overinitiation replication ofthe escherichia coli ...frequency of chromosomal replication affect...

JOURNAL OF BACTERIOLOGY, Feb. 1989, p. 674-683 Vol. 171, No. 20021-9193/89/020674-10$02.00/0Copyright C 1989, American Society for Microbiology

Overinitiation of Replication of the Escherichia coli Chromosomefrom an Integrated Runaway-Replication Derivative of Plasmid R1

ROLF BERNANDER, ANDREW MERRYWEATHER, AND KURT NORDSTROM*

Department of Microbiology, Biomedical Center, University of Uppsala, Box 581, S-751 23, Uppsala, Sweden

Received 19 July 1988/Accepted 24 October 1988

A 16-base-pair fragment, deletion of which completely inactivated oriC, was replaced by a temperature-dependent runaway-replication derivative (the copy number of which increases with temperature) of the IncFllplasmid R1. The constructed strains were temperature sensitive, and flow cytometry revealed a severalfoldincrease in the DNA/mass ratio following shifts to nonpermissive temperatures. The cell size distribution wasbroader in the constructed strains relative to that in the wild type because of asynchrony between thechromosome replication and cell division cycles. This difference was more pronounced for counterclockwiseinitiation of chromosomal replication, in which small DNA-less cells and long filaments were abundant.Following a temperature shift the cell size distributions became even more broad, showing that changes in thefrequency of chromosomal replication affect cell division and emphasizing the interplay between these twoprocesses.

In the Escherichia coli cell cycle, the period of chromo-some replication, the C period, is directly followed by theperiod required for nucleoid segregation, septum formation,and cell division, the D period (9). These processes arestrictly coordinated with respect to growth of the cell, suchthat initiation of bidirectional chromosomal replication fromthe unique origin oriC (3, 34) occurs only once per cycle, ata specific cell mass or volume (11). This ensures thatcomplete chromosomes can be segregated to each daughtercell at cell division, while it prevents the synthesis ofredundant genetic material. Despite extensive research, themechanisms that regulate and coordinate initiation of chro-mosomal replication and cell division are unclear. It hasbeen suggested that cell division is triggered by an eventduring the termination of DNA replication (14, 19, 42, 43).Alternatively, cell division may be triggered when cellsreach a certain critical length. If this length is attained at thesame time as chromosomal replication is terminated, the twoprocesses become coordinated without the need for a directlink between them (12, 13). It is clear, however, thatinitiation of chromosome replication and cell division arecoregulated in a negative fashion by at least two independentsystems (8), such that DNA damage or a block in chromo-some replication results in the inhibition of cell division.Furthermore, defective nucleoid segregation also affects celldivision, and results in aberrant septum positioning in thecell (12, 20).The identification of elements important for the control of

replication of several plasmid replicons has relied on theisolation of copy number mutants (for reviews, see refer-ences 31 and 37). However, such elements have not beenrevealed during attempts to isolate mutations in the genescontrolling chromosomal replication of E. coli. Instead,chromosomal copy number mutations have been mapped togenes encoding subunits of RNA polymerase (rpoB andrpoC [35, 41]), an enzyme that does not show the specificityexpected from an element which regulates initiation ofDNAreplication. Overinitiation of chromosomal replication hasbeen observed for strains harboring certain dnaA genemutations, but only under conditions involving temperature

* Corresponding author.

shifts (17, 18, 22). Furthermore, overexpression of the dnaAgene also increases chromosomal initiation frequency (1, 6,32, 45). However, all these manipulations of the dnaA generesult only in limited and transient effects on the rate ofchromosomal initiation.As a novel approach to gain insight into the mechanisms

connecting chromosomal DNA replication and growth of thebacterial cell, we constructed strains in which the initiationfrequency of chromosomal replication could be varied inde-pendently of the constraints imposed by normal cell cycle-related control mechanisms. This was achieved by substitut-ing a 16-base-pair (bp) fragment essential for the oriCfunction for the mini R1 plasmid pOU71 (described in detailbelow), the initiation frequency of which could be controlledin a graduated manner by changing the temperature.We report the construction and characterization of strains

with this novel control of chromosomal replication. Weshow that these strains contain more DNA at increasedgrowth temperatures, and we describe the effects of theseincreased temperatures on growth characteristics and cellsize distribution.

MATERIALS AND METHODSBacterial strains and plasmids. The E. coli K-12 strains and

plasmids used in this study are described in Table 1. PlasmidpOU71 consists of the X cI857 repressor allele and X PRcloned upstream of the R1 basic replicon (24). At 30°C, thisplasmid has the normal R1 copy number (about one perchromosome equivalent), but with increasing temperaturethe X cI857 repressor activity gradually decreases, resultingin increased transcription from A PR into the R1 basicreplicon. This directs an increased synthesis of RepA, therate-limiting protein necessary for the initiation of R1 repli-cation (27). In this way it was possible to increase thefrequency of DNA replication gradually in a controlledmanner. Plasmid pOU420 contains the PstI F1 fragment fromthe R1 basic replicon (30) cloned into the PstI site of pBR325(J. E. L. Larsen, unpublished data). Downstream of thecopA gene the XcI857 temperature-sensitive repressor geneand the A PR promoter were cloned; they were oriented suchthat transcription from A PR opposed that from the constitu-tive copA promoter. Therefore, at low temperatures, when

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OVERINITIATION OF E. COLI CHROMOSOME REPLICATION 675

TABLE 1. E. coli strains and plasmids used in this studya

Designation Genotype, phenotype Reference or source

StrainsEC1005 metBi nalA relAI spoTi xr F- 15EC::71CW EC1005; AoriC::pOU71, Rep-Td, initiating clockwise. This studyEC::71CC EC1005; AoriC::pOU71, Rep-Td, initiating counterclockwise. This studyHB101 hsdS20 (r- m-) recAJ3 ara-14 proA2 lacYl galK2 rpsL20 xyl-5 mtl-i 25

supE44 X- F-UD671 ara argE A(pro-lac) gyrA rpoB rpsL666 F- 36

PlasmidspBR322 Apr Tcr 5pBR325 Apr Tcr Cmr 4pBS1 Apr Cmr 10.5-kbp HindIII insert from E. coli terminus region 2pCW1 and 2 Apr, Rep-Td This studypCC1 and 2 Apr, Rep-Td This studypGW71 Apr, A PR and X cI857 gene deleted from pOU71, R1 basic replicon E. G. H. Wagner (unpublished data)pHP45fQ Apr, Smr 33pLK4 Apr AoriC 23pOU71 Apr, Rep-Td 24pOU420 Apr Cmr copA(Ts) J. E. L. Larsen (unpublished data)pRB20 Sur 16-bp oriC deletion This studypRB103 Apr Sur, Rep-Td This studypRB107 Apr Sur, Rep-Td This studyRSF1010 Smr Sur 16p71CW Apr Smr, Rep-Td This studyp71CC Apr Smr, Rep-Td This study

a Abbreviations: Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Kmr, kanamycin resistant; Smr, streptomycin resistant; Sur, sulfonamide resistant;Tcr, tetracycline resistant; Rep-Td, replication temperature dependent; (Ts), temperature-sensitive; kbp, kilobase pairs.

the cI857 protein repressed A PR, this high-copy-numberplasmid synthesized large amounts of CopA RNA. How-ever, with increasing temperature, the repressing activity ofc1857 protein was reduced, and the resulting increase incountertranscription from X PR reduced synthesis ofCopA ina graduated fashion to a virtual shutoff at 42°C.Media and growth conditions. Bacteria were grown in

Luria broth or M9 medium (25) supplemented with 0.2%glucose. Complete minimal medium was M9 medium sup-plemented with 0.2% glucose and 0.2% Casamino Acids(Difco Laboratories, Detroit, Mich.). The appropriate aminoacids (50 pug/ml) were added when required. Unless other-wise stated, antibiotics were used at concentrations of 100,g/ml (streptomycin and sulfonamide) or 50 ,ug/ml (chloram-phenicol, ampicillin, and kanamycin).DNA technology. DNA isolation and cloning, radioactive

labeling of DNA, agarose gel electrophoresis, Southernblotting, and hybridizations were performed essentially asdescribed by Maniatis et al. (25). Chromosomal DNA wasisolated by a modification of the procedure described byMarmur (26). Autoradiographs were scanned by using adual-wavelength scanner (model CS-930; Shimadzu).P1 transduction. Isolation of transducing phage and trans-

ductions were performed as described previously (29). A lowtemperature (30°C) was used in transductions involving the AcI857 allele.

Construction of strains EC::71CW and EC::71CC. Toexchange the oriC fragment for pOU71 by in vivo recombi-nation, recombinant plasmids were constructed in whichpOU71 was flanked by the oriC sequences adjacent to the16-bp BglII fragment to be replaced. The source of theseoriC sequences was plasmid pLK4, which comprises a2.5-kbp BglII chromosomal origin fragment (positions -470to +2006 according to reference 7) containing a 16-bpdeletion between the BglII sites (positions +23 to +38)cloned into the BamHI site of pBR322 (Fig. 1). Since both

pOU71 and pLK4 confer Apr as their sole resistance marker,pLK4 was cleaved with EcoRI and PvuII and ligated to the7.3-kbp EcoRI-PvuII fragment of RSF1010, thereby replac-ing most of the pBR322 sequence, including the Apr gene,with the RSF1010 basic replicon and the sulfonamide resis-tance gene, thus forming plasmid pRB20. Plasmid pOU71,which was linearized with BamHI, was then inserted intothe unique BglII site of pRB20 located at the 16-bp oriC dele-tion. Recombinant plasmids were chosen in which pOU71initiated either clockwise (pRB103) or counterclockwise(pRB107) relative to the flanking chromosomal DNA. Bothrecombinant plasmids were cleaved with SnaBI and NruI,thus removing the RSF1010 sequence, and ligated to the 2.1kbp SmaI fragment from pHP45fQ. This so-called fl fragmentharbors the aad gene, the product of which mediates resis-tance to streptomycin. The resultant plasmids p71CW andp71CC therefore comprise pOU71, initiating clockwise andcounterclockwise, respectively, flanked by chromosomaloriC sequences (positions -413 to +22 and +39 to +2006)and carrying the aad gene outside these oriC sequences (Fig.1).Plasmids pCW71 and pCC71 were introduced into the

chromosome by homologous recombination, as describedpreviously (28).

Reisolation of plasmids from chromosomal DNA. Chromo-somal DNA from strains EC::71CW and EC::71CC wascleaved with NruI, self-ligated, and used to transformHB101 cells to Apr, with recombination between isolatedplasmid and host chromosomal sequences being limited bythe recA mutation carried by this strain. Since pOU71contains no NruI recognition sequences, the resultant plas-mids should carry the entire plasmid sequence flanked byapproximately 1.16 and 2.37 kbp of chromosomal DNA tothe left and right, respectively (Fig. 2).

Determination of relative plasmid copy number at differenttemperatures. EC1005 cells were transformed to Apr by each

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676 BERNANDER ET AL.

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FIG. 1. Construction of plasmids. The bars represent the plasmids that were used in the construction of plasmids p71CW and p71CC. Notethat plasmids pRB107 and p71CC (not shown) are identical to pRB103 and p71CW, respectively, except that the orientation of the pOU71sequence (indicated by a box with a bold outline) is reversed. Relevant regions are indicated as follows: horizontal lines, chromosomal oriCsequences spanning positions -470 to +22, denoted oriCL; vertical lines, chromosomal oriC sequences spanning positions +39 to +2006,denoted oriCR; solid box, plasmid origin of vegetative replication, denoted oriV; solid arrow, vegetative origin of pOU71, the arrow indicatesthe direction of fork movement, denoted oriRi; diagonal hatch, R1 basic replicon; checkered, X cI857 repressor gene and X PR promoter;light shading, RSF1010 sequences; heavy shading, genes encoding resistance to ampicillin, sulfonamide, and streptomycin (aad). Restric-tion enzyme recognition sites are denoted as follows: B, BamHI; Bg, BgIII; E, EcoRI; N, NruI; P, PvuII; 5, SnaBI; Sm, SmaI; a slashbetween two restriction enzyme designations indicates that the hybrid restriction site is no longer recognized by either of the indicatedenzymes.

plasmid to be tested, and the resultant strains were grown inLuria broth containing 50 jig of ampicillin per ml at 30'C toa density of 60 Klett units before they were diluted into freshmedium prewarmed to 30, 38, and 420C. After 2 h, 1-mlsamples were withdrawn and novobiocin was added to 500pg/ml, to prevent further DNA replication, before 2-1.dportions of appropriate dilutions were spotted onto twonitrocellulose filters. Cells were lysed, and the immobilizedDNA was hybridized to probes taken either from the chro-mosomal terminus region (pBS1) or the pOU71 basic repli-con. Autoradiographs were scanned, and the relative copynumber of the plasmids at different temperatures was deter-mined. Similarly treated EC1005 strains harboring eitherp71CW or p71CC were used as positive controls, andpGW71, a derivative of pOU71 which lacks the X c1857 geneand A PR promoter, was used as a negative control.Flow cytometry. Bacteria were prepared for flow cytome-

try as described previously (39) except that cells were notwashed before they were fixed in ethanol (final concentra-tion, 70%). Flow cytometry was performed with a flowcytometer (model Argus; Skatron). For comparison of dif-ferent samples, each sample was prerun together with a

population of stationary-phase EC1005 cells, to compensatefor differences in staining efficiencies. The control popula-tion was placed at a fixed position in the histogram, andthe appearance of the sample population was compared

with that of the same sample run without stationary-phasecells.

RESULTS

Strain construction. In order to control chromosomalinitiation frequency with the temperature-inducible pheno-type conferred by plasmid pOU71, it was necessary both toinsert pOU71 into the chromosome and to inactivate thechromosomal origin. This was achieved by replacing a 16-bpBglII fragment, which was situated at the left extreme of theminimal chromosomal origin and essential for its function,by pOU71 (Fig. 2). Such a strategy not only mimics normalchromosomal initiation as closely as possible, by ensuringthat replication of the chromosome, although initiated atpOU71, starts within oriC, but also preserves most of theoriC sequence, thus maintaining regions of possible impor-tance for cell cycle coordination.To select for isolates in which the oriC fragment was

exchanged for pOU71 by in vivo recombination, a positiveselection strategy was used, as described previously (28).This technique required the construction of recombinantplasmids p71CW and p71CC, in which pOU71 was orientedto initiate clockwise and counterclockwise, respectively,and flanked by the oriC sequences adjacent to the chromo-

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OVERINITIATION OF E. COLI CHROMOSOME REPLICATION

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HBIPH Bg P NBg Bg BBBgBHPI V I I I ) *Y 6iV<I

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FIG. 2. Physical map of origin regions in strains EC1005, EC::71CW, and EC::71CC. The top broad, solid line represents chromosomalDNA around the minimal origin of wild-type strain EC1005. The scale below denotes the nucleotide sequence numbering, according to Buhkand Messer (7). The two lower broad, solid lines represent the plasmid pOU71 DNA replacing the 16-bp BgIII fragment (positions +23 to +38)of the minimal chromosomal origin in strains EC::71CW and EC::71CC, respectively. Relevant genes (stippled boxes, with the arrow denotingthe direction of translation), transcripts (solid arrows), and the RI plasmid origin (open arrow representing the direction of fork movement)are indicated. The precise extent of transcription distal to the repA gene from the X pp and pCopB promoters is unknown, as denoted by thebroken lines. Restriction enzyme recognition sites are denoted as follows: B, BamHI; Bg, BgIII; E, EcoRI; H, HindIII; P, PstI; N, NruI; a

slash between two restriction enzyme designations indicates that the hybrid restriction site is no longer recognized by either of the indicatedenzymes.

somal target site (see above). P1 transduction was used totransfer the integrated plasmids from UD671, the intermedi-ate strain required for the positive selection procedure, intostrain EC1005. Isolates EC::71CW and EC::71CC, whichwere derived from the integration of p71CW and p71CC,respectively, were chosen for further analysis. It should benoted that during this transfer a reduced ampicillin concen-

tration (20 Vg/ml) and minimal medium were required toselect for strain EC::71CC but not strain EC::71CW. Fur-thermore, growth of EC::71CC in Luria broth was subse-quently shown to result in increased filamentation comparedwith growth in complete minimal medium (data not shown).We therefore suggest that the growth of EC::71CC may bemore imbalanced than that of EC::71CW in medium support-ing high growth rates.

Verification of the correct site of integration. The firstevidence suggesting that pOU71 was integrated into the oriCregion in strains EC::71CW and EC::71CC was the cotrans-duction of the Apr gene, carried by pOU71, with chromo-somal genes asnA and ilvD located close to oriC. To confirmthe location of the integrated pOU71, chromosomal DNAisolated from EC1005, EC::71CW, and EC::71CC was

cleaved separately with BamHI, BglII, HindIII, and PstI andsubjected to Southern blotting analysis. Hybridization withan oriC-specific probe revealed major hybridizing fragmentsappropriate for the replacement of the 16-bp BglII oriCfragment by pOU71, with the concomitant loss of both BglII

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FIG. 3. Southern blotting analysis of chromosomal DNA. Chro-mosomal DNA isolated from strains EC1005 (EC), EC::71CW(CW), and EC::71CC (CC) was digested separately with BamHI,BgIII, HindIlI, and PstI. Samples containing 1 fig of DNA were

subjected to gel electrophoresis in 0.7% agarose prior to transfer tonitrocellulose, as described in the text. Immobilized DNA was

hybridized to an 802-bp oriC-specific probe extending between theSacl (position -375) and ClIa (position +427) restriction sites.Fragments that hybridized poorly to the probe, as a consequence ofa limited region of homology but which were clearly visible on more

heavily exposed autoradiographs (data not shown), are indicated byarrowheads. The nature of the novel unexpected fragments in BglII-and HindIII-digested DNA (indicated by dots) is discussed in thetext. The scale represents approximate fragment sizes in kilobasepairs.

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TABLE 2. Temperature effect on plasmid copy number

Ratio between hybridization to plasmid andPlasmida chromosomal probes atb:

300C 38'C 420C

pGW71 1 1 0.7p71CW 1 14 93pCW1 1 12 67pCW2 1 12 123p71CC 1 11 107pCC1 1 14 90pCC2 1 14 116

a Strain EC1005 was used as the host for all plasmids. Plasmids isolatedfrom the chromosomes of strains EC::71CW and EC::71CC are denoted pCWand pCC, respectively.

b The ratio was determined 2 h after the temperature was shifted. The ratioof hybridization between the plasmid and chromosomal probe found at 300C isset to 1 for each plasmid, and the ratios at 38 and 420C were normalized to thisvalue (see text for details).

sites (Fig. 3). Interestingly, minor hybridizing fragmentswere also observed in digests of EC::71CW and EC::71CCchromosomal DNAs. We believe that these minor frag-ments, which represent only a small proportion of thehybridizing DNA, arise as the result of stalled forks at theunidirectional R1 origin. It is important, however, to stressthat with all enzymes tested, the major hybridizing frag-ments are compatible with the correct replacement of the16-bp BglII oriC sequence with pOU71 in both orientations.

Reisolation of plasmids from chromosomal DNA. To exam-ine whether the plasmids inserted into the chromosomeretained their temperature-inducible initiation frequency, R1replicons were reisolated from the chromosomes ofEC::71CW and EC::71CC, as described above. Restrictionmapping of several of these plasmids (pCW1 and pCW2 fromEC::71CW, and pCC1 and pCC2 from EC::71CC) confirmedthat pOU71 was located and oriented correctly within oriC inboth strains, and that oriC-flanking sequences not containedin the original plasmids (p71CW and p71CC) before integra-tion were present at the expected locations. This argues thatthe recombination event did not grossly perturb the chromo-somal DNA surrounding the site of integration.

If no alterations occurred in these plasmids during theintegration and subsequent reisolation, they should haveretained their temperature-inducible initiation frequency.We therefore determined their relative copy numbers atdifferent temperatures (see above). At 30°C all plasmidswere shown to be present at an approximately equal copynumber (data not shown). However, those plasmids carryingthe X c1857 repressor gene and A PR promoter increased incopy number about 10-fold over 2 h at 38°C and about100-fold at 42°C, whereas the control plasmid pGW71showed no significant change in copy number (Table 2).There was essentially no difference between the response ofthe original plasmids and that of those isolated followingintegration, nor between those in which pOU71 was orientedclockwise or counterclockwise. We therefore conclude thatthe temperature inducibility of plasmid copy number isunaffected by either the insertion into the bacterial chromo-some or the subsequent reisolation.The only active origin in EC::71CW and EC::71CC is that

of plasmid pOU71. To be able to attribute unequivocally anyeffects of increased temperature to overinitiation at theinserted pOU71 origin, it was important to demonstrate thatthis was the only origin capable of replicating the chromo-some of the constructed strains. To show this we used

plasmid pOU420 (described in detail above), which harborsthe gene encoding the antisense RNA (CopA) that is respon-sible for negatively regulating R1 copy number. Transcrip-tion was arranged such that at 30'C large amounts of CopARNA were synthesized, inhibiting replication from R1 repli-cons present in the same cell, but with increasing tempera-ture CopA synthesis was reduced in a graduated fashion tonegligible levels at 420C.

Strains EC::71CW and EC::71CC were transformed sep-arately with equal amounts of pOU420 and pBR325. It wasfound to be necessary to allow only 30 min for geneexpression during transformation at 30TC, since longer peri-ods resulted in cell death at all temperatures (data notshown), presumably because of the effect of CopA. Thetransformation mixtures were plated equally onto pre-warmed complete minimal medium plates containing 50 ,ugof chloramphenicol per ml and incubated overnight at dif-ferent temperatures. At 370C large numbers of transformantswere obtained with both plasmids. In contrast, however, at30'C very few colonies were formed by either strain whenthey were transformed with pOU420 (less than 1% of thoseresulting from transformation with pBR325). Thus, synthesisof large amounts of CopA RNA, which results in essentiallycomplete inhibition of initiation from an R1 origin, allowslittle or no survival of EC::71CW or EC::71CC, therebyconfirming that pOU71 is the sole active origin in thesestrains.

Overinitiation of chromosomal replication in strainsEC::71CW and EC::71CC. Our chief aim in constructingstrains EC::71CW and EC::71CC was to study the effects ofoverinitiation on various aspects of cell growth and physiol-ogy. It was therefore crucial to ask whether these strainsindeed increased initiation of chromosomal replication as thetemperature was increased.Flow cytometry was used to measure cell size and DNA

content, which were determined as light scattering andfluorescence, respectively, for cultures growing exponen-tially at 300C or shifted to 38 and 42°C (Fig. 4). After a shiftfrom 30 to 38°C, the DNA content of EC::71 CW increased,while the cell size distribution did not broaden greatly,indicating that the cells must have a higher DNA/mass ratioat 38°C. Following a shift to 420C, both cell size and DNAcontent increased, making overall changes in the DNA/massratio difficult to assess. However, since the increase in DNAappeared to exceed that of cell size, it is probably that theDNA-mass ratio was also increased at this temperature. Inpopulations of EC::71CC, both cell size and DNA contentincreased significantly at both elevated temperatures. Sincethe DNA content was greatly increased in all size classes ofcells relative to that of cells grown at 30°C, it is extremelylikely that the DNA:mass ratio was also significantly ele-vated at both 38°C and 42°C. Therefore, it is clear thatelevated temperatures increase the DNA content of strainsin which chromosomal replication is initiated by pOU71.Growth and viability of strains EC::71CW and EC::71CC.

To be able to use the constructed strains in studies of thechromosome replication and cell division cycles, we had toanalyze their general properties. The most important ofthese were their grown and viability at different tempera-tures.During exponential growth in complete minimal medium

at 30°C, the generation time for both EC: :71CW andEC::71CC (measured as 70 and 80 min, respectively) wassubstantially longer than that of the wild-type strain (50 min).In order to study how increased temperature affected growthrate and viability, EC1005, EC::71CW, and EC::71CC were

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42tCFIG. 4. DNA content (fluorescence) and cell size (light scatter) distributions for strains EC1005 (A), EC::71CW (B), and EC::71CC (C).

Samples were taken 2 h after temperature shifts from 30TC to the temperature indicated below the histogram. Numbers indicate instrumentrecording channels. For reference, two light-scatter channels and two fluorescence channels are indicated by lines in the histograms. Eachhistogram represents 20,000 cells, exclusive of DNA-less cells (no fluorescence), which were not recorded.

grown at 30'C in complete minimal medium for 5 h beforethey were shifted to temperatures of 30, 36, 37, 38, and 420Cfor 6 h. As expected, the growth rates of all strains increasedafter the temperature shift (Fig. 5), but only at 30 and 360Cwas this higher growth rate sustained by all strains for 6 h. Incontrast, at temperatures above 360C the growth rate ofEC::71CW and EC::71CC started to decline after an initialincrease, with the onset of this decline being both more rapidand pronounced with increasing temperature. StrainEC::71CC was more temperature sensitive than EC::71CW,exhibiting a more rapid response at all temperatures above360C, such that in 6 h following a shift to 420C only one massdoubling occurred.At all temperatures, both EC::71CW and EC::71CC con-

tinued to increase in optical density, albeit at a very slowrate, over the entire 6-h period studied, despite the rapidonset of the decline in growth rate at elevated temperatures.This slow but significant growth was also noted at interme-diate temperatures in classical tests of colony-forming abil-ity. To determine the relative colony-forming ability at

different temperatures, strains EC1005, EC::71CW, andEC::71CC were grown in complete minimal medium at 30'Cto the mid-exponential phase. Appropriate dilutions in thesame medium were then plated onto prewarmed completeminimal medium plates and incubated at 30, 38, and 420C. Asexpected, the wild-type strain had approximately the sameviability at all temperatures (Table 3). However, althoughthe viability of EC::71CW was similar at 30 and 380C, growthat 420C reduced the number of colonies formed by about105-fold. Strain EC: :71CC showed a similar decrease at420C, but it also exhibited a 50-fold decrease in viability at38°C, relative to that at 30°C. This was in keeping with thepreviously observed increased temperature sensitivity ofEC::71CC relative to that of EC::71CW.Growth of EC::71CC at 38°C was very slow, taking over

48 h to form small colonies. Thus, although incubation at38°C seriously affected these cells, resulting in a very slowgrowth rate, many of them still formed small colonies. Incontrast, the few colonies formed at 42°C appeared afterovernight incubation. Cells from such colonies, although

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FIG. 5. Growth characteristics during temperature shifts. Strains EC1005, EC::71CW, and EC::71CC were grown in complete minimalmedium for 5 h at 30°C, prior to dilution to 1:10 in prewarmed medium at 30, 36, 37, 38, and 42°C. Incubation was continued at thesetemperatures for 6 h after the shift. Cultures were maintained in the mid-exponential growth phase by appropriate dilution with prewarmedmedium. Values for optical density, which were monitored throughout with a Klett-Summerson colorimeter at 640 to 700 nm, were adjustedappropriate to the dilution of the cultures and are presented as a log-linear plot against time. The vertical line denotes the time of temperatureshift from 30°C to the temperature indicated above each panel.

temperature resistant, retained a broad cell size distributionand grew more slowly than wild-type cells (data not shown),suggesting that chromosomal replication is still initiated bypOU71. Such isolates therefore presumably carry mutationswhich suppress the lethal effect of high temperatures.

Cell size distribution. At 30TC the variation around themean cell size for the population of EC: :71CW cells was onlya little greater than that for the wild type, whereas that forEC::71CC cells was much larger, suggesting that growth ismore perturbed when pOU71 initiates chromosomal replica-tion in the counterclockwise orientation rather than wheninitiation occurs first in the clockwise direction (Fig. 4). Thedistributions of DNA content were also broader at 30TC inthe constructed strains compared with those in the wild type,such that cells of any given size class contained variableamounts of DNA.

It is apparent from the histograms of populations of cellsshifted from 30 to 38 or 42TC that both EC::71CW andEC::71CC showed an increase in cell size distribution withincreasing temperature. This increase was only slight inpopulations of EC::71CW that were shifted to 380C for 2 h,but increased to a much greater extent following a shift to42TC. However, populations of EC::71CC cells showed anincreased abundance of elongated cells at both elevatedtemperatures. The flow cytometry histograms only showcells which contained DNA; microscopic examination re-vealed the presence of small DNA-less cells in populationsof both EC::71CW and EC::71CC (Fig. 6). In cultures ofEC::71CC these cells were readily observed at 30'C andincreased in number dramatically following a shift to 38 or

TABLE 3. Relative frequency of CFU at different temperatures

Relative colony-forming ability ata:Strain

300C 38°C 42°C

EC1005 1.0 0.8 1.0EC::71CW 1.0 1.1 2.3 x 10-5EC::71CC 1.0 b 3.9 x 10-5

a Values are normalized to the number of colonies at 30'C, which was set to1.0 for each strain (see text for details).b-, After prolonged incubation (>2 days), small colonies appeared at a

relative frequency of 0.02.

Ac_

B /

FIG. 6. Phase-contrast (left) and fluorescence micrographs(right) of strains EC1005 (A), EC::71CW (B), and EC::71CC (C).Micrographs were taken 5 h after a temperature shift from 30 to380C, to accentuate the difference in the number of minicellsproduced by each strain. Fluorescence microscopy was performedby the same DNA-staining technique described previously for flowcytometry (39). Arrows indicate presumptive DNA-less cells (nofluorescence). Bar, 10 p~.

I+ EC100S 300C 360Co EC::?ICW* EC::7lcC1 111A~I

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420C, indicating that there was an effect on cell division.However, although these small cells were present in culturesof EC::71CW at all temperatures, they were much lessabundant than in EC::71CC (data not shown).

DISCUSSIONBy exploiting the well-understood initiation control sys-

tem of plasmid R1, we have described the construction andcharacterization of strains in which the effect of increasedchromosomal initiation frequency on various aspects of thecell cycle can be studied. This was achieved by using in vivorecombination to replace a small, essential region of thechromosomal origin, oriC, by an R1 derivative pOU71, theinitiation frequency of which is temperature dependent.Since plasmid R1 replicates unidirectionally (10), strainswere constructed in which pOU71 initiates chromosomalreplication in either a clockwise or counterclockwise orien-tation. For both these strains, it was demonstrated thatpOU71 is positioned in the expected orientation at theleft-hand end of the inactivated minimal chromosomal ori-gin, as shown by transduction experiments and Southernblotting analysis, and that R1 replicons reisolated from thesestrains retain the temperature-dependent initiation fre-quency. Furthermore, the inserted plasmid origin was shownto be absolutely required for chromosomal replication ofthese strains.The characteristics of the two strains can be summarized

as follows. First, the DNA content of both strains increasedwith increasing temperature, as expected if pOU71 retainedits replication properties when it was inserted into oriC.Second, growth and viability were strongly affected bytemperature. At a low temperature (300C), growth andmorphology were essentially the same as those for previ-ously described strains replicating from an integrated R1plasmid (23). A high temperature (420C) was lethal for bothstrains, while an intermediate temperature (380C) allowedgrowth at reduced rates. Third, the cell size distributionincreased for both strains as the temperature was increased.Fourth, the characteristics of the strains with the R1 repliconin opposite orientations were different. The cell size distri-bution of strain EC::71CC was broader at all temperatures,and it displayed a greater temperature sensitivity than didstrain EC::71CW.There are several possible explanations for the observed

temperature sensitivities of the strains. The most obvious isthat a greatly increased gene dosage results in metabolicdisturbances that cause reduced viability. Also, there mayexist an upper limit to the DNA concentration that can betolerated by the cell. Interestingly, viable mutants with anincreased chromosomal initiation frequency only increasethe chromosomal copy number by two- to fourfold (35, 41).A similar increase has been reported following the stimula-tion of chromosomal initiation by overproducing DnaAprotein (45). Thus, a three- to fourfold increase in the copynumber of the chromosome may be inhibitory to cell growth,and these new constructs could exceed this critical value ata high temperature. A third possibility is that with increasedinitiation rates these strains form complex nucleoid struc-tures, which cannot be resolved at cell division, and there-fore lead to cell death. In accordance with this is the largenumber of filaments seen at 42°C for both strains and thepersistence of elongated cells in stationary-phase cultures(data not shown), suggesting that cell division cannot resolvethese cells even after long periods.The effects on cell size distributions that were observed

also deserve comment. We believe that the increase in cell

size distribution at a low temperature in both strains, ascompared with that of strain EC1005, results from therandom timing of chromosomal replication within the cellcycle. This occurs when chromosomal replication is initiatedfrom an integrated R1 replicon rather than from oriC (23). Asimilar broadening of cell size distribution (and DNA con-tent) observed both in populations of cells harboring certainmutant recA or dnaA alleles (39, 40) and in cells displayingthe constitutive stable DNA replication phenotype (44) hasbeen attributed to asynchronous initiation of chromosomalreplication or to degradation of aborted or stalled replicationforks (38). An elevated temperature results in the increasedincidence of elongated cells and very small DNA-less cells inpopulations of both EC: :71CW and EC: :71CC. Interestingly,temperature-sensitive gyrB mutants also form elongatedcells at restrictive temperatures (12). This is due to thepresence of unresolvable nucleoids in the center of thesecells which block cell division since, in the absence ofnucleoid segregation, septum formation cannot occur at thenormal position. In some cases, septa form at aberrantlocations along the cell, resulting in small DNA-less cells.This has also been observed in cells harboring the parD-gyrAdouble mutation (20, 21). Since both EC::71CW andEC::71CC contain increased amounts of DNA at highertemperatures, it is likely that complex nucleoid structuresare formed, with similar effects on septum formation andlocalization.

Preliminary marker frequency analyses (data not shown)give similar results to those obtained with other strains inwhich the chromosome is replicated from an R1 replicon(23). These results suggest that EC::71CC is strongly biasedtoward unidirectional replication, whereas EC: :71CW isessentially bidirectional. We believe this is the major reasonfor the differences observed between strains EC::71CW andEC::71CC. An increased temperature sensitivity is to beexpected for a unidirectionally replicating chromosome,since it contains two free chromosome ends for each roundof replication. It is probable that a new initiation, before theprevious round of replication has been completed, would beblocked or would give rise to chromosome breaks, thereforecausing decreased viability when the initiation frequency isincreased. Also, the elongation time of chromosome repli-cation should be considerably longer in a unidirectionallyreplicating strain compared with that in a strain replicatingbidirectionally. In such a strain, replication therefore moreoften has not been completed at the time of normal celldivision, which could lead to aberrant septum localization.This could explain the increased frequency of elongated cellsand filaments found in EC::71CC compared with those foundin EC::71CW. It has also been suggested that replication ofthe terminus region may trigger cell division (14, 42, 43).Since for a unidirectionally replicating chromosome thisevent occurs prior to completion of the round of replication,the cell may be forced to form a septum at an aberrantposition. The high incidence of both DNA-less cells andelongated cells in populations of EC::71CC is also in accor-dance with this suggestion. Finally, the gene dosage is moredisturbed in a unidirectionally replicating strain, which mayreduce the growth rate and result in increased cell death athigher temperatures, when this imbalance is further in-creased. An interesting question is how bidirectional repli-cation is induced in EC::71CW. This cannot be due to eventswithin the integrated R1 replicon, since EC::71CC behavesdifferently. At present we assume that the second fork isassembled as the replisome initiated at the R1 origin tra-verses the remainder of oriC clockwise of pOU71.

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To our knowledge this is the first demonstration of con-trollable and extensive overreplication of a bacterial chro-mosome. We show that overinitiation has profound effectson the physiology of the cells and is ultimately lethal. Byusing temperatures in the range of 30 to 38TC, the effects ofsustained and moderate but nonlethal overinitiation of chro-mosomal replication on the frequency of cell divisions can bestudied. This should allow us to determine whether there isa direct coupling between the chromosome replication cycleand the cell division cycle, an important feature of one classof models for cell division control. It has been suggested thatgenes regulating early steps in cell division could be inducedby replication of the terminus region of the chromosome (14,42, 43). With the use of lac fusions it should be possible toinvestigate whether their expression in these systems isindeed affected by increased replication as the temperatureis increased. The two strains that we describe differ withrespect to the directionality of chromosome replication.Strain EC::71CC can be used to study how unidirectionalreplication of the chromosome affects the bacterial cell cycleand physiology. In strain EC: :71CW primary initiationevents are presumably uncoupled from the generation of thesecond fork at oriC, and this can be exploited in order tostudy how bidirectional replication is initiated. In conclu-sion, our systems provide a novel approach to the study ofseveral important questions concerning cell cycle coordina-tion.

ACKNOWLEDGMENTS

We gratefully acknowledge the excellent technical assistance ofLena Moller and thank Stuart Austin, Gerhart Wagner, and PollyWeller for critical reading of the manuscript.

This work was supported by the Swedish Natural Science Re-search Council and the Swedish Cancer Society. A.M. is therecipient of a postdoctoral fellowship from the European MolecularBiology Organisation.

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