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Key Words ATPase, biological clock, receiver domain, kai, signal transduction
Abstract Cyanobacteria such as Synechococcus elongatus PCC 7942 exhibit 24-h rhythms of gene expression that are controlled by an endogenous circadian clock that is mechanistically distinct from those described for diverse eukaryotes. Genetic and biochemical experiments over the past decade have identified key components of the circadian oscillator, input pathways that synchronize the clock with the daily environment, and output pathways that relay temporal information to downstream genes. The mechanism of the cyanobacterial circadian clock that is emerging is based principally on the assembly and disassembly of a large complex at whose heart are the proteins KaiA, KaiB, and KaiC. Signal transduction pathways that feed into and out of the clock employ protein domains that are similar to those in two-component regulatory systems of bacteria.
CONTENTS INTRODUCTION THE CYANOBACTERIA THE CIRCADIAN CLOCK A CYANOBACTERIAL CIRCADIAN CLOCK THE SYNECHOCOCCUS ELONGATUS PCC 7942 CIRCADIAN CLOCK The Model System: S. elongatus PCC 7942 The S. elongatus PCC 7942 Clock Proteins: KaiA, KaiB, KaiC TRANSCRIPTIONAL CONTROL IN THE S. ELONGATUS TIMING MECHANISM KAI OSCILLATOR INPUT: CIKA, LDPA, AND PEX KAI OSCILLATOR OUTPUT: SASA, CPMA, AND GROUP TWO SIGMA FACTORS DISCUSSION kai Genes and Circadian Clock Evolution A MODEL FOR S. ELONGATUS PCC 7942 TIMEKEEPING FINAL COMMENTS
A consequence of Earth's rotation about its axis once every 24 h is that the majority of organisms that populate her are subject to daily fluctuations in light and temperature. As a result, inhabitants of this planet, from bacteria to humans, have optimized their existence by evolving mechanisms to adjust to and anticipate daily changes in the environment (38, 130). Chronobiologists have developed various model systems to investigate circadian (24-h rhythmic) phenomena; this review focuses on the circadian mechanism that has evolved within the cyanobacteria, focusing on the model organism Synechococcus elongatus PCC 7942. A brief review of the cyanobacteria as a group and the broader implications of clock gene orthologs among these fascinating and widespread bacteria are discussed, as well as the present state of knowledge concerning the prokaryotic biochemical mechanism of timekeeping. In its revelation, the timing mechanism of the cyanobacterial circadian clock is unfolding as an intricate relationship between protein-protein interactions, protein modifications, and novel twists to traditional bacterial signal transduction pathways. Many players involved in every aspect of the S. elongatus circadian clock--from environmental input, to temporal output, and the oscillator itself--have been identified, but proposed models raise many questions that are still unresolved.
Cyanobacteria are a fascinating and exceptionally diverse group of photoautotrophic prokaryotes. Their genetic lineage is evidently among the oldest on Earth, as fossilized cyanobacterium-like organisms are present in 3500 Ma-old conglomeratic Apex chert (17, 145, 146). Oxygenic photosynthesis originated in those antiquated cyanobaeteria, and that distinctive metabolic activity was paramount to the creation of our present day oxygen-enriched atmosphere (80). There is remarkable genetic diversity among extant cyanobacteria as exemplified by comparing the mol% G + C content of genomes. For example, Nostoc species strain PCC 7524 has 39% G + C, Synechococcus elongatus (PCC 6301/7942) has 55% G + C, and Cyanobium species strain PCC 6707 has a genome with nearly 70 mol% G + C (158). The morphological diversity among cyanobacteria is also intriguing. Numerous species, including those within the Aphanacapsa, Chroococcus, Merismopedia, Synechocystis, and Synechococcus genera, grow as ovoid- or rod-shaped unicells ranging in diameter from 0.4 to 40 [micro]m (173). Some of these unicellular species can live as single ceils but also may remain, after cell division, in tightly grouped cell aggregates (127, 129). Unicellular species likely regulate this lifestyle choice based upon prevailing environmental conditions (129). These cell aggregates often appear highly organized, perhaps reflecting an underlying social order (53, 127). Other cyanobacterial species, such as those from the genera Anabaena, Lyngbya, Scytonema, Stigonema, Tolypothrix, and Trichodesmium, are long, thin, multicellular filaments commonly surrounded by a mucilaginous sheath. They are typically about 10 [micro]m in diameter and can be several hundred micrometers long (55, 149). Many filamentous species can form differentiated ceils including hormagonia (motile fragment of a cyanobacterial filament), akinetes (resting cyanobacterial spores), and terminally differentiated ceils called heterocysts, which develop under nitrogen-limited conditions and essentially function as anaerobic chambers for nitrogen fixation (46, 49, 180).
As would be expected from their long evolutionary history, genetic diversity, and morphological malleability, cyanobacteria are found in nearly every habitat that sunlight penetrates. Amazingly, a cave-dwelling Gloeocapsa species survives under light intensities as low as 1 lux (~0.02 [micro]mol photon [m.sup.-2] [s.sup.-1]) (27). Thermophilic cyanobacterial species have been isolated from geothermal hot springs throughout the world and have maximum growth temperatures ranging from 50[degrees] to 74[degrees]C (1, 45, 113, 114, 121). Mesophilic species are ubiquitous and have been isolated from most dry land ecosystems, including karst and travertine regions. They also flourish in benthic, limnetic, lotic, and pelagic fresh- and saltwater habitats (6, 21, 28, 42, 99, 123, 124, 126, 136, 143). Aquatic species, including Oscillatoria agardhii, Aphanizomenon flos-aquae, and Microcystis aeruginosa, find "a place in the sun" by regulating their buoyancy and relative position in the water column via production of proteinacous gas vesicles (7, 30, 31, 171). Aphanothece halophytica, Dactylococcopsis salina, Microcoleus chthonoplastes, and Spirulina major, among many other cyanobacterial species, are halotolerant if not true halophilic organisms. These or closely related species have been isolated from practically every known hypersaline environment (18, 32, 41). Psychrophilic species, such as Nodularia harveyana, Phormidum frigidum, and Rivularia minutula, are typically the predominant life forms in their low-temperature surroundings. Their seemingly inhospitable habitats include the tundra, ice shelves, glacial moraines, and polar desert soils of both the Arctic and the Antarctic regions (132, 148, 172). Both cold and hot desert cyanobacteria have an uncommon and astonishing ability to withstand multiple rounds of desiccation and subsequent rehydration (13, 131). As a general survival strategy in harsh environments, cyanobacteria have evolved to resourcefully create their own macroscopic, insular environments; these include such complex ecosystems as coastal tidal, hot spring and hypersaline microbial mats, the environmentally essential (and extremely sensitive) cryptobiotic desert crusts, and even fresh- and saltwater blooms (58, 115, 166).
The range of both growth rates and metabolic activities in cyanobacteria are also noteworthy. Typical freshwater Synechococcus species have doubling times of several hours, whereas cyanobacterial populations in the cold, oligotrophic, dry deserts of Antarctica may have doubling times of nearly 10,000 years (43, 116). Carbon dating in these polar regions supports one implication of this slow growth rate estimate by showing that living cyanobacterial cells can be over 1000 years old (16). Clearly, many interesting survival strategies have evolved in the cyanobacteria, and not all of them are passive. Cylindrospermopsis raciborskii, Hapalosiphon fontinalis, Hormothamnion enteromorphoides, Umezakia natans, and most of the aforementioned genera make a varieity of cyanotoxins as secondary metabolites (8, 60, 61, 72, 90, 181). These toxic metabolites are species-specific alkaloids, macrolides, and short linear or cyclic peptides that can be cytotoxic, hepatotoxic, or even neurotoxic to many organisms including mammals (35).
The primary metabolic activities such as photosynthesis, carbon and nitrogen fixation, and de novo vitamin and cofactor biosynthesis that have allowed cyanobacteria to inhabit practically every environment have also made them common participants in symbiotic associations. Species of the Calothrix, Cylindrospermum, Fischerella, and Nostoc genera form endophytic, epiphytic, and true symbiotic relationships with numerous plants, fungi, sponges, and protists (26, 34, 54, 59, 76, 137, 162, 176). It is generally accepted that modern plastids (including the land plant chloroplasts) evolved from a free-living cyanobacterium after its sequestration by a primordial eukaryotic-like cell (23, 98). The success of this particular 1 to 2 billion-year-old endosymbiotic event was evidently unique, as all extant plastids are considered monophyletic (34). Primary plastids, those directly descended from that first cyanobiont, still reside in rhodophyte, chlorophyte, and glancocystophyte algae (34). Evolutionary relationships among cyanobacteria and these plastids remain an intriguing and not well-resolved area of study (22, 23, 98, 107, 119, 134, 153, 163, 164).
Given their exceptional variety of form, faculty, and function--a spectrum that has only been hinted at in this short introduction--what biological properties help define the cyanobacteria? Cyanobacteria have an intracytoplasmic, thylakoid membrane used to house their photosynthesis machinery (77). [If exception makes the rule, then consider Gloeobacter violaceous PCC 7421, whose photosystems are located within the cytoplasmic membrane (138).] They also harbor both photosystem I and photosystem II and, thus, use water as a reductant during oxygenic photosynthesis (44, 183, 184). Cyanobacteria absorb light energy for photosynthesis by synthesizing and utilizing the chlorophyll a molecule, phycobiliproteins, and accessory phycobilin pigments like phycoerythrin, allophycocyanin, and phycocyanin (19, 103). High concentrations of these latter two pigments often make the organisms appear greenish-blue, leading to their previous designation as "blue-green algae" and current moniker of cyanobacteria. However, not all are blue-green in color, and the broadly distributed prochlorophyte species use chlorophylls a and b as antenna pigments and do not elaborate phycobilin antennae (83, 128). In addition to the accepted photosynthetic traits, we speculate here that the presence of circadian clock genes, and presumably a functional circadian clock, are also general cyanobacterial characteristics (96).
THE CIRCADIAN CLOCK
The circadian clock is an endogenous cellular mechanism that allows organisms to temporally regulate gene expression and thereby regulate complex biological processes as a function of time (38, 40, 130). Temporal organization of cellular functions allows organisms to capitalize on the environmental condition at any given time of day. General conceptual models for circadian systems involve three basic elements. The circadian oscillator is the entity directly responsible for keeping time on an approximately 24-h scale. Input pathways are the conduits for entrainment, a process that uses the daily reception of environmental stimuli to reset the circadian clock to local time every day. Output pathways couple timing information from the oscillator to downstream genes or other targets and allow the expression of subsequent circadian behaviors (Figure 1A, see color insert).
[FIGURE 1 OMITTED]
Three main characteristics define rhythms of behavior as a circadian process, as distinct from oscillations that are driven by environmental or specific metabolic cycles (Figure 1B). The first intrinsic characteristic is that circadian rhythms persist under constant environmental conditions, i.e., in the absence of a light/dark (LD), temperature, or humidity cycle, demonstrating an endogenous source for the rhythmicity. The persistent oscillation in the artificial constant condition is designated the free-run, and the period (peak-to-peak or trough-to-trough duration) of the free-running rhythm is characteristic of the timing mechanism of an individual organism's intrinsic clock. Second, circadian systems must be sensitive to an environmental stimulus to achieve entrainment with the sidereal day. The periods of free-running circadian rhythms are rarely 24 h precisely in length; daily entrainment is imperative so as not to fall out of phase with the light and dark cycles of an exactly 24-h day. A final criterion that distinguishes a circadian process is that the timing mechanism maintains its intrinsic periodicity at different constant temperatures within the organism's physiological range, a feature known as temperature compensation. This is characterized by the observation that at different ambient temperatures the rate of the timing mechanism varies only slightly, in contrast to simple biochemical reactions whose rates are greatly affected by changes in temperature (38, 40, 130).
These general characteristics of circadian rhythms are useful for chronobiologists in interpreting rhythmic observations. However, as rules, they are oversimplifications. Intrinsic periods vary depending on environmental conditions such as light intensity, as formalized in Aschoff's Rule (38). In general, diurnal organisms have shorter free-running periods as light Intensity increases, and the converse holds for nocturnal species (Figure 1C). Temperature compensation is often misinterpreted as temperature insensitivity. Circadian clocks are indeed sensitive to temperature via their input pathways. A temperature cycle can be as strong an entrainment cue as an LD cycle in some organisms (93). However, the biochemical mechanism of the oscillator accommodates changes in temperature, such that gross changes in period do not occur as would be observed for the rate of an enzymatic reaction over a similar range of degrees. Some buffeting occurs to keep the clock within an entrainable range no matter which climate on earth the organism finds itself inhabiting.
A CYANOBACTERIAL CIRCADIAN CLOCK
Physiological studies regarding the temporal separation of cyanobacterial nitrogen fixation and oxygenic photosynthesis were among the first to suggest that cyanobacteria have endogenous timing mechanisms (50). Many filamentous (multicellular) diazotrophic cyanobacteria, such as Anabaena sp. strain PCC 7120, develop microaerobic heterocysts to spatially separate oxygen-sensitive nitrogen fixation from oxygen-evolving photosynthetic metabolism (79, 175). The discovery of temporal separation arose from the question of how unicellular species, lacking the option of differentiation, could balance these seemingly incompatible processes. During the mid-1980s, a body of evidence …