Chromosome Structuring Limits Genome Plasticity in Escherichia coli

Chromosome organizations of related bacterial genera are well conserved despite a very long divergence period. We have assessed the forces limiting bacterial genome plasticity in Escherichia coli by measuring the respective effect of altering different parameters, including DNA replication, compositional skew of replichores, coordination of gene expression with DNA replication, replication-associated gene dosage, and chromosome organization into macrodomains. Chromosomes were rearranged by large inversions. Changes in the compositional skew of replichores, in the coordination of gene expression with DNA replication or in the replication-associated gene dosage have only a moderate effect on cell physiology because large rearrangements inverting the orientation of several hundred genes inside a replichore are only slightly detrimental. By contrast, changing the balance between the two replication arms has a more drastic effect, and the recombinational rescue of replication forks is required for cell viability when one of the chromosome arms is less than half than the other one. Macrodomain organization also appears to be a major factor restricting chromosome plasticity, and two types of inverted configurations severely affect the cell cycle. First, the disruption of the Ter macrodomain with replication forks merging far from the normal replichore junction provoked chromosome segregation defects. The second major problematic configurations resulted from inversions between Ori and Right macrodomains, which perturb nucleoid distribution and early steps of cytokinesis. Consequences for the control of the bacterial cell cycle and for the evolution of bacterial chromosome configuration are discussed

Long-Range Chromosome Organization in E. coli: A Site-Specific System Isolates the Ter Macrodomain

The organization of the Escherichia coli chromosome into a ring composed of four macrodomains and two less-structured regions influences the segregation of sister chromatids and the mobility of chromosomal DNA. The structuring of the terminus region (Ter) into a macrodomain relies on the interaction of the protein MatP with a 13-bp target called matS repeated 23 times in the 800-kb-long domain. Here, by using a new method that allows the transposition of any chromosomal segment at a defined position on the genetic map, we reveal a site-specific system that restricts to the Ter region a constraining process that reduces DNA mobility and delays loci segregation. Remarkably, the constraining process is regulated during the cell cycle and occurs only when the Ter MD is associated with the division machinery at mid-cell. The change of DNA properties does not rely on the presence of a trans-acting mechanism but rather involves a cis-effect acting at a long distance from the Ter region. Two specific 12-bp sequences located in the flanking Left and Right macrodomains and a newly identified protein designated YfbV conserved with MatP through evolution are required to impede the spreading of the constraining process to the rest of the chromosome. Our results unravel a site-specific system required to restrict to the Ter region the consequences of anchoring the Ter MD to the division machinery

The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain

International audienceThe Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼-¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed

Wie die Made im Speck? Fredsbesættelsen 1940-1943 belyst gennem samarbejdet mellem Esbjerg politi og værnemagten

Wie die Made im Speck? The Peaceful Occupation of Denmark 1940-1943 Seen in the Light of Cooperation Between the Police in Esbjerg and the Wehrmacht »Sie fühlten sich wie die Made im Speck,« wrote the head of the Gestapo in Esbjerg of the German troops in the town. Danish historians of the German occupation of Denmark have also noted the comfortable life that the peculiar occupation regime in Denmark afforded the Wehrmacht compared to conditions in other occupied countries. The present article provides a more complex picture of the Wehrmacht's situation on the so-called »whip cream front.« It focuses on conditions from the viewpoint of the German authorities charged with administering the occupation regime at both the local and national levels in cooperation with the Danish authorities. The main source for depicting these conditions is internal German correspondence, especially confidential messages to and from the commander of the German troops in Denmark, General Erich Lüdke. Some of these documents, used here for the first time, are essential for understanding how the so-called peaceful occupation was experienced and managed by the German authorities charged with its implementation. Since maintenance of law and order on the part of both occupier and occupied was crucial to the occupation regime, their cooperation is also assessed from a contemporary Danish viewpoint, namely, as it was experienced by the chief constable of Esbjerg, Børge Hebo. It was his task, whenever German interests were involved, to solve police problems together with the local German commander. It was paramount for the Danish government to keep legal jurisdiction in Danish hands. The police, therefore, played an essential, yet vulnerable role, since assurance of law and order was the very basis of the peace occupation. The abilities of Esbjerg's chief constable to negotiate and mediate between the German military and the population were put to the test shortly after the beginning of the occupation. Because of its strategic importance throughout the war the town was filled with a large number of German troops, resulting almost immediately in brawls with the male population. For the first few months the cooperative effort to keep things calm ran smoothly, but then it became increasingly marked by complaints on the part of the German commander about the hostile behaviour of the population. The commander found in Hebo an intrepid counterpart who managed to manoeuvre deftly within the confines of the occupation rules, evidencing considerable virtuosity in exploiting any lack of clarity regarding police jurisdiction. He was able to perform his function as intermediary between the occupation forces and the population without losing the trust of the latter through concessions to the former. Complaints about the inadequacy of his measures in dealing with the people of Esbjerg were frequently lodged with the negotiators at the national level. Because of the German personnel problem and the lack of clarity in the agreement on police cooperation the Danish police had authority vis-à-vis the Wehrmacht that would seem incompatible with the relation of occupied to occupier. This is a factor that helps to understand how Hebo managed. A circumstance that facilitated his task was the attitude of Esbjerg's commander, who compared to commanders in other towns seems to have developed a greater appreciation of the importance of maintaining peaceful conditions and dependence on the efforts of the Danish police. It is probable that in Esbjerg as in other places of strategic importance the Germans had assigned a commander who understood Danish conditions. Like Esbjerg's commander the military commander at the national level, Erich Lüdke, endeavoured to uphold amicable cooperative relations and preserve the status quo. The German plenipotentiary ( Reichsbevollmächtigter ) Renthe-Fink supported this policy and what it entailed in the way of futile efforts to enforce internal discipline. There was clearly considerable uncertainty in some segments of the military about how to play the role of peaceful occupier, resulting among other things in persistent Danish complaints, thus weakening the bargaining position of the German authorities. The German negotiators let the Danish authorities understand that they were strongly dissatisfied with the efforts of the police, while in communications with Berlin there was praise for Danish efforts along with futile attempts to call attention to problems caused by internal conflicts over jurisdiction, rivalries, and irresponsibility among the occupation authorities. In acknowledgment of the problems with the troops Berlin was presented with arguments for extending the jurisdiction of the Danish police over members of the Wehrmacht. The Reich authorities, however, were deaf to these arguments, and the foundation of the peaceful occupational regime gradually eroded. A crucial problem in maintaining stability was the lack of unified German policy ( Einheitlichkeit ), creating a difficult situation for the Germans in charge of negotiations. The lack of a common attitude and uniform conduct sprang in part from disagreements on jurisdiction between competing authorities in the German hierarchy. The chain of command was apparently so unclear that all those involved seemed to define their own rules of cooperation with the Danish authorities. Both the air force and the navy created obstacles to the endeavours of Lüdke, Renthe-Fink, and the latter's successor Werner Best to coordinate, improve, and impose discipline on the German effort to preserve the peaceful occupation. Nor was there much evidence of ability or will on the part of the highest authorities in Berlin to see the advantages for Germany of upholding the status quo. By September 1943 German confidence in the ability and will of the Danish police to solve cases of sabotage, spying, and destabilizing activity was gone, and the Gestapo took over. The agreement, which the Danish Government under protest had concluded with the German occupation force to guarantee the safety of military personnel in return for not infringing on the sovereignty and neutrality of the Danish state, was now broken by both parties. Seen through the eyes of German soldiers assignment to occupied Denmark may well have felt like »die Made im Speck,« but for the Germans in charge of negotiations the mission was marked by a futile struggle within their own camp to explain and reach consensus on how to play that odd game with no clear rules inaugurated by the peculiar conditions of a regime of peaceful occupation. Translated by Michael Wolf

<i>E</i>. <i>coli</i> nucleoid positioning and chromosome segregation.

<p>(A) The extent of the excised segment is indicated on the diagram below the map of the NSL-Ori region (segments a, b and c). (B) Montage of merged pictures of parS^P1 (green), DAPI staining (red) and phase-contrast micrographs (grey) of MG1655 cells upon excision of chromosomal DNA segments a, b and c carrying <i>oriC</i> and a <i>parS</i> (Ori-5 or Ori-6). Control sample in the absence of excision is presented on the top panels. (C) Montage of merged pictures of MG1655 cells upon excision of chromosomal DNA segments carrying <i>oriC</i> and the <i>parS</i> tag (Ori-5 and Ori-6; indicated in green), together with a <i>parS</i>^<i>T1</i> (indicated in yellow) on the remnant part either in Ori region (panel a) or in Ter region (panel b) or expressing a MatP-mCherry fusion protein (panel c), HU-mCherry staining (red; panel a) and phase-contrast micrographs (grey). Control sample in the absence of excision is presented on the top panels. (D) Montage of merged pictures of <i>parS</i>^<i>P1</i> Ori-6 (green), DAPI staining (red) and phase-contrast micrographs (grey) of MG1655 cells upon excision of chromosomal DNA segment b carrying <i>oriC</i> and the <i>parS</i> tag (Ori-6) in the presence of cephalexin. Control sample in the absence of excision is presented. Black arrowheads indicate excised rings carrying Ori markers. (E) Time-lapse experiment representing the dynamics of <i>parS</i>^<i>P1</i> Ori-6 tag upon excision of segment b. Montage of merged pictures of MG1655 cells upon excision of chromosomal DNA segments carrying <i>oriC</i> and the <i>parS</i> tag (Ori-6) and expressing HU-mCherry. Positioning of the focus was observed for 300 min with 10 min intervals. Black arrowheads indicate chromosome remnants. Scale bar indicate 2 μm.</p

Effect of the inactivation of the MaoP-<i>maoS</i> system.

<p>(A) Travelled distance of different markers in WT strain and a <i>maoP</i> deletion derivative. Columns indicate the mean value with standard deviation calculated for 30 independent foci. The x axis represents the chromosomal genetic map (position in Mb from <i>thrABC operon</i>). The MDs (Ori, Right, Left and Ter) and the NS regions are indicated above the graph. (B) Colonies of strains carrying a normal (white colonies) or an inverted configuration (blue colonies) upon DNA inversion between Ori and Right MDs in a WT and <i>maoP</i> genetic background. (C) Positioning of chromosomal marker Ori-3 in a WT (left panel) and in a <i>maoP</i> mutant (right panel) observed in 400 cells. Cells are sorted for length, ascending from top to bottom. In the heat maps blue corresponds to low and red to high intensity. The diagram represents the relative position of the foci as a function of the cell size.</p

Strains used in excision experiments.

<p>Strains used in excision experiments.</p

Identification of determinants required for Ori MD structuring.

<p>(A) Effect of the presence of <i>maoP in trans</i> or the insertion of <i>maoS</i> at an ectopic position on deletions spanning the <i>hdfR-yifE</i> region (from coordinates 3944752–3948043) on Ori-3, Ori-4 and NSR-2 mobility. The extent of the region remaining upon the deletion is indicated. Mobility of markers Ori-3, Ori4 and NSR-3 are indicated; the loss of DNA constraint is indicated by “-” and DNA constraint indicated by “+”. (B) Programmed excision of chromosomal segments by site-specific recombination. The two <i>att</i> sites are integrated in the chromosome in the same orientation. Excisive recombination promoted by Int+Xis results in the excision and circularization of the intervening segment carrying <i>att</i>P or <i>att</i>B (depending on the order of <i>att</i>L and <i>att</i>R sites in the chromosome). <i>att</i>L and <i>att</i>R sites are flanked by the 5’ and 3’ parts of <i>lacZ</i>, respectively. The excised segment is devoid of replication origin and is not replicated. Integrative recombination between <i>att</i>P and attB occurs at very low frequency preventing fusion of the two molecules. (C) Travelled distance of Ori markers upon excision of different segment in the Ori region. The ori Td-3 segment carries <i>maoS</i> and <i>maoP</i> indicated by a red square. Mobility of markers Ori-3, Ori4 or Ori-6 are indicated; the loss of constraints is indicated by “-”and DNA constraint indicated by “+”.</p

Strains used in inversion experiments.

<p>Strains used in inversion experiments.</p

Exclusion from the nucleoid of large DNA rings looped out from the Ori region of the chromosome.

<p>Montage of merged pictures of <i>parS</i>^P1 (green), DAPI staining (red) and phase-contrast micrographs (grey) of FBG150 cells upon excision of chromosomal DNA segments carrying the <i>parS</i> tag. The extent of the excised segment is indicated on the diagram above the micrographs (segments a, b and c). Control sample in the absence of excision is presented on the top lane (no excision). Black arrowheads indicate Ori markers present on excised rings. Scale bar indicate 2 μm.</p