Etude de la ségrégation de la région terminale du chromosome d'Escherichia coli

Les bactéries utilisent la polarité de la réplication, de l'origine vers la région terminale de leur chromosomes circulaires, pour contrôler la dynamique de l'ADN durant le cylce cellulaire. Chez Esherichia coli, la ségrégation commence par une migration active des régions origines suivit progressivement par le reste du chromosome. Les dernières étapes de la ségrégation ont été activement étudiées dans le cas dimères de chromosome formées par recombinaison homologue au cours de la réplication. Pour résoudre ces structures, la protéine associée au divisome qui transloque l'ADN FstK est nécessaire. FtsK pompe les chromosomes en direction du site dif en utilisant la polarité des motifs KOPS (FtsK Orienting Polar Sequence). Des expériences sur l'étude de la recombinaison entre sites dif ont suggérées que FtsK n'est active que pour résoudre les dimères de chromosome. En utilisant un système de deux couleurs pour visualiser des paires de loci dans des cellules vivantes, nous montrons que la résolution spaciale des loci des deux chromosomes frères est orienté de manière précise de l'origine vers le site dif. Par ailleurs, la ségrégation ordonnée dans une région de 200kb autour du site dif, dépend de l'activité orienté de FtsK mais pas de la présence de dimères ou leur résolution. La ségrégation médiée par FtsK nécessite la protéine MatP qui retarde la ségrégation de la région autour du site dif jusqu'à la fin du cycle cellulaire. Nous concluons que FtsK ségrège activement la région terminale du chromosome d'Escherichia coli, qu'il soit sous forme monomérique ou dimérique, et ce de manière précise et ordonnée. Nos résultats sont cohérant dans un modèle où FtsK permet le relâchement de la cohésion médiée par MatP et / ou l'interaction avec le divisome de la région terminale, de manière ordonnée selon les KOPSBacteria 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 manne

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 KOPSrecognition 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 step

The terminal region of the <it>E. coli </it>chromosome localises at the periphery of the nucleoid

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>E. coli </it>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.</p> <p>Conclusions</p> <p>Our approach allows to reliably observing the positioning of chromosome loci along the width of <it>E. coli </it>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.</p