Rapid and sequential movement of individualchromosomal loci to specific subcellular locationsduring bacterial DNA replicationPatrick H. Viollier*, Martin Thanbichler*, Patrick T. McGrath*, Lisandra West, Maliwan Meewan, Harley H. McAdams,and Lucy Shapiro†Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305Contributed by Lucy Shapiro, April 13, 2004The chromosomal origin and terminus of replication are preciselylocalized in bacterial cells. We examined the cellular position of 112individual loci that are dispersed over the circular Caulobactercrescentus chromosome and found that in living cells each locushas a specific subcellular address and that these loci are arrayed inlinear order along the long axis of the cell. Time-lapse microscopyof the location of the chromosomal origin and 10 selected loci in theorigin-proximal half of the chromosome showed that during DNAreplication, as the replisome sequentially copies each locus, thenewly replicated DNA segments are moved in chronological orderto their final subcellular destination in the nascent half of thepredivisional cell. Thus, the remarkable organization of the chro-mosome is being established while DNA replication is still inprogress. The fact that the movement of these 10 loci is, like thatof the origin, directed and rapid, and occurs at a similar rate,suggests that the same molecular machinery serves to partitionand place many, if not most, chromosomal loci at defined subcel-lular sites.Bacterial chromosomes are not static structures. They un-dergo dynamic topological changes during DNA replication,segregation, and transcription (1–5). For bacteria with circularchromosomes, replication initiates at a single origin of replica-tion (ori) and proceeds bidirectionally toward the terminus ofreplication (ter) (6). During replication, the DNA double helix isunwound locally, introducing compensatory superhelicity andentanglements that are relieved by the action of topoisomerases(1). Before newly replicated DNA can be segregated, the twosister chromosomes are unlinked by topoisomerases, resolvases,and recombinases (2, 7, 8). After the completion of replicationand segregation, each half of the predivisional cell contains onesister chromosome. The mechanism whereby the chromosomesare moved, positioned, and finally restructured before celldivision is poorly understood.Fluorescence in situ hybridization (FISH) has been used tovisualize distinct chromosomal loci in fixed bacterial cells (9). Inaddition, a technique to label chromosomal loci in live cells hasbeen developed by using a lac repressor GFP hybrid protein(LacI-GFP) that binds to arrays of lac operator (lacO) sequencesinserted at specific sites on the chromosome (10–12). By usingthis labeling technique along with time-lapse fluorescence mi-croscopy (FM), both the Escherichia coli and the Bacillus subtilisori have been shown to move rapidly toward the cell poles onceDNA replication has initiated (13–15).Chromosome replication and segregation is coordinated withother events during the cell cycle, such as polar morphogenesisand cell division. In Caulobacter, DNA replication initiates onceand only once per cell cycle and proceeds bidirectionally from asingle origin (16, 17). Caulobacter cell division is asymmetric,yielding a replicative stalked cell and a nonreplicative swarmercell with polar pili and a polar flagellum (18–20). DNA repli-cation is blocked in the swarmer cell, as in the eukaryotic G1phase cell. Chromosome replication can only initiate once theswarmer cell has differentiated into a stalked cell. This transitionis thus analogous to the eukaryotic G1-to-S transition. Swarmercells are separable from stalked cells by density gradient cen-trifugation and, once separated out and resuspended in freshmedium, progress synchronously through the cell cycle. Inswarmer cells, the ori is located at the pole that bears the pili andthe flagellum (Fig. 1D). After the initiation of DNA replication,a second copy of ori appears at the opposite cell pole (21). At theend of the cell cycle, as DNA replication ceases, the two copiesof ter are observed near the division plane. In support of thisobservation, immunocytological experiments in which newlyreplicated DNA was localized by using antibodies to the thymineanalog BrdUrd showed that DNA replicated early in the cellcycle was found near the cell poles. As the cell cycle progressed,newly replicated DNA was located at increasing distance fromthe cell poles, progressively closer to the plane of division, wherethe DNA replicated last was observed (22).Whereas the positioning of the ori and ter has been extensivelystudied in E. coli, B. subtilis, and Caulobacter, little is knownabout the movement and deposition of specific interveningchromosomal loci during the cell cycle. Two loci between the oriand ter have been shown to occupy distinct subcellular positionsin live B. subtilis cells (23). Analysis of 22 chromosomal loci byFISH in fixed E. coli cells has similarly provided evidence for theorderly arrangement of the chromosome (24). Here, we describea comprehensive analysis of the arrangement of ⬎100 chromo-somal sites in live cells and explore the arrangement of a subsetof these sites in relation to one another as cells progress throughthe cell cycle. We demonstrate that, like the ori, newly replicatedloci rapidly move in an orderly fashion to their specific addressin the incipient daughter cell. Thus, the remarkable organiza-tion of the chromosome is established while DNA replication isongoing.Experimental ProceduresBacterial Strains and Growth Conditions. Caulobacter crescentusCB15N and derivatives were grown in M2G minimal or peptone-yeast extract containing 0.02% glucose complex medium. Thecomposition of M2G and peptone-yeast extract, protocols forbacteriophage ⌽Cr30-mediated generalized transductions, plas-mid mobilization from E. coli to Caulobacter by conjugation,and synchronization by Percoll density gradient centrifugationAbbreviations: CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; FISH, fluo-rescence in situ hybridization; FROS, fluorescent repressor-operator system; FM, fluores-cence microscopy; IFM, immunofluorescence microscopy; LacI, lac repressor; TetR, tetrepressor.See Commentary on page 9175.*P.H.V., M.T., and P.T.M. contributed equally to this work.†To whom correspondence should be addressed at: Department of Developmental
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