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Polar Localization of the Replication Origin and Terminus in Escherichia Coli Nucleoids During Chromosome Partitioning

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Polar Localization of the Replication Origin and Terminus in Escherichia Coli Nucleoids During Chromosome Partitioning Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Polar localization of the replication origin and terminus in Escherichia coli nucleoids during chromosome partitioning Hironori Niki1 and Sota Hiraga Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto 862-0976, Japan We show the intracellular localization of the Escherichia coli replication origin (oriC) and chromosome terminus during the cell division cycle by FISH. In newborn cells, oriC is localized at the old-pole-proximal nucleoid border and the terminus at the new-pole-proximal nucleoid border. One copy of replicated oriC migrates rapidly to the opposite nucleoid border. These oriC copies are retained at both nucleoid borders, remaining at a constant distance from each cell pole. The terminus segment migrates from the nucleoid border to midcell and is retained there until the terminus is duplicated. The origin, terminus and other DNA regions show three migration patterns during active partitioning of daughter chromosomes. [Key Words: Cell division cycle; FISH; mitotic-like apparatus; oriC; positioning; segregation] Received December 29, 1997; revised version accepted February 4, 1998. Escherichia coli cells have a 4.6-Mb circular chromo- cated chromosomes do not separate from each other but some (Blattner et al. 1997) that is folded and organized in remain together at midcell. When protein synthesis re- a compact form called the nucleoid. During the cell di- sumed, the chromosomes moved from midcell to cell vision cycle, replication of the chromosomal DNA ini- quarter positions without cell elongation. This suggested tiates bidirectionally in a specific chromosome region, that rapid movement of chromosomes was responsible oriC. The two replication forks meet at the chromosome for chromosome partitioning in bacteria (Hiraga 1990; region opposite oriC. In this region, the progress of rep- Hiraga et al. 1990). Bipolar localization of the chromo- lication forks traveling clockwise and counter-clockwise some segment near oriC was visualized in Bacillus sub- is inhibited at specific DNA sequences, ter, in a direc- tilis (Webb et al. 1997) and E. coli (Gordon et al. 1997). tion-specific manner (Hill 1996). Thus, this region is rep- LacI-binding sequences were inserted into the chromo- licated at the end of a round of chromosome replication some near oriC, and the green fluorescent protein (GFP) and is called the replication terminus. The replicated fused to LacI was detected in living cells. Fluorescent chromosomal DNA molecules are physically separated foci of the GFP–LacI fusion protein bound to the chro- by the processes of decatenation and/or recombination, mosomal site near oriC showed that one copy of the and then each chromosome is partitioned with high fi- replication origin region moved to the site of new pole delity into daughter cells (for review, see Hiraga 1992; formation, near the site of cell division, following repli- Løbner-Olesen and Kuempel 1992; Rothfield 1994; Wake cation at or near the cell poles. In addition, bipolar lo- and Errington 1995). The partitioning mechanism is calization of the Spo0J protein was also observed by im- poorly understood in prokaryotic cells. Recently, bipolar munofluorescence microscopy or Spo0J–GFP in B. sub- localization of specific chromosome regions during the tilis (Glaser et al. 1997; Lin et al. 1997). Spo0J binds to a cell division cycle was reported, and this movement sug- DNA site in the origin-proximal third of the chromo- gested that a mitotic-like apparatus was involved in some (Lin et al. 1997) and colocalizes with the oriC re- chromosome partitioning in bacteria (Glaser et al. 1997; gion (Lewis and Errington 1997). A spo0J mutant causes Lin et al. 1997; Mohl and Gober 1997; Webb et al. 1997; production of a significant proportion of anucleate cells Wheeler and Shapiro 1997). and is blocked at the onset of sporulation (Ireton et al. Donachie and Begg (1989) and Hiraga et al. (1990) ob- 1994). These results suggested that Spo0J is involved in a served that after inhibition of protein synthesis, repli- mitotic-like apparatus responsible for the migration of the oriC region, and the binding site for Spo0J is a cis- 1Corresponding author. acting site for partitioning, like a eukaryotic centromere. E-MAIL [email protected]; FAX 96-371-2408. In Caulobacter crescentus, ParB binds to an AT-rich se- 1036 GENES & DEVELOPMENT 12:1036–1045 © 1998 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/98 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Migration of the origin and terminus in E. coli quence, parS, downstream of the parAB operon, which is throughout the cell division cycle and that E. coli does located within 80 kb of the replication origin (Mohl and not have a mitotic-like apparatus for rapid chromosome Gober 1997). The ParA and ParB proteins are localized at movement during partitioning. cell poles in one stage of the cell cycle, and overproduc- In contrast to the above conclusion, we show here sta- tion of ParA and ParB inhibits both cell division and tistical results by FISH indicating that one copy of the chromosome partitioning. Thus, ParA and ParB may duplicated oriC region migrates rapidly along the nucle- function as components of a mitotic-like apparatus, and oid from one border to another. We also show a different the parS site is the only candidate for a centromere that dynamic migration pattern of the terminus region of the has been sequenced in a bacterial chromosome. chromosome. By simultaneous use of DNA probes cor- Partition defective mutants that produce anucleate responding to different chromosomal regions and labeled cells upon cell division were isolated in E. coli (Hiraga et with different fluorescent compounds, in situ hybridiza- al. 1989). The mukF, mukE, and mukB genes form an tion revealed cell cycle-dependent nucleoid organiza- operon, and null mutants of each gene show a severe tion. We propose a model for the positioning of daughter defect in chromosome partitioning and cell viability chromosomes. (Niki et al. 1991; Yamanaka et al. 1996). The mukB gene codes for a filamentous protein of 170 kD whose struc- ture resembles that of motor proteins such as myosin or Results kinesin (Niki et al. 1992). MukB is presumably respon- sible for generating motive force during chromosome Migration of the oriC DNA segment across partitioning. In B. subtilis, the SpoIIIE protein that lo- the nucleoid calizes at the center of the division septum promotes Bidirectional chromosome replication is initiated at chromosome segregation into the prespore during sporu- oriC, which is a minimal 245-bp sequence (Oka et al. lation (Wu and Errington 1994, 1997). The origin-proxi- 1980) located at 84 min on the E. coli chromosome. To mal third of the chromosome is segregated into the pre- analyze the intracellular localization of oriC by FISH, we spore compartment by Spo0J, localized at the cell poles used two kinds of DNA segments as hybridization during sporulation (Glaser et al. 1997), and the rest of the probes (Fig. 1). The oriC–gid DNA probe was prepared chromosome is translocated to the prespore by SpoIIIE. from a 6.3-kb DNA segment containing the oriC and gid In addition to the bipolar behavior of the chromosome genes. The asn–rbs DNA probe was prepared from about origin copies, the F plasmid also shows bipolar migration 13 kb of the oriC-flanking DNA segment containing the of replicated plasmid DNA molecules, which are ac- asn to rbs genes, but not oriC. There is an ∼2-kb gap tively partitioned into daughter cells on cell division between the minimal oriC region and the left border of (Gordon et al. 1997; Niki and Hiraga 1997). The F plas- the asn–rbs DNA segment. Cells of strain CSH50 grow- mid is partitioned to daughter cells with high fidelity ing exponentially in minimal glucose medium (doubling owing to two trans-acting genes, sopA and sopB, and a time 55 min) were fixed and hybridized with either probe cis-acting site, sopC (Ogura and Hiraga 1983). An F plas- (Fig. 2A,B). mid molecule having the sopABC partitioning system is One or more apparent fluorescent foci were observed localized at midcell in newborn cells. The replicated in more than 97% of cells using the oriC–gid probe. Cells plasmid molecules migrate in opposite directions to cell with one, two, three, and four foci were 27%, 63%, 5.4%, positions one-fourth and three-fourths of the cell length and 4.6% of total cells having fluorescent foci, respec- and are tethered at these positions until the cell divides. tively. In short cells with a single focus, the focus was Therefore, each daughter cell receives at least one F plas- localized at a pole-proximal nucleoid border (Fig. 2A, row mid DNA molecule. P1 plasmid shows the same behav- a). Two foci in short cells were either closely localized ior during the cell division cycle (Gordon et al. 1997). near one nucleoid border (Fig. 2A, row b) or separated Interestingly, Soj, in an operon with spo0J, and ParA in from each other (Fig. 2A, rows c–e). In long cells, two foci C. crescentus, belong to the ATPase family including the were localized near the two borders of a nucleoid (Fig. plasmid-encoded F-SopA and P1-ParA proteins, essential 2A, rows f–h). Similar results were obtained with the for plasmid partitioning. B. subtilis Spo0J and C. cres- asn–rbs DNA probe (Fig. 2B), indicating that one copy of centus ParB are members of a family that includes F- the replicated oriC-flanking region, like oriC, also mi- SopB and P1-ParB.
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