumulated large amounts of glycogen. The transition from photosynthetic to nitrogen fixation phase correlated with degradation of glycogen and a respiration burst. When enough ATP and NADH/NADPH accumulated and intracellular oxygen was lowered by respiration, the cells start fixing nitrogen.

Earlier studies of Cyanothece were performed mostly on 12-h light/12-h dark grown cells and also on cells grown under continuous light and short light–dark periods (21). Invariably, the nitrogenase genes in the 35-gene cluster demonstrated the most consistent circadian behavior (13, 14, 21). Interestingly, the nitrogenase genes were among the large majority of genes displaying a circadian rhythm even in experiments in which some other genes oscillated with a 12-h period. The observation of gene oscillation with a 12-h period led to an earlier proposal of an ultradian rhythm (17). Here, we probed expression of the nifB and nifH genes; both were clearly modulated by the ultradian rhythm, a feature consistent with the ultradian modulation of the nitrogenase activity (Fig. 5). The ultradian modulation of the nitrogenase genes supports the notion that the ultradian rhythm is, under favorable conditions, dominant in all aspects attributed earlier exclusively to circadian control. In this sense, it is highly relevant to ask if the ultradian and circadian processes are not reflecting a single control mechanism (23).

To clarify the interrelationship between the ultradian and circadian rhythms, we measured temperature dependence of the respective periods (Fig. 4). The circadian period was shown to be well temperature-compensated (Q₁₀ ∼1), whereas the ultradian period was found to be strongly temperature-dependent (Q₁₀ = 1.55). We conclude that the ultradian metabolic oscillation represents a process that is truly independent from the circadian cycle.

The ultradian rhythms were replaced or dominated by circadian rhythms when the cell irradiance was lowered either by self-shading in high cell density (Figs. 1 and 2) or by dimming the irradiance incident at the photobioreactor cuvette (Fig. 3). In SI Materials and Methods, we show an experiment that supports the conclusion that the ultradian rhythm is largely suppressed when photosynthesis and growth are not saturated by light (Fig. S1A).

The ultradian rhythm may also depend on fast growth that requires both high light and high CO₂. However, we show in Fig. S1B that the growth rate was maximal at ca. 33 °C and declined toward 36 °C and 39 °C. This is a qualitatively contrasting temperature dependence compared with that of the ultradian period that declined monotonously between 27 °C and 39 °C. We conclude that the growth rate is not a driver of the ultradian phenomena. This conclusion was further supported by the demonstration (Fig. S2) that the change of the specific growth rate by increasing the sparging gas CO₂ concentration in the 700–1,000 ppm range was small compared with the much steeper decrease of the ultradian period.

It appears that two independent mechanisms of temporal separation of photosynthesis and nitrogen fixation are possible in Cyanothece: the ultradian rhythm that prevails when photosynthesis is saturated by light and CO₂ and the circadian rhythm that dominates when the rate of photosynthesis is limited.

Interestingly, the experiments reported here also shed light on an earlier observation that the circadian patterns of photosynthesis and nitrogen fixation often appeared even during the first entrainment period, a feature that was hard to understand without the context shown in Fig. 1. The experimental cultures used to be inoculated with batch cultures that were 10–14 d old and, thus, closely resembled ca. >200-h cells in Fig. 1 that were probably already poised for the circadian regimen before inoculation. It was the photobioreactor that permitted us to work under a variety of environmental conditions; we can now assimilate the information from these different experiments.

On the Homology Between the Metabolic Rhythms in Cyanothece and in Saccharomyces cerevisiae.

Aerobically growing, dense continuous cultures of S. cerevisiae exhibit autonomous energy-metabolite ultradian oscillations of ca. 2–5 h periods that are observed under low nitrogen and low carbon conditions (18, 24–26). Although yeast are eukaryotic organisms that separate incompatible processes in different compartments, they also use temporal separation to carry out incompatible metabolic reaction and further alleviate futile cycles (27). A difference between the ultradian oscillations in yeast and in Cyanothece relates to the culture density. In contrast to yeast, the Cyanothece ultradian oscillations do not occur in high cell density that leads to self-shading of the penetrating light and to a reduction of the ultradian oscillatory amplitudes. This difference is only due to the role of light penetration through the culture that is critical for cyanobacteria and irrelevant to yeast.

However, several principal homologous features of metabolic oscillations of Cyanothece and S. cerevisiae can be found despite their largely different prokaryotic and eukaryotic character. The yeast oscillations, similar to the oscillations reported here for Cyanothece, consisted of two distinct phases: the anabolic, carbohydrate-storing phase (reductive in the yeast and photosynthetic in the cyanobacterium) and the catabolic (oxidative in the yeast and N₂ fixing in the cyanobacterium). The ultradian oscillations of dissolved O₂ and pH (CO₂) were observed in both organisms, accompanied by accumulation of carbohydrates in the anabolic/photosynthetic phase that was concluded by a burst of respiration, (see refs. 18 and 24 for yeast). Also, cell division occurred only in the anabolic/photosynthetic phase (see ref. 18 for yeast). In yeast, many genes participating in the regulation of metabolic processes were oscillating with the same ultradian period as the energetic-metabolic oscillations. In Cyanothece, the nitrogenase-related genes nifB and nifH were also clearly modulated by the ultradian rhythm, a feature consistent with the ultradian modulation of the nitrogenase activity. The homology between the metabolic ultradian oscillations in yeast and in cyanobacteria suggests that both organisms may have the same strategy of benefiting from the ultradian cycling in separating the mutually exclusive metabolic processes, anabolic or photosynthetic and catabolic or nitrogen-fixing reactions, and avoiding futile cycles (18, 27).

The finding of the ultradian metabolic cycle of Cyanothece also resonates with the recent report (2) that highlighted significant differences among six Cyanothece strains subjected to identical external cues. The differences were interpreted as indicating importance of metabolic signals that are specific for each strain rather than pure regulation by the circadian clock. An important question to ask in future research relates to the role of such metabolic signals and of the ultradian cycle in natural conditions. Cyanothece is a benthic organism that lives in mats or clusters on rocks in intertidal areas (28). Ultradian synchronization of alternating oxygenic and microoxic conditions in the Cyanothece-containing biofilms can be considered as a potentially important supplement to the circadian control in adjusting to actual temperature, light, and carbon availability in the environment.

Materials and Methods

Growth Conditions, Real-Time Measurements, and Sampling.

The Cyanothece sp. strain ATCC 51142 (28) was obtained from Jana Stöckel (Washington University, St. Louis), and maintained in complete ASP2 medium (29, 30) and buffered with TAPS and TAPSO as described earlier (16).

For growing and real-time monitoring of cultures, we used flat panel photobioreactors (FMT-150, Photon Systems Instruments), which were described in detail previously (19). Unless specified otherwise, the culture temperature was stabilized at 36.0 ± 0.3 °C, irradiance was 130–160 μmol photons m⁻²⋅s⁻¹ of red light (∼627 nm) supplemented with 25 μmol photons m⁻²⋅s⁻¹ of cool white or blue light (∼455 nm). A high concentration of dissolved CO₂ was generated by sparging with 0.5% CO₂.

For diurnal entrainment, the culture grown previously on a shaker in an Erlenmeyer flask was inoculated in a nitrate-free ASP2 medium in the photobioreactor with alternating 12-h light and 12-h dark periods. After the entrainment lasting at least 5 d, the lights were switched on continuously.

For ultradian metabolic cycles induced by removal of nitrate from the medium, the culture was first grown in photobioreactors in ASP2 medium with nitrate until the late exponential or early linear phase. The cells were harvested by centrifugation and resuspended in nitrate-free ASP2 medium. Cells continued to grow in continuous light without nitrate in batch or in turbidostat mode (OD₆₈₀ = 0.7). Aliquots for cell counting; quantification of chlorophyll, carbohydrate storage, total carbon, and total nitrogen content; and measurement of nitrogenase activity were collected every 2–2.5 h for 24–54 h. Concentrations of dissolved oxygen and CO₂ were measured by the photobioreactor in situ every minute as described in ref. 31.

Analytical Measurements.

Estimation of chlorophyll, carbohydrate, carbon, and nitrogen content and cell counts were done as described previously (16).

The nitrogenase activity was determined using the standard acetylene reduction method: Culture aliquots of 15 mL were incubated in gas-tight 40-mL volume glass vials with 8 mL of acetylene for 2 h in the light and temperature parameters that were used for growing. Then, 4 mL of headspace gas were assayed by gas chromatography. Potential ethylene contamination of acetylene was checked in 4 mL of gas measured immediately after the addition of the acetylene.

Quantitative RT-PCR scans were performed in a Mini-Opticon System (Bio-Rad). Normalized relative quantities (NRQ) and SEs of relative quantities were determined according to previous work (32). Expression levels of 16S and rpnA genes were used as endogenous controls for normalizing the abundance of nifB and nifH transcripts. Detailed information about RNA extraction, RT-PCR conditions is available in (SI Materials and Methods and Table S1).