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MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves.

Danilova O, Reyes-Lamothe R, Pinskaya M, Sherratt D, Possoz C - Mol. Microbiol. (2007)

Bottom Line: We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole.Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells.We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

ABSTRACT
The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.

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Polar positioning of ori1 in mukB and mukB topA10 cells. A. Snapshot analysis of ori1 positioning in different strains as indicated (wt: IL02; mukB: OS27; mukB topA10: OS47; topA10: OS70). For about 500 cells of each strain, cells were first classified into the five types shown in the schematic. Types A–C correspond to non-septating cells with one ori1 focus (type A), two ori1 foci closely spaced (type B) and two ori1 foci well separated (type C); type D corresponds to septating cells with segregated two sister ori1 foci, and type abn corresponds to all the other cells judged abnormal. Cells were divided along the longitudinal axis in six equal parts: left pole, left quarter, mid-cell left, mid-cell right, right quarter and right pole. The ori1 position (histograms) was classed in either polar (black), quarter (dark grey) or mid-cell (light grey). The predominant position was represented by schematics on the right-hand side of the histograms. Three types of abnormal cells were distinguished: *lacking ori1; **containing two ori1 foci in the same cell half and ***containing more than two ori1 foci. B. Time-lapse analysis of ori1 in mukB cells. Examples of successful (top) and unsuccessful (bottom) segregation are illustrated. The arrows indicate the position of the sister ori1 foci at the time of division (dashed line). Images were taken every hour.
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fig01: Polar positioning of ori1 in mukB and mukB topA10 cells. A. Snapshot analysis of ori1 positioning in different strains as indicated (wt: IL02; mukB: OS27; mukB topA10: OS47; topA10: OS70). For about 500 cells of each strain, cells were first classified into the five types shown in the schematic. Types A–C correspond to non-septating cells with one ori1 focus (type A), two ori1 foci closely spaced (type B) and two ori1 foci well separated (type C); type D corresponds to septating cells with segregated two sister ori1 foci, and type abn corresponds to all the other cells judged abnormal. Cells were divided along the longitudinal axis in six equal parts: left pole, left quarter, mid-cell left, mid-cell right, right quarter and right pole. The ori1 position (histograms) was classed in either polar (black), quarter (dark grey) or mid-cell (light grey). The predominant position was represented by schematics on the right-hand side of the histograms. Three types of abnormal cells were distinguished: *lacking ori1; **containing two ori1 foci in the same cell half and ***containing more than two ori1 foci. B. Time-lapse analysis of ori1 in mukB cells. Examples of successful (top) and unsuccessful (bottom) segregation are illustrated. The arrows indicate the position of the sister ori1 foci at the time of division (dashed line). Images were taken every hour.

Mentions: Tracking of the ori1 locus, located 15 kb counterclockwise of oriC, by fluorescent repressor-operator sites (FROS; Lau et al., 2003), revealed abnormal positioning and segregation of sister origins in mukB mutants. In wild-type cells growing at 22°C, without overlapping replication cycles, our snapshot data (Fig. 1A) were consistent with the segregation pathway previously described (Wang et al., 2006). ori1 is always close to mid-cell in newborn cells and after duplication at mid-cell, sister origins migrate in opposite directions towards the quarter positions, where they remain until cell division. In contrast, snapshots of mukB mutants showed that duplicated ori1 foci were most often positioned close to opposite old poles (types C and D; ∼70% of 2×ori1 cells), with focus duplication being inferred to occur either at an old pole or at mid-cell (type B).


MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves.

Danilova O, Reyes-Lamothe R, Pinskaya M, Sherratt D, Possoz C - Mol. Microbiol. (2007)

Polar positioning of ori1 in mukB and mukB topA10 cells. A. Snapshot analysis of ori1 positioning in different strains as indicated (wt: IL02; mukB: OS27; mukB topA10: OS47; topA10: OS70). For about 500 cells of each strain, cells were first classified into the five types shown in the schematic. Types A–C correspond to non-septating cells with one ori1 focus (type A), two ori1 foci closely spaced (type B) and two ori1 foci well separated (type C); type D corresponds to septating cells with segregated two sister ori1 foci, and type abn corresponds to all the other cells judged abnormal. Cells were divided along the longitudinal axis in six equal parts: left pole, left quarter, mid-cell left, mid-cell right, right quarter and right pole. The ori1 position (histograms) was classed in either polar (black), quarter (dark grey) or mid-cell (light grey). The predominant position was represented by schematics on the right-hand side of the histograms. Three types of abnormal cells were distinguished: *lacking ori1; **containing two ori1 foci in the same cell half and ***containing more than two ori1 foci. B. Time-lapse analysis of ori1 in mukB cells. Examples of successful (top) and unsuccessful (bottom) segregation are illustrated. The arrows indicate the position of the sister ori1 foci at the time of division (dashed line). Images were taken every hour.
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Related In: Results  -  Collection

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fig01: Polar positioning of ori1 in mukB and mukB topA10 cells. A. Snapshot analysis of ori1 positioning in different strains as indicated (wt: IL02; mukB: OS27; mukB topA10: OS47; topA10: OS70). For about 500 cells of each strain, cells were first classified into the five types shown in the schematic. Types A–C correspond to non-septating cells with one ori1 focus (type A), two ori1 foci closely spaced (type B) and two ori1 foci well separated (type C); type D corresponds to septating cells with segregated two sister ori1 foci, and type abn corresponds to all the other cells judged abnormal. Cells were divided along the longitudinal axis in six equal parts: left pole, left quarter, mid-cell left, mid-cell right, right quarter and right pole. The ori1 position (histograms) was classed in either polar (black), quarter (dark grey) or mid-cell (light grey). The predominant position was represented by schematics on the right-hand side of the histograms. Three types of abnormal cells were distinguished: *lacking ori1; **containing two ori1 foci in the same cell half and ***containing more than two ori1 foci. B. Time-lapse analysis of ori1 in mukB cells. Examples of successful (top) and unsuccessful (bottom) segregation are illustrated. The arrows indicate the position of the sister ori1 foci at the time of division (dashed line). Images were taken every hour.
Mentions: Tracking of the ori1 locus, located 15 kb counterclockwise of oriC, by fluorescent repressor-operator sites (FROS; Lau et al., 2003), revealed abnormal positioning and segregation of sister origins in mukB mutants. In wild-type cells growing at 22°C, without overlapping replication cycles, our snapshot data (Fig. 1A) were consistent with the segregation pathway previously described (Wang et al., 2006). ori1 is always close to mid-cell in newborn cells and after duplication at mid-cell, sister origins migrate in opposite directions towards the quarter positions, where they remain until cell division. In contrast, snapshots of mukB mutants showed that duplicated ori1 foci were most often positioned close to opposite old poles (types C and D; ∼70% of 2×ori1 cells), with focus duplication being inferred to occur either at an old pole or at mid-cell (type B).

Bottom Line: We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole.Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells.We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

ABSTRACT
The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.

Show MeSH
Related in: MedlinePlus