circadian programs in cyanobacteria: adaptiveness and mechanism

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August 16, 1999 13:0 Annual Reviews AR089-13

?Annu. Rev. Microbiol. 1999. 53:389–409

Copyright c© 1999 by Annual Reviews. All rights reserved

CIRCADIAN PROGRAMS IN CYANOBACTERIA:Adaptiveness and Mechanism

Carl Hirschie Johnson1 and Susan S. Golden21Department of Biology, Vanderbilt University, Nashville, Tennessee 37235;e-mail: [email protected];2Department of Biology, Texas A&M University,College Station, Texas 77843; e-mail: [email protected]

Key Words clock, fitness,kai, competition, luciferase,Synechococcus

■ Abstract At least one group of prokaryotes is known to have circadian regulationof cellular activities—the cyanobacteria. Their “biological clock” orchestrates cellularevents to occur in an optimal temporal program, and it can keep track of circadiantime even when the cells are dividing more rapidly than once per day. Growth compe-tition experiments demonstrate that the fitness of cyanobacteria is enhanced when thecircadian period matches the period of the environmental cycle. Three genes have beenidentified that specifically affect circadian phenotypes. These genes,kaiA, kaiB, andkaiC, are adjacent to each other on the chromosome, thus forming a clock gene cluster.The clock gene products appear to interact with each other and form an autoregulatoryfeedback loop.

CONTENTS

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390What Is a Circadian Program?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390Discovery of Circadian Programs in Prokaryotes. . . . . . . . . . . . . . . . . . . . . . . . 390

Circadian Programs in Cyanobacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391Nitrogen Fixation/Photosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391Luciferase Reporters Illuminate a Path for Genetic Analyses. . . . . . . . . . . . . . . . 392The Cyanobacterial Clock Is Unperturbed by Rapid

Cell Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393Global Orchestration of Gene Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

Are These Clock Programs Adaptive?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396Competition as a Test of Fitness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396

Roles of Light and Dark. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398Genetic Dissection of This “Clockwork Green”. . . . . . . . . . . . . . . . . . . . . . . 399

Thekai Clock Gene Cluster. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399Other Clock Genes?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

Evolutionary Aspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402

0066-4227/99/1001-0389$08.00 389

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?390 JOHNSON ■ GOLDEN

Evolution of thekaiCGene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402Are There Circadian Timepieces in Other Prokaryotes?. . . . . . . . . . . . . . . . . . . . 403

Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406

INTRODUCTION

What Is a Circadian Program?

Circadian rhythms are endogenous biological programs that time metabolic and/orbehavioral events to occur at optimal phases of the daily cycle. They have threediagnostic characteristics. The first is that in constant conditions, the programsfree-run with a period that is∼24 h in duration. The second is that, in an appropri-ate environmental cycle (usually a light-dark and/or temperature cycle), the rhythmwill take on the period of the environmental cycle, that is, circadian rhythms willentrain to the environmental cycle. The final characteristic is that the period of thefree-running rhythm is nearly the same at different constant ambient temperatureswithin the physiological range; that is, circadian rhythms are temperature compen-sated. It is these three characteristics that define circadian rhythms, not the detailsof their biochemical mechanisms. Indeed, questions of considerable interest arewhether circadian mechanisms have evolved more than once and, if so, whethercompletely different biochemical processes have been harnessed to the task indifferent organisms. The fascination of circadian rhythms is how a biochemicalmechanism can keep time so precisely over such a long time constant (∼24 h) atdifferent ambient temperatures.

Before 1985, it was believed that circadian programs were exclusively a propertyof the eukaryotic domain (24). It was a reasonable assumption that rapidly growingprokaryotes would not have circadian organization, because it was thought thatan endogenous timekeeper with a period close to 24 h would not be useful toorganisms that divide more rapidly than once every 24 h, as do many prokaryotes.The assumption might be stated as, “Why have a timer for a cycle that is longerthan your lifetime?” Although intuitive, this conclusion is flawed. It is basedon the presumption that a bacterial cell is equivalent to a sexually reproducingmulticellular organism, which it is not. A bacterial culture is more like a massof protoplasm that grows larger and larger and incidentally subdivides. A mothercell does not die to make daughter cells; sheis the daughter cells. From thisperspective, it is reasonable that a 24-h temporal program could be adaptive torapidly dividing protoplasm if the fitness of that protoplasm changes as a functionof daily alterations in the environment (light intensity, temperature, etc).

Discovery of Circadian Programs in Prokaryotes

The proposal that prokaryotes might have circadian programs is not new. In the past35 years, bothEscherichia coliandKlebsiella aerogeneswere proposed to exhibitcircadian behavior (15, 54), but these studies were not persuasive (see below). Thatprokaryotic cells, either unicellular or multicellular, were too simple to express

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?CIRCADIAN PROGRAMS IN CYANOBACTERIA 391

circadian behavior became a dogma, despite the fact that there were almost nopublished reports of rigorous tests of the proposition (24).

Studies in the late 1980s on cyanobacteria began to change this mind-set. Itwas not researchers who were interested in the presence of circadian timekeepersin prokaryotes who initiated this research, but those who attempted to resolvehow some unicellular (or non-heterocystous, filamentous) cyanobacteria could fixnitrogen. Why was this an apparent dilemma? Photosynthesis evolves oxygen, andoxygen strongly inhibits the nitrogenase enzyme. How then can a photosyntheticunicellular organism fix nitrogen? Nitrogen fixation appeared to be doomed by theessential process that provides cellular energy. An imaginative idea to reconcilethese incompatible processes surfaced in the 1980s; photosynthesis and nitrogenfixation could be separated in time—photosynthesis in the day and nitrogen fixationat night [reviewed by Golden et al (12)]. The first data suggesting that this “temporalseparation” could be a metabolic program controlled by a circadian clock werefrom the non-heterocystous, filamentous cyanobacteriumOscillatoria sp. (52).Those authors found a nocturnal rhythm of nitrogenase activity that persisted incontinuous light (LL). Shortly thereafter, temporal separation of photosynthesis(in the day) and nitrogenase activity (in the night) was demonstrated in the marineunicellular cyanobacteriaSynechococcusspp. Miami BG 43511 and 43522 (39).This pattern continued in LL for at least 3 days, but these authors preferred tointerpret their data in terms of regulation by the cell division cycle rather than bya circadian clock (39).

Huang and coworkers were apparently the first to clearly recognize that cyano-bacteria were exhibiting circadian rhythms, and, in a series of publications begin-ning in 1986, they demonstrated all three salient properties in the same organism,the unicellular freshwaterSynechococcussp. RF-1 (6, 7, 13, 16–20). These pio-neers studied the rhythm of nitrogen fixation and of amino acid uptake in thiscyanobacterium, and were also the first to report the isolation of mutants affect-ing these processes (20). Another ground-breaking study was that of Sweeney &Borgese (55), who were the first to demonstrate temperature compensation of adaily rhythm in the marine cyanobacteriumSynechococcussp. WH7803.

We now know of circadian programs expressed in a number of cyanobacterialspecies. In addition to those already mentioned, circadian rhythms have beendemonstrated inSynechococcussp. strain PCC 7942 (32 and below), and thegeneraSynechocystis(2, 3),Anabaena(T Kondo & M Ishiura, unpublished data;35),Cyanothece(47, 48),Trichodesmium(5, 8, 42), and possiblyProchlorococcus(49). What about other prokaryotes? This issue is addressed later in this review.

CIRCADIAN PROGRAMS IN CYANOBACTERIA

Nitrogen Fixation/Photosynthesis

The yin/yang rhythms of nitrogen fixation and photosynthesis fit the expectationthat a major role of circadian timers is to temporally program metabolic eventsto occur at optimal phases of the environmental cycle. In addition to the work

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on Oscillatoria spp. andSynechococcusspp. Miami BG 43511/43522 and RF-1,another series of studies that supports the hypothesis of temporal separation hasinvestigated the marine unicellular cyanobacteriumCyanothecesp. strain ATCC51142 (9, 47, 48). A remarkable feature of this nitrogen-fixing cyanobacterium isthe presence of large carbohydrate granules within the cells, which are easily vi-sualized by electron microscopy. These granules accumulate progressively duringdaytime photosynthetic activity and dissipate during nocturnal nitrogen fixation(47).

There is, however, a counter-example to the temporal separation hypothesisamong nitrogen-fixing cyanobacteria: the non-heterocystous, filamentous cyano-bacteriumTrichodesmiumspp. fixes its nitrogen during the daytime, simultane-ously with photosynthesis (5, 42). How doTrichodesmiumspp. accomplish thisfeat? We don’t know, but it underscores the fact that cyanobacteria are capableof fixing nitrogen in the presence of oxygen and therefore that other mechanismsmust exist for lowering oxygen in the vicinity of nitrogenase (11). For example,Synechococcussp. RF-1 increases its aerobic respiration rate whenever nitrogenfixation is under way (14). Enhanced respiration may be an additional mechanismfor depleting oxygen in the vicinity of nitrogenase, even in cyanobacteria thatseparate photosynthesis and nitrogen fixation in time.

How does the circadian clock regulate the nitrogenase output rhythm? InSyne-chococcussp. RF-1,Cyanothecespp., andTrichodesmiumspp., there are circadianrhythms of nitrogenase messenger RNA (mRNA) abundance. As expected fromthe phasing of nitrogen fixation, inSynechococcussp. RF-1 andCyanothecespp.,the peak of nitrogenase mRNA abundance is nocturnal (9, 17), whereas inTri-chodesmiumspp., it is diurnal (8). These rhythms of mRNA abundance presumablydrive circadian rhythms of nitrogenase abundance and activity.

Luciferase Reporters Illuminate a Path for Genetic Analyses

Our interest in cyanobacteria as a model system for studying circadian programsstemmed from the genetic advantages that some cyanobacteria offer (12, 24, 33).The rhythms of nitrogen fixation, amino acid uptake, and carbohydrate content arereproducible, but the labor-intensive nature of the assays would dismay any butthe strong-hearted from using this type of rhythm for a mutant screen [althoughthe Huang group was undaunted and succeeded in isolating some mutants (see20)]. To reap the benefits that a genetically tractable prokaryote would offer, wesearched for a cyanobacterium that is amenable to molecular/genetic analyses andexhibits circadian rhythms of a parameter that can be assayed continuously formany cycles by an automated system.

The unicellular freshwater cyanobacteriumSynechococcussp. strain PCC 7942was a good candidate for this approach. Although PCC 7942 does not fix nitrogen,it has many advantages for genetic analyses: It is transformable by circular orlinear DNA, recombines at homologous sites, can receive DNA by conjugationfrom E. coli at high efficiency, can express reporter genes, and has a genome that

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is smaller than that ofE. coli (12). Based on these characteristics, many genetictools were developed (1).

We created a circadian reporter strain ofSynechococcussp. strain PCC 7942by transforming it with a construct in which theVibrio harveyi luciferase geneset luxAB is expressed under the control of the promoter for aSynechococcusphotosystem II gene,psbAI(32). This reporter strain was named AMC149. Theluminescence rhythm expressed by AMC149 in liquid cultures or from singlecolonies on agar medium is easily assayed by automated monitoring systems basedon either photomultiplier tubes (1, 32) or CCD (charge coupled device) cameras(29, 33). The luminescence pattern conforms to all three salient properties of cir-cadian rhythms: persistence in continuous conditions (LL) with a period closeto 24 h, entrainability by light/dark (LD) signals, and temperature compensation(32). The luminescence rises during the day and falls during the night. We foundthat the luminescence rhythm is an accurate reporter ofpsbAIgene expression(36), confirming our expectation that this rhythm reflects circadian control overthe promoter of thepsbAIgene.

The Cyanobacterial Clock Is Unperturbedby Rapid Cell Division

Cultures of AMC149 that are growing with doubling times as rapidly as one divi-sion every 6–10 h continue to exhibit circadian rhythms ofpsbAIgene expression(31, 40). The amplitude of the luminescence patterns reflectingpsbAIpromoteractivity is a function of the stage of the growth cycle for colonies on agar or forliquid cultures. For example, in early log phase, the luminescence patterns displaya clear circadian rhythm that grows in amplitude exponentially (Figure 1). This

Figure 1 Rhythmic gene expression in the unicellular cyanobacteriumSynechococcussp.strain PCC 7942. Generalized circadian patterns ofpsbAIpromoter activity monitored byluminescence of the reporter strain AMC149 are shown in three different phases of the growthcycle: exponential, sustained, and senescent.

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exponential-phase pattern can be precisely modeled by assuming only two essen-tial components: (a) a circadian rhythm ofpsbAIpromoter activity in every celland (b) an exponential increase in the number of cells in the culture (31). After thegrowth rate of the culture slows, the luminescence pattern stabilizes into a circa-dian pattern of consistent amplitude (sustained phase). It is in this growth phasethat the circadian oscillator can be most easily assayed. As the culture ages, therhythm slowly damps (senescent phase). The damping observed in the senescentphase probably results from nutrient depletion (including what is very importantto the photosynthetic cyanobacteria—diminution of light intensity by high celldensities).

Not only can the cyanobacterial clock keep track of circadian time in exponen-tially dividing cells, it can also gate cell division. InSynechococcussp. strain PCC7942 growing in LD cycles, division occurs only during the day. In LL, however,division is blocked by the circadian clock in the early subjective night phases andis allowed in the subjective day and late subjective night (40). Therefore, not onlydoes the clock keep track of circadian time in cells that divide two or three timesa day, it controls when that division is allowed (gate open) or forbidden (gateclosed). Using flow cytometry, we determined that the DNA replication rate andthe growth in size of each individual cell is not rhythmic over the circadian cycle,but it is the timing of division that is controlled by the circadian program (40). Inthe photosynthetic marine prokaryote genusProchlorococcus, there also appearsto be circadian control of division in rapidly growing cells (49). Together, thesedata highlight the fallacy of the bias described in the second paragraph of thisreview; cellular events can waltz to a circadian andante even when the cells arerapidly dividing in allegro.

Global Orchestration of Gene Expression

How many genes are controlled by the circadian clock in cyanobacteria? InSyne-chococcussp. RF-1, Huang et al (16) conducted a study of protein syntheticpatterns as a function of circadian time and found>10 polypeptides that exhibitcircadian rhythms of translation and are expressed in a variety of phase relation-ships. We therefore wondered how extensive circadian control of gene expressionis in Synechococcussp. strain PCC 7942 and devised a strategy to globally searchfor rhythmic control over promoters (38).

The bacterial luciferase gene set (luxAB) was inserted into theSynechococcussp. strain PCC 7942 genome so as to achieve random insertions ofluxABthrough-out the chromosome (38). We screened the luminescence expression patterns fromthe∼800 clones whose luminescence was bright enough to be easily monitored.Unexpectedly, the luminescence expression patterns of essentially all of these 800colonies manifested clear circadian rhythmicity. These rhythmic colonies exhibiteda range of waveforms and amplitudes, and they also showed at least two predom-inant phase relationships. We defined Class-1 genes as those whose expressionpeaks at the end of the day and Class-2 genes as those peaking at the end of the

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Figure 2 Luminescence patterns of the promoter activity of Class-1 and Class-2 genesas assayed with theluxAB reporter. The upper trace is from the Class-1 genepsbAI,whereas the bottom trace is from the Class-2 genepurF. Data were recorded in constantlight (LL), but the times of expected or subjective night are shown by the gray bars alongthe abscissa (from 37).

night (38). This promoter-trap experiment shows that circadian programming ofgene expression is pervasive in cyanobacteria.

Our original luminescent strain, which was genetically engineered with thepromoter ofpsbAI, is a Class-1 gene (peak at dusk; trough at dawn), as shown inFigure 2. The transcriptional activity of the reverse-phase Class-2 genes, however,is maximal at about dawn and minimal at about dusk (Figure 2). We have identifiedone of the Class-2 genes aspurF, which encodes a key regulatory enzyme in thede novo purine synthetic pathway, glutamine PRPP amidotransferase (37, 38).It is intriguing that glutamine PRPP amidotransferase is sensitive to oxygen, asis nitrogenase inSynechococcussp. RF-1, and that both of the genes encodingthese enzymes are expressed in the night or Class-2 phase. Therefore, even thoughSynechococcussp. strain PCC 7942 does not fix nitrogen, we hypothesize that theClass-2 expression pattern of thepurF gene is related to the oxygen sensitivityof glutamine PRPP amidotransferase and that it is another example of the same“temporal-separation” program exhibited by the expression of nitrogenase in someof the nitrogen-fixing cyanobacteria (25, 37).

Based on our results with the random promoter-trap experiment, we formu-lated a model that incorporates both nonspecific circadian control and circadian

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regulation by specifictransfactors (38). In the simplest version of the model, therewould be one (or a few) types of Class-1-specificcis elements turned on duringthe day by a Class-1-specifictrans factor. A different set of Class-2-specificciselements would be turned on at night by a Class-2-specifictransfactor, and so on.Because of the large number of genes that are apparently influenced by the clock incyanobacteria, however, it seems unlikely that each of them is controlled by a spe-cific regulatory factor. Some global factor(s) could be involved as well. In supportof our model for regulatory pathways that are specific for subsets of cyanobacte-rial genes, we discovered a gene whose altered expression significantly lowers theamplitude of the luminescence rhythm driven by some promoters (including thatof psbAI), but not of luminescence rhythms driven by other promoters (such asthat ofpurF). This gene encodes a sigma70-like transcription factor,rpoD2, andis a member of a family of sigma factor genes inSynechococcussp. strain PCC7942 (58). ApparentlyrpoD2 is a component of an output pathway of the circadianoscillator that affects the rhythmic expression of a subset of clock-controlled genesin Synechococcussp. strain PCC 7942.

ARE THESE CLOCK PROGRAMS ADAPTIVE?

Competition as a Test of Fitness

Although it is logical that circadian programming of the steps of gene expression,metabolic reactions, cell division, etc, could be adaptive, no rigorous test of theproposition had been performed in any organism, either eukaryotic or prokaryotic,prior to our recent work with cyanobacteria (41). We tested the value of circadianprogramming to reproductive fitness in cyanobacteria by using wild-type and mu-tant strains that exhibit different free-running periods (21, 33). The strains we usedwere C22a (period∼23 h), wild-type (period∼25 h), and C28a (period∼30 h).Both C22a and C28a have point mutations in thekaiC gene (see 21 and below).All three strains grew in pure culture at essentially the same rate in LL and inLD cycles, so there did not appear to be a significant advantage or disadvantageto having different circadian periods when the strains were grown in single-straincultures (41).

However, when we mixed different strains together and grew them in com-petition with each other, a remarkable pattern emerged that depended on (a) theendogenous period of each strain and (b) the period of the LD cycle. We testeddifferent LD regimens that had equal amounts of light and darkness but in whichthe frequency of the LD cycle differed: a 22-h cycle (LD 11:11), a 24-h cycle (LD12:12), and a 30-h cycle (LD 15:15). In each case, the strain whose period mostclosely matched that of the LD cycle eliminated the competitor (41). Poor fitnesswas not necessarily associated with mutant phenotypes—in fact, the mutant strainseasily defeated the wild-type if the mutant period was a better match to the LDcycle. These results were obtained for batch liquid cultures that were diluted every8 days and for cultures maintained at constant cell density in a turbidostat (41).

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Figure 3 Kinetics of competition between wild-type (period∼25 h) and the mutantstrain C28a (period∼30 h). Raw data (open-symbol points and heavy lines) of com-petitions in LD 12:12 vs LD 15:15 are compared with the results of modeling (thinlines) using the equation shown in the panel. (In LL, both strains do equally well.)Terms: w, relative fitness (wwt for wild type, wC28afor C28a);p, fraction of a givenstrain in the mixed population (pt for generationt; pt+1 for generation t+ 1). Ordinateis the percentage of colonies in the population that are C28a; abscissa is the estimatednumber of generations (from 41).

Estimation of the selective pressure by taking more time points suggested thatthe selective coefficient was surprisingly strong. As shown in Figure 3, a simplemodel that assumes (a) the selective pressure to be constant and (b) a lag in theinitiation of selection suggested to a first approximation that the relative fitnessof the less successful strain could be as low as 0.7– 0.8 (wwt ∼ 0.7 in LD15:15,wC28a∼ 0.85 in LD12:12). Because the growth rates of the strains in pure cultureare not different by a factor of 20–30%, the modeling result depicted in Figure 3indicates that we are observing a case of soft selection in which the poorer fitnessof inferior genotypes is most obvious under competition. We cannot rule out thatsmall, presently unmeasurable differences exist between the growth rates of thesestrains in pure culture, but soft selection seems to be the predominant mechanismresponsible for the strong selection under competition. The results are unlikelyto be caused by an unrelated mutation that is deleterious for growth because (a)mutant strains can outgrow wild types in the appropriate combination of biologicaland environmental periods and (b) a genetic test confirmed that only the differ-ences between thekaiCalleles of these strains are responsible for the competitiveadvantage/disadvantage (41).

How do the victorious strains win? Other than demonstrating that soft selec-tion is operating, we do not yet know the mechanism of the selection. We do

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know that the phasing ofpsbAIgene expression is disrupted within strains in non-optimal LD cycles (41). This is consistent with the idea that the circadian programorders cellular processes to optimally match environmental cycles; when this or-der is disturbed, fitness is reduced. We conclude that the circadian pacemaker incyanobacteria confers a significant competitive advantage when the period of theclock matches that of the environmental cycle, thus achieving optimal phasingof cellular events. This is the first rigorous demonstration in any organism of anadvantage conferred by a circadian system to fitness.

ROLES OF LIGHT AND DARK

One of the big three properties of circadian clocks is their ability to be entrained tothe environmental cycle. Entrainment means that the period of the biological clockbecomes equal to that of the environmental cycle. Light and dark signals are usuallyconsidered to be the primary signals that set the phase of circadian clocks. In someorganisms, the photopigments involved in circadian entrainment are known, suchas phytochromes, rhodopsins, or cryptochromes (10, 23, 43, 51, 53, 56). In manycases, however, the identity of the relevant circadian photopigments remains amystery (23, 43). The photopigments mediating the entrainment of cyanobacterialclocks fall into the latter group.

In Synechococcussp. strain PCC 7942, we have attempted to glean clues asto the identity of clock photopigments by action spectroscopy. Our preliminarydata suggest that blue and red light are most effective in setting the phase of thecyanobacterial clock, whereas green and far-red light are ineffective (T Kondo& C Johnson, unpublished observations). The phasing effect of red light was notreversed by far-red light, nor was the effect of blue light reversed by red light [asreported for light-regulated gene expression by Tsinoremas et al (57)]. The spec-trum does not coincide with that expected for photosynthesis in cyanobacteria, nordoes it fit with the behavior of a classically acting phytochrome. InSynechococcussp. RF-1, red light of 680 nm was also found to be an effective phasing agent,and far-red light of 730 nm did not reverse red-light phasing (7). These data sug-gest that there are specific, unknown pigments that are the eyes of the clock ofSynechococcusspp. strains PCC 7942 and RF-1.

Because almost all tests for circadian rhythmicity in cyanobacteria have usedLL as the constant condition, it is appropriate to ask whether this circadian clockrequires the presence of light to run. Most cyanobacteria are obligate photoau-totrophs, includingSynechococcussp. strain PCC 7942. In constant darkness (DD),all luciferase reporter strains derived from PCC 7942 show rapidly damped os-cillations in DD. Is this because the clock has stopped, or is it possible that thecentral timekeeper is still running and the outputs are turned off as an energy-saving device? One line of inquiry to answer this question inSynechococcussp.strain PCC 7942 used light pulses that can reset the phase of the clock as probesof the pacemaker’s phase in DD. The data suggested that the clock continued torun in this photoautotroph, even in DD (30).

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Is the presence of light a necessity for the unimpeded precession of the clock,or is it merely that a certain metabolic rate must be maintained for the clockto express itself? This question has been addressed in cyanobacterial speciesthat can grow heterotrophically. After a period of adaptation,Synechocystissp.strain PCC 6803 andCyanothecespp. can grow heterotrophically: on glucose forSynechocystisand on glycerol forCyanothece. Using thednaK::luxAB reporterstrain ofSynechocystissp. growing on glucose, Aoki & coworkers were able toshow that the luminescence rhythm persisted for many cycles in DD (3). Similarly,the rhythms of nitrogenase activity and carbohydrate content persisted robustly for>4 days inCyanothecespp. grown on glycerol (48). Therefore, in these speciesof cyanobacteria it is clear that the clockwork can run in DD if metabolism ismaintained and that the clock is not directly light dependent. It seems likely that,in the absence of other energy sources, DD is perceived as a signal to shut downunnecessary processes. But the central clock of photoautotrophic cyanobacteriaappears to have favored status and continues to run in DD unlinked to its energy-guzzling outputs—rather like an automobile engine idling in neutral gear.

GENETIC DISSECTION OF THIS “CLOCKWORK GREEN”

The kai Clock Gene Cluster

Using thePpsbAI::luxABreporter strain (AMC149) described above, we screened>500,000 clones ofSynechococcussp. strain PCC 7942 that had been treated withthe mutagen ethylmethanesulfonate. Over 100 mutants exhibiting various circadianphenotypes, including arhythmia, altered waveforms, and atypical periods (rangingbetween 14- and 60 h) were isolated (33). Most of these mutants grow apparentlyas well as the wild type and exhibit no other obvious phenotype besides circadiananomalies.

Efficient rescue of mutant phenotypes is possible inSynechococcusspp. bythe introduction of libraries of wild-typeSynechococcusDNA, and we succeededin rescuing>30 mutants of various phenotypes. DNA fragments from severalrescued mutants complemented other mutant phenotypes, including short-period,long-period, and arhythmic phenotypes. These rescue experiments allowed usto pinpoint a cluster of three adjacent genes, namedkaiA, kaiB, andkaiC (21;kai means “rotation” or “cycle” in Japanese). All of the mutants that have beencomplemented so far can be rescued by a plasmid carrying the entirekaiABCcluster, and 19 mutations were mapped by DNA sequencing to the threekai genes.All are missense mutations resulting from single nucleotide exchanges. Most ofthe mutations are recessive (rescue by wild-type DNA is complete), but a few aresemidominant, such as that of the 60-h-period mutant C60a. Each of the threegenes has at least two clock mutations mapped to it, and the largest gene,kaiC, hasmany mutations that include all the possible clock phenotypes: short period, longperiod, low amplitude, and arhythmia. No significant similarity was found amongthekaigenes and any other previously reported genes in prokaryotes or eukaryotes,except that there is a possible homolog of thekaiCgene among unidentified open

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reading frames in the genomic sequences of archaebacteria (see below). Moreover,there are two P-loop motifs in thekaiCgene. This motif, [G or A]XXXXGK[T orS], is a GTP/ATP nucleotide-binding region (46). ThekaiABCcluster appears to bea clock-specific region of the chromosome in cyanobacteria, because deletion ofthe entire cluster or of any one of thekai genes separately does not affect viability(in single-strain cultures), but does cause arhythmicity (21).

Figure 4 summarizes much of our knowledge about the relationships amongthese components (21). Promoter activities were found in the upstream regions ofboth thekaiA andkaiB genes. ThekaiA promoter gives rise to a monocistronic

Figure 4 Feedback model for the circadian oscillator ofSynechococcussp. strain PCC7942. Feedback of KaiA (positive) and a possible KaiABC complex (negative) on thekaiBCpromoter is depicted. ThekaiA gene is transcribed as a single transcript, whereas thekaiBandkaiCgenes are cotranscribed. Both thekaiAandkaiBCpromoters are rhythmically active(figure modified from 21). Because the KaiA, KaiB, and KaiC proteins interact, a multimericcomplex is shown (for convenience, homodimers are shown interacting to form a largercomplex).

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kaiAmRNA, whereas thekaiBpromoter produces a dicistronickaiBCmRNA. BothkaiAandkaiBCtranscripts are rhythmically abundant. Inactivation of any singlekaigene abolished these rhythms and loweredkaiBC promoter activity. Continuousoverexpression ofkaiCrepressed thekaiBCpromoter (negative feedback), whereaskaiA overexpression enhanced it (positive feedback). Overexpression of thekaiCgene for a few hours reset the phase of the rhythms. Consequently, the level ofKaiC expression is directly linked to the phase of the oscillation.

One clue to the mechanism of this clockwork is that the Kai proteins appear tointeract. Yeast two-hybrid and in vitro binding assays indicate that the KaiA, KaiB,and KaiC proteins interact both homotypically and heterotypically (22). One longperiod mutant exhibits an altered heterotypic interaction between KaiA and KaiB;this result suggests that inter-Kai contact is important to the clock mechanism(22). Using a novel method based on resonance energy transfer to assay protein-protein interactions, we have confirmed that KaiB polypeptides interact (59). Takentogether, these results suggest that there is negative feedback control ofkaiCexpression by the KaiC protein to generate a circadian oscillation in cyanobacteriainvolving protein-protein interactions and that KaiA sustains the oscillation byenhancingkaiCexpression.

The model is still very preliminary, however. It is likely that this feedbackmodel—which borrows many of its features from clock models of eukaryotes—is oversimplified. For example, the model implies that the Kai protein levels arerhythmic and that changes in Kai protein levels within the physiological rangewill elicit phase resetting. Mutations of the genes encoding Kai proteins that affectprotein-protein interactions should alter circadian properties. Does KaiC bind ATPand/or GTP, and, if so, does modification of the P-loop motif disrupt normal time-keeping? Are there other molecular players, such as Kai-interacting transcriptionalfactors? These and other predictions demand extensive testing.

Thekai genes apparently encode products that are crucial for the activity of aclockwork in cyanobacteria. But is this the only clock mechanism in cyanobacterialcells? This question cannot be dismissed lightly, because multiple clock mech-anisms have been discovered in the unicellular eukaryotic algaGonyaulaxspp.(44). So far, no concrete evidence indicates the presence of more than one clockmechanism inSynechococcusspp. For example, most mutations that affect the pe-riod of one reporter strain similarly affect the period of other reporter strains; forexample, the periods of thePpsbAI::luxAB, PpurF::luxAB, andPkaiBC::luxABreporter strains respond similarly to mutations inkaiABC (21; C Johnson & SGolden, unpublished data). Nevertheless, we must remain alert to the possibilitythat new data may indicate that even in simple prokaryotic unicellular organisms,multiple circadian oscillators could coexist.

Other Clock Genes?

One of the questions posed in the previous section was whether there are moregenes involved in the circadian mechanism ofSynechococcussp. strain PCC 7942.Although we have achieved saturation mutagenesis with ethylmethanesulfonate,

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we do not have enough pieces to fit the puzzle together yet. For example, iffeedback regulation of thekai cluster is crucial, then what are the transcriptionfactors? None of the Kai proteins has a DNA-binding motif. It is possible that a Kaiprotein complex interacts with a general transcription factor. We might not havediscovered this factor in our screens to date if it is an essential transcription factorthat confers a lethal phenotype when mutated. To identify other components in thecircadian clockwork, imaginative new screens (and possibly new mutagens) needto be tested. For example, transposon mutagenesis has identified a histidine proteinkinase gene whose inactivation causes a short-period phenotype (O Schmitz & SSGolden, unpublished observations). Another tactic is to search for Kai-interactingproteins by a yeast two-hybrid (or other) screen.

Yet another strategy to uncover new genes that encode components of the clock-work is to isolate extragenic suppressors. One suppressor that emerged from theinitial attempts to rescue clock mutations is the period-extender gene,pex (34).When the copy number of thepexgene is increased,pexlengthens the period ofwild-type and other strains by 2 h. Disruption ofpexshortens the period by 1 h,and overexpression lengthens the period by up to 3 h. No meaningful homologsto pexwere detected in DNA or protein databases (34). Screens that have beenspecifically designed to find other extragenic suppressors (e.g. by mutation of clockmutants to find altered phenotypes) have not yielded non-kai candidates, but thehunt continues.

EVOLUTIONARY ASPECTS

Evolution of the kaiC Gene

We have found strong candidates for homologs to thekaiCgene in the genomic seq-uences of the archaeaMethanobacterium thermoautotrophicum(50),Methanococ-cus jannaschii(4), Pyrococcus horikoshii(27), andArchaeoglobus fulgidus(28).As shown in Figure 5 in which the archaebacterial sequences are aligned roughlyend to end with those of thekaiCgene from the cyanobacteriaSynechococcussp.(21) andSynechocystissp. (26), the amino acid sequence identities between thecyanobacteria and these archaebacteria range between 25.7% and 34.2% over theentire length of the proteins. More important, there is conservation of the P-loopmotif GXXXXGK[T or S], which is a GTP/ATP nucleotide-binding region (46) andis indicated on Figure 5 by the double underlines. This motif appears to be highlyconserved among thekaiC genes of the cyanobacterial species and the putativekaiChomologs of the archaea. ThekaiChomolog ofMethanococcussp. is shorterthan the others, and the P-loop motif appears only once in theMethanococcussequence, whereas it appears twice in the sequences of all the other genes.

Prompted by considerations relating to the endosymbiotic theory of the originof eukaryotic organelles, we searched the databases for possiblekai homologs ineukaryotes. At present, no encouraging candidates have appeared. In particular,there are no apparent homologs in the chloroplast genomes of tobacco or other

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higher plants, nor is there any significant hybridization betweenkai DNA and thechloroplast genome of the eukaryotic algaChlamydomonas(Y Xu, S Surzycki &CH Johnson, unpublished observations). Therefore, there are no data that yet en-courage the view that the clockwork that evolved in cyanobacteria was transferredto eukaryotes by endosymbiosis or another mechanism of gene transfer.

Are There Circadian Timepieces in Other Prokaryotes?

As discussed in the first section of this review, it used to be thought that prokary-otic organization and lifestyle were incompatible with circadian clocks. As ad-dressed herein, we now realize that circadian programming is an integral part ofcyanobacterial organization and that this temporal program has adaptive value.But what about non-photosynthetic prokaryotes? This is a challenging question,with momentous implications for understanding the early evolution of circadianrhythmicity. The previous suggestion thatE. colimight have a circadian clock (15)was based on an old study (45) in whichE. coli cells were grown in rich nutrientmedium in an apparatus whose temperature control was almost certainly poor.Therefore, the possible daily trends noted by Halberg & Conner (15) are likely tobe merely a result of a diurnal cycle of temperature, to which the growth rate ofE. coli would be exquisitely sensitive. (Incidentally, there are no kai homologs inthe genome ofE. coli.) A later study that was more careful to control temperaturepurported to discover circadian rhythms of growth rate inKlebsiellaspp. (54), buta circadian trend is not obvious from the raw data and requires extensive statisticalanalyses to uncover. Moreover, the only circadian property addressed in that studywas an∼24-h oscillation; the other salient properties—temperature compensationand entrainment—were neglected. At this writing, there is no persuasive evidencethat circadian clocks reside in prokaryotes other than cyanobacteria.

Nonetheless, we think that there is a good chance that circadian clocks will befound in other eubacteria for which a daily timekeeper will enhance fitness. Thekeys are to find the proper conditions and to choose an appropriate parameter tomeasure. What about the third great domain of biology, theArchaea? Despite thefact that the ecological niche occupied by some archaebacteria, e.g. the halobac-teria, is not very different from that occupied by cyanobacteria [i.e. both live inan aqueous habitat and derive their energy from the daily sunlight cycle (in thecase of halobacteria, under anaerobic conditions)], there is no direct evidence thatarchaebacteria have invented an endogenous daily clock. In fact, it is not clearthat anyone has yet looked seriously for circadian clocks in archaebacteria. Theexistence of putativekaiC homologs inArchaeaencourages a concerted hunt forclocks, but we must also caution that theArchaeain which akaiC homolog hasbeen found are methanogens and/or extremophiles that inhabit environments forwhich an endogenous 24-h clock would not appear to be adaptive (e.g. deep-seamethane vents, hot springs, etc). Discovering circadian clocks in archaebacteriacould help us to understand much about the evolution of circadian rhythms.

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CONCLUDING REMARKS

Just as fruitfly pupae metamorphose and eclose as winged adults, so has the circa-dian clock field undergone a dramatic transformation in the past 10 years. Muchof that transformation is caused by the rapid progress on identifying clock com-ponents inDrosophila melanogaster, Neurospora crassa, plants, and mammals.More subtle transformations, but no less profound, include the dislodging of sev-eral entrenched dogmas, including the id´ee fixe that prokaryotes were too simpleto have complex timepieces and that rapidly growing cells cannot keep track ofcircadian time. Studies of cyanobacteria have single-handedly disposed of thesedogmas and have also provided the first rigorous demonstration of the fitness valueof circadian programming. We expect that, with the shattering of the prokaryoticbarrier, new insights into the distribution, mechanism, adaptiveness, and evolutionof circadian clocks will be forthcoming.

ACKNOWLEDGMENTS

We thank the members of our laboratories who allowed us to refer to their re-sults before publication and to our long-term collaborators, Dr. Takao Kondo andDr. Masahiro Ishiura, for a delightful and productive collaboration. Our researchhas been supported by grants from the NIMH (MH43836 and MH01179 to CHJ)and NSF (MCB-9219880 and MCB 9513367 to CHJ; MCB-9311352 and MCB-9513367 to SSG). NSF and JSPS supported a joint USA-Japan Co-operative Pro-gram grant for travel among our labs and those of Drs. Kondo and Ishiura (NSFINT9218744 and JSPS BSAR382). The Human Frontier Science Program fundscollaborative research of our labs with those of Drs. Kondo and Ishiura (grant#RG-385/96).

Visit the Annual Reviews home page at http://www.AnnualReviews.org

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Annual Review of Microbiology Volume 53, 1999

CONTENTSTransformation of Leukocytes by Theileria and T. annulata , Dirk Dobbelaere, Volker Heussler 1

Addiction Modules and Programmed Cell Death and Antideath in Bacterial Cultures, Hanna Engelberg-Kulka, Gad Glaser 43

Wolbachia Pipientis : Microbial Manipulator of Arthropod Reproduction, R. Stouthamer, J. A. J. Breeuwer, G. D. D. Hurst 71

Aerotaxis and Other Energy-Sensing Behavior in Bacteria, Barry L. Taylor, Igor B. Zhulin, Mark S. Johnson 103

In Vivo Genetic Analysis of Bacterial Virulence, Su L. Chiang, John J. Mekalanos, David W. Holden 129

The Induction of Apoptosis by Bacterial Pathogens, Yvette Weinrauch, Arturo Zychlinsky 155

Poles Apart: Biodiversity and Biogeography of Sea Ice Bacteria, James T. Staley, John J. Gosink 189

DNA Uptake in Bacteria, David Dubnau 217Integrating DNA: Transposases and Retroviral Integrases, L. Haren, B. Ton-Hoang, M. Chandler 245

Transmissible Spongiform Encephalopathies in Humans, Ermias D. Belay 283

Bacterial Biocatalysts: Molecular Biology, Three-Dimensional Structures, and Biotechnological Applications of Lipases, K-E. Jaeger, B. W. Dijkstra, M. T. Reetz

315

Contributions of Genome Sequencing to Understanding the Biology of Helicobacter pylori , Zhongming Ge, Diane E. Taylor 353

Circadian Rhythms in Cyanobacteria: Adaptiveness and Mechanism, Carl Hirschie Johnson, Susan S. Golden 389

Constructing Polyketides: From Collie to Combinatorial Biosynthesis, Ronald Bentley, J. W. Bennett 411

Giant Viruses Infecting Algae, James L. Van Etten, Russel H. Meints 447Mechanisms for Redox Control of Gene Expression, Carl E. Bauer, Sylvie Elsen, Terry H. Bird 495

Intercellular Signaling During Fruiting-Body Development of Myxococcus xanthus , Lawrence J. Shimkets 525

Clostridial Toxins as Therapeutic Agents: Benefits of Nature's Most Toxic Proteins, Eric A. Johnson 551

Viruses and Apoptosis, Anne Roulston, Richard C. Marcellus, Philip E. Branton 577

The Cytoskeleton of Trypanosomatid Parasites, Keith Gull 629

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circadian programs in cyanobacteria: adaptiveness and mechanism

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