Coordination between Chromosome Replication, Segregation, and Cell Division in Caulobacter crescentus

Progression through the Caulobacter crescentus cell cycle iscoupled to a cellular differentiation program. The swarmer cellis replicationally quiescent, and DNA replication initiatesat the swarmer-to-stalked cell transition. There is a very shortdelay between initiation of DNA replication and movement ofone of the newly replicated origins to the opposite pole ofthe cell, indicating the absence of cohesion between the newlyreplicated origin-proximal parts of the Caulobacter chromosome.The terminus region of the chromosome becomes located at theinvaginating septum in predivisional cells, and the completelyreplicated terminus regions stay associated with each otherafter chromosome replication is completed, disassociating verylate in the cell cycle shortly before the final cell divisionevent. Invagination of the cytoplasmic membrane occurs earlierthan separation of the replicated terminus regions and formationof separate nucleoids, which results in trapping of a chromosomeon either side of the cell division septum, indicating thatthere is not a nucleoid exclusion phenotype.

INTRODUCTION

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Introduction

Reliable transmission of genetic material to cellular offspringis a fundamental challenge for all organisms. It requires DNAreplication, disentanglement of the replicated DNA molecules,efficient segregation of sister chromosomes into the two daughtercells, and coordination of these processes with other cell cycleevents, like cell division. Most bacteria possess a single circularchromosome, and DNA replication is normally initiated at a singleorigin of replication (ori) (33). DNA replication proceeds bidirectionallytoward the terminus (ter) region, where the two replicationforks meet, resulting in termination of DNA replication. Severaldeterminants involved in sister chromosome separation are locatedin the terminus region (31). Studies of the subcellular localizationof specific chromosomal regions and components of the replicationmachinery have shown that bacterial chromosomes are spatiallyorganized within the cell in a dynamic fashion that changesduring DNA replication and segregation (13-15, 17, 19, 25, 28,29, 36, 37, 48-51). Individual loci on the circular chromosomeoccupy specific positions within the cell and move rapidly andwith direction during the DNA segregation process (37, 48).The specific localization patterns are different for differentgenera and growth conditions, but the fact that bacteria localizetheir origin and terminus regions of the chromosome at specificpositions indicates that there are mechanisms that ensure correctorganization and subcellular placement of the chromosome.

The spatial organization of the chromosome and the DNA replicationapparatus has been well characterized in Caulobacter crescentus(19, 22, 48). Caulobacter exists as morphologically and functionallydistinct cell types, and cellular differentiation is an integralpart of its cell cycle (21, 44, 45). Asymmetrical cell divisionresults in two different progeny cells, a replicative stalkedcell and a nonreplicative swarmer cell. In the G₁-arrested swarmercell, the origin-proximal region of the chromosome is locatednear the flagellated pole, the terminus-proximal part of thechromosome is located near the opposite pole of the cell, andregions in between are located in the cell with a linear correlationbetween their positions on the chromosome and their locationsin the cell (19, 22, 48). Thus, regions close to the originon the genetic map are also located close to the origin withinthe cell. After a specific time, the swarmer cell differentiatesinto a stalked cell by shedding the flagellum, synthesizinga stalk at the same pole, and initiating chromosome replication.One of the replicated origins moves to the opposite pole ofthe cell, while the other stays at the original pole (19, 48).Loci along the arms of the chromosomes are sequentially movedin the order that they are replicated to their final destinationsin the predivisional cell (22, 48). Cell division results information of a replicationally quiescent swarmer cell and astalked cell, which initiates a new round of DNA replicationimmediately after cell division. The temporal coordination betweenDNA replication and separation of the origin and terminus regionsof the chromosome in Caulobacter has not been characterized.

In Escherichia coli, separation and movement of the origin regionsmay be delayed compared to when they are replicated, suggestingthat newly replicated regions of the chromosome cohere to eachother (2, 18, 28, 46). Some studies have indicated that cohesionis absent or the period of cohesion is brief (8, 25, 28, 43),but other studies have shown that the period of cohesion isquite long and is observed during most of the S phase (2, 18,46). Separation of the fully replicated terminus regions occurslate in the cell cycle, shortly before cell division (2, 25,28, 29). Thus, separation of the replicated terminus regionsis delayed significantly compared to completion of DNA replication.

Both E. coli and Bacillus subtilis use a mechanism called nucleoidocclusion to ensure that cell division does not take place untilchromosome segregation has been completed, thereby avoidingcatastrophic breakage of the chromosome by division throughthe nucleoid (35). In both organisms, a DNA-binding proteinassociated with the bulk of the chromosomal DNA inhibits assemblyof the cell division apparatus in the vicinity of the nucleoid(3, 54). This prevents initiation of cell division until thedaughter chromosomes are separated from each other, forminga DNA-free region or a region with a low concentration of DNAin the middle of the cell. The Caulobacter genome does not containobvious homologs of the previously characterized nucleoid occlusionproteins, and it is not known if nucleoid occlusion actuallytakes place in Caulobacter.

In this work, I examined the coordination between chromosomesegregation and cell cycle events like DNA replication and celldivision in Caulobacter by simultaneously characterizing theDNA replication status using flow cytometry and cellular eventslike origin and terminus localization or cell division usingfluorescence microscopy in cells progressing synchronously throughthe cell cycle. The results showed that there was not cohesionof the newly replicated origin-proximal regions of the chromosomebut there was a significant delay in separation of the replicatedterminus regions. Cell division is initiated before final separationof the daughter chromosomes in Caulobacter, implying that thereis not nucleoid occlusion.

MATERIALS AND METHODS

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Materials and Methods

Strains. To create strains with specific regions of the chromosome taggedwith cyan fluorescent protein (CFP)-LacI, the xylX::pHPV472allele expressing the LacI-CFP chimeric protein from a xylose-induciblepromoter (48) was moved into the synchronizable wild-type strainCB15N (also designated NA1000) (12) by bacteriophage ${Phi}$ Cr30-mediatedgeneral transduction (9) with a lysate prepared from strainMO6 (48), resulting in strain RBJ176. A lacO array cassette(25) inserted at position 3,973,878 in the Caulobacter chromosomewas transduced into the RBJ176 strain with a ${Phi}$ Cr30 lysate preparedfrom strain MPO17 (48), resulting in strain RBJ185. This strainhas the origin-proximal part of the chromosome tagged with CFP-LacI.A lacO array cassette inserted into the terminus region of theCaulobacter chromosome (position 1,968,285) was transduced intoRBJ176 with a ${Phi}$ Cr30 lysate prepared from strain ML133 (48), resultingin strain RBJ200. In this strain the terminus region of thechromosome is tagged with CFP-LacI.

Growth conditions. Strains were grown in peptone-yeast extract (PYE) medium (9)at 30°C until they reached an optical density at 450 nmof 0.1 to 0.2. When relevant, expression of the LacI-CFP proteinwas induced by adding 0.2% xylose, and 50 µM isopropyl-ß-Dthiogalactoside (IPTG) was added to reduce binding of the repressorprotein. After 30 to 45 min of induction, the cells were synchronizedas described previously (47). The isolated swarmer cells wereresuspended in PYE (containing 50 µM IPTG for the LacI-CFP-expressingstrains), incubated with shaking at 30°C, and allowed tosynchronously proceed through the cell cycle. Samples were collectedevery 10 min for a total of 100 min and fixed for either flowcytometry or microscopy.

Flow cytometry analysis of DNA replication. Samples used for flow cytometry were fixed using cold 70% ethanolas described previously (52) at the same times and from thesame cultures as the samples used for fluorescence microscopy.Batch cultures were fixed either without any additional treatmentor after addition of 60 µg/ml rifampin and 36 µg/mlcephalexin, followed by incubation at 30°C for 3 h to allowongoing rounds of replication to be completed. Staining usingmithramycin and ethidium bromide and flow cytometry was performedas described previously (30), using an A10-Bryte flow cytometer(Apogee Flow Systems Ltd.). Data from synchronized cultureswere analyzed using the programs AFSCtrl (version 1.61; ApogeeFlow Systems Ltd.) and Microsoft Excel 2002 (Microsoft Corporation).The percentage of the chromosome replicated was determined basedon the average channel number of the flow cytometry DNA signal.To determine the percentage of cells that had initiated DNAreplication, a normal distribution was fitted to the DNA histogramof isolated swarmer cells, where $~$ 98% of the cells had a singlenonreplicating chromosome. This normal distribution was fittedto the left parts of the one-chromosome peaks in the DNA histogramsfrom the early times during the synchrony. The number of cellsin the fitted one-chromosome peak and the total number of cellswere used to calculate the percentage of cells that had initiatedDNA replication. Similarly, a normal distribution was fittedto the two-chromosome peak in a DNA histogram from a sampletaken at 90 min, where gating based on the cell size (lightscatter) was used to remove the DNA signals from divided cells.This two-chromosome peak normal distribution was fitted to theright parts of the two-chromosome peaks in the DNA histogramsfrom the late times of the synchrony. Control experiments showedthat the flow cytometer gave a signal during data acquisitionfor all the samples from a single experiment that was sufficientlystable to make this type of comparison between different samplesvalid. The percentage of cells that divided was determined bygating the light scatter and DNA fluorescence flow cytometrydata in order to determine the percentage of the cells thatwere small and had a low DNA content at the late times duringthe synchrony. Cytograms were displayed, and DNA histogramsfor mixed cultures of exponentially growing cells were analyzedas described previously (34), using the program WinFlow (version3.0; Flemming G. Hansen, The Technical University of Denmark,Lyngby, Denmark).

Microscopy. Synchronized Caulobacter cells were fixed by addition of 2.5%paraformaldehyde and 30 mM NaPO₄ buffer (pH 7.5) and incubationfor at least 1 h at 0°C. The fixation solution was removedby gently spinning the cells. Identical localization patternswere obtained using live and fixed cells. The cells were immobilizedusing a thin layer of agarose as described previously (16).When relevant, 2 µg/ml N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridiniumdibromide (FM4-64) was added to the agarose to fluorescentlylabel the cytoplasmic membrane, or 1 µg/ml 2-(4-amidinophenyl)-6-indolecarbamidinedihydrochloride (DAPI) was added to stain the chromosomal DNA.Similar nucleoid staining patterns were obtained using livecells and cells fixed using formaldehyde, methanol, or osmiumtetroxide, as well as with cells stained with the DNA-specificdyes DAPI, propidium iodide, and SYTO16. Phase-contrast andfluorescence images were acquired using a Leica DM5000B microscopewith a x100 HCX PL APO NA 1.4 objective and a Leica DFC350FXcooled charge-coupled device camera controlled through FW4000software (version 1.2.1; Leica Microsystems). Images were processedusing ImageJ (Wayne S. Rasband, National Institutes of Health,Bethesda, MD) and Photoshop CS (Adobe). The different cell typesand localization patterns were counted manually using ImageJand the Cell Counter plug-in (Kurt De Vos, Columbia University,New York, NY). At least 300 cells were counted for each datapoint.

RESULTS

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Results

Cytological tools for characterizing chromosome organization. The coordination between DNA replication, chromosome movement,and cellular morphology in synchronized Caulobacter cells wascharacterized using LacI-CFP-expressing strains with a cassettecontaining tandem lac operator arrays integrated in the origin-or terminus-proximal region of the chromosome (48). The intracellularlocation of the tagged region of the chromosome was visualizedbased on the fluorescence signal from the LacI-CFP chimera bindingto the lacO cassette. Cell morphology was examined both by phase-contrastmicroscopy and by staining the cytoplasmic membrane using thefluorescent lipophilic styryl dye FM4-64 (39). The chromosomereplication status and size of the cells were simultaneouslycharacterized using flow cytometry. Only a single operator cassettewas integrated in the strain, the expression of the fluorescentrepressor was kept at the minimum level required to detect thesignal, and repressor binding was reduced by adding an intermediateamount of the LacI inducer IPTG. Under these conditions andwith the cassette inserted 43 kbp anticlockwise from the replicationorigin or in the terminus region of the chromosome, no effecton cell cycle progression, DNA contents, or cell size was observedusing flow cytometry (Fig. 1A to D). High-level production ofthe fluorescent repressor protein, use of a cassette insertionsite closer to the origin, or simultaneous use of two differentoperator cassettes resulted in detectable cell cycle defects(data not shown), so these conditions were not used in thiswork.

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FIG. 1. Analysis of the cell cycle in tagged and wild-type Caulobacter cells using flow cytometry. (A to D) Cytograms showing the sizes and DNA contents of Caulobacter cells with specific regions of the chromosome labeled using CFP-LacI. Orthogonal light scatter is proportional to cell size, and fluorescence is proportional to the DNA content of the cells. (A and B) Cytograms of strain RBJ185 in which a cassette containing tandem repeated lac operators is integrated near the origin. (C and D) Cytograms of strain RBJ200 in which the lac operator cassette is integrated in the terminus region of the chromosome. (A and C) Strains in the absence of expression of the CFP-LacI protein. (B and D) Cytograms of the same cultures after induction of CFP-LacI as described in Materials and Methods. Approximately 100,000 cells were analyzed. (E and F) Analysis of the cell cycle in exponentially growing wild-type Caulobacter cells using computer simulations of DNA histograms obtained by flow cytometry. (E) DNA distribution pattern (solid circles) of wild-type strain CB15N treated with rifampin to block initiation of replication and cephalexin to block cell division. Ongoing rounds of DNA replication continue, resulting in accumulation of cells with either one or two fully replicated chromosomes. The data were used to determine the means and standard deviations of normal distributions fitted to the one- and two-chromosome peaks. (F) DNA distribution pattern (solid circles) of a mixed untreated exponentially growing CB15N culture. Simulations of the cells in the G₁, S, and G₂/M phases are indicated by thin black lines. The thick gray curve represents the sum of the simulated cells in different stages of the cell cycle. The means and standard deviations for the one- and two-chromosome peaks obtained from rifampin-treated cells (A) were used in the simulation to determine the proportions of cells in the different cell cycle stages.

To compare the durations of the G₁, S, and G₂/M phases of thecell cycle observed using synchronized tagged cells with thedurations in batch cultures of nonsynchronized exponentiallygrowing wild-type cells, computer simulations of DNA histograms(34) were performed (Fig. 1E and F). The same growth conditionswere used for the synchronized cultures and the exponentiallygrowing cultures. Simulation of the cell cycle in exponentiallygrowing nonsynchronized cells resulted in an 18- ± 2-minG₁ phase; the length of the S phase was 50 ± 2 min, andthe G₂/M phase was 27 ± 3 min long. The cell cycles insynchronized cultures of tagged strains were characterized byperforming a flow cytometry analysis of samples taken at differenttimes during the cell cycle (see Materials and Methods). Thetime of occurrence of an event in a synchronized culture isthe time at which 50% of the cells have entered the correspondingstage (2). Initiation of DNA replication took place $~$ 16 min intothe cell cycle of synchronized tagged strains, replication proceededat approximately the same rate until the entire chromosome hadbeen replicated at $~$ 64 min, and the cells divided approximately95 min into the cell cycle (Fig. 2B, 3B, and 4B). Thus, thecell cycle timing of the tagged strains in synchronized cultureswas essentially the same as the cell cycle timing in a batch-grownculture of the parent wild-type strain. Therefore, the CFP-LacItagging of the chromosome and the synchronization procedureused in this work did not appear to significantly affect cellphysiology.

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FIG. 2. Intracellular location of the origin-proximal region of the chromosome during the Caulobacter cell cycle. (A) Phase-contrast and fluorescence microscopy images of RBJ185 cells with the origin-proximal part of the chromosome tagged using CFP-LacI. The cells were synchronized, and samples were removed at different times after incubation of the isolated swarmer cells in PYE growth medium. The top panel shows overlays of phase-contrast and CFP fluorescence (cyan) images. The middle panel shows fluorescence signals from the dye FM4-64, which specifically binds to the cytoplasmic membrane. The lower panel shows overlays of the fluorescence signals of the cytoplasmic membrane dye (red) and the CFP fluorescence (cyan) marking the position of the origin-proximal region of the chromosome. Scale bar = 2 µm. (B) DNA replication and cell division status of the synchronized culture determined using flow cytometry. The green circles indicate the percentage of cells that have initiated DNA replication, the yellow triangles indicate the progression of DNA replication, the blue diamonds indicate the percentage of cells that have completed chromosome replication, and the red squares indicate the percentage of cells that have divided. (C) Quantification of the abundance of cells with different origin localization patterns. The yellow triangles indicate cells with a single origin focus in the pole-proximal 15% of the cell, the green squares indicate cells with a single origin focus in other parts of the cell, the blue diamonds indicate cells with two bipolarly located origin foci, and the red circles indicate cells with two origin foci with other localization patterns (e.g., one focus at a pole and one focus near the middle of the cell). (D) Data from panels B and C combined. The yellow triangles indicate cells with a single pole-proximal origin focus, the green circles indicate cells that have initiated DNA replication, the blue diamonds indicate cells with two origin foci, and the red squares indicate cells that have divided.

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FIG. 3. Intracellular location of the chromosomal terminus region during the Caulobacter cell cycle. (A) Fluorescence and phase-contrast microscopy images of RBJ200 cells with the terminus region tagged using CFP-LacI progressing synchronously through the cell cycle. The top panel shows overlays of phase-contrast and CFP fluorescence (cyan) images. The middle panel shows the FM4-64-labeled cytoplasmic membrane. The lower panel shows overlays of the fluorescence signals of the cytoplasmic membrane dye (red) and the CFP fluorescence (cyan) marking the position of the chromosome terminus region. Scale bar =2 µm. (B) DNA replication and cell division status of the synchronized culture. The green circles indicate cells that have initiated DNA replication, the yellow triangles indicate the progression of DNA replication, the blue diamonds indicate cells that have completely replicated the chromosome, and the red squares indicate cells that have divided. (C) Quantification of cells with different terminus localization patterns and the presence of an invagination at the future division site. The green circles indicate smooth cells with a single terminus focus, the red triangles indicate cells with a single terminus focus that have a visible constriction at the future division site, showing that the cell division process has been initiated, and the blue squares indicate constricted cells with two separated terminus foci. No smooth cells with two terminus foci were observed. (D) Quantification of the location of the terminus focus in predivisional cells at different times during the cell cycle. Blue indicates cells in which the terminus focus is located in the flagellated part of the predivisional cell, green indicates predivisional cells in which the terminus focus is located at the invaginating septum, and red indicates predivisional cells with the terminus focus in the stalked part of the cell. (E) Phase-contrast and cytoplasmic membrane staining images of early and late predivisional cells having different membrane invagination patterns. The white arrows indicate cells in which a clear invagination of the cytoplasmic membrane could be seen using the cytoplasmic membrane dye but no invagination of the cell was visible by phase-contrast microscopy. The yellow arrows indicate cells with the cytoplasmic membrane invaginated and a mild indentation at the future division site that could be observed by phase-contrast microscopy. The red arrows indicate late predivisional cells in which phase-contrast microscopy showed a very deep invagination at the future division site and the cytoplasmic membrane stain indicated that separate compartments were formed in the two parts of the cell.

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FIG. 4. Nucleoid morphology during the Caulobacter cell cycle. (A) Phase-contrast and fluorescence microscopy images of wild-type CB15N cells with the chromosomal DNA stained using DAPI. The cells were synchronized, and cell samples were removed at different times during incubation of the isolated swarmer cells in growth medium. The top panel shows phase-contrast images, and the middle panel shows the FM4-64 signal from the cytoplasmic membrane. The lower panel shows the fluorescence signal from DAPI binding to DNA. Scale bar = 2 µm. (B) DNA replication and cell division status of the synchronized culture. The circles indicate the cells that have initiated DNA replication, the triangles indicate the progression of DNA replication, the diamonds indicate cells that have completely replicated the chromosome, and the squares indicate the cells that have divided. (C) Quantification of cells with different nucleoid morphology patterns and the presence of a constriction at the future division site. The circles indicate smooth cells with a single nucleoid, the triangles indicate cells with a single bilobed nucleoid that have a visible indentation at the future division site, and the squares indicate constricted cells with two separated nucleoids. No smooth cells with a single bilobed nucleoid or two separate nucleoids were observed.

Timing of origin movement. The timing of origin movement was compared to the timing ofinitiation of DNA replication by synchronizing a LacI-CFP-expressingstrain with a lac operator cassette inserted 43 kbp anticlockwisefrom the replication origin. Four different localization patternsof the origin-proximal part of the chromosome were observed(Fig. 2). Nonreplicating swarmer cells had a single origin focuslocated in the flagellated-pole-proximal 15% of the cell. Shortlyafter initiation of DNA replication, one of the newly replicatedorigins moved to the opposite pole of the cell, while the otherorigin copy remained at its previous location, resulting information of cells with a bipolar origin localization pattern.Transiently, during the part of the cell cycle in which initiationof DNA replication took place, a few cells with a single nonpolarorigin focus or two asymmetrically located foci, with at leastone of the foci in the central part of the cell, were observed(Fig. 2C). These cells were most likely in the process of movingone of the newly replicated origin regions and therefore wereintermediates in the DNA segregation process. The low abundanceof cells with origin localization patterns that reflected intermediatesin the origin segregation process indicated that origin movementwas a fast process, as previously observed using time-lapsemicroscopy (48). When the timing of initiation of DNA replicationwas compared with the timing of appearance of cells with twoseparated origin foci (Fig. 2D), a very short 2- to 4-min delaybetween initiation of DNA replication and origin movement wasobserved.

Terminus movement and separation. The coordination between cell cycle progression, terminus separationor movement, and cell division was examined using a strain withthe terminus region of the Caulobacter chromosome tagged withLacI-CFP. In nonreplicating swarmer cells, the terminus regionof the chromosome is initially located near the pole oppositethe origin (20, 48). During the DNA replication process, theterminus region moved from the pole opposite the stalk to nearmidcell (Fig. 3). Even after completion of DNA replication,the majority of the cells had only a single terminus focus (Fig.3B and C). Only in deeply constricted late predivisional cellswere two separated terminus foci observed, and the low frequency( $~$ 5%) of cells with two separated terminus foci indicated thatthe life span of the cellular stage with separated termini wasquite short. Similar terminus localization patterns and frequenciesof cells with separated termini were observed in wild-type Caulobactercells using fluorescence in situ hybridization (data not shown).Thus, the completely replicated terminus regions of sister chromosomesstayed associated with each other for an extended period (20to 30 min) after chromosome replication was completed. The terminusregions disassociated shortly before the final cell separationstep in the cell division process. The intracellular locationof the terminus focus in predivisional cells varied significantly(Fig. 3D). In early predivisional cells, the terminus focuswas located primarily in the flagellated part of the cell orat the invaginating septum. In late predivisional cells, theterminus was located primarily at the septum or in the stalkedcompartment of the cell. The high proportion of predivisionalcells with the terminus region located at or very close to theinvaginating septum indicated that the terminus was activelytrapped or localized at the future division site.

Chromosome trapping by the invaginating septum. Cell division and chromosome segregation have to be coupledin order to avoid the formation of nucleoid-free cells or chromosomebreakage at cell division. To investigate the coordination betweenchromosome segregation and cell division, the number of cellswith any sign of constriction at the future division site inphase-contrast microscopy images was determined (Fig. 3). Thisrevealed that initiation of cell constriction, an early stepin the cell division process, took place at approximately thesame time in the cell cycle as completion of chromosome replication.Simultaneous staining of the cytoplasmic membrane using thefluorescent dye FM4-64 and examination of cell morphology usingphase-contrast microscopy revealed that the cell constrictionprocess involves several distinct steps. At 60 min, cells withvisible ingrowth of the cytoplasmic membrane but no sign ofindentation as determined by phase-contrast microscopy wereobserved (Fig. 3E). The bright signal from the membrane dyeat the invaginating septum reflected the fact that two membraneswere located close to each other, which resulted in a more intensesignal. Cells with constriction visible both by cytoplasmicmembrane staining and phase-contrast microscopy were also observed(Fig. 3E). Late in the cell cycle, very deeply constricted cellswere observed (Fig. 3E). Cytoplasmic membrane staining indicatedthat separate compartments were present in the two halves ofthe late predivisional cells. The vast majority of the constrictedcells had only a single terminus focus (Fig. 3A and C), showingthat cytoplasmic membrane invagination took place well beforeseparation of the replicated sister chromosome termini.

To examine coordination between cell division and nucleoid separation,the chromosomal DNA in synchronized wild-type cells were stainedusing the DNA-specific fluorescent dye DAPI (Fig. 4). ChromosomalDNA was present in the entire cell at all stages during thecell cycle, and no distinct nucleoids and chromosome-free regions,like those observed in E. coli and B. subtilis (42, 56), werepresent in Caulobacter. Simultaneous staining of the cytoplasmicmembrane and the chromosome showed that initiation of invaginationof the cytoplasmic membrane in Caulobacter takes place overthe nucleoid. Pinched cells had a single bilobed nonseparatednucleoid with significant DNA staining at the invaginating septum.Only deeply constricted late predivisional cells had completelyseparated nucleoids in which no DNA staining was observed atthe division site (Fig. 4). Thus, most of the Caulobacter celldivision processes take place before complete separation ofthe replicated chromosomes. Only the last step in the cell divisionprocess, final cell separation, takes place after separationof the sister chromosome terminus regions (Fig. 3) and completesegregation of the nucleoids (Fig. 4).

DISCUSSION

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Discussion

Generation of viable offspring is very complicated, involvingmany different processes that all have to be coordinated witheach other both temporally and spatially. Despite recent advances,the mechanisms by which different bacterial cell cycle eventsare coordinated have not been well characterized (5, 32). Caulobacteris particularly well suited for the study of bacterial cellcycle control. The unique characteristics of this organism includeasymmetric cell division that results in progeny with distinctmorphological features and fates (45), which makes it easierto track the intracellular location and movement of cell cycleproteins and chromosomal regions. Synchronized cell populationscan be obtained without perturbing the normal physiology ofthe cell (Fig. 1), which allows examination of morphology, proteinand DNA localization, and gene expression at defined stagesof the cell cycle. Additionally, clear G₁, S, and G₂/M phasesof the cell cycle can be identified since initiation of replicationtakes place only once per cell cycle. However, in most of theprevious work in which different events during the Caulobactercell cycle were examined, only a single process was examinedat a time. When the results of different laboratories or evendifferent papers from the same research group are compared,very different data for timing of cell cycle events and durationof the entire cell cycle are found, making it difficult to comparethe data for timing of different cell cycle events to each other.In this work, growth conditions were standardized to ensureequivalent values for timing of cell cycle events in differentstrains and for experiments done on different days. Additionally,multiple types of data were simultaneously acquired from synchronizedCaulobacter cultures to obtain information about several differentcell cycle events.

In E. coli newly replicated origin-proximal regions of the chromosomemay cohere to each other, resulting in a delay in separationand movement of the origin regions (2, 18, 28, 46). The temporalextent of the period of cohesion is debated; according to somestudies the period of cohesion is brief or absent (8, 25, 28,43), but other studies have shown that the period of cohesionis quite long, taking place during most of S phase (2, 18, 46).Previous studies of chromosome organization in Caulobacter didnot examine the temporal relationship between DNA replicationand movement of the origin-proximal part of the chromosome,since only origin movement was characterized (19, 48). By simultaneouslyexamining DNA replication using flow cytometry and the intracellularlocation of the origin-proximal part of the chromosome usingLacI-CFP binding to a lac operator cassette inserted near ori,the coordination between these processes was characterized (Fig.2). Within 2 to 4 min of initiation of DNA replication, cellswith two separated origin foci were observed. The lac operatorcassette used to visualize the intracellular location of theorigin-proximal region was inserted 43 kbp from the origin,which was expected to be replicated approximately 1 min afterinitiation of DNA replication. Additionally, because of thelimited resolution of light microscopy, origin foci had to moveat least 0.2 µm from each other before separate foci couldbe observed. With the previously observed rate of origin movement(48), this could result in up to a 1-min delay between initiationof movement and the time when separated origin foci could beobserved by light microscopy. Thus, the very short time betweeninitiation of DNA replication and separation of the replicatedorigins showed that no cohesion between the newly replicatedorigin-proximal regions of the sister chromosome takes placein Caulobacter. Alternatively, the cohesion period is very short,less than 2 min. The newly replicated DNA regions appear tostay associated for only enough time to allow homology-directedrepair of replication errors.

In contrast, there appears to be a very significant delay, atleast 20 min, between replication of the terminus region ofthe Caulobacter chromosome and the appearance of two separatedterminus foci in the cells (Fig. 3). This indicates that theterminus regions of sister chromosomes either cohere to eachother or for other reasons remain associated with each otherfor an extended time. The separation of the replicated terminusregions occurs late in the cell cycle, shortly before the finalstep in the cell division process. A significant delay betweencompletion of DNA replication and separation of the fully replicatedsister chromosome terminus regions has also been observed inE. coli (2, 25, 28, 29). The potential causes of the delay interminus separation include formation of a chromosome dimerby homologous recombination and catenation of the sister chromosomes.XerCD-mediated site-specific recombination at the dif recombinationsite located in the terminus is responsible for converting achromosome dimer to monomers (26), and topoisomerases, primarilytopoisomerase IV, are responsible for decatenation (11). Bothof these activities are activated by FtsK, a DNA translocaselocated at the tip of the invaginating septum that has functionsboth in transporting DNA and in cell division (1, 7, 10, 55).Directional information provided by short repeated sequenceswith a biased orientation that change abruptly at dif may helpFtsK determine the direction that its substrate has to be pumped(4, 6, 38). Directed DNA movement by FtsK may be involved inpositioning the terminus at the division site. The multiplefunctions of FtsK may have a role in coupling DNA segregationand cell division temporally and spatially. The XerCD, topoisomeraseIV, and FtsK proteins are highly conserved in Caulobacter, makingit likely that similar mechanisms are employed in terminus localizationand separation.

During vegetative growth E. coli and B. subtilis ensure thatcell division does not take place until chromosome segregationhas been completed using a specific protein associated withthe bulk of the chromosomal DNA (3, 54). This protein inhibitsinitiation of cell division, thereby preventing assembly ofthe cell division machinery in the regions of the cell wherethe chromosome is located. When the daughter chromosomes areseparated from each other, forming a DNA-free region in themiddle of the cell, cell division is allowed. This mechanismfor preventing cell division until completion of chromosomesegregation is termed nucleoid occlusion (35). Assembly of theFtsZ ring at midcell, the earliest known step in the cell divisionprocess, takes place during the S phase in Caulobacter (41).Invagination of the cytoplasmic membrane in Caulobacter is observedat approximately the same time that DNA replication is completed,much earlier than separation of the replicated terminus regionsand formation of two separate nucleoids (Fig. 3 and 4). Thus,a chromosome is trapped on either side of the cell divisionseptum, implying that there is not nucleoid occlusion of celldivision in Caulobacter. A similar trapping of the chromosomeby an invaginating septum is observed during sporulation inB. subtilis, in which an asymmetric cell division event trapsabout one-third of the chromosome in the prespore compartment;the rest of the chromosome is then transported into the smallprespore compartment by the SpoIIIE protein (53). However, whenDNA replication is inhibited in Caulobacter, FtsZ rings formprimarily at polar regions with little or no chromosomal DNA(40). Thus, assembly of the cell division apparatus in Caulobactermay have a preference for, but not an absolute requirement for,assembling in regions of the cell with low DNA contents.

Cytoplasmic membrane invagination takes place approximately20 min before separation of the progeny cells, the final stepof the cell division process (Fig. 3). Simultaneous visualizationof ingrowth of the cytoplasmic membrane using the fluorescentdye FM4-64 and invagination of the outer part of the cell wallusing phase-contrast microscopy indicated that the two eventsare uncoupled. Invagination of the cytoplasmic membrane occursbefore invagination of the outer parts of the cell wall, andseveral different stages in the cell constriction process canbe observed by light microscopy (Fig. 3E). Faster constrictionof the cytoplasmic membrane than of the outer membrane was alsorecently observed by cryoelectron tomography (23). The Caulobacterlate predivisional cell is compartmentalized, since cytoplasmicand membrane proteins cannot diffuse between the nascent progenycell compartments (23, 24). At this stage during the cell cycle,the cytoplasmic membrane is very deeply invaginated, and insome cells there is complete separation into separate compartments(Fig. 3) (23). It is likely that the septum formed by cytoplasmicmembrane invagination is responsible for preventing diffusionbetween the two cell parts. Such compartmentalization by a septumis observed during sporulation in B. subtilis (27). Additionalhigh-resolution analysis, using a method like cryoelectron tomography,of cells during different stages of cell division is requiredto obtain a detailed understanding of how and when the innerand outer membranes are closed off, how this is coupled to separationof the terminus region of the chromosome, and if it is involvedin formation of a diffusion barrier between the two parts ofthe cell.

ACKNOWLEDGMENTS

I thank Anders Løbner-Olesen, Ole Skovgaard, and ToveAtlung for critical reading of the manuscript and Flemming G.Hansen for help using the WinFlow program.

This work was supported by grants from The Danish Natural SciencesResearch Council and The Carlsberg Foundation.

FOOTNOTES

* Mailing address: Department of Life Sciences and Chemistry, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark. Phone: 45 46742471. Fax: 45 46743011. E-mail: rbjensen{at}ruc.dk.

REFERENCES

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