ldpA Encodes an Iron-Sulfur Protein Involved in Light-Dependent Modulation of the Circadian Period in the Cyanobacterium Synechococcuselongatus PCC 7942

We generated random transposon insertion mutants to identifygenes involved in light input pathways to the circadian clockof the cyanobacterium Synechococcus elongatus PCC 7942. Twomutants, AMC408-M1 and AMC408-M2, were isolated that respondedto a 5-h dark pulse differently from the wild-type strain. Thetwo mutants carried independent transposon insertions in anopen reading frame here named ldpA (for light-dependent period).Although the mutants were isolated by a phase shift screeningprotocol, the actual defect is a conditional alteration in thecircadian period. The mutants retain the wild-type ability tophase shift the circadian gene expression (bioluminescent reporter)rhythm if the timing of administration of the dark pulse iscorrected for a 1-h shortening of the circadian period in themutant. Further analysis indicated that the conditional short-periodmutant phenotype results from insensitivity to light gradientsthat normally modulate the circadian period in S. elongatus,lengthening the period at low light intensities. The ldpA geneencodes a polypeptide that predicts a 7Fe-8S cluster-bindingmotif expected to be involved in redox reactions. We suggestthat the LdpA protein modulates the circadian clock as an indirectfunction of light intensity by sensing changes in cellular physiology.

INTRODUCTION

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Introduction

Light is a major environmental signal that synchronizes circadianrhythms in various organisms to coordinate the daily oscillationsof biological phenomena with the sidereal day. The input pathwaysthat receive and transmit environmental signals to the centralcircadian oscillator of circadian systems vary among organisms,particularly in the mechanisms of perception of light signals.In mammals, light that sets the circadian clock is receivedby membrane-associated opsin proteins in the eyes that carryretinal chromophores (15, 39). In Drosophila, both opsin-basedphotopigments and the blue light photoreceptor cryptochromefunction as photoreceptors for input pathways (39). In Neurospora,the putative transcription factors WC-1 and WC-2, which interactwith FRQ, a critical element of the circadian system, are necessaryfor light perception (10, 25). In higher plants, phytochromesand cryptochrome transduce light signals to the clock (38).

Cyanobacteria are the only prokaryotes demonstrated thus farto have bona fide circadian systems that have the propertiesof those in eukaryotic organisms (13). In various cyanobacterialgenera, the circadian clock has been shown to regulate suchdiverse biological activities as nitrogen fixation (14), aminoacid uptake (8), and gene expression (22, 24). Several genesrelated to circadian clock function have been identified inthe unicellular cyanobacterium Synechococcus elongatus PCC 7942,a strain for which many genetic tools are available. These includethe kaiA, kaiB, and kaiC genes, essential components for circadianrhythms (16).

One basic aspect of a circadian rhythm is the free-running period(FRP), which is the duration of the oscillation, peak to peakor trough to trough, under constant environmental conditions(4). Several loci in S. elongatus are known to affect the FRPunder conditions of constant light and temperature. Many allelesof the kaiA, kaiB, and kaiC genes affect the FRP (16). An extracopy of the pex gene, which encodes a protein of unknown function,extends the circadian period by about 2 h (23). The sasA gene,which encodes a histidine protein kinase, amplifies circadianoscillation and may be a major conduit for temporal informationout of the clock to the genes it controls (17). The residualrhythmicity in an sasA mutant has a short FRP. The only componentknown to be involved in an input pathway for resetting the clockis the histidine protein kinase CikA, which belongs to the bacteriophytochromefamily of proteins (32). Mutants defective in cikA have a shortFRP.

In this study, we identified the ldpA (light-dependent period)gene of S. elongatus PCC 7942 as encoding a new component ofan input pathway to the cyanobacterial clock. Its predictedproduct is a previously undescribed protein that carries iron-sulfurcluster-binding motifs. Disruption of ldpA significantly attenuatedthe ability of S. elongatus to modulate the FRP under differentlight intensities. Our results suggest that the circadian clockis modulated by the redox state in cyanobacteria, acting atleast in part through the iron-sulfur protein LdpA.

MATERIALS AND METHODS

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Materials and Methods

Strains and culture conditions. The bacterial strains and plasmids used in this study are listedin Table 1. All Synechococcus strains were grown in modifiedBG-11 medium (BG-11 M) (6) or on BG-11 M agar plates under continuouslight (100 microeinsteins m^-2 s^-1) at 30°C. Spectinomycin(5 µg ml^-1), chloramphenicol (7.5 µg ml^-1), kanamycin(5 µg ml^-1), or gentamicin (1 µg ml^-1) was usedas needed for selection of S. elongatus transformants.

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TABLE 1. Strains and plasmids used in this study

Transposon mutagenesis. A modified Tn5 transposon was introduced into wild-type AMC408by conjugal transfer from Escherichia coli of pAM1037, a derivativeof pRL1058, as described previously (20). AMC408 carries a PpurF::luxABreporter that exhibits class 2 circadian bioluminescence rhythms(24). Restriction enzymes and modifying enzymes were purchasedfrom Promega and used as directed by the manufacturer. DNA flankingthe transposon was recovered by extracting genomic DNAs fromexconjugants, digesting them with KpnI, circularizing them withT4 DNA ligase, and introducing them as plasmids into E. coliDH10B by electroporation (2, 20). Km^r plasmids were recovered,and nucleotide sequences of the genomic regions flanking thetransposons were determined by cycle sequencing (dye terminatorcycle sequencing ready reaction; ABI PRISM; PE Applied Biosystems,Foster City, Calif.) with primers AMO134 and AMO285, which arecomplementary to each end of the transposon.

Sequence analysis and Fe-S motif determination. Sequence motif determination was performed by using multipleapproaches. The full-length LdpA sequence was first used tosearch against the nonredundant protein database and the proteinstructural database (PDB) at the National Center for BiotechnologyInformation by using BLAST (1) (http://www.ncbi.nlm.nih.gov/BLAST/).In this search, we used the BLOSUM45 amino acid substitutionmatrix, with a gap opening penalty of 11, an extension penaltyof 1, an Expect value of 10, and a word size of 3. A more sensitivedatabase search using a Bayesian algorithm (37) was carriedout with the BALSA program (http://bayesweb.wadsworth.org/balsa/balsa.html).Sequence motifs were further searched for the LdpA aminoacidsequence by using the FingerPRINTScan (http://www.bioinf.man.ac.uk/fingerPRINTScan/),eMOTIF Search (http://motif.stanford.edu/emotif/emotif-search.html),Block (http://blocks.fhcrc.org/blocks/blocks_search.html), andInterPro (http://www.ebi.ac.uk/interpro/) servers. Structuralfold prediction was done by using a threading server, BIOINBGU(http://www.cs.bgu.ac.il/ $~$ bioinbgu/form.html). Multiple-sequencealignment of LdpA with three ferredoxins with known 7Fe-8S structureswas done by using the T-COFFEE server (http://www.ch.embnet.org/software/TCoffee.html),followed by manual refinement. To assess the statistical significanceof sequence similarities between LdpA and 7Fe-8S ferredoxins,we used an alignment-independent program, PRSS, that calculatesthe probability of similarities of randomly shuffled and unshuffledsequences by using a distance matrix Monte Carlo procedure (29).The analysis was done in the Biology Workbench web server (http://workbench.sdsc.edu/).The gap-opening penalty was set at 12, and the gap-extendingpenalty was set at 2, and 1,000 global shuffles were performedby using the BLOSUM50 scoring matrix.

Construction of plasmids for complementation and disruption of ldpA. An intact copy of ldpA was reconstructed from the transposoninsertion-containing alleles recovered from AMC408-M1 and AMC408-M2.We excised a 3.7-kb EcoRI-HpaI fragment that includes the Nterminus-encoding and upstream flanking regions of ldpA frompAM2180 (Km^r plasmid recovered from AMC408-M2; Fig. 2) and insertedit into EcoRI- and SmaI-digested pBluescriptII SK+ to createpAM2182. The 2.3-kb HpaI-BglII fragment, which encodes the C-terminalportion and downstream flanking region of ldpA, was excisedfrom pAM2181 (Km^r plasmid recovered from AMC408-M1) and insertedinto SmaI- and BamHI-digested pBluescriptII SK+ to create pAM2183.After complete digestion of pAM2182 with EcoRI and its partialdigestion with PstI, a 2.9-kb EcoRI-PstI fragment was clonedinto EcoRI- and PstI-digested pAM2183 to create pAM2184. pAM2184contains the entire open reading frame (ORF) and the upstreamand downstream flanking regions of ldpA. The 1.3-kb PvuII fragmentthat contains ldpA was excised from pAM2184 and cloned intoEcoRV-digested pBluescriptII SK+ to create pAM2185. A 1.7-kbPvuII fragment in which ldpA is downstream of the lacZ promoterwas excised from pAM2185 and cloned into SmaI-digested pAM1706to create pAM2186, a vector that targets inserted genes to anS. elongatus locus called neutral site II (NSII; GenBank accessionno. U44761). In this plasmid, ldpA is inserted upstream of theluxCDE genes (present to direct synthesis of the aldehyde substrateof the luciferase reporter) but transcribed divergently. Therecreated transposon mutants AMC694 and AMC695 were transformedwith pAM2186 to create complemented strains AMC696 and AMC697,respectively.

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FIG. 2. Physical map of the ldpA region of the S. elongatus chromosome and plasmids used to reconstruct intact and disrupted ldpA alleles. Restriction sites are indicated by the following abbreviations: E, EcoRI; Pv, PvuII; Ps, PstI; An, AlwNI; Bg, BglII; Hp, HpaI. Insertion sites of the transposon and Gm^r cassette are indicated by filled and open triangles, respectively. Broken lines indicate regions derived from the transposon. Plasmids pAM2180 and pAM2181 were recovered by restriction digestion and circularization from genomic DNA of mutants AMC408-M1 and AMC408-M2, respectively.

For directed disruption of ldpA, pAM2185 was partially digestedwith AlwNI and made blunt ended by using T4 DNA polymerase.Into the linearized plasmid, we inserted a 2.0-kb PvuII-SmaIfragment from pAM2055 that includes a gentamicin resistance(Gm^r)-encoding gene to create pAM2188. We transformed wild-typePpurF::luxAB reporter strain AMC408 and short-period (22 h)mutant AM548 with pAM2188 to create AMC664 and AMC665, respectively.Complete segregation of the mutant allele was confirmed by PCRwith primers 5'-AGAACTTCGGGATGGGC-3' and 5'-GAACGTCTAACAGGACG-3',which cross the insertion site of the gene encoding Gm^r.

Assay of bioluminescence rhythms. The original Tn5 insertion mutants were identified by bioluminescencemonitoring with a Packard TopCount luminometer (2). Briefly,mutant exconjugants grown on BG-11 M agar in continuous light(LL) were inoculated into BG-11 M and incubated on a rotaryshaker for 2 weeks. Cyanobacterial suspensions (10 µl)at an optical density at 750 nm (OD₇₅₀) of 0.7 were used toinoculate BG-11 M agar pads in 96-well sample plates. Theseplates were incubated under standard LL conditions for 24 hand then subjected to a 12-h dark interval to synchronize theclocks of all of the cells in the population prior to bioluminescencemonitoring on the TopCount luminometer. Illumination aroundthe TopCount stackers results in a light gradient over the 96-wellmeasuring plates, such that outer wells (columns 1 and 12) receivea fluence of approximately 225 microeinsteins m^-2 s^-1 and theinner wells (columns 6 and 7) receive approximately 50 microeinsteinsm^-2 s^-1.

For measurement of the bioluminescence rhythm under turbidostaticcultivation conditions, 150-ml cyanobacterial suspensions withan OD₇₃₀ of 0.7 to 1.0 were used to inoculate 1.4 liters ofBG-11 M in flat bottles. The cultures were bubbled with airunder standard LL conditions and incubated until the OD₇₃₀ reached0.25. The cultures were then subjected to a 12-h dark intervalto synchronize their circadian rhythms. Cell density was monitoredby measurement of infrared light penetration into the cell suspension.An increase in OD₇₃₀ triggered a switch that activated a pumpto add fresh medium and keep an OD₇₃₀ of 0.25. An aliquot (24ml) was withdrawn every hour, and bioluminescence was measuredby a photomultiplier tube apparatus. The output of the photomultipliertube was amplified electronically and read by a computer analog-to-digitalconverter. The reading was averaged over the measurement periodand plotted on the ordinate in millivolts as bioluminescence.Phase shifts and FRPs were calculated by regression proceduresas described previously (32).

RESULTS

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Results

Screening for light input mutants. We generated random transposon mutations in a PpurF::luxAB bioluminescentreporter strain of S. elongatus PCC 7942 (AMC408) and screenedfor mutants affected in light input pathways to the circadiansystem. In all screenings, a 12-h dark interval or a temperaturecycle was used for synchronization of the circadian rhythm first,and then photic signals (5- or 12-h dark interval) were appliedat specific times of the day that induce a circadian phase shiftin the wild type. We chose exconjugants that showed phases differentfrom that of the wild-type strain after the second dark pulseas candidate input mutants.

We used three screening protocols to accommodate mutants thatmight or might not be synchronized initially by photic signals.All of the protocols used a 5-h dark pulse that, for wild-typeS. elongatus, would reset the relative phasing of circadianrhythms by as much at 10 h, depending on the stage in the circadiancycle at which it was administered (32). The protocols wereas follows: (i) a 12-h dark interval to synchronize rhythms,followed by a transfer to LL and a 5-h dark pulse that started53 h after the transfer to LL (56 mutant candidates among 700exconjugants); (ii) a temperature cycle of 12 h at 40°C,12 h at 30°C, and 12 h at 40°C in LL, followed by a12-h dark interval that began 36 h after the end of the last40°C pulse (234 mutant candidates among 2,600 exconjugants);and (iii) a temperature cycle of 12 h at 40°C, 12 h at 30°C,and 12 h at 40°C in LL, followed by a 5-h dark pulse thatbegan 53 h after the end of the last 40°C pulse (93 mutantcandidates among 700 exconjugants). From these candidate inputmutants, we chose five exconjugants that reproducibly showedphases different from that of the wild-type strain after thephase-resetting dark pulse and recovered the transposons andflanking DNA as Km^r plasmids. The mutants were recreated bytransforming AMC408 with the plasmids that had been linearizedby cleavage with the restriction enzyme used for their recovery.Two mutants, AMC408-M1, which was originally isolated by protocol3, and AMC408-M2, isolated by protocol 1, retained the phenotypesexpected for input mutants (Fig. 1). These mutants also hada slightly shorter free-running circadian period than the wild-typestrain under these screening conditions (Fig. 1).

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FIG. 1. Traces of circadian rhythms of bioluminescence from PpurF::luxAB reporter strains. Symbols: diamonds, wild type; triangles, mutant AMC408-M1; squares, mutant AMC408-M2. Hour zero in LL indicates the time point of the end of the 12-h dark interval used for initial synchronization. The black bar indicates the time of application of a 5-h dark pulse for phase resetting.

Identification of transposon insertion sites. To identify the sites of mutations in AMC408-M1 and AMC408-M2,we determined the nucleotide sequences of the genomic regionsflanking the transposons. Nucleotide sequencing revealed thatthe two strains carry independent insertions in the same ORF,as shown in Fig. 2. We designated this ORF the ldpA gene onthe basis of phenotypic characteristics described below.

Translation of the ldpA ORF and comparison to similar sequencesin public databases suggested that the start codon is a GTGand that LdpA is a soluble protein of 352 amino acid residues.Putative ldpA homologs (percent amino acid identity) are encodedin the genomes of Synechocystis sp. strain PCC 6803 (sll0031,49%), Anabaena sp. strain PCC 7120 (alr2308, 44%), Nostoc punctiformeATCC 29133 (44.0%), marine Synechococcus sp. strain WH8102 (43%),and Prochlorococcus marinus strain MED4 (35%), indicating conservationamong diverse cyanobacteria.

Determination of an LdpA Fe-S motif. The deduced amino acid sequence of LdpA was first searched againstthe National Center for Biotechnology Information nonredundantdatabase by using a gapped BLAST (1). Seven significant databasehits (E < 10^-6) were recovered that were all annotated ashypothetical proteins from recently sequenced cyanobacterialgenomes. A similar BLAST search against a more restricted databasefor protein structures (PDB) was done that generated no significanthits. However, the best hit (PDB code 1H98) was a ferredoxinwith 3Fe-4S and 4Fe-4S centers from Thermus thermophilus (Table2). A more sensitive database search was conducted with a Bayesianalgorithm (37) that identified significant hits as a ferredoxinfrom Clostridium acidurici (PDB code 1FCA) with two 4Fe-4S centersand another from Azotobacter vinelandii with 7Fe-8S centers(Table 2).

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TABLE 2. Determination of Fe-S center types in LdpA by using multiple sequence motif search methods

To determine more accurately the type of Fe-S centers likelyto be present in LdpA, we analyzed the sequence by using multiplemotif prediction programs that have different sensitivitiesfor certain domains because of intrinsic differences in algorithmsand database content (Table 2). Indeed, our searches indicatedthe presence of 4Fe-4S, 3Fe-4S, and 7Fe-8S types of clusterswith significant statistical support. Furthermore, a structuralfold recognition analysis carried out for LdpA generated a mostsignificant hit for a 7Fe-8S ferredoxin (PDB code 5FD1 fromA. vinelandii) and second most significant hit for an 8Fe-8Sferredoxin (PDB code 1CLF from C. pasteurianum). The resultdemonstrates a high probability that the LdpA protein adoptsa tertiary structure similar to that of the 7Fe-8S ferredoxinin A. vinelandii. In reviewing all of the available evidence,the consensus appears to be that LdpA has two Fe-S centers,one for 3Fe-4S and another for 4Fe-4S. A more refined sequencealignment between LdpA and proteins with known 7Fe-8S centers(3, 7, 26) showed full conservation of all Cys residues at theputative Fe-S center domain (Fig. 3). Statistical support (Pvalues) for each pairwise comparison (29) between LdpA and known7Fe-8S proteins is shown in Table 2. The entropy-based analysiswithout influence of the alignment shows that the sequencesare indeed significantly similar, with 100% of the comparisonshaving P values smaller than 0.001, which is significant enoughto establish a direct homologous relationship.

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FIG. 3. Comparison of the deduced amino acid sequence encoded by the ldpA gene of S. elongatus PCC 7942 and ferredoxins with known 7Fe-8S centers. LdpA, GenBank accession no. AY136759; T. thermophilus ferredoxin, SwissProt accession no. P03942; Bacillus schlegelii ferredoxin, SwissProt accession no. Q45560; A. vinelandii FdI, SwissProt accession no. P00214. Only the Fe-S center domain is shown. Conserved Cys residues are highlighted, and corresponding Fe-S centers are indicated by brackets. The PDB codes for the ferredoxins from A. vinelandii, T. thermophilus, and B. schlegelii are 1F5B, 1H98, and 1BC6, respectively. Pairwise sequence similarities between LdpA and other ferredoxins were assessed by an alignment-independent PRSS test. Statistical significance (P values) for the pairwise sequence comparison is indicated on the right of the sequence alignment.

Insertional inactivation of ldpA and complementation of the mutant phenotype by a wild-type allele. We created a Gm^r interruption allele of ldpA by introducingthe drug resistance cartridge into an AlwNI site that is nearthe insertion site of the transposon in mutant AMC408-M2. Thisreconstructed mutant had the short-period phenotype observedin the original transposon mutants (Table 3). To confirm thatldpA alone is responsible for the phenotype, we introduced awild-type ldpA gene into ldpA Tn5 mutants. For this experiment,we used strains AMC694 and AMC695, which carry the M1 and M2Tn5 mutations in a background with a reporter configurationdifferent from that of AMC408. A heterologous promoter, P_lacZ,was provided in the vector because sequence data suggested thatldpA is downstream of a thioredoxin reductase gene with whichit might be cotranscribed; thus, the ldpA fragment might nothave its own promoter (data not shown). As shown in Table 3,the FRPs of both AMC694 and AMC695 were restored to the wild-typeFRP by the introduction of ldpA (AMC696 and AMC697, respectively).We concluded that loss of function of ldpA is responsible forthe short-period phenotypes in the collection of ldpA mutants.

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TABLE 3. FRPs of ldpA mutants and complemented derivatives

Characterization of the light input phenotype of ldpA mutants. To elucidate the presumed input pathway defect of the mutants,we performed a series of phase-resetting experiments. A darkpulse administered at different times in the circadian cyclereproducibly causes phase shifts that differ with respect tomagnitude and direction in S. elongatus (32). We compared therelationships between the timing of the 5-h dark pulse relativeto the circadian cycle and the resultant phase shift in thewild type and in ldpA mutants. The results are plotted as phaseresponse curves (PRCs) in Fig. 4. The shapes of the PRCs ofAMC694 and AMC695 are almost identical to that of the PRC ofthe wild-type strain. This indicates that both mutants retainthe wild-type ability to shift the phase of the circadian rhythm.

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FIG. 4. PRCs for 5-h dark pulses in the wild-type strain (solid circles) and ldpA mutants AMC694 ( ${circ}$ ) and AMC695 ( ${triangleup}$ ). PRCs for duplicate samples of each strain are shown. Circadian time (CT) is a normalization for the difference between the circadian periods of the strains; 1 circadian h = 1/24 of a full circadian cycle. Positive values represent phase advances, and negative values represent phase delays.

The FRPs of ldpA mutants were shorter than that of the wild-typestrain by approximately 1 h under light conditions of 25 microeinsteinsm^-2 s^-1 (Table 3). Light is known to affect circadian systemsnot only with respect to phase resetting but also by influencingthe FRP (4). Generally, increasing light intensity shortensthe FRP in diurnal organisms and phototrophic organisms, includingunicellular algae (9) and higher plants (34). As shown in Fig.5A, the FRP of wild-type S. elongatus decreases with increasinglight intensity by about 1 h. However, ldpA mutants show nosubstantive change in period over this range of light intensities(Fig. 5A).

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FIG. 5. Effect of white light fluence on the FRP. Panels A and B show the FRPs of the wild type (A) and C22a (B) (•) and those genetic backgrounds with ldpA disrupted ( ${circ}$ ). Panels C and D show the FRPs of C22a (C) and C22a with ldpA disrupted (D) with ( ${triangleup}$ , ${blacktriangleup}$ ) and without DCMU addition ( ${circ}$ , •). DCMU was added to media at a final concentration of 0.2 µM at the beginning of a 12-h dark interval. Error bars indicate standard deviations (n = 8).

To examine this phenotype in more detail, we inactivated ldpAin the short-period mutant C22a (22 h), which has a point mutationin the kaiC gene (16). The FRP of C22a is more sensitive tothe change in light intensity than is the wild-type strain,varying by about 3 h over the light gradient tested (Fig. 5B).Inactivation of ldpA decreased the sensitivity of the periodto light intensity in C22a as well (Fig. 5A). These resultssuggest that the primary effect of inactivation of ldpA is attenuationof the ability to increase the period length with decreasinglight intensity, not a defect in the ability to phase shiftin response to a dark pulse. This specific phenotype led usto name the locus light-dependent period gene A.

Effect of a photosynthesis inhibitor on the circadian period. The expectation that LdpA carries Fe-S clusters suggests involvementin electron transfer and redox reactions (5). One possibilityis that LdpA mediates a redox signal from the photosyntheticapparatus to the circadian oscillator. To test this, we examinedthe effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU),an inhibitor of the photosystem II reaction center, on the free-runningcircadian period. If the putative LdpA protein transmits photosyntheticinformation, addition of DCMU would be expected to have a smallereffect on the FRP of an ldpA mutant than on that of a wild-typestrain. As shown in Fig. 5C, addition of a sublethal concentrationof DCMU increased the FRP in C22a but did not block sensitivityto different light intensities. DCMU also increased the FRPof the C22a-ldpA mutant. The period of the C22a-ldpA mutantwas still shorter than that of C22a in the presence of DCMU(Fig. 5D). These results indicate that photosynthetic activitycontributes to the light input pathways that modulate FRP, butthe putative LdpA protein is not the sole component that transmitslight intensity signals to the circadian clock.

Disruption of ldpA had pleiotropic effects. The ldpA disruptantsgrew slightly more slowly than the wild type, with a doublingtime of 7.3 h instead of the 6.1 h of the wild-type strain undera light fluence of 34 microeinsteins m^-2 s^-1. ldpA mutants werealso yellowish green in color because of a phycocyanin contentthat was only 50 to 70% of that of the wild-type strain. Becausea slower growth rate would lead to a lower cell density andgreater light penetration into the culture, we wanted to excludethe possibility that the short-period phenotype of ldpA mutantsis an indirect effect of the slow growth rate. We compared theFRPs of an ldpA mutant and the wild-type strain under turbidostaticculture conditions in which the OD₇₃₀ was maintained at 0.25.The mutant showed a consistently shorter period (24.2 h) thanthe wild-type strain (25.2 h) under 16 microeinsteins of whitelight m^-2 s^-1.

DISCUSSION

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Discussion

Mutants identified by a screen for circadian-resetting defectsrevealed the involvement of a previously undescribed Fe-S proteinin the modulation of the circadian period of S. elongatus. Theprimary defect in the mutants is not an inability to reset perse, as is seen in mutants that lack the CikA histidine proteinkinase (32). Rather, ldpA mutants are insensitive to the periodlengthening under low light fluence that occurs in the wild-typestrain. As a result, even under a gradient of light intensities,ldpA mutants always exhibit an FRP that is at the short endof the spectrum observed for the wild type. Thus, at the highestlight intensities we use routinely in the laboratory, the mutantand wild-type strains have equivalent FRPs (Fig. 5). The screenfor phase-resetting mutants used a monitoring setup in whicha light gradient was present across the 96-well plates; we donot know the fluence received by the controls with which thesemutants were compared when they were originally identified.Our current understanding of the phenotype suggests that theywere running with a 1-h shorter FRP than the controls, suchthat the timing of the resetting dark pulse fell at a slightlydifferent point during the circadian cycle for the wild typeand the ldpA mutants. With these data in mind, we now pair mutantsand controls in the same columns of sample plates for screening(27). Despite the serendipitous identification of these mutantsin the resetting screen, ldpA does seem to be involved in aninput pathway to the clock but affecting a parametric (recurrentperiod-modulating) rather than nonparametric (single phase-resetting)aspect. Identification of LdpA provides a means by which togain insight into the biological mechanisms behind Aschoff'srule, the observation that circadian periods tend to decreasewith an increase in light intensity (4).

Several Arabidopsis thaliana mutants have conditional FRP changesdepending on the light intensity. Deficiency in phyA (defectin one of the phytochrome photoreceptors) causes an exaggeratedincrease in FRP relative to that of the wild type in responseto decreased red light fluence (34). A cry1 (cryptochrome bluelight receptor) mutation similarly affects the response to decreasingblue light (34). The gi-1 weak allele of GI, which encodes amembrane-spanning protein, shortens the FRP and reduces therate of period lengthening with decreasing fluence (28). Conversely,mutation of ZTL lengthens the FRP and increases the rate ofperiod lengthening with decreasing light intensity (35). Amongthese, the phyA mutant also has a resetting defect, taking alonger time that the wild type for re-entrainment under 10-hlight-10-h dark cycles (34).

In contrast to phyA or cry1 Arabidopsis mutants, disruptionof ldpA decreased the rate of change in FRP over a range oflight intensities. This is most similar to Arabidopsis GI mutants(28). It is likely that the putative LdpA protein has a functionthat represses the light sensitivity of the circadian clocksuch that it tends to run slower at decreased light intensity.Another mutation that causes a short-period phenotype in S.elongatus PCC 7942 is in pex, a gene that encodes a proteinof unknown function (23). We expect that cyanobacteria haveseveral input pathways that modulate the FRP, as do plants.

Addition of a sublethal concentration of the photosystem IIinhibitor DCMU also increased the FRP. This suggests that photosynthesiscan be a part of the light input pathway of the circadian systemin cyanobacteria. This is consistent with the finding that pulsedaddition of a lethal concentration of DCMU at various timesduring the circadian cycle resets the rhythm similarly to adark pulse (K. Okamoto and T. Kondo, unpublished results), asshown here for one time point (Fig. 6). In the eukaryotic algaLingulodinium polyedrum (formerly Gonyaulax polyedra), a sublethalconcentration of DCMU shortens the FRP (30). DCMU also inhibitsphase shifting in response to a light pulse in L. polyedrum(19) and in Chlamydomonas reinhardtii (18). Thus, a connectionbetween photosynthetic electron transport and light input pathwaysof the circadian system is a common characteristic among somephototropic organisms.

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FIG. 6. Resetting of circadian rhythms by DCMU. Cyanobacterial suspensions (3 ml) with an OD₇₃₀ of 0.3 were exposed a synchronizing 12-h dark interval, and then 1 µM DCMU (A) or 0.05% ethanol (solvent used for DCMU; B) was added. After incubation for 12 h under standard light conditions, cells were collected by centrifugation, washed twice with fresh BG-11 M, and suspended in 3 ml of BG-11 M. Samples (10 µl) were inoculated onto sample plates, and circadian rhythms were measured. The interval of DCMU treatment is indicated by the gray bar in panel B.

The deduced sequence of LdpA predicts that it is a 7Fe-8S proteinsimilar to some ferredoxins (3, 7, 26). This suggests that LdpAis involved in reduction, oxidation, or electron transfer (5).This biochemical function is consistent with a role that repressesthe sensitivity of the circadian clock to light. We proposethat photosynthetic activity (favors reduction) negatively regulatesthe activity of LdpA, repressing the sensitivity of the circadianclock to light. This model can explain the observation thatthe short-period phenotype of ldpA mutants is prominent onlyat low light intensity (Fig. 5).

Alternatively, the putative LdpA protein may function as a redoxsensor unrelated to light per se (5). In the SoxR protein ofE. coli, oxidative stress changes the redox state in the [2Fe-2S]cluster and leads to a change in its activity as a transcriptionfactor (11, 12). In the FNR protein, exposure to oxygen causesthe conversion of [4Fe-4S] to [2Fe-2S] and this conversion causesa change in protein conformation that represses its transcriptionfactor activity (21). The cellular redox state was recentlysuggested to modulate the mammalian circadian system (31). Thereduced phycocyanin content in an ldpA mutant, like the shortperiod, is typical of the phenotype of wild-type S. elongatuscells incubated at a high light intensity. However, in the mutant,the antenna pigment does not increase when cells are incubatedunder low-light conditions (data not shown). Recent reportsindicate that responses to a high light intensity and nutrientlimitation in this organism share pathway components, indicatingan integration of photoreception and metabolic signals (33,36). LdpA may lie within such a pathway. Whether the connectionis direct or indirect, LdpA seems to adjust the cyanobacterialclock to changes in the light environment.

ACKNOWLEDGMENTS

We thank S. Canales, J. L. Ditty, N. Ivleva, H. Min, and D.Bell-Pedersen for helpful comments regarding the manuscript.

This work was supported by National Science Foundation grantMCB-9982852 to S.S.G.; grants 11233203 and COE 13CE2005 to T.K.from the Japanese Ministry of Education, Culture, Sports, Science,and Technology; and a JSPS fellowship to M.K.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843-3258. Phone: (979) 845-9824. Fax: (979) 862-7659. E-mail: sgolden{at}tamu.edu.

Present address: University of Tokyo, Meguro, Tokyo 153-8902,Japan.

REFERENCES

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