JOURNAL OF BACTERIOLOGY, May 1990, p. 2313-2319 Vol. 172, No. 50021-9193/90/052313-07$02.00/0Copyright © 1990, American Society for Microbiology

Relationship between DNA Cycle and Growth Rate inSynechococcus sp. Strain PCC 6301

BRIAN J. BINDER* AND SALLIE W. CHISHOLMRalph M. Parsons Laboratory, 48425, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 29 August 1989/Accepted 15 January 1990

Flow cytometry was used to examine cell cycle regulation in Synechococcus sp. strain PCC 6301 under avariety of growth conditions. The DNA frequency distributions of exponentially growing and dark-blockedpopulations confirmed that this cyanobacterium contains multiple chromosome copies even at very slow growthrates. Furthermore, the presence of major peaks corresponding to other than 2" chromosome copies stronglysuggests that DNA replication is initiated asynchronously. Although this suggestion is at odds with the standardformulation of the procaryotic cell cycle model, it is similar to recent observations of asynchrony in Escherichiacoli replication mutants.

The coordination ofDNA replication with cell growth anddivision is a fundamental requirement of all microorganisms.The empirical rules by which such coordination is achievedin Escherichia coli are well known, although the underlyingmolecular mechanisms remain the subject of active research(11, 12, 16). While the basic characteristics of the chromo-somes and replicatory machinery in cyanobacteria appear todiffer little from those in E. coli (13, 17), the regulation ofreplication and cell division in this group of organismsappears to have some unique features which have not to datebeen reconciled with the classical procaryotic paradigm (13).

Perhaps the most widely reported observation concerningcyanobacterial cell cycles is the presence of high genomecopy numbers in these cells, observed in a number of strainsunder a variety of conditions (25, 29, 36). While in E. coli thechromosome copy number in any particular cell ranges from1 to 2 in cultures growing relatively slowly and is greaterthan 2 only at generation times shorter than the chromosomereplication time (C) plus the postreplication period (D) (1, 11,16), the mean per cell DNA content of Synechococcus sp.strain PCC 6301 (formerly Anacystis nidulans) is reported tobe as high as 6 chromosome equivalents at generation timesapproximating the estimated length of C + D in this organ-ism (25).

In addition to these high chromosome copy numbers,there is some evidence that regulation of chromosome rep-lication in cyanobacteria may differ in some respects fromthat in E. coli. In the latter case, replication is initiatedsimultaneously at all chromosome origins within any partic-ular cell (9, 11, 33). As a consequence of such synchronousinitiation, the number of origins (or pairs of replication forks)at any time over the cell cycle will be 2n (where n = 0, 1, 2,or 3) (12). Skarstad et al. (32) have confirmed that this is infact the case for wild-type E. coli but have also shown thatsome mutants can contain unexpected (#2n) origin numbers.Pouphile et al. (M. Pouphile, S. Brown, and M. Lefort-Tran,Biol. Cell 61:14A, 1987) presented evidence that the cyano-bacterium Synechococcus sp. strain PCC 6311 may containsimilarly unexpected chromosome numbers and suggestedthat these reflect asynchronous initiation in this strain. Incontrast, we recently found no evidence for such asyn-chrony in another Synechococcus strain (1).Numerous workers have used synchronously dividing

* Corresponding author.

cultures of Synechococcus sp. strain PCC 6301 to study thecyanobacterial cell cycle (2, 4-6, 18, 20). (Note that synchro-nous division among cells within a population should not beconfused with synchronous initiation of replication within asingle cell, as discussed above.) In general agreement withthe behavior of slowly growing E. coli (12, 16), DNAsynthesis has been found in these studies to be restricted toa certain portion of the cell cycle, although the position ofthat portion within the cycle has not been well defined (13).These studies rely on the assumption that the behavior of asynchronized population as a whole reflects the behavior ofthe cell cycle within a given cell during balanced, asynchro-nous growth. However, the behavior of the experimentalcultures in some cases belies this assumption. Herdman etal. (20), for example, reported that after the induction ofsynchrony (with a dark period and cessation of CO2 supply),a portion of the population could divide only once and thenapparently became inviable. The authors hypothesized thatthis effect was related to the stage of the cell cycle occupiedby each cell at the onset of darkness. In this case, therefore,effects of the synchronizing treatment on cell cycle physiol-ogy itself make unambiguous interpretation of the resultsdifficult (13).An alternative experimental approach involves flow cyto-

metric discrimination and analysis of unperturbed compo-nent subpopulations from an asynchronous culture growingunder defined conditions. This sort of analysis has been usedto study eucaryotic cells for some time and has morerecently been applied to E. coli (32-34), which was found toconform to the predictions of the procaryotic cell cyclemodel (33). Likewise, using this approach we have recentlyshown that cell cycle regulation in the relatively slow-growing unicellular marine cyanobacterium Synechococcussp. strain WH-8101 is consistent with the slow growth caseof a generalized Cooper-Helmstetter model (1).

In order to reconcile our observation of a "well-behaved"cell cycle in Synechococcus sp. strain WH-8101 with previ-ous reports of "aberrant" behavior in Synechococcus sp.strain PCC 6301 and related strains, we now extend our flowcytometric analysis to the latter species. We have examinedthe DNA frequency distributions for exponential-phase anddark-blocked populations of Synechococcus sp. strain PCC6301 growing at different rates and confirm that cells of thisspecies contain high chromosome copy numbers under awide variety of conditions. In addition, the DNA distribu-

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2314 BINDER AND CHISHOLM

tions of both exponentially growing and dark-blocked pop-ulations strongly suggest that initiation of chromosome rep-lication is asynchronous in this species.

MATERIALS AND METHODS

Synechococcus sp. strains PCC 6301 and WH-8101 wereprovided by John Waterbury (Woods Hole OceanographicInstitution). Unless otherwise indicated, experimental cul-tures were grown at 38°C on medium C (23) supplementedwith 10 mM bicarbonate and bubbled with 2.5% CO2 in air(pH -7.8). Light levels were adjusted by varying the posi-tion of culture tubes with respect to the light bank (Sylvaniacool-white fluorescent bulbs) and by application of up tothree layers of nylon window screening. The maximum lightintensity employed was 200 microeinsteins m-2 s-1 (PAR;measured with a Biospherical Instruments scalar irradiancemeter).

Culturing was semicontinuous; batch cultures were care-fully monitored and reinoculated periodically during theexponential growth phase to ensure that balanced growthprevailed. In all cases, cultures were maintained for at least10 generations under the specified experimental conditionsprior to analysis. Culture growth was monitored by directmicroscopic cell counts, electronic particle counts (CoulterCounter model ZM), or in vivo fluorescence (7). Growthrates measured by these three methods were indistinguish-able.Samples for balanced growth DNA distributions were

taken during the mid-exponential growth phase, at celldensities not exceeding -6 x 106 cells per ml. For dark-blocked samples, cultures in mid-exponential phase werewrapped in aluminum foil and incubated (under otherwiseunchanged conditions) for the equivalent of three generationtimes prior to sampling.Samples containing a total of approximately 108 cells were

centrifuged (10,000 x g, 15 min), suspended in a smallvolume (<0.5 ml) of supernatant, and added to 10 ml ofice-cold methanol with a 24-gauge syringe. These sampleswere stable for at least 12 months at 4°C. For DNA staining,portions of these methanol-fixed cells were centrifuged(11,000 x g, 5 min), washed once in phosphate-bufferedsaline (PBS, pH 7.5), and then suspended in PBS plusHoechst 33342 (0.1 ,g/ml final concentration). Samples werestained and analyzed in groups of six to eight; as a stainingcontrol, a standard sample was processed with each group(see below).

Stained samples were analyzed on a Coulter Epics-V flowcytometer equipped with a 6-W argon-ion laser. The laserwas tuned to 365 nm (300 mW output) and focused with a33-mm spherical quartz lens. Hoechst fluorescence wasmeasured between 418 and 590 nm. Unstained cells showedinsignificant fluorescence in this band. Microscopic exami-nation of the cells comprising peaks in the DNA frequencydistributions (as sorted by the flow cytometer) revealedinsignificant numbers of clumps or chains; thus, these dis-tributions are assumed to reflect the true distribution ofper-cell DNA content within a population.DNA frequency distributions of Synechococcus sp. strain

PCC 6301 remained stable over the course of the exponentialgrowth phase within a culture (Fig. 1B to F) and amongsuccessive culture transfers (not shown). As the growth rateof this culture declined, the mean per-cell fluorescencedropped (Fig. 1G to I) and resolved into three distinct peaks,the modes of which were in the ratio 1:1.5:2 (Fig. 1I). Thislast sample proved convenient for relating Hoechst fluores-

cence (as measured by the flow cytometer) to genomeequivalents. We consider it unlikely (though not impossible)that cells would be arrested with precisely 1.5 chromosomecopies, as this distribution implies. Therefore, our workinghypothesis is that these peaks in fact represent 2, 3, and 4genome copies, respectively, and we scale the relative DNAaccordingly in all the distributions presented here. Thisassignment of genome numbers is supported by colorimetricanalysis of bulk DNA (by the Burton diphenylamine method[10]): the DNA per genome in Fig. 11 (as derived from themean DNA per cell measured colorimetrically on a compa-rable sample and the mean genome equivalents per cellcalculated from the DNA frequency distribution) was 4.4 fg,in rough agreement with the published Synechococcus sp.strain PCC 6301 genome size of 3.5 to 3.8 fg (19, 21). Ourassignment of genome numbers is further supported by theconsistent appearance of peaks corresponding to integerchromosome copy numbers in the distributions scaled ac-cording to our hypothesis.

In order to account for drift in instrument performanceand for variations in staining, we used portions of the sampleshown in Fig. 11 as an external standard, which was ana-lyzed on the flow cytometer immediately before and afterevery experimental sample. The positions of the peaks inthis standard sample were used to normalize the experimen-tal distributions, while the coefficient of variation of thesepeaks provided an indication of general instrument perfor-mance.

It is unlikely that the cellular DNA contents we measuredhere are significantly affected by nonchromosomal DNA.Although Synechococcus sp. strain PCC 6301 is known tocarry two plasmids, together these represent only 2% of thegenome size (24, 30). Thus, extremely high copy numberswould be required before plasmid DNA could significantlyalter the DNA frequency distribution of a population.

RESULTS

Examination of the DNA frequency distribution of bal-anced, asynchronous populations of cyanobacteria in con-stant light and after a period of darkness can yield informa-tion about the timing and regulation of cell cycle stages inthese organisms (1). In preparation for such a study, weexamined in detail the time course of dark-induced changesin the DNA distribution in a single Synechococcus sp. strainPCC 6301 culture growing at near its maximum growth rate(,u = 0.2 h-1) (Fig. 2). A short time after the onset ofdarkness, cell division ceased and the broad unimodal DNAdistribution characteristic of the exponential phase at thisgrowth rate (Fig. 1) began to resolve into peaks (Fig. 2B toD). After 3 h of darkness the population was composedlargely of cells with 3, 4, 5, and 6 genome copies; few cellswith intermediate amounts of DNA (i.e., in the process ofreplication) were in evidence (Fig. 2E). In contrast to thecase when the population gradually entered stationary phase(Fig. 1), these peaks were not shifted toward less DNA percell, but rather appeared to be derived directly from theexponential-phase distribution. In both cases, the DNAdistributions included multiple peaks corresponding to otherthan 2' chromosome copies.

Continued incubation in the dark had no effect on theshape of the DNA distribution, although a slight drift upwardin the peak modes was evident (Fig. 2E to G). The constancyof the shape of the distribution makes it likely that thisincrease reflects a change in staining efficiency of thesesamples rather than a true change in DNA content. Based on

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DNA CYCLE AND GROWTH RATE IN SYNECHOCOCCUS SP.

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(B to I) Relative cell number versus per-cell DNA content for population at times specified by the respective letter in panel A. Culture wasgrown at 38°C at a light intensity of -200 microeinsteins m-2 s-1; growth rate was 0.20 h-1.

this time course, we chose a dark treatment corresponding tothree doubling times (-10 h for this particular culture) as ourstandard dark-block treatment.Upon restoration of light to this culture, cell division

resumed at its previous rate, and the DNA distributionreturned to a single broad peak. The lack of synchronous celldivision upon release of dark-blocked Synechococcus cellsinto the light contrasts with some of the results of others (2,3, 18, 20), but the experimental conditions were not identicalin these cases.Having established the conditions under which balanced

growth and dark-blocked DNA frequency distributionscould be reliably obtained (Fig. 1 and 2), we could proceed toexamine these distributions in Synechococcus cells growingat a range of light-limited growth rates (Fig. 3). For balanced-growth (i.e., exponentially growing) populations, as growthrate decreased there was a systematic trend toward peakscorresponding to lower copy numbers and toward sharperdefinition of these peaks (i.e., toward smaller proportions ofcells with intermediate DNA content). Thus, at IL = 0.19 h-peaks 3, 4, and 5 were evident but poorly defined, at IL =0.07 h-1 peaks 2, 3, and 4 were dominant, and at ,u = 0.02h-1, the slowest growth rate analyzed, peaks 2 and 3comprised virtually the entire population (Fig. 3C, E, andG). These changes in the DNA distributions of balanced-growth cultures reflected the changes we observed above ina single plateauing culture over time (compare Fig. 1G, H,

and I with Fig. 3C, D, and F). The mean cellular DNAcontent calculated from these distributions increased lin-early with growth rate (Fig. 3H). Note that at every growthrate examined, peaks corresponding to other than 2" chro-tnosome copies were in evidence.As was the case above for the fast-growing culture (Fig.

2), darkness resulted in a resolution of the balanced-growthdistributions into their component peaks without an appar-ent change in the range of copy numbers present (Fig. 3B toD). At lower growth rates, darkness had no significant effecton the shape of the DNA distribution; in these instances, thebalanced-growth distributions were already composed ofhighly resolved peaks (Fig. 3E to G).These DNA distributions are dramatically different from

those presented by Armbrust et al. (1) for Synechococcus sp.strain WH-8101. In order to determine whether this differ-ence was the result of the different growth temperaturesemployed, we examined a culture of Synechococcus sp.strain PCC 6301 growing at 25°C at a growth rate of -0.07h-1 (Fig. 4A and B). To our surprise, the apparent copynumber in these cultures was even higher than in 38°Ccultures growing at comparable rates (compare Fig. 4A andB with 3E); the dark-blocked population resolved into mul-tiple peaks corresponding to 2 to 8 chromosome copies andhigher (Fig. 4B). Note that Synechococcus sp. strain WH-8101 growing at the same temperature and roughly the samerate showed only the expected ln and 2n peaks (Fig. 4C).

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DISCUSSION

Some aspects of cell cycle regulation in Synechococcussp. strain PCC 6301 remain unreconciled with the procary-otic paradigm derived from studies with E. coli. The DNAfrequency distributions in exponentially growing and dark-blocked Synechococcus sp. strain PCC 6301 populationsinclude anomalous DNA peaks corresponding to other than2' chromosome copies. In addition, these distributions con-firm previous observations that cells of this species containmultiple chromosome copies even at generation times muchlonger than the presumed length of C + D.The prototypical cell cycle model for procaryotes was

formulated by Cooper and Helmstetter (11), based on studiesof E. coli. By combining this model for DNA replication (asa function of cell age) with an age distribution for a popula-tion under given conditions, the expected per-cell DNAdistribution for that population can be derived (1, 33). In thecase of slow growth (generation time [td] > C + D), cellsinherit a single chromosome copy (and therefore a singlereplication origin), and replication of that chromosome isusually preceded and always followed by a gap (i.e., a periodwith no DNA synthesis) (12, 16). As a consequence of suchgaps in the cell cycle, cells of different ages within thepopulation may have the same DNA content; this is reflectedby distinct peaks in the DNA frequency distribution of thatpopulation (1). The DNA distribution of slowly growingpopulations at steady state should therefore be bimodal

(given sufficient measurement precision), with peaks at 1 and2 chromosome equivalents.

In the fast-growth case of the model (td < C + D), cellsinherit greater than 1 chromosome equivalent. The amountof DNA inherited is a function of the length of C and Drelative to td; in the case of E. coli growing at its maximalrate (td of -20 min, C of -40 min, and D of -20 min), cellsinherit approximately 3 chromosome equivalents (and fourorigins). Prior to cell division, new rounds of replication areinitiated synchronously at all origins, and one set of oldrounds is completed. When C > td, cells inherit partiallyreplicated chromosomes and synthesis must occur through-out the cell cycle (i.e., there are no gaps). Therefore, nodistinguishable peaks should be present in the steady-stateDNA frequency distribution (1, 33). In contrast, when D >td, cells inherit 2 or more complete chromosomes, DNAsynthesis is limited to discrete portions of the cell cycle(assuming C < td), and distinct peaks in the DNA frequencydistribution may be present. Although D appears never to begreater than td in E. coli, it can be in other species (14, 22, 26,27).

Cellular DNA content in Synechococcus sp. strain PCC6301 in the present study reached as high as 6 genomeequivalents at the fastest growth rate examined (td = 3 h;Fig. 3B) and was never less than 2 chromosome equivalentseven at the slowest growth rate (td = 35 h; Fig. 3G).Furthermore, at all but the highest growth rate examined,

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(solid lines) and dark-blocked (dotted lines) populations of Synecho-coccus sp. strain PCC 6301 at different light-limited growth rates.Cultures were grown in constant light at 38°C and sampled inmid-exponential phase. For the dark-block, mid-exponential-phasecultures were incubated in the dark for periods equivalent to 3generation times. (A) Growth rate versus light intensity. Culturemedium contained 10 mM NaHCO3 and was bubbled with 2.5%C02, as described in text (circles), or was buffered at the samepH (7.8) with 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (triangles). (B to G) DNA distributions at thegrowth rates and light intensities indicated by the respective lettersin panel A. For ease of comparison among treatments, the DNAscale was normalized so that the mean distance between peakswithin a given distribution corresponded to 1 genome equivalent.(H) Mean relative DNA per cell (calculated from the exponentialgrowth distributions in panels B to G) versus growth rate.

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grown at 25°C. (A) Relative cell number versus per-cell DNAcontent for Synechococcus sp. strain PCC 6301 growing exponen-tially at ,u = 0.07 h-1. (B) Same culture as in panel A, dark-blockedfor 25 h. (C) Synechococcus sp. strain WH-8101 growing at ,u 0.06h-'. DNA scale normalized as described in the legend to Fig. 3.

the balanced-growth DNA frequency distributions containeddistinct peaks (Fig. 3C to G), indicative of gaps in DNAsynthesis. These data strongly suggest that in Synechococ-cus sp. strain PCC 6301, C + D> td at all the growth ratesexamined (since cells always appear to inherit greater than 1genome equivalent), that C < td for growth rates less than0.19 h-' (since distinct peaks are present in the DNAfrequency distributions), and that D > td at these growthrates (since cells inherit 2 or more genome equivalents and C< td)Our observations of multiple genome copies in Synecho-

coccus sp. strain PCC 6301 are consistent with previous datafor this and other cyanobacterial species (13). For example,Roberts et al. (29) found that Synechococcus sp. strain PCC7002 cells had 3 genome copies even when growing atgeneration times of 20 h. Mann and Carr (25) likewisepresented evidence for multiple copies in Synechococcus sp.strain PCC 6301 at relatively slow growth rates, although theincrease in DNA content observed with increasing growthrate was more dramatic in that study than we found here(Fig. 3H). These authors estimated C + D in this species tobe approximately 3 h for cells with a td of -26 h, aconclusion that is inconsistent with their own data indicatingrelatively high cellular DNA contents at slow growth rates,as well as with the data we present here. Full reconciliationof these data with the Cooper-Helmstetter model must awaitfurther quantitation of the relevant cell cycle parameters inSynechococcus sp. strain PCC 6301.An implicit feature of the Cooper-Helmstetter model is

that chromosome replication is initiated synchronously at allorigins within any given cell (9, 11). This assumption pre-dicts that cells will contain 2" origins (where n = 0, 1, 2, 3,... etc.) at all times, regardless of td, C, or D. As gaps inDNA replication are taken to result from the completion of around of replication prior to the next initiation, it follows that

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2318 BINDER AND CHISHOLM

cells within such gaps should contain 2' chromosome equiv-alents. This assumption has recently been tested and foundto hold for E. coli (32), but it is clearly violated in the presentcase; the DNA frequency distributions of Synechococcus sp.strain PCC 6301 cultures growing under a variety of condi-tions contained peaks corresponding to other than the pre-dicted 2" chromosome equivalents (Fig. 1 to 4).Although at odds with the behavior expected in wild-type

E. coli, the presence of anomalous peaks in the DNAdistributions presented here for Synechococcus sp. strainPCC 6301 bear a striking resemblance to those reported forcertain E. coli mutants after treatment with rifampin orchloramphenicol (31, 32, 34). These inhibitors prevent theinitiation of new rounds of DNA replication in E. coli (byinhibiting RNA and protein synthesis, respectively), butpermit the completion ("run out") of those rounds of repli-cation already in progress. After this treatment, therefore,cells contain only complete chromosome copies, the numberof which corresponds to the number of replication originspresent at the time of inhibition. As predicted, the DNAfrequency distribution of wild-type E. coli after inhibition iscomposed largely of peaks corresponding to cells with 1, 2,4, or 8 genome equivalents (depending on the original growthrate) (32). On the other hand, when the same treatment isapplied to certain replication-related mutants, the resultingdistributions'include peaks corresponding to other chromo-some copy numbers (e.g., 3, 5, and 6) as well (31, 34). Thislatter pattern has been termed the asynchrony phenotype,reflecting the view that such anomalous DNA peaks are theresult of asynchronous initiation of replication at the originspresent within a cell (32).The asynchrony phenotype is detectable in E. coli popu-

lations only after inhibition of new initiation and run-out ofreplication. As previous observations indicate that darknesscan have just such an effect on cyanobacteria (1, 18, 20), wehypothesize that the anomalous peaks (i.e., those corre-sponding to other than 2n chromosome equivalents) weobserved in dark-blocked Synechococcus sp. strain PCC6301 populations are in fact analogous to those observed ininhibitor-treated E. coli replication mutants. Thus, whenexponentially growing cultures are subjected to darkness,rounds of replication which have already been initiatedcontinue to completion, but no new initiations occur. Somedivision clearly occurs after the onset of darkness (Fig. 2A),but this appears to be insufficient to significantly deplete thehigher peaks or enrich the lower ones in most cases (Fig. 3Bto G).The presence of distinct peaks in the DNA frequency

distributions of balanced-growth (nonblocked) populationsof Synechococcus sp. strain PCC 6301 implies that replica-tion episodes in cells of these populations may be separatedby gaps. Thus it appears that initiation is asynchronous tosuch an extent that some chromosomes are fully replicatedbefore others are initiated. This is particularly evident invery slowly growing populations, which largely comprisecells within one of three replicatory gaps, containing 2, 3, or4 chromosomes (Fig. 3E to G). Darkness has no significanteffect on the DNA frequency distributions of these popula-tions because so few cells are in the process of replication atany given time. The extent to which these multiple gapsoccur regularly within every cell cycle or are instead theprobabilistic result of "random" initiation during the cyclecannot be addressed at present.

In any steady-state population, cellular DNA contentsshould range over a factor of 2. This is because newly borncells must contain half as much DNA as cells just prior to

division. Although the balanced-growth distributions pre-sented here for Synechococcus sp. strain PCC 6301 conformin large part to this expectation, a portion of the populationsappear to fall outside the predicted bounds (Fig. 2 to 4). Thiswas particularly apparent in the 25°C cultures, in whichcellular DNA contents ranged from 2 to greater than 10genome equivalents (Fig. 4). This phenomenon may berelated to the asynchrony phenotype: asynchronous initia-tion might result in an effective uncoupling between cellchromosome replication and cell division, so that some cellsare born with less than the expected DNA content. Depend-ing on the details of cell cycle regulation (which for thestrongly asynchronous case presented here are unknown),this situation might lead to broadening in the DNA (and/orage) distribution of the population (9). Alternatively, thisfeature of the DNA frequency distributions could result froma highly variable D period; variability in this parameter hasoften been found to be higher than that in other cell cycleparameters (8, 22).Among previous studies of cell cycle regulation in Syn-

echococcus sp. strain PCC 6301, there is little indication ofasynchronous initiation. In a number of studies involvingsynchronized cultures, DNA synthesis appears to be contin-uous within a specific (though large) portion of the cell cycle(2-6, 20). However, the resolution obtained in these studieswas clearly limited. Division is by definition instantaneous inany particular cell, yet it occupied 50% of the generationtime of the synchronized cultures in many of these studies. Itis obvious, therefore, that the extrapolated length of otherphases of the cell cycle will be subject to great uncertaintyand that the multiple gaps for which we have evidence couldhave been obscured.

Skarstad and Boye (31) discuss three models which mightexplain the asynchrony phenotype in E. coli, based on trulyasynchronous initiation, abortive initiation, and selectivechromosome degradation. Although we assumed in theforegoing discussion that initiation was truly asynchronous,we cannot at present distinguish between these-alternativesfor Synechococcus sp. strain PCC 6301.DNA frequency distributions of Synechococcus sp. strain

WH-8101 display none of the unusual characteristics de-scribed here for Synechococcus sp. strain PCC 6301 (1). Therange of growth rates examined in both studies overlapped,and although different growth temperatures were employed,these differences apparently cannot account for the discrep-ancy (Fig. 4). Although both strains are currently classifiedas Synechococcus spp., they fall into distinct "strain clus-ters" within that grouping and should therefore not beconsidered congeners (35). Thus, it is not inconceivable thatthe cell cycle might be regulated differently in each. It isimportant' to note that there are indications of the asyn-chrony phenotype in Synechococcus sp. strain PCC 6311(Pouphile et al., Biol. Cell, 1987) and Synechococcus sp.strain PCC 7942 (this study, data not shown), both consid-ered to be independent isolates of the same species as strainPCC 6301 (15, 28, 35, 37). Therefore, the DNA distributionsobserved here do not merely reflect aberrant behavior in ourparticular Synechococcus culture. The extent to which thisregulatory pattern occurs among the other Synechococcusclusters, and in other cyanobacterial groups generally, re-mains to be elucidated.

ACKNOWLEDGMENTSWe thank Rob Olson, Ginger Armbrust, Jim Bowen, and John

Waterbury for their helpful discussions and advice. We also thankan anonymous reviewer for critical and extremely helpful remarks.

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DNA CYCLE AND GROWTH RATE IN SYNECHOCOCCUS SP. 2319

This research was supported in part by National Science Foun-dation grant OCE-8614488 and Office of Naval Research contractsN00014-84-C-0278 and 87-K-0007.

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