Coordination of Growth, Chromosome Replication/Segregation, and Cell Division in E. coli.

Bacterial cells growing in steady state maintain a 1:1:1 relationship between an appropriate mass increase, a round of DNA replication plus sister chromosome segregation, and cell division. This is accomplished without the cell cycle engine found in eukaryotic cells. We propose here a formal logic, and an accompanying mechanism, for how such coordination could be provided in E. coli. Completion of chromosomal and divisome-related events would lead, interactively, to a "progression control complex" (PCC) which provides integrated physical coupling between sister terminus regions and the nascent septum. When a cell has both (i) achieved a sufficient mass increase, and (ii) the PCC has developed, a conformational change in the PCC occurs. This change results in "progression permission," which triggers both onset of cell division and release of terminus regions. Release of the terminus region, in turn, directly enables a next round of replication initiation via physical changes transmitted through the nucleoid. Division and initiation are then implemented, each at its own rate and timing, according to conditions present. Importantly: (i) the limiting step for progression permission may be either completion of the growth requirement or the chromosome/divisome processes required for assembly of the PCC; and, (ii) the outcome of the proposed process is granting of permission to progress, not determination of the absolute or relative timings of downstream events. This basic logic, and the accompanying mechanism, can explain coordination of events in both slow and fast growth conditions; can accommodate diverse variations and perturbations of cellular events; and is compatible with existing mathematical descriptions of the E. coli cell cycle. Also, while our proposition is specifically designed to provide 1:1:1 coordination among basic events on a "per-cell cycle" basis, it is a small step to further envision permission progression is also the target of basic growth rate control. In such a case, the rate of mass accumulation (or its equivalent) would determine the length of the interval between successive permission events and, thus, successive cell divisions and successive replication initiations.

Mechanisms for chromosome segregation.

Bacteria face the problem of segregating their gigantic chromosomes without a segregation period restricted in time and space, as Eukaryotes do. Segregation thus involves multiple activities, general or specific of a chromosome region and differentially controlled. Recent advances show that these various mechanisms conform to a "pair and release" rule, which appears as a general rule in DNA segregation. We describe the latest advances in segregation of bacterial chromosomes with emphasis on the different pair and release mechanisms.

The bacterial nucleoid: nature, dynamics and sister segregation.

Recent studies reveal that the bacterial nucleoid has a defined, self-adherent shape and an underlying longitudinal organization and comprises a viscoelastic matrix. Within this shape, mobility is enhanced by ATP-dependent processes and individual loci can undergo ballistic off-equilibrium movements. In Escherichia coli, two global dynamic nucleoid behaviors emerge pointing to nucleoid-wide accumulation and relief of internal stress. Sister segregation begins with local splitting of individual loci, which is delayed at origin, terminus and specialized interstitial snap regions. Globally, as studied in several systems, segregation is a multi-step process in which internal nucleoid state plays critical roles that involve both compaction and expansion. The origin and terminus regions undergo specialized programs partially driven by complex ATP burning mechanisms such as a ParAB Brownian ratchet and a septum-associated FtsK motor. These recent findings reveal strong, direct parallels among events in different systems and between bacterial nucleoids and mammalian chromosomes with respect to physical properties, internal organization and dynamic behaviors.

FtsK actively segregates sister chromosomes in Escherichia coli.

Bacteria use the replication origin-to-terminus polarity of their circular chromosomes to control DNA transactions during the cell cycle. Segregation starts by active migration of the region of origin followed by progressive movement of the rest of the chromosomes. The last steps of segregation have been studied extensively in the case of dimeric sister chromosomes and when chromosome organization is impaired by mutations. In these special cases, the divisome-associated DNA translocase FtsK is required. FtsK pumps chromosomes toward the dif chromosome dimer resolution site using polarity of the FtsK-orienting polar sequence (KOPS) DNA motifs. Assays based on monitoring dif recombination have suggested that FtsK acts only in these special cases and does not act on monomeric chromosomes. Using a two-color system to visualize pairs of chromosome loci in living cells, we show that the spatial resolution of sister loci is accurately ordered from the point of origin to the dif site. Furthermore, ordered segregation in a region ∼200 kb long surrounding dif depended on the oriented translocation activity of FtsK but not on the formation of dimers or their resolution. FtsK-mediated segregation required the MatP protein, which delays segregation of the dif-surrounding region until cell division. We conclude that FtsK segregates the terminus region of sister chromosomes whether they are monomeric or dimeric and does so in an accurate and ordered manner. Our data are consistent with a model in which FtsK acts to release the MatP-mediated cohesion and/or interaction with the division apparatus of the terminus region in a KOPS-oriented manner.

A defined terminal region of the E. coli chromosome shows late segregation and high FtsK activity.

BACKGROUND: The FtsK DNA-translocase controls the last steps of chromosome segregation in E. coli. It translocates sister chromosomes using the KOPS DNA motifs to orient its activity, and controls the resolution of dimeric forms of sister chromosomes by XerCD-mediated recombination at the dif site and their decatenation by TopoIV. METHODOLOGY: We have used XerCD/dif recombination as a genetic trap to probe the interaction of FtsK with loci located in different regions of the chromosome. This assay revealed that the activity of FtsK is restricted to a ∼400 kb terminal region of the chromosome around the natural position of the dif site. Preferential interaction with this region required the tethering of FtsK to the division septum via its N-terminal domain as well as its translocation activity. However, the KOPS-recognition activity of FtsK was not required. Displacement of replication termination outside the FtsK high activity region had no effect on FtsK activity and deletion of a part of this region was not compensated by its extension to neighbouring regions. By observing the fate of fluorescent-tagged loci of the ter region, we found that segregation of the FtsK high activity region is delayed compared to that of its adjacent regions. SIGNIFICANCE: Our results show that a restricted terminal region of the chromosome is specifically dedicated to the last steps of chromosome segregation and to their coupling with cell division by FtsK.

The terminal region of the E. coli chromosome localises at the periphery of the nucleoid.

BACKGROUND: Bacterial chromosomes are organised into a compact and dynamic structures termed nucleoids. Cytological studies in model rod-shaped bacteria show that the different regions of the chromosome display distinct and specific sub-cellular positioning and choreographies during the course of the cell cycle. The localisation of chromosome loci along the length of the cell has been described. However, positioning of loci across the width of the cell has not been determined. RESULTS: Here, we show that it is possible to assess the mean positioning of chromosomal loci across the width of the cell using two-dimension images from wide-field fluorescence microscopy. Observed apparent distributions of fluorescent-tagged loci of the E. coli chromosome along the cell diameter were compared with simulated distributions calculated using a range of cell width positioning models. Using this method, we detected the migration of chromosome loci towards the cell periphery induced by production of the bacteriophage T4 Ndd protein. In the absence of Ndd production, loci outside the replication terminus were located either randomly along the nucleoid width or towards the cell centre whereas loci inside the replication terminus were located at the periphery of the nucleoid in contrast to other loci. CONCLUSIONS: Our approach allows to reliably observing the positioning of chromosome loci along the width of E. coli cells. The terminal region of the chromosome is preferentially located at the periphery of the nucleoid consistent with its specific roles in chromosome organisation and dynamics.