The Rolex and the Hourglass: a Simplified Circadian Clock in Prochlorococcus?

Many cyanobacteria have a sophisticated circadian clock based on an elegant biochemical system requiring only three protein components: KaiA, KaiB, and KaiC (2). However, genome sequencing has revealed that the most abundant cyanobacterium of all, the marine cyanobacterium Prochlorococcus marinus, possesses KaiB and KaiC but lacks KaiA (9). In this issue, Axmann et al. show that the Prochlorococcus KaiC protein in vitro possesses an enzymatic activity suggesting that it could be a functioning component of a timing mechanism (1). Those authors also provide convincing evidence that Prochlorococcus once had KaiA but lost it relatively recently. They suggest that compensating mutations of KaiC allow the KaiBC system to retain some timing function in the absence of KaiA. Prochlorococcus appears not to have a robust circadian clock, but it may have a timing mechanism that runs for one diurnal cycle (4). Axmann et al. suggest that KaiB and KaiC form the basis for this simplified clock in Prochlorococcus (1). Their study raises fascinating questions about the function, origins, and evolution of a circadian clock in cyanobacteria and other phototrophs.

Why do cyanobacteria need a clock?

As phototrophs, cyanobacteria are absolutely dependent on sunlight. Their biochemistry and physiology must be attuned to the availability of solar energy. Key metabolic functions (e.g., oxygenic photosynthesis and nitrogen fixation) and major steps in the cell cycle (DNA replication and cytokinesis) may all be restricted to particular times of the day (13). One could imagine a primitive phototroph that controlled its physiology simply according to immediate circumstances. However, a circadian clock confers the crucial advantage that it allows the organism to anticipate future changes in light intensity: it can prepare for dawn before the sun comes up, and it can prepare for nightfall before the sun goes down. The most-studied cyanobacterial circadian clock is that of the model freshwater cyanobacterium Synechococcus sp. strain PCC7942 (2). This cyanobacterium, like many others, has a true circadian rhythm that persists for several days even in the absence of external cues after the cells are switched to growth in continuous weak light (2).

Biochemistry of the “classical” circadian clock of cyanobacteria.

The central timing mechanism of Synechococcus sp. strain PCC7942 is based on oscillations in the phosphorylation state of KaiC, induced by interactions with KaiB and KaiA (2). An extraordinary feature of this clock is that it works even in vitro. When KaiA, KaiB, and KaiC are placed into a test tube with a supply of ATP, sustained and temperature-compensated oscillations in the phosphorylation state of KaiC, with a period of approximately 24 h, ensue (6). The roles of KaiA and KaiB in this system are to modulate autophosphatase and autokinase activities of KaiC: KaiA promotes autokinase activity, while KaiB promotes autophosphatase activity. The way in which these interactions lead to circadian oscillations is still under discussion (2). It is likely that the period of the clock is set by another enzymatic activity of KaiC, a very weak ATPase activity (2, 11). In vivo, there are further “input” interactions that set the clock to the appropriate time zone and “output” interactions through which the clock controls chromosome compaction and patterns of gene expression (2). Biochemical and genetic approaches both show that all three Kai proteins are absolutely required for the central timing mechanism (2, 6).

Prochlorococcus is evolving fast.

Prochlorococcus marinus is a key player in the ecology of the planet and probably the most abundant photosynthetic organism on earth (8). It is the dominant phototroph in huge areas of the open ocean, predominantly nutrient-poor regions in the tropical and subtropical oceans (8, 9, 13). It is found from the surface down to depths of 100 to 200 m, with different ecotypes adapted to different depths. It has particularly small cells (about 0.5 μm in diameter) and a small genome with about 1,700 to 2,200 protein-encoding genes, depending on the strain (9). It might be expected that a highly successful organism with a simple phototrophic lifestyle living in a relatively stable environment would also be rather stable in evolutionary terms. Remarkably, however, genome sequencing of Prochlorococcus and its relatives revealed that Prochlorococcus is evolving into new niches right now. Prochlorococcus strains are clearly under intense selective pressure to reduce their genome size and have been through a recent process of genome reduction. This has involved the loss of perhaps several hundred genes, some so recently that their remnants can still be detected in the genome (9). The current work by Axmann et al. (1) and previous work by Holtzendorff et al. (4) provide convincing evidence that kaiA is a case in point. All sequenced Prochlorococcus strains retain kaiB and kaiC, but none has an intact kaiA gene. Nevertheless, several Prochlorococcus strains have remnant kaiA genes, suggesting a stepwise loss of kaiA during genome streamlining (1, 4).

Physiology and biochemistry of the Prochlorococcus diurnal cycle.

There is strong evidence for a diurnal cycle in several Prochlorococcus ecotypes, with DNA replication and cytokinesis occurring strictly once per day and always at the same time (13). However, it is less clear to what extent the diurnal cycle is under the control of a circadian clock. The best available data come from a previously reported study of Prochlorococcus sp. strain PCC9511 (4), which is genetically almost identical to strain MED4, used by Axmann et al. (1). Holtzendorff et al. performed a classic circadian experiment with Prochlorococcus sp. strain PCC9511 and its larger-genomed relative, Synechococcus sp. strain WH8102 (4). Cells were entrained to a diurnal cycle and then switched to growth in continuous weak light. Traditional indicators of the cyanobacterial diurnal cycle were monitored: DNA replication, cytokinesis, and mRNA levels for several key genes. Synechococcus sp. strain WH8102 (which retains KaiA) displayed circadian rhythms, in which 24-h oscillations remained detectable for several cycles after the switch to continuous light. In contrast, Prochlorococcus rhythms do not appear to persist after the switch to continuous light. Despite this, there is evidence for diurnal timing, for instance, with kaiB and kaiC mRNA levels peaking just before dawn (4). These results are consistent with a circadian timer that runs for about 24 h but no longer (Fig. 1). Unfortunately, Prochlorococcus is a very difficult organism to manipulate genetically (12). Therefore, Axmann et al. took the biochemical route, overexpressing the Prochlorococcus KaiB and KaiC proteins and studying them in vitro. They show that Prochlorococcus KaiC retains a feature that is thought to be important for circadian timing in Synechococcus: it has a weak ATPase activity that is modulated by KaiB. In Synechococcus sp. strain PCC7942, it is probably this activity that sets the period of the clock (2, 11). However, the Prochlorococcus KaiBC system does not show any potential for generating oscillations in KaiC phosphorylation. Instead, KaiC is permanently phosphorylated due to a constitutive autokinase activity. There is no sign of a competing KaiB-stimulated autophosphatase activity (1). Does the simplified system act only as a 24-h timer, more like an hourglass than a clock (Fig. 1)?

FIG. 1.

Tentative scheme for the control of the Prochlorococcus diurnal cycle by a 24-h timer triggered at sunrise. Circadian information was taken from Holtzendorff et al. (4), obtained for Prochlorococcus marinus PCC9511 in culture.

Why is an hourglass good enough for Prochlorococcus?

Prochlorococcus lives in a relatively simple, unchanging environment where it is unlikely to encounter unpredictable deep shade such as what freshwater cyanobacteria might experience due to vegetation or shifting sediments. Furthermore, it is not found at high latitudes (8) and therefore does not experience large changes in day length. In such an environment, it may be sufficient to have an hourglass. An organism that can rely on getting a time check once a day (most likely at dawn) will not need a clock that runs for longer than 24 h (Fig. 1). It may prove significant that the strains used by Axmann et al. (1) and Holtzendorff et al. (4) are high-light, near-surface ecotypes. It would be interesting to check whether deeper-living ecotypes (which will get a much weaker and less reliable time check at dawn) also have only 24-h timers.

Origins and evolution of clock genes in cyanobacteria and other phototrophs.

KaiC is clearly related to RecA, a universal prokaryotic DNA repair enzyme (2, 3, 10). KaiC retains DNA-binding activity, suggesting that the clock may have originated from a regulatory circuit controlling DNA recombination or chromosome compaction in order to minimize UV damage (10). Interestingly, connections between DNA damage, DNA repair, and circadian timing are now becoming apparent in otherwise very different eukaryotic circadian clocks (see reference 7, for example). While the full KaiABC system appears to be found only in cyanobacteria, genome sequencing projects have unearthed KaiBC pairs in other phototrophs only very remotely related to cyanobacteria, for example, purple bacteria and the green nonsulfur bacterium Chloroflexus (3, 5). If these organisms have KaiB and KaiC, why do they not have KaiA too? Purple bacteria and Chloroflexus have relatively large genomes; they do not appear to have been subjected to the drastic genome streamlining that has taken place in Prochlorococcus. One study suggested that cyanobacterial kaiB and kaiC were dispersed into other bacteria by horizontal gene transfer (3), but there are alternatives (Fig. 2). Note the interesting possibility that the simplified Prochlorococcus KaiBC timer may be a “throwback” to an ancient, widespread bacterial system.

FIG. 2.

Tentative proposal for the sequence of evolutionary events that generated KaiBC and KaiABC timing systems in phototrophic bacteria. (For relevant discussion of the question, see references 1, 3, 4, and 10). Another view is that the KaiBC system evolved in the cyanobacterial clade and then spread to other phototrophs by horizontal gene transfer (3). However, that work also proposed the origin of KaiA in cyanobacteria after the divergence of Prochlorococcus and marine Synechococcus species (3), which now seems implausible in the light of our current understanding of cyanobacterial evolutionary relationships and the presence of remnant kaiA genes in some Prochlorococcus strains (1, 4). The question of the origin and spread of KaiBC and KaiABC systems needs further examination.

Future directions for research.

Much remains to be learned about circadian rhythms and the roles of KaiB and KaiC in Prochlorococcus. Unfortunately, Prochlorococcus is a very difficult organism for mutagenesis and molecular genetics. Some genetic tools are becoming available, however (12), so there is hope of progress in this direction. Studies of the phosphorylation state of Prochlorococcus KaiC in vivo would also be interesting. A broader question concerns the role of KaiB and KaiC in purple bacteria and Chloroflexus. Little is known of the circadian biology of these phototrophic organisms, apart from an indication of circadian timing in a purple bacterium (5). Do they have either a 24-h timer or a robust circadian clock, and is timing provided by a KaiBC system? Further phylogenetic studies may give new insights into the origins of their kai genes: are they an ancient, conserved bacterial system, or have they spread from cyanobacteria by horizontal gene transfer?