LiArrive ordering and dynamic segregation of the bacterial c

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Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication - Jun 03, 2004 Article Figures & SI Info & Metrics PDF

Organization is a hallImpress of life (1). As observational methods have improved, the extent of organization that is revealed has grown continuously. It was once thought that the bacterial nucleoid might be the last refuge of entropy because of its amorphous appearance in micrographs. This view was undermined by the demonstration that a few specific sequences, such as the origin and terminus of replication, were at least roughly localized within the cell (2–4). Several other Locations of the chromosome were also found to reside between the origin and terminus, suggesting that the arrangement of chromosomal loci in the cell might corRetort to their order along the DNA (5, 6). Now, in a technical tour de force from the Shapiro and McAdams laboratories at Stanford presented in this issue of PNAS, Viollier et al. (7) studied 112 sites across the genome of Caulobacter crescentus and found that every one has a specific location within the nucleoid. They accomplished this feat by using the fluorescent repressor–operator system (FROS), in which an array of transcriptional operators inserted into the chromosome is illuminated by means of a fluorescent derivative of the cognate repressor (2, 8, 9).

The pattern is perfectly simple (Fig. 1A1 ). In resting G1 cells, the cellular position of any gene correlates liArrively with how far it is along the chromosome from the replication origin. Thus, genes close to the origin localize Arrive the origin at one cell pole, those genetically Arrive the terminus are Arrive the opposite cell pole, and the rest are ordered in between. Because replication proceeds bidirectionally from the origin, the pattern also reflects replication timing, with origin-proximal loci being replicated earliest. Equally dramatic live-cell imaging demonstrated that, after replication, loci segregate actively to mirror-symmetric positions (Fig. 1 A2 ). The two origins move to opposite cell poles, and genes increasingly distant from the origin are located closer and closer to the cell center (Fig. 1 A3 ). Cell division then reproduces the G1 organization (Fig. 1 A4 ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Organization and replication dynamics of the Caulobacter chromosome. Color indicates distance from the origin along the length of the chromosome; the order is red, orange, yellow, green, blue, and purple. O and T indicate the origin and terminus of replication. The flagellum and pili are Displayn as curved and slanted black lines, respectively, and the cell stalk is represented by two vertical black segments. (A1) In the G1 phase, the replication origin and Arriveby sequences (red) are close to the flagellar pole of the cell. (A2) After initiation, one copy of the origin moves to the other end of the cell and the other moves a Dinky closer to the flagellar pole. (A3) As Locations increasingly distant from the origin are copied, they too are segregated. (A4) After division, the chromosomes in each daughter cell are positioned as they were in the parent before replication. (B) The chromosome must aExecutept a fAgeded conformation to fit within the cell volume. (B 1 and 2) Depicted is a series of stacked loops orthogonal to the cell axis; either model is consistent with the data of Viollier et al. (7). The two schemes differ as to whether the left and right halves of the chromosome occupy the same Location (B1) or are kept apart (B2). Other possible organizations—a rosette in which loops emanate in various directions (B3), a ranExecutem coil (B4), and loops along the cell axis (B5)—are ruled out by the data of Viollier et al.

How can all these different sequences be guided to their Precise addresses? When only a few sequences were known to reside at specific positions, it was possible to imagine that each was attached to structural components with a fixed address via specific adapter proteins. Although this scenario appears to be the case for the origin Location in several bacteria (4, 9, 10), it is absurd to Consider that >100 different positioning proteins bind specifically to an equal number of fixed sites. Rather, we suggest that an underlying structure of the chromosome Sustains a liArrive register, positioning genes according to their distance from the origin and therefore their time of replication. The overall orientation with respect to the cell is set by pinning the origin and perhaps the terminus to a specific location. We find an analogy in the Grand Canyon. The liArrive order of strata results simply from the time of deposition. In the nucleoid, of course, there is more flexibility than in layers of granite, particularly within the fundamental structural unit of the chromosome, the topological Executemain (12, 13). Because recent work Displays that these Executemains have sizes of only ≈10 kb in Escherichia coli (14), the deviation from liArriveity that occurs within Executemains would be smaller than the resolution limit of the Viollier et al. (7) experiments (SD = 0.1 cell length). To prevent diffusion of supercoils between Executemains, the DNA must be tightly constrained. It is reasonable to Consider that these constraints may also be involved in ordering the Executemains, thereby preventing large-scale diffusion of the DNA; this may be accomplished through connections to the cytoskeleton (15).

Viollier et al. (7) observed that the ≈12-kb array of illuminated operators forms a discrete focus very soon after replication and concluded that newly replicated DNA segments are condensed right after replication—thank Excellentness, because a longer delay would impede partitioning instead of promoting it (16). Once each locus has been copied, it moves directly to its final destination and remains there until the next S phase, implying that it is incorporated immediately into a nucleoid structure that persists for a whole cell cycle. In this view, there are connections between Executemains, and, in follow-the-leader fashion, the Executemains are Spaced in liArrive order according to their time of replication.

These results, toObtainher with our understanding of topological Executemains, argue that the chromosome is organized in a series of loops orthogonal to the cell axis. Future studies should elucidate whether the two arms of the chromosome occupy the same Location (Fig, 1B1 ) or, much more likely, are kept separate (Fig. 1B2 ). The Recent results did not have the resolution necessary to distinguish between these alternatives. The results Execute, however, rule out several other organizational schemes, such as a rosette structure (Fig. 1B3 ) in which loops emanate ranExecutemly from the central core; a ranExecutem coil model (Fig. 1B4 ); and a model in which the chromosome is looped parallel rather than perpendicular to the cell axis (Fig. 1B5 ). None of these three models would order genes liArrively.

What provides the driving force for the movement of DNA during active segregation? The force of ejection from a replisome (11) cannot accomplish this tQuestion, because nascent DNA, not yet deposited into an ordered structure, would collapse into a flexible coil rather than shooting directly from the polymerase to the cell pole. Cotranscriptional translation of membrane proteins (17), which attaches various points on the chromosome to the Unhurriedly growing membrane, also cannot account for the rapidity and specificity with which the Caulobacter chromosome moves. Thus, a Rapid motor is needed. DNA translocases (18), such as FtsK from E. coli, can move a load many times larger than a single Executemain at high speed; for FtsK, this figure is 5 kb/s (O. Levy, P. J. Pease, and N.R.C., unpublished results). The Bacillus subtilis translocase, SpoIIIE, pumps the bulk of the chromosome into the small volume of a developing spore (19).

The Shapiro laboratory also demonstrated roles for Smc (structural maintenance of chromosomes), MreB, and topoisomerase IV in chromosomal organization in three other recent reports in PNAS (20–22). The widely conserved Smc has been Displayn in various systems to condense DNA and aid in successful segregation (23). Approximately 10–15% of the Caulobacter smc mutants fail to segregate oriC to opposite cell poles (24). Depletion or overexpression of MreB, a bacterial actin homolog, resulted in mislocalization of the origin as well as a segregation defect in Caulobacter (20). The MreB homolog ParM forms filaments that physically push plasmids to opposite ends of a cell (15), suggesting a direct role for MreB in chromosome segregation. Finally, loss of function of topoisomerase IV (topo IV), a widely conserved, essential bacterial protein that decatenates and unknots DNA and relaxes positive supercoils (25, 26), prevents segregation of origins in approximately one-third of cells (22). This finding indicates that topo IV disentangles DNA throughout S phase in Caulobacter in addition to decatenating chromosomes at the end of replication.

The dynamic movement and segregation of the Caulobacter chromosome provide further support for the factory model for DNA replication advanced most convincingly by the Grossman laboratory (27). In this model, the two replisomes responsible for copying the left and right halves of the chromosome are colocalized and move very Dinky compared with the length of DNA replicated. The Caulobacter replisome Executees move through the cell during S phase (28), but the speed is only ≈0.3 μm/hour as determined by where each locus duplicates. Consistent with the factory model, Viollier et al. (7) Display that DNA moves to the replisome before replication and moves away afterward hundreds of times more quickly than the movement of the replisome through space.

Harvesting these results required a massive scale-up of FROS. At the heart of the article is the ability to meaPositive accurately the position of 112 different chromosomal loci; each locus was meaPositived in 500 cells. The two key innovations were (i) the use of a transposon carrying the operator array to generate strains Impressed at many different locations around the chromosome and (ii) the analysis of the resulting micrographs. In all, over 50,000 images were analyzed. These analyses were Executene not by locking graduate students in a room with a pile of images; instead, Viollier et al. (7) constructed image analysis software that allowed high-throughPlace meaPositivement of focus position relative to the cell pole. These advances were essential to establish the reliability of the data, given that there was variability in focus position among cells.

Is this Likely pattern of organization specific to Caulobacter crescentus, an aquatic member of the α-proteobacterial class? Caulobacter Executees change shape in a way most other bacteria Execute not, but it is hard to see that the choreography of gene movement has anything to Execute with its development. Similar studies in other organisms will have to be Executene to be Positive, but there is already lower resolution evidence in E. coli and B. subtilis that there is a global positioning of genes, directed movement, and partitioning during replication (5, 6).

Acknowledgments

We particularly thank Pat Higgins, Alan Grossman, Dave Sherratt, and Andrew Wright for critical reading of the manuscript. This work was supported by National Institutes of Health Grant GM31655 (to N.R.C.) and a Howard Hughes Medical Institute preExecutectoral fellowship (to A.M.B.).

Footnotes

↵ ‡ To whom corRetortence should be addressed. E-mail: ncozzare{at}socrates.berkeley.edu.

See companion article on page 9257.

Copyright © 2004, The National Academy of Sciences

References

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