PMID:18410497

Citation	Espeli, O, Mercier, R and Boccard, F (2008) DNA dynamics vary according to macrodomain topography in the E. coli chromosome. Mol. Microbiol. 68:1418-27
Abstract	The organization of the Escherichia coli chromosome has been defined genetically as consisting of four insulated macrodomains and two less constrained regions. Here we have examined the movement of chromosomal loci by tracking fluorescent markers in time-lapse microscopy during a complete cell cycle. Analysing the positioning, the segregation pattern and the motility of markers allowed us to show that the dynamic behaviour of loci belonging to various macrodomains and less constrained regions is radically different. In macrodomains constraints on mobility are apparent whereas in non-structured regions, markers exhibited a greater motility that may explain their ability to interact with flanking macrodomains. Following replication, duplicated markers belonging to macrodomains show a colocalization step and this landmark is not apparent in non-structured regions. Chromosome segregation occurs in three steps: first, the origin-proximal half of the chromosome consisting of the Ori macrodomain and the two non-structured region segregates concomitantly in a short period of time. Second, the Right and Left macrodomains segregate progressively following the genetic map. Third, the Ter macrodomain is rapidly segregated before division, after a significant period of colocalization. Macrodomain territories defined as cellular spaces occupied by the different macrodomains can be identified.
Links	PubMed Online version:10.1111/j.1365-2958.2008.06239.x
Keywords	Chromosome Segregation; Chromosomes, Bacterial; DNA Replication; DNA, Bacterial/chemistry; DNA, Bacterial/genetics; Escherichia coli/cytology; Escherichia coli/genetics; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Microscopy; Nucleic Acid Conformation
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Significance

Points of the Paper:

• Chromosome Segregation Occurs in 3 steps:

1. Origin-proximal regions separate after a short period of time (Ori MD, NSR, and NSL)
2. Rt. And Lt. MD separate following the genetic map
3. Ter MD rapidly segregates shortly before division

• Length of cohesion time is MD-specific
• MD territories are identified- “Home” positions
• Movement towards “Home” positions is dependent upon domain

-symmetrical for Ori and Ter MD -asymmetrical for Rt, Lt MDs and NS regions

Figures Details

Figure 1A- map of tags
Figure 1B- Average number of foci seen correlates to the map position/ MD
Multiple plateau regions of 1 focus Rt. And Lt. MD- not a linear decrease
Figure 1C- # of Observed Foci vs. # of Expected Foci
MD foci< Expected Foci
NS foci ~ Expected Foci
Cohesion in the MD regions, but not in NS

Figure 2A- time lapse photography exceeding the duration of the cell cycle (Movie S1H)

Figure 2B- Individualization occurs over 2 successive cell cycles
1st- close to Ori last- close to dif
NS loci separate at the same time as the Ori MD, but are replicated later
Rt. And Lt. MD segregate linearly along the genetic map
Ter MD- multiple foci(Movie S1H- Ter-2)

SD overlaps division, so 2 foci are present in a newborn cell

Figure 3A- Plotting the Movements of the Individual Foci within Mother/ Daughter cell
Green Line- individualization time
Red Line- division

Segregation Process contains 2 phases:
1. Process of segregation per se
2. Period when the foci are steady at the home position
“Home” Position- area defined by the SD of the home position of the averaged patterns
MD- regular and progressive segregation to future home position
NS- irregular segregation pathway (but they segregating accurately before division)
a. abrupt movements
b. opposite to segregation direction
c. sister foci merge for long times after individualization
d. foci can migrate to poles

Ori MD- occur symmetrically close to the quarter positions- both foci at the same speed
Ter MD- strictly symmetrical segregation pattern- foci moved at different speeds
Rt. And Lt. MD- not localized symmetrically around midcell- moved at different speeds

Figure 3B- Averages of data in Figure 3A
NS Regions- segregation roughly similar to Rt. Or Lt. MD segregation

Mobility of the Chromosomal Loci
Figure 4A- Follow the movement of foci for 50 minutes
Green Star- point of individualization
Red Star- end of the trajectory

Useful Materials and Methods

• Time lapse microscopy
• parS tags inserted into genome and bound by ParB-GFP

Calculations

• Asymmetry Index

1-[(distance to midcell of the closest home position)\(distance to midcell of the furthermost postion)] AI- lower the number, the more symmetrical the movement

• Number of Expected Loci

Expected Foci Calculation (Helmstetter 1996) N=2^[1-x)C+D/τ]
N= number of copies of a locus
x= portion of the C period when the locus is replicated
C= duration of the replication (min)
D= length of the period b/t the end of replication and division (min)
τ= generation time (min)

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