genome streamlining results in loss of robustness of the circadian clock in the marine...

1. To whom all correspondence should be addressed: Laurence Garczarek, Station Biologique, BP 74, 29682 Roscoff cedex,France; e-mail: garczare@sb-roscoff.fr.

Cyanobacteria are the simplest photosyntheticorganisms and only prokaryotes that have beendemonstrated so far to possess a circadian clock (fora review on early cyanobacterial clock research, seeGolden et al., 1997). The cyanobacterial clock has

been shown to confer an adaptive fitness to the dielchanges of light and darkness (Ouyang et al., 1998;Roussel et al., 2000; Mori and Johnson, 2001). The cir-cadian clock induces rhythmic oscillation to diversecellular processes that are defined by a period length

Genome Streamlining Results in Loss of Robustness of the Circadian Clock in the Marine

Cyanobacterium Prochlorococcus marinus PCC 9511

Julia Holtzendorff,* Frédéric Partensky,* Daniella Mella,* Jean-François Lennon,*

Wolfgang R. Hess,† and Laurence Garczarek*,1

*Université Pierre et Marie Curie (Paris 06) and Centre National de la Recherche Scientifique (UMR 7144), Station Biologique, France, †Freiburg University,

Institute of Biology II, Experimental Bioinformatics, Freiburg, Germany

Abstract The core oscillator of the circadian clock in cyanobacteria consists of3 proteins, KaiA, KaiB, and KaiC. All 3 have previously been shown to beessential for clock function. Accordingly, most cyanobacteria possess at least 1copy of each kai gene. One exception is the marine genus Prochlorococcus, whichwe suggest here has suffered a stepwise deletion of the kaiA gene, together withsignificant genome streamlining. Nevertheless, natural Prochlorococcus popula-tions and laboratory cultures are strongly synchronized by the alternation ofday and night, displaying 24-h rhythms in DNA replication, with a temporalsuccession of G1, S, and G2-like cell cycle phases. Using quantitative real-timePCR, we show here that in Prochlorococcus marinus PCC 9511, the mRNA levelsof the clock genes kaiB and kaiC, as well as a few other selected genes includingpsbA, also displayed marked diel variations when cultures were kept under alight-dark rhythm. However, both cell cycle and psbA gene expression rhythmsdamped very rapidly under continuous light. In the closely relatedSynechococcus sp. WH8102, which possesses all 3 kai genes, cell cycle rhythmspersisted over several days, in agreement with established cyanobacterial mod-els. These data indicate a correlation between the loss of kaiA and a loss ofrobustness in the endogenous oscillator of Prochlorococcus and raise questionsabout how a basic KaiBC system may function and through which mechanismthe daily “lights-on” and “lights-off” signal could be mediated.

Key words Prochlorococcus, marine Synechococcus, circadian clock, kaiA, cell cycle, geneexpression

of about 24 h, the persistence of the rhythmic oscilla-tion under free-running conditions (such as continu-ous illumination), and “temperature compensation,”that is, that period length is constant under a range oftemperatures. Circadian rhythms in cyanobacteriawere first evidenced in Synechococcus RF-1, where a 12-h dark block entrains a rhythmic diurnal dini-trogen fixation pattern that persists for at least 4 dayswith decreasing magnitude in continuous light(Grobbelaar et al., 1986). Using the model strainsSynechococcus elongatus PCC 7942 (Kondo et al.,1993), Thermosynechococcus elongatus BP-1 (Onai et al., 2004), and Synechocystis sp. PCC 6803 (Aoki et al.,1997), it was actually shown that the endogenoustiming mechanism generates and maintains a 24-hperiodicity to global gene expression patterns (Liu et al., 1995), as well as cell division (Mori et al., 1996)and chromosome compaction (Smith and Williams,2006). In all these organisms, the central circadianoscillator is formed by 3 clock proteins, KaiA, KaiB,and KaiC, all essential to the generation of circadianrhythms (Kondo et al., 1994; Ishiura et al., 1998).

Since the discovery of the kai operon (Kondo et al.,1994), models of the cyanobacterial clock are in con-stant evolution. The core oscillator is fully functional invitro. Incubation of KaiA, KaiB, and KaiC from S. elon-gatus PCC 7942 in the presence of ATP results in a cir-cadian rhythm in the KaiC phosphorylation state(Nakajima et al., 2005) and suggests that oscillationoccurs solely as a result of interactions between the 3 Kai proteins, driving rhythmic changes in thephosphorylation level of KaiC. This protein is phos-phorylated at 2 sites, first on a threonine residue, then on a serine. Subsequently, the threonine isdephosphorylated, resulting in a form of KaiC that isonly serine-phosphorylated (S-KaiC). Then, the serineis dephosphorylated and KaiC returns to an unphos-phorylated state. The protein KaiA is essential for thisprocess, since this cyclic phosphorylation and dephos-phorylation becomes oscillatory through a double-negative feedback loop that toggles between 2 statesdepending on whether the concentration of free KaiAis high or low. In particular, S-KaiC binds stoichiomet-rically to both KaiA and KaiB (Rust et al., 2007). Theformation of the KaiABC complex prevents KaiA fromactivating KaiC phosphorylation. Thus, when S-KaiCconcentration is high, KaiA is sequestered by S-KaiCand KaiB, and KaiC dephosphorylation predominates;when S-KaiC concentration is low, KaiA is released andKaiC phosphorylation is activated.

Although the presence of a circadian clock hasbeen demonstrated in only a few freshwater strainsat the molecular level, it is generally assumed that allcyanobacteria possess such a clock. However,genome analysis of several different strains ofProchlorococcus, the smallest and most abundant pho-tosynthetic organism on earth, has suggested thatmembers of this genus may lack the kaiA gene,whereas kaiBC are present (Rocap et al., 2003).Natural populations of Prochlorococcus are highlysynchronized by the daily alternation of light anddarkness, displaying diel pattern of cell cycling andgene expression (Garczarek et al., 2001; Holtzendorffet al., 2001; Holtzendorff et al., 2002), and this couldbe taken as a hint for the occurrence of a circadianclock. However, preliminary physiological studiestackling this hypothesis have provided only confus-ing evidence for such a clock (Jacquet et al., 2001b).

In the present article, we use gene expression ofthe psbA gene (encoding the D1 protein of the photo-system II reaction center core), in addition to cellcycle studies, as a marker to test the presence of cir-cadian oscillation under free-running conditions incontinuous cultures of P. marinus PCC 9511 and we compare this to the situation in the closely relatedmarine Synechococcus sp. WH8102 that possesses all 3 kai genes. Although the psbA gene is a widelyaccepted marker for studying the cyanobacterial cir-cadian rhythm (Kondo et al., 1993), the cell cycle ofProchlorococcus lends itself to the same purpose dueto its eukaryotic-like phase separation. The G1-like(occurrence of 1 chromosome), S-like (DNA replica-tion), and G2-like (occurrence of 2 chromosomes)phases can be easily monitored by flow cytometry(Binder and Chisholm, 1995). For the sake of simplic-ity, the cell cycle phases will be named G1, S, and G2hereafter.

In synchronized cell populations, the cell cyclephases are restricted to certain times during the light-dark (LD) cycle and cells divide only once per day(Vaulot et al., 1995). This bimodal cell cycle has beenstudied extensively in Prochlorococcus, but it has alsobeen observed in some marine Synechococcus strains(Sweeney and Borgese, 1989; Binder and Chisholm,1995; Binder, 2000; Jacquet et al., 2001a). In contrast,the commonly used (freshwater) model strains oftenpossess multiple chromosomes that replicate asyn-chronously and divide several times per day (Binderand Chisholm, 1990; Mori et al., 1996; Mori andJohnson, 2001).

188 JOURNAL OF BIOLOGICAL RHYTHMS / June 2008

MATERIALS AND METHODS

Cyclostat Design and Cell Sampling from theContinuous Cultures

All our culture experiments were run in a custom-made “cyclostat,” a modification of the illuminationsystem described by Bruyant et al. (2001). In brief, itconsisted of 2 vertical rows of 10 fluorescent U-shapedtubes each (OSRAM Dulux L 2G11, 55 W/12–950, day-light) located on both sides of the water tank, and con-nected to dimmable, electronic ballasts (OSRAM,Quicktronic, QT1 55/230–240 DIM). Culture flaskswere set in a 50-L glass tank filled with water kept at22 °C by a cooling bath (GE Healthcare) and placedbetween the 2 rows of neon tubes. The whole setupwas enclosed in a ventilated cupboard to avoid any light contamination during the dark period. Thephotoperiod length and shape as well as maximumgrowth irradiance were computer controlled usingcustom-made software written in Visual Basic 6.0. Thesame software also controlled a sampler automaticallycollecting every hour 2 mL volumes (maintained inthe dark at 4 °C for a maximum of 6 h before fixationwith 0.2% glutaraldehyde (Sigma Aldrich), freezing inliquid nitrogen, then transfer to –80 °C) for flowcytometry analyses.

All cultures were grown in PCRS11 medium(Rippka et al., 2000), and synchronized by exposureto LD cycle for at least 2 weeks before shifting themunder continuous irradiance.

Experimental Design

A preliminary, short-term experiment in batch cul-ture, mainly aiming at studying gene expression ofselected genes during LD cycles, was performed with2-L axenic cultures of P. marinus PCC 9511 grown at22 °C under an LD cycle with light irradiances vary-ing in a sinusoidal way between 0 and 500 μmolquanta.m–².s–1. The last dilution with fresh PCRS11medium was made 2 days before sampling started.During the experiment, 80 mL were sampled at 2-hintervals for RNA extraction and cell cycle analysis.

A second, long-term experiment aiming at studyingcell cycle and psbA expression of P. marinus PCC 9511under free-running conditions was made with contin-uous input of fresh PCRS11 medium, to maintain cellsin exponential growth throughout the samplingperiod. For this experiment, PCC 9511 cells were

grown in 8-L polycarbonate flasks (Nalgene) under 12-h:12-h LD rhythms with growth irradiance varyingfrom 0 to 1000 μmol quanta.m–².s–1 before being shiftedto a constant illumination of 350 μmol quanta.m–².s–1,corresponding to a similar integrated light dose per 24-h period as the modulated LD cycle. Flasks wereequipped with 5 separate sterile tubing for 1) airexchange (through a sterile 0.2-μm filter), 2) cell sam-pling for flow cytometry, 3) cell sampling for RNA, 4)addition of fresh culture medium, and 5) elimination ofexcess culture volume to a waste bottle. By daily adap-tation of the dilution rate to the cell density (with anaverage rate of 1.2 mL/min), an average cell density(before cell division) of about 1.1 108 cells mL–1 wasmaintained throughout the experiment. Sampling forRNA (150 mL from each replicate culture) was per-formed manually every 3 h from 0600 to 1800 h andevery 4 h from 1800 to 0600 h into 50-mL tubes thatwere immediately centrifuged for 10 min at 4 °C at 10,000 rpm (7,267 × g). The supernatant was dis-carded and cells were resuspended in 1 mL of freshPCRS11 medium, centrifuged for 1 min at 12,000 rpm(15,294 × g), and resuspended again in 0.5 mL of Trizol(Invitrogen), then frozen at –80 °C until extraction.During dark phases, cells were manipulated underfaint green light (i.e., inactinic light).

A third, long-term experiment was made to studythe cell cycling of Synechococcus sp. WH8102 underfree-running conditions. Duplicate 750-mL culturesof this strain were grown in 2-L polycarbonate flasks(Nalgene) under modulated 12-h:12-h LD rhythms,varying from 0 to 150 μmol quanta.m–² s–1 beforebeing shifted to continuous illumination of 55 μmolquanta.m–².s–1.

Flow Cytometry

Flow cytometric analysis was performed asdescribed in Marie et al. (1999). For cell cycle analy-sis, cells were diluted in 10 mM Tris (pH 8) digestedwith DNA-free RNaseA for 1 h at 37 °C, prior tostaining for 1 h at room temperature with SYBR-Green I, Molecular Probes. Samples were analyzedusing a FACScan (Beckton Dickinson, San Diego, CA)with a flow rate of 25 mL.min–1 for 4 min to analyzeapproximately 20,000 cells. Samples were collected aslistmode files and analyzed using Cytowin 4.1(Vaulot, 1989). The percentages of the cell cyclephases were further computed using MCYCLE (P.S.Rabinovitch, Phoenix Flow Systems, San Diego, CA).

Holtzendorff et al. / THE CIRCADIAN CLOCK OF PROCHLOROCOCCUS LACKS ROBUSTNESS 189

RNA Extraction, Reverse Transcription, andQuantitative PCR

Frozen cells in Trizol were thawed for 15 min in awater bath set at 65 °C, incubated for 15 min andcooled to room temperature. Cell suspension was thenmixed for 5 min. with 0.1 mL of chloroform. Phaseseparation was done by centrifugation at 9,000 × g for15 min at 4 °C. The aqueous phase was submitted toa DNase treatment using Qiagen columns followingthe manufacturer’s instructions.

Primers for reverse transcription and real-time PCR(RT-PCR; Table 1) were designed using Primer Express(Applied Biosystem, v2.0). The cDNA was obtained byreverse transcription of 100 ng of RNA and 4 pmol ofthe reverse primer. RNA was denatured for 10 min at70 °C, before a mix of SuperScriptII (Gibco-BRL), 1×reaction buffer, 10 μM dithiothreitol (DTT), and 0.25mM of each dNTP was added. The reaction was incu-bated at 42 °C for 50 min followed by cDNA denatura-tion at 72 °C. RT-PCR was done on a BiosystemsGeneAmp 5700 (Applied Biosystems) using the SYBRGreen PCR master mix (Applied Biosystems) and 10-to 10,000-fold dilutions of the reverse transcripts.Reactions cycles were as follows: 10 min at 95 °C fol-lowed by 40 cycles of 15 sec at 95 °C and 1 min at 60 °C.Data were normalized using the ΔΔCT method asdescribed in the Applied Biosystems user bulletin #2(http://dna-9.int-ed.uiowa.edu/RealtimePCRdocs/Compar_Anal_Bulletin2.pdf).

RESULTS

Reduction of the kai Operon in Prochlorococcus

Although Prochlorococcus was previously describedas missing the kaiA gene (Rocap et al., 2003), the nowavailable data from more than 20 genome sequences

of this genus and of the closely related marineSynechococcus spp. indicates a stepwise deletion of kaiA.All marine Synechococcus have an intact kaiABC operonon the reverse complementary strand upstream of theribosomal protein operon starting with rplU (rpl21),similar to the freshwater model strain S. elongatus PCC7942 (Fig. 1). In contrast, in all Prochlorococcus but 2 strains, a dicistronic kaiBC operon is present at a genomic location that is otherwise syntenic.Intriguingly, Prochlorococcus spp. MIT9313 and MIT9303contain at the expected position a reading frame thatmay be a relic kaiA truncated to ~15% of its original size,or a kaiA pseudogene (Fig. 1). As judged by their posi-tion in a 16S rRNA-based phylogenetic tree (Fig. 1), aswell as in a consensus tree based on the Prochlorococcuscore genome (Kettler et al., 2007), MIT9313 andMIT9303 strains form the earliest-branching cladewithin the genus Prochlorococcus. Furthermore, thesestrains stand out by their genome sizes (2.4 and 2.6Mbp, respectively; Rocap et al., 2003; Kettler et al.,2007), which are similar to that of marine Synechococcus(Palenik et al., 2003), whereas other Prochlorococcus havebeen subjected to a strong genome streamlining(Dufresne et al., 2005; Kettler et al., 2007). It is also worthnoting that in both strains, several conserved aminoacid motifs (for instance, ALFAA) are located upstreamof the truncated kaiA reading frame, separated from itby 2 stop codons (Fig. 1B). At last, the rplU-kaiA inter-genic spacer is much longer in these strains (458nucleotides [nt] in MIT9313, compared to 238 nt in S. elongatus PCC 7942, or only 146 nt in P. marinusMED4). We conclude that the kaiA gene is most likely inthe process of being lost in this group of cyanobacteria.

Thus, 2 questions arise: What are the functional con-sequences of a natural kaiA deletion that must haveoccurred at a relatively short evolutionary time scale?And if functional, what adaptive processes have takenplace in the circadian system of Prochlorococcus as a con-sequence?

Expression of kai Genes in Synchronized P. marinus PCC 9511 Cells

To study patterns of kai gene expression, a synchro-nized P. marinus PCC 9511 batch culture was sampledevery 2 h during 1 LD cycle for flow cytometric analy-sis and RNA extraction. From late night until lateafternoon, the culture remained in the G1 phase (Fig.2A). Chromosome replication was initiated 2.5 hbefore sunset and reached a maximum around the LDtransition, followed by a maximum of cells in the G2phase 4.5 h after sunset. Most cells divided during thefirst 9 h of the dark period, as shown by the increase in

Table 1. Primers used for RT-PCR analysis.

Primer Sequence

ftsZ Fw CTTGGGGCAGGGGGCAATCCAAGftsZ Rev TAACTCCTCCCATACCC

psbA Fw TTTCCAGGCAGAGCACAACApsbA Rev ATCCTCCGAACATACCTGCAA

psaB Fw GATCAGGTATTACCGAAGCAATGApsaB Rev AATGATATAAGCCTGAATAAGCTATATT

pcb Fw GGGCTGGTAATTCAGGTGTAGCTpcb Rev CCCTGCATGAGCGACATG

rbcL Fw GCCAAGAAGGTGTCCCAAGArbcL Rev TCGACCAAGTACCAGTAGAAGATTCA

NOTE: RT-PCR = reverse transcription and real-time PCR.

the percentage of cells in G1 during this period. Underthese conditions, kaiB and kaiC mRNA levels closelyfollowed each other (Fig. 2B), increasing during thenight and reaching their maxima toward the end of thedark period with mRNA amounts displaying 5- to 10-fold changes. In a previous study in P. marinus MED4,a strain genetically quasi-identical to PCC 9511(Rippka et al., 2000), the transcription initiation site ofkaiB and kaiC genes was mapped to a single promoterin front of kaiB (Vogel et al., 2003). Together with theexpression data obtained in the present study, thisindicates that both genes are probably transcribed inthe form of a dicistronic operon. The minor differencesobserved in gene expression patterns may result fromslightly different posttranscriptional stability of kaiBand kaiC mRNAs.

We also analyzed the expres-sion of the purF gene, encoding apurin synthetase involved inDNA synthesis. Maximum purFmRNA amounts were detectedat the end of the light period,coinciding with the S phase (Fig.2C). For control, we tested theexpression of dnaA, encoding theinitiator protein of chromosomereplication, and psbA, coding the D1 protein (Fig. 2C). Asshown previously, dnaA mRNAamounts reached a maximumduring the replication phase(Holtzendorff et al., 2001) andpsbA mRNA levels were highestat noon (Garczarek et al., 2001).

Sigma factors have beenshown to be involved in the out-put of the circadian clock in S.elongatus PCC 7942 (Tsinoremaset al., 1996; Nair et al., 2002). Twogenes encoding potential sigmafactors in MED4, PMM1629 andPMM1697, displayed mRNAmaxima at or near the LD and DLtransitions, respectively (Fig. 2D),suggesting their possible rele-vance for the regulation of genesexpressed either during light ordark phase. Although PMM1629might be a homolog to the Syne-chocystis sp. PCC 6803 sigmafactor sigB (sll0306) since tran-scripts of both genes substantiallyaccumulate upon entry into thedark phase (Gill et al., 2002) and

cluster in phylogenetic analysis (data not shown), itwas not possible to make a similar assignment forPMM1697.

Prochlorococcus Cell Cycling and psbA GeneExpression under Free-Running Conditions

As master regulator, the Kai oscillator also controlsparts of the cell cycle in S. elongatus and determinesphases where replication and division are allowed orblocked (circadian gating; Mori et al., 1996; Mori andJohnson, 2001). Therefore, we used the distinct G1, S,and G2 phases of the Prochlorococcus cell cycle as amarker to test for the circadian clock activity underfree-running conditions and analyzed in parallel the

PCC7942 : AFFADVPVTKVVEIHM ELMDEFAKKLRVEGRSEDILLDYRLTLIDV IA HLCEMYRRSI PRET*MIT9303 : ALFAD* SIT * VVEIHIDLIDAFWQQFKLELHERDSS SGVLFALIDLS NHLWVMLQRWLSAEVPL SLISISSDLEDASEMQL *MIT9313 : ALFA A* SIT * FVEIHMDLIDAFWQQFRLELHER ESSPGVLFALLNLS KHLWAMLQHSLSAEVPL R*

(aa 284;

( 44 nt kaiB )

(−4 nt kaiB)

P. marinus

MED4/PCC 9511

Prochlorococcus

marinus SS120

Prochlorococcus sp.

MIT9313

Prochlorococcus sp.

MIT9303

Synechococcus sp.

WH7803

Synechococcus sp.

WH8102

Synechococcus sp.

PCC 7942

458rplUrpmA kaiC

460rplUrpmA kaiC

kaiA238

rplUrpmA kaiB kaiC

kaiArplUrpmA kaiB kaiC

rplUrpmA kaiC

90 nt kaiB)

Figure 1. Organization of the kai gene region in different cyanobacterial strains. (A) On righthand side, the kai operon is located head-to-head to genes coding for the ribosomal proteinsrplU (rpl21) and rpmA (rpl27) in all marine picocyanobacteria, like in the model freshwaterstrain Synechococcus elongatus PCC 7942. Whereas all Synechococcus possess a tricistronickaiABC operon, the kaiA gene (bold letters on dark gray background) has been deleted fromall Prochlorococcus, except in strains MIT9313 and MIT9303 in which it is present as a truncatedgene or a pseudogene. Numbers correspond to the lengths in nucleotides (nt) of the rplU-kaiAor rplU-kaiB intergenic spacers. The hypothetical protein-coding gene P9303_05421 annotatedin the MIT9303 rplU-kaiA intergenic region is drawn in light gray. Phylogenetic relationshipsbetween the 7 strains are indicated on left-hand side, by a 16S rRNA neighbor joining treebased on a 1469-nt full-length alignment and performed using the program MEGA 3.1. (B)Comparison of the C-terminal part of KaiA from model strain S. elongatus PCC 7942 (aminoacids 223–284) to the Prochlorococcus MIT9303 and MIT9313 sequences deduced from theirtruncated kaiA genes. The likely start of translation is at a GTG in both strains (the corre-sponding valine is labeled with an arrow). Asterisks indicate stop codons; the distance from thekaiA stop codon to the start of the kaiB gene is given in nt. In MIT9303, possible translationalcoupling is evidenced by the 4-nt overlap between the kaiA aUGA stop and the kaiB AUGastart. This figure is based on genomic information with the following GenBank entries:NC_007604 (PCC7942), NC_005070, and NC_009481 for WH8102 and WH7803; NC_005072,NC_005071, NC_005042, and NC_ 00882 for Prochlorococcus MED4/PCC 9511, MIT9313, SS120,and MIT9303, respectively.

expression of psbA. Continuous cultures were moni-tored for 3 days under LD then shifted to continuouslight (hereafter LL) in the afternoon of day 4. All overthe experiment, cell densities varied between 1.1 and2.2 × 108 cells mL–1. Figure 3A and 3B shows the cellcycle analysis. In both replicates under LD, the Sphase maximum occurred at the LD transition, fol-lowed by a peak of cells in G2, 4 to 5 h later. The shiftto LL in the afternoon of day 4 resulted in an increaseof the height and broadness of the following S phasepeak. From day 5 on, cell cycle rhythms vanished andno further S phase peak was detected at virtual duskor any other time during the following 3 days. Theaverage percentage of cells in the S phase over 24 hincreased from 15% to 30% upon the shift, indicatingthat under LL more cells were able to replicate. Sincecultures were constantly diluted, we indirectly esti-mated their growth rate under LD as 0.65 d–1, basedon the changes in the percentage of cells in theactive phases of the cell cycle (μCC; Carpenter andChang, 1988; Jacquet et al., 2001a). Though we couldnot apply this method under LL, it must be notedthat cells remained in slow-growing mode (sensu

Armbrust et al., 1989), that is,they never exhibited multipleDNA peaks.

From the same cultures, RNAwas sampled every 3 h duringthe light and every 4 h duringthe dark phase for 3 days beforeand 4 days after the shift. UnderLD, the psbA expression curve(Fig. 3C) followed that of thelight intensity, with a distinctpeak at noon as shown previ-ously (Garczarek et al., 2001,and Fig. 1C). The shift to LL wasaccompanied by a prolongationof the psbA expression into thevirtual night, after which thepsbA expression lost any rhythmand dropped to a constant level.Detection of fluorescence para-meters was not possible due tothe low Chl a content of the cells at high light. However, the gene expression profiles agreedwell with the cell cycle pattern;neither of them continued tooscillate under free-runningconditions.

To further confirm the appar-ent absence of circadian rhythm under LL, comple-mentary experiments were realized using batchcultures of P. marinus PCC 9511 grown at 22 °C and 25 °C under modulated light rhythms with a maxi-mum of 300 μmol quanta m–2 s–1 then shift to LL (110μmol quanta m–2 s–1). Though cells remained in expo-nential growth during the 3 days following the shift,none of these experiments gave rise to a persistence ofcell cycle rhythmicity under free-running conditions(data not shown).

Cell Cycling under Free-Running Conditions inthe kaiABC-Possessing Synechococcus sp. WH8102

To compare the results observed in P. marinusPCC 9511 to the situation in a close relative that pos-sesses all 3 kai genes, we analyzed the distribution of thecell cycle phases under free-running conditions in themarine cyanobacterium Synechococcus sp. WH8102 (Fig.4). Since WH8102 turned out to grow poorly in the veryhigh light conditions used for PCC 9511, we applied a 12:12-h LD cycle with a maximum of 150 μmol

Prochlorococcus marinus PCC9511

Time (h)

06 09 12 15 18 21 00 03 06 09 12

06 09 12 15 18 21 00 03 06 09 12 06 09 12 15 18 21 00 03 06 09 12

06 09 12 15 18 21 00 03 06 09 12

kaiBkaiC

psbAdnaApurF

PMM1629PMM1697

Irradiance% G1

% G2% S

.m−2

.s−1

Figure 2. Cell cycle and gene expression of a 12:12-h LD-synchronized Prochlorococcus mar-inus PCC 9511 culture with irradiances (dotted line) varying in a sinusoidal way between 0and 500 μμmol quanta.m–².s–1 (or μμE.m–².s–1). White and black bars indicate light and dark peri-ods. (A) Percentage of cells in the G1, S, and G2-like phases of the cell cycle. (B) Relative vari-ation of kaiB and kaiC mRNA levels. (C) Relative variation of purF, dnaA, and psbA mRNAlevels. (D) Relative variations of the mRNA level of putative sigma factors genes (corre-sponding to locus tags PMM1629 and PMM1697 in P. marinus MED4).

quanta m–² s–1 during the sinusoidal light phase to 2replicate batch cultures for several weeks before shift-ing them to a continuous illumination of 55 μmolquanta m–² s–1.

Cultures were monitored for 3 days under LDbefore being shifted to LL in the afternoon of day 3.Under LD, WH8102 grew at a μCC of about 1.6 j–1 andshowed a similar cell cycle as P. marinus PCC 9511 with

bimodal chromosome frequency distribution. In bothreplicates under LD, the S phase maximum occurred 2h before dusk (Fig. 4A). Unlike Prochlorococcus,WH8102 displayed about 10% of cells in the S phasethroughout the day, except at noon when this percent-age dropped down to 0. Cultures did not display asharp peak of cells in the G2 phase, but containedabout 30% to 40% of G2 cells nearly throughout the day(Fig. 4B). This percentage dropped significantly onlyduring the S phase peak.

Under free-running conditions, oscillations of theS phase were observed during 4 consecutive days.However, the amplitude of the S phase peak drasti-cally dropped and the peak broadened. The percent-age of cells in S phase never dropped down to 0 asunder LD. Cells remained in exponential growthover the course of the experiment. However, thegrowth rate of the strain increased under LL and thepercentage of G2 cells dropped whereas the numberof the G1 cells increased. Thus, many G2 cells were

Time (h)

Prochlorococcus marinus PCC9511

Irradiance

% G1% G2

06 18 06 18 06 18 06 18 06 18 06 18 06 18 06 0618

.m−2

.s−1

−2.s

.m−2

.s−1

Figure 3. Cell cycle and gene expression of an LD-synchronizedProchlorococcus marinus PCC 9511 culture under free-runningconditions. Replicate cultures (open symbol and filled symbol)were acclimated for 2 weeks to a 12:12-h LD cycle with light irra-diances (dotted line) varying in a sinusoidal way between 0 and1000 μμmol quanta.m–².s–1 (or μμE.m–².s–1) before being shifted tocontinuous light (350 μμmol quanta.m–².s–1). The graph shows thedistribution of the cell cycle phases and psbA expression levelunder LD (day 1 to 4), and after shifting to continuous light (day5 to 8). (A) Percentage of cells in the S phase (triangle). (B)Percentage of cells in the G1 (circle) and G2 (inversed triangle)phases. (C) Relative variation of psbA mRNA amounts. The dot-ted line indicates the growth irradiance (right axis). White andblack bars indicate light and dark periods; gray bars indicate vir-tual night during the continuous illumination.

Time (h)

Synechococcus sp. WH8102

Irradiance% S

% G1% G2

.m−2

.s−1

−2.s

06 18 06 18 06 18 06 18 06 18 06 18 06 0618

Figure 4. Cell cycle of an LD-synchronized Synechococcus sp.WH8102 culture under free-running conditions. Replicate cul-tures (open symbol and filled symbol) were acclimated for 2weeks to a 12:12-h LD cycle with irradiances (dotted line) vary-ing in a sinusoidal way between 0 and 150 μμmol quanta.m–².s–1

(or μμE.m–².s–1) before being shifted to continuous light (55 μμmolquanta.m–².s–1). Distribution of the cell cycle phases of 2 repli-cate cultures. (A) S phase; (B) G1 (circles) and G2 (inversed tri-angles) phases. White and black bars indicate light and darkperiods; gray bars indicate virtual night during the continuousillumination.

apparently light-limited under LD and could notcomplete division, whereas under LL a larger frac-tion of the cells proceeded through a full divisioncycle every day.

DISCUSSION

Reduction of the kai Operon in Prochlorococcus

Cyanobacteria are the only prokaryotes known todate to have a fully operational circadian clock. In allmodel strains studied to date, this clock is based on acore oscillator consisting of 3 proteins, KaiA, KaiB,and KaiC (Kondo et al., 1994; Ishiura et al., 1998). InProchlorococcus, however, no kaiA gene is annotatedin any of the 11 strains for which total genome infor-mation is currently available. However, occurrence ofunusually long kaiB-rplU intergenic spacers in thelow-light-adapted Prochlorococcus strains MIT9313and MIT9303 led us to discover a truncated kaiAreading frame in this region, indicating an evolution-ary intermediate between the 3- and the 2-gene formof the kai operon (Figs. 1 and 5). A truncated kaiAgene has also been found in some filamentouscyanobacteria (Uzumaki et al., 2004). As observedhere for Prochlorococcus strains MIT9313 andMIT9303, those KaiA proteins are truncated from theN-terminus and thus correspond to the C-terminalpart of full-length KaiA proteins. In contrast, even theshortest one, KaiA of Anabaena PCC 7120, is with 102

amino acids still longer than the predictedProchlorococcus KaiA (i.e., 54 and 71 residues forMIT9313 and MIT9303, respectively). The most strik-ing difference, however, is the replacement or dele-tion of most of the 23 conserved amino acid positionsthat the truncated KaiA from Anabaena PCC 7120 wasfound to share with all longer KaiA proteins(Uzumaki et al., 2004). Hence, this situation inProchlorococcus is fundamentally different from filamen-tous cyanobacteria or from the archaic Gloeobacter vio-laceus, which has no kai genes altogether (Nakamuraet al., 2003). The scheme in Figure 5 illustrates thatthe loss of kaiA in Prochlorococcus went alongside anintensive genome reduction process (Dufresne et al.,2005) and thus might be the result of genome stream-lining.

Cell Cycle and Gene Expression in DifferentCyanobacteria

In Prochlorococcus cells synchronized by an LDcycle, the chromosome replication always takes placetoward the end of the light phase, no matter the lightor temperature conditions (Vaulot et al., 1995, anddata not shown). Thus, an endogenous mechanismmust set a time frame for the initiation of the S phaseand once initiated, a round of replication is alwayscompleted. However, the exact time for DNA synthe-sis initiation is seemingly under the control of exter-nal factors. In culture, cells that grow at highirradiance or temperature replicate earlier than cellsgrown at low irradiance or temperature (data notshown). In contrast, in nature, the occurrence of UVradiation in the upper layer has been suggested todelay the initiation of replication, explaining thatdeep populations, although living in colder waters,replicate earlier than populations from surfacewaters (Vaulot et al., 1995). When LD synchronizedpopulations are shifted to LL, the ongoing replicationphase peak is broadened (Fig. 3), indicating that dueto the longer illumination time, more cells are able toinitiate replication. This suggests that in addition tothe endogenous mechanism setting the time framefor replication initiation, the cells need to have a cer-tain cell size or energy status.

In contrast to Prochlorococcus, many freshwatercyanobacteria such as Synechococcus sp. PCC 6301(Binder and Chisholm, 1995) or S. elongatus PCC 7942(Kondo et al., 1997) possess multiple chromosomecopies that are replicated nonsynchronously duringthe entire light phase and cells can divide severaltimes during 1 day. Accordingly, differences in the

WH8102 kaiABC (2.4 Mb)

(1.75 Mb) SS120

Synechococcus

(2.4 Mb) MIT9313

Prochlorococcus

(2.7 Mb) S. elongatus PCC 7942 kaiABC

(1.65 Mb) MED4

Marine cyanobacteriaFreshwater cyanobacteria

Common ancestor

kai(A)BC

Figure 5. Evolution scheme of the kai operon in cyanobacteria.This model shows the separation of the marine lineagesSynechococcus and Prochlorococcus from a common ancestor andthe stepwise reduction of the kaiA reading frame that occurredonly in the Prochlorococcus lineage, alongside a drastic genomestreamlining.

timing of the expression of cell cycle-related genescan be found. For example, the purF gene reaches inS. elongatus PCC 7942 maximal promoter activity atthe end of the night (Liu et al., 1996; Min et al., 2004),coinciding with replication starting in the morningand lasting throughout the entire day (Mori et al.,1996). In contrast, in P. marinus PCC 9511 expressionof purF, as well as of dnaA, occurs during a shortertime frame and is peaking toward the end of the lightphase, concomitantly with the shorter S phase. In thiswork, we also demonstrated that the timing of kaigene expression (peak toward the end of the night)differs from that found in S. elongatus PCC 7942. Inthis strain, transcription of the kai genes starts at sun-rise and reaches maximum levels at sunset (Xu et al.,2000) or in the early dark phase (Imai et al., 2004).This different timing alone should not interfere withfunctioning of the circadian clock, as has beendemonstrated in PCC 7942 (Tomita et al., 2005), butled to the conclusion that no transcription-translationfeedback is necessary in the cyanobacterial core clockwork. Therefore, the clock genes are clearly differ-ently regulated in Prochlorococcus compared to S.elongatus. Since in the absence of LD cycle, geneexpression and cell cycle rhythms vanished quasiimmediately in Prochlorococcus, we further concludethat the presence of kaiB and kaiC clock genes alone isnot sufficient for maintaining robust circadianrhythms in Prochlorococcus.

Before this study, there was no information aboutthe cell cycle of synchronized Synechococcus sp.WH8102 cells. Like P. marinus PCC 9511, WH8102cells display a bimodal cell cycle when grown underan LD cycle. However, phases were not as distinctlyseparated as in PCC 9511, where one can observe aclear succession of G1, S, and G2 phases. In WH8102,about 10% of the cells remained in S, and even morestriking, the culture always showed a high number ofcells in G2, dropping only transiently during the Sphase peak. The number of Synechococcus cells in G2also dramatically dropped upon shift into LL, indi-cating that these cells were likely light-limited underLD and blocked in G2. Such a blockage in the G2phase has never been observed in Prochlorococcus,which rather remains in G1 (Jacquet et al., 2001b).Thus, the marine Synechococcus sp. WH8102 seems topossess different cell cycle check points thanProchlorococcus. Most importantly in the context ofthe current study, WH8102 displayed a clear oscilla-tion of replication phase timing under free-runningconditions (though not as marked and persistent asin well-studied model cyanobacteria such as

Synechococcus sp. PCC 7942; see, e.g., Golden et al.,1997). This supports the idea that it is indeed the lossof kaiA that is responsible for the absence of theseoscillations and therefore the loss of robustness of thecircadian rhythm in Prochlorococcus.

Evidence for Rudimentary Clock Function inProchlorococcus

In a previous study using P. marinus MED4, Jacquetet al. (2001b) observed some minor oscillations of thepercentage of cells in S and G2 phases after shifting anLD-entrained culture to LL. This is seemingly at oddswith our results on PCC 9511 (Fig. 3), which we con-firmed under different irradiances and temperatures(data not shown). However, the range of variationsreported by these authors in the free-running periodwas only around 7% to 8% for the cells in S phase andless than 5% for the G2 cells, that is, much lower thanthose observed for our control, kaiA-containing strainSynechococcus sp. WH8102 (15% in S phase, 18% in G1,and 8% in G2; Fig. 4). So we think these oscillationswere likely not significant. Based on our findings, oneis tempted to conclude that Prochlorococcus does notpossess a circadian clock. Indeed, it is well known thatmany physiological phenomena are regulated by LDcycles independent of an internal clock. However, sev-eral features in Prochlorococcus rather suggest the pres-ence of a clock working on a 24-h base: 1) naturalProchlorococcus populations and cultures under LDcycles display 24-h rhythms of gene expression and cellcycling; 2) profiles of gene expression during the darkphase indicate an anticipation of timing, for instance,kaiBC and of PMM1629 mRNAs peaks in the dark with4-h advance to the lights-on signal (Fig. 2B, D); and 3)most genes involved in clock functioning besides kaigenes are present and well conserved in allProchlorococcus strains, despite the genome streamlin-ing process. Among these genes, some encode proteinsknown to mediate output from the primary timekeeper, including the adaptive sensor output kinaseSasA (Iwasaki et al., 2000), the SasA-interactingresponse regulator RpaA (Takai et al., 2006), and thecircadian phase modifier CpmA (Katayama et al.,1999). In MED4, homologs of these genes arePMM1077, PMM0128, and PMM1278, respectively.This suggests that the bimolecular KaiBC system inthis organism is functional and does generate an out-put signal. In contrast, there is no homolog for LabA,required for negative feedback regulation of KaiC(Taniguchi et al., 2007) in any Prochlorococcus. All thesedata are compatible with the idea that a core oscillatory

mechanism and output apparatusis functional in Prochlorococcus,but working in an hourglass-likefashion rather than as a self-sustained oscillator. This hypoth-esis cannot be checked by a directgenetic approach because of theabsence of a genetic system in P. marinus PCC 9511 or MED4,making the construction of nullmutants impossible, but it couldbe verified in future studies byinactivating kai genes in strains of the closely related marineSynechococcus genus, for example,in WH8102.

Work in the model cyanobac-teria S. elongatus PCC 7942, Ther-mosynechococcus elongatus BP-1,and Synechocystis sp. PCC 6803has given rise to refined mod-els of the cyanobacterial clock(Woelfle and Johnson, 2006;Miyoshi et al., 2007; Mori et al.,2007; van Zon et al., 2007; Yodaet al., 2007). There is strong bio-chemical evidence and modelsagree in the fact that the KaiAprotein is essential for circadianclock function (Nishiwaki et al.,2007; Rust et al., 2007). Therefore,it appears cumbersome howeven a rudimentary cyanobacter-ial clock should function withoutKaiA and additional adaptationsmay be postulated: To be func-tional, Prochlorococcus KaiC pro-teins must have biochemicalproperties that differ from theKaiC of model cyanobacteria. Since biochemical assayson model cyanobacteria have demonstrated that KaiCmainly remains unphosphorylated in the absence ofKaiA, a higher ability for autophosphorylation of theProchlorococcus KaiC might, for instance, be expected.

It is worth noting the deep dichotomy in phyloge-netic analyses between KaiC from all marineSynechococcus and Prochlorococcus strains MIT9303 andMIT9313, and KaiC from all other Prochlorococcus (Fig.6), since that correlates with the presence or absence ofat least a residual kaiA gene. Direct sequence compar-isons show that there are 6 amino acid positions whereKaiC from all Prochlorococcus (including the truncated

kaiA-possessing MIT9313 and MIT9303) deviate fromall other KaiC, namely, those corresponding to S. elon-gatus Gly-213, Asn-389, Ser-416, Ala-466, Ile-490, andSer-493, which are replaced in Prochlorococcus spp. byThr/Ala/Ser, Leu, Ala, Arg, Phe, and Ala, respectively.There are also 2 additional positions specific to kaiA-lacking Prochlorococcus, namely, those corresponding toS. elongatus Asn-131 and Ser-158, which are replaced bySer/Thr and Tyr, respectively. It will be interesting totest possible functional consequences in future bio-chemical or genetic experiments.

Our data are compatible with a rudimentary circa-dian clock in Prochlorococcus that functions on a daily

Freshwatercyanobacteria andother model strains

MarineSynechococcus

All Prochlorococcuswithout kaiA

Synechococcus CC9605

Synechococcus BL107Synechococcus CC9902

Synechococcus RS9917Synechococcus WH7805

Synechococcus WH7803Synechococcus RS9916

Synechococcus CC9311Synechococcus RCC307

Synechococcus WH5701Prochlorococcus MIT9303Prochlorococcus MIT9313

Prochlorococcus MIT9211Prochlorococcus NATL2AProchlorococcus NATL1A

Prochlorococcus MIT9515Prochlorococcus MIT9312Prochlorococcus MIT9301Prochlorococcus AS9601Prochlorococcus MIT9215

Synechococcus PCC7942Acaryochloris marina

Thermosynechococcus elongatus BP1Anabaena sp. PCC7120

Synechocystis PCC6803

Prochlorococcuswith truncated kaiA

Synechococcus WH8102

Prochlorococcus SS120Prochlorococcus PCC9511/MED4

Figure 6. Phylogenetic tree of KaiC proteins from all available marine Synechococcus andProchlorococcus and some other selected cyanobacteria. A multiple sequence alignment wasmade with full-length KaiC sequences from 28 taxa (529 amino acid positions). The identityamong these protein sequences ranged from 71% to 97%. Phylogenetic trees were constructedby applying different methods, including maximum parsimony, minimal evolution, andneighbor-joining, as integrated in the MEGA 3.1 software (Kumar et al., 2004). Gaps weredeleted and bootstrap analysis of 10,000 resamplings with the optimality criterion set to min-imum evolution yielded the values shown at the respective nodes if >>65%. All treeingapproaches yielded very similar relationships and identical tree topologies. A neighbor-joining tree is shown here. P. marinus PCC 9511/MED4 and Synechococcus sp. WH8102 are inboldface letters and boxed. GenBank accession numbers for Prochlorococcus strains: MED4:NP_893459.1; MIT9515: YP_001011817.1; MIT9301: YP_001091752.1; AS9601: YP_001009933.1;MIT9215: YP_001484770.1; MIT9312: YP_397936.1; NATL2A: YP_292107.1; NATL1A:YP_001015589.1; MIT9211: ZP_01004882.1; SS120: NP_875814.1; MIT9303: YP_001016561.1;MIT9313: NP_895245.1; Synechococcus strains: PCC 7942: YP_400233.1; WH7805:ZP_01122950.1; RS9916: ZP_01470888.1; CC9605: |YP_382418.1; RS9917: |ZP_01079510.1;WH7803: YP_001225687.1; WH8102: NP_896645.1; WH5701: ZP_01084034.1; CC9311:YP_731418.1; BL107: ZP_01468509.1; CC9902: YP_376561.1; RCC307: YP_001228080.1;Thermosynechococcus elongatus BP-1: NP_681273.1; Anabaena PCC 7120: NP_486926.1;Acaryochloris marina: Q6L8L1; Synechocystis sp. PCC 6803: NP_442950.1.

basis and has lost its robustness. The presence orabsence of KaiA may make the difference between aself-sustained timer and an hourglass. Indeed, evenfor a minimalist genome-containing organism suchas Prochlorococcus, which thrives in a resource-poorenvironment, fine-tuning of gene expression isimportant. Apparently, a rudimentary clock that isunder a more direct control of external factors is suf-ficient to generate the required output signal on a 24-h basis. If so, one very important question remainingto solve is how the synchronization between thisinternal oscillator and the external and internal factorsis achieved. Also in the seemingly well-functioningclock system of marine Synechococcus, no orthologs ofknown input proteins such as the circadian inputkinase CikA (Schmitz et al., 2000) and the light-dependent protein A, a mediator of the metabolicstate (Ivleva et al., 2005) can be found. Thus, it is anopen question for all marine picocyanobacteria howdaily signals such as lights-on and lights-off are inte-grated with their fully functional (Synechococcus) orresidual (Prochlorococcus) clock. Future research maybe directed on determining the precise biochemicalproperties of Prochlorococcus KaiC, and on elucidat-ing the linkage between the cellular redox statusand/or the daily light/dark signals.

ACKNOWLEDGMENTS

We thank Anne Peyrat, Emilie Didierjean, and JensReinhardt for help with sampling, Dominique Mariefor advice on flow cytometry, and Jens Reinhardt andAkira Peters for critical reading of the manuscript. Wethank the EU project Margenes, the Marine GenomicsEurope Flagship project “Synchips,” the FreiburgInitiative in Systems Biology, and the “RégionBretagne” (program “IMPALA“) for financial support.

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