Not so long ago, chronobiologists did not think that bacteria expressed circadian rhythms. These timing systems regulate myriad biological processes, including metabolism and gene expression, helping organisms adapt to environmental changes within the course of each solar day that involve shifts from light to dark as well as temperature and humidity cycles. Before the mid-1980s, investigators assumed that endogenous timekeepers with periods close to 24 h would not be useful to microorganisms, many of which divide more than once every 24 h.

That assumption might be stated as, "Why produce a timekeeper for a cycle that is longer than your lifetime?" This view holds each bacterial cell as the equivalent of an independent multicellular organism. However, another way to envision bacterial cells in a colony is as a mass of protoplasm that grows and only incidentally subdivides. From this perspective, a 24-h program for a microbial colony could be viewed as providing the means for this protoplasm as a whole to adapt to regular changes in its environment.

Consider photosynthetic cyanobacteria, whose metabolic links to the day-night cycle make them good candidates for having circadian programs. Indeed, during the mid-1980s, several research groups reported that particular cyanobacterial species express daily rhythms of photosynthesis and nitrogen fixation in both lightdark (LD) cycles and in constant light (LL). For example, Tan-Chi Huang and collaborators at Academia Sinica, Taipei, Taiwan, were the first to recognize that the unicellular freshwater cyanobacterium Synechococcus sp. RF-1 exhibits all three of the salient characteristics of circadian rhythms (see box at left).

Circadian Rhythms of Gene Expression and Cell Division in Cyanobacteria

Inspired by Huang, my colleagues and I began to study circadian rhythms in microorganisms while collaborating with several other research groups, including those led by Takao Kondo and Masahiro Ishiura at Nagoya University in Japan, and Susan Golden at Texas A&M University (now at the University of California in San Diego). Our early breakthrough was the application of luciferase reporters for the analysis of circadian programs in cyanobacteria.

For example, Golden and her collaborators had genetically transformed Synechococcus elongatus with a luciferase reporter (luxAB) under the control of a promoter for a gene encoding a component of the photosynthesis apparatus (psbAI). Together we assayed rhythmic gene expression noninvasively by following "glowing" cells in batch cultures (Fig. 1A). Meanwhile, Kondo and Ishiura developed a high-throughput screening system for isolating mutants whose rhythmic luminescence patterns vary. Later, Irina Mihalcescu at Universite´ Joseph Fourier-Grenoble I in St-Martin d'He`res, France, and Stanislas Leibler at Rockefeller University in New York, N.Y., determined that even single cyanobacterial cells exhibit exquisite luminescence rhythms (Figs. 1B & 1C).

The luminescence rhythms expressed by these transformed S. elongatus cells also fulfill all three key criteria of circadian rhythms (see box). Thus, several Synechococcus species exhibit circadian rhythmicity, displacing the dogma that microrganisms lack this capability. Prokaryotes were finally welcomed into the "circadian club." Cell divisions as frequent as once per 5-6 hours do not stop S. elongatus from keeping track of 24-h cycles, despite predictions of biologists that cells doubling faster than once per 24 hours would not express circadian clocks. Thus, the clock keeps ticking when a cell divides, with the two daughter cells retaining appropriate phasing (Fig. 1C).

Not only is the S. elongatus clock unperturbed, it apparently regulates the timing of cell division. In constant light when photosynthetic S. elongatus cells might be expected to divide rapidly and continuously, cell division is forbidden in the early night, and this forbidden phase recurs rhythmically (Fig. 1D). Therefore, not only does its circadian system keep an accurate pace in cells that divide two or three times per day, it rhythmically controls when cell division is allowed or forbidden. Apparently cyanobacteria simultaneously track two timing processes that express different periods, a circa- 24-h clock and cell division, which can be much faster.

How many genes do circadian clocks control? In eukaryotes, 10-20% of the genome is rhythmically expressed, as gauged by rhythms of mRNA abundance. Rather than measure mRNA abundances, my group along with those of Kondo and Golden monitored promoter activity with the luciferase reporter. We inserted the luxAB cassette into the S. elongatus genome randomly throughout the chromosome to identify promoter/enhancer regions that could turn on luciferase expression. Time-course analyses of the resulting luminescent cyanobacterial colonies indicated that the activities of essentially all promoters are rhythmically regulated (five examples are shown in Fig. 1A). Some of this measured difference between cyanobacteria and eukaryotes may be due to the fact that we assayed promoter activities, whereas typical eukaryotic studies have measured mRNA abundance.

It is not known how this global gene regulation in cyanobacteria is coupled to its central clockwork. Possible mechanisms include rhythmic activation of trans factors or rhythmic changes in the topology of the cyanobacterial chromosome. Indeed, supercoiling evidence is consistent with the chromosome of S. elongatus  undergoing massive circadian changes in topology (Fig. 1E). Further, DNA-binding dyes indicate that the chromosome rhythmically compacts and relaxes (Fig. 1F). We have suggested that the core circadian clockwork regulates gene expression by controlling chromosomal topology that, in turn, modulates torsion-sensitive transcription.

How Does the Microbial Circadian Clockwork Tick?

Using the S. elongatus luciferase reporter system to screen for clock gene mutants, Kondo and Ishiura in Japan, my group in Tennessee, and Golden's in Texas isolated several hundred mutants with altered circadian clock phenotypes. These mutants helped us to identify a three-gene cluster, kaiA, kaiB, and kaiC, with the name coming from the Japanese word kai, meaning rotation or cycle number. These genes encode the proteins KaiA, KaiB, and KaiC that are essential for clock function in S. elongatus and which constitute its core circadian oscillator. We found no significant similarity among the kai genes or between them and any reported genes in eukaryotes. However, there are potential homologs for the kai genes within genomes of many other eubacteria and archaea.

In the early years of analyses, the data on the cyanobacterial clock mechanism supported the hypothesis of a transcription-translation feedback loop, in which clock proteins regulate the activity of their own promoters through a process that resembles circadian clock loops of eukaryotes. Subsequently, however, several lines of evidence converged to indicate that transcription and translation were not necessary for circadian rhythms in this bacterium. The most dramatic demonstration was that the three purified Kai proteins can reconstitute a temperature- compensated circadian oscillation in vitro for at least 10 days, alternatively phosphorylating and dephosphorylating the clock protein KaiC (Fig. 2A) without any transcription and translation, reflecting the process that occurs in vivo (Fig. 2B). This example currently is the only one in which a circadian clock can be reconstituted in vitro from molecular components. Although the Kai proteins are competent to make this posttranslational oscillator, precisely how it is coupled to other components of this microbial circadian system is not known.

Visualizing the Kai Proteins, the Gears of the Microbial Circadian System

The cyanobacterial system is the only circadian system for which the molecular structures of all core component proteins have been solved. KaiC forms a hexamer that resembles a double doughnut with a central pore that is partially sealed at one end (Fig. 2C). This protein contains 12 ATP-binding sites. The residues in KaiC that are phosphorylated during the in vitro rhythm have been identified. Meanwhile, KaiA has two major domains and forms dimers in which the N-terminal domains are swapped with the C-terminal domains. KaiB was successfully crystallized from three different species of cyanobacteria and forms dimers or tetramers (Fig. 2C).

The three-dimensional structures of these proteins are helping us to understand how they interact and to develop models depicting how they are phosphorylated rhythmically. Briefly, KaiA associates with KaiC, promoting sequential autophosphorylation of KaiC on specific residues. Once KaiC becomes hyperphosphorylated, its conformation changes, allowing it to associate with KaiB and switch into an autophosphatase state. While KaiC dephosphorylates, its monomers exchange among the hexamers, synchronizing their phosphorylation status. When KaiC becomes hypophosphorylated, it dissociates from KaiB, and the cycle begins anew (Fig. 2D).

Gauging the Fitness Advantage of the Elaborate Clock System

Arhythmic strains of cyanobacteria (S. elongatus), fungi (Neurospora), and insects (Drosophila) grow and reproduce efficiently in the lab. Therefore, microorganisms and insects growing under laboratory conditions do not require their circadian clock systems.

Although circadian timekeepers are thought to enhance the fitness of organisms growing under natural conditions, Synechococcus is one of the few organisms in which that hypothesis has been rigorously tested. Thus, cyanobacterial strains that express particular circadian properties such as rhythmicity/ arhythmicity and altered periods, were mixed to determine which strain would out-compete its counterparts under different environmental conditions (Fig. 3A).

In such growth tests, strains with a functioning biological clock out-compete arhythmic strains in environments that have a rhythmic light/dark cycle, whereas in environments in which the light is held constant, rhythmic and arhythmic strains grow at comparable rates (Fig. 3B; sometimes the arhythmic strain even wins slowly). Further, among rhythmic strains with different periods, the strains whose endogenous period most closely matches the period of the environmental cycle out-compete strains whose period does not match that of the environment (Fig. 3C). Therefore, in rhythmic environments, the fitness of cyanobacteria is improved when the clock is operational and when its circadian period is consonant with the period of the environmental cycle.

Such testing provides the first rigorous demonstrations of a fitness advantage conferred by a circadian system, and our experiments inspired a later study in the plant Arabidopsis that used the same type of competition/mutant protocol. In S. elongatus, the mechanistic basis for these competition results is not known. The observation that various S. elongatus strains grow robustly in pure culture but can be outcompeted in mixed cultures suggests to us that cells communicate within mixed cultures, perhaps via secreted factors. In any case, cyanobacteria can benefit from their daily clock system, even though they divide more rapidly than once daily.

We are still faced with many questions to address, including why the period is 24 h and what maintains it at different temperatures; how the oscillator is entrained; how the in vitro oscillator is related to the in vivo system; whether rhythmic changes in chromosomal topology are part of the regulatory pathway or a consequence of rhythmic regulation; whether S. elongatus cells with circadian rhythms depend on secreted factors to out-compete otherwise comparable cells; and whether other types of bacteria have circadian systems that depend on all or some of the kai genes. The study of circadian clocks in bacteria has been fertile ground to till, and it will certainly continue to yield abundant harvests of new insights.

ACKNOWLEDGMENTS

I thank Takao Kondo, Susan Golden, Masahiro Ishiura, and members of their laboratory groups; who, together with our group, began the study of circadian clocks in S. elongatus and continue to make seminal contributions to this fascinating and unpredictable subject. I also thank my colleagues at Vanderbilt University who have helped to bring our understanding of the S. elongatus circadian system to its current level, especially Yao Xu, Tetsuya Mori, Mark Byrne, Martin Egli, Ximing Qin, Rekha Pattanayek, Sabuj Pattanayek, Phoebe Stewart, Dewight Williams, and Mark Woelfle.

The cyanobacterial clock project in my lab was supported by grants from the National Institute of General Medical Sciences at the National Institutes of Health, the National Science Foundation, and the Human Frontiers Science Program.

SUGGESTED READING

Ditty, J. L., S. R. Mackey, and C. H. Johnson (ed.). 2009. Bacterial circadian programs. Springer, New York.

Ishiura, M., S. Kutsuna, S. Aoki, H. Iwasaki, C. R. Andersson, A. Tanabe, S. S. Golden, C. H. Johnson, and T. Kondo. 1998. Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria. Science 281:1519-1523.

Johnson, C. H., M. Egli, and P. L. Stewart. 2008. Structural insights into a circadian oscillator. Science 322:697-701.

Kondo, T., C. A. Strayer, R. D. Kulkarni, W. Taylor, M. Ishiura, S. S. Golden, and C. H. Johnson. 1993. Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria. Proc. Natl. Acad. Sci. USA 90:5672- 5676.

Liu, Y., N. F. Tsinoremas, C. H. Johnson, N. V. Lebedeva, S. S. Golden, M. Ishiura, and T. Kondo. 1995. Circadian orchestration of gene expression in cyanobacteria. Genes Dev. 9:1469-1478.

Mackey, S. R., and S. S. Golden. 2007. Winding up the cyanobacterial circadian clock. Trends Microbiol. 15:381-388.

Mori, T., and C. H. Johnson. 2001. Independence of circadian timing from cell division in cyanobacteria. J. Bacteriol. 183:2439-2444.

Mori, T., D. R. Williams, M. O. Byrne, X. Qin, H. S. Mchaourab, M. Egli, P. L. Stewart, and C. H. Johnson. 2007. Elucidating the ticking of an in vitro circadian clockwork. PLoS Biology 5:e93.

Nakajima M., K. Imai, H. Ito, T. Nishiwaki, Y. Murayama, H. Iwasaki, T. Oyama, and T. Kondo. 2005. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science 308:414-415.

Woelfle, M. A., Y. Ouyang, K. Phanvijhitsiri, and C. H. Johnson. 2004. The adaptive value of circadian clocks: an experimental assessment in cyanobacteria. Curr. Biol. 14:1481-1486. 

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