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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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Colocalization of MukB with the origin region.A. Colocalization is indicated as a number (%) on black lines linking MukB and each given loci positioned on the circular map of the E. coli chromosome (ori1, R2, ter2 loci from OS18 and OS29, OS69, OS19 cells respectively). Colocalization (%) of ter2 with ori1 in IL05 cells is indicated on the purple line.B. Snapshot analysis of MukB-GFP and ori1 positioning. Images of representative wild-type cells (OS18; see histogram for legend). Histogram representing the proportion of the different MukB patterns for each cell type (A–D, Fig. 2). Green bars correspond to MukB/ori1 colocalization, and patterns 1, 2 and 3 correspond to one, two or three MukB foci colocalizing with one ori1 focus. When cells contains two ori1 foci, each ori1 focus can colocalize either with one or two MukB, generating pattern 1/1, 1/2 or 2/2. Grey bars and pattern 0 correspond to the absence of colocalization of one ori1 focus with MukB, including cells containing one ori1 focus or two ori1 foci (patterns 0/0, 0/1 and 0/2 are included in pattern 0). Pattern ‘a’ corresponds to cells with an extra MukB focus distant from the ori1/MukB colocalizing foci.C. Time-lapse tracking simultaneously MukB and ori1 (without or with ori1 duplication, left and right respectively; OS18 cells). The number of MukB foci per cluster (bottom panel) and the time intervals between images (below panels) are indicated.D. Representation of the cell-cycle dynamics of ori1 and MukB deduced from snapshot and time-lapse analysis.
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fig03: Colocalization of MukB with the origin region.A. Colocalization is indicated as a number (%) on black lines linking MukB and each given loci positioned on the circular map of the E. coli chromosome (ori1, R2, ter2 loci from OS18 and OS29, OS69, OS19 cells respectively). Colocalization (%) of ter2 with ori1 in IL05 cells is indicated on the purple line.B. Snapshot analysis of MukB-GFP and ori1 positioning. Images of representative wild-type cells (OS18; see histogram for legend). Histogram representing the proportion of the different MukB patterns for each cell type (A–D, Fig. 2). Green bars correspond to MukB/ori1 colocalization, and patterns 1, 2 and 3 correspond to one, two or three MukB foci colocalizing with one ori1 focus. When cells contains two ori1 foci, each ori1 focus can colocalize either with one or two MukB, generating pattern 1/1, 1/2 or 2/2. Grey bars and pattern 0 correspond to the absence of colocalization of one ori1 focus with MukB, including cells containing one ori1 focus or two ori1 foci (patterns 0/0, 0/1 and 0/2 are included in pattern 0). Pattern ‘a’ corresponds to cells with an extra MukB focus distant from the ori1/MukB colocalizing foci.C. Time-lapse tracking simultaneously MukB and ori1 (without or with ori1 duplication, left and right respectively; OS18 cells). The number of MukB foci per cluster (bottom panel) and the time intervals between images (below panels) are indicated.D. Representation of the cell-cycle dynamics of ori1 and MukB deduced from snapshot and time-lapse analysis.

Mentions: Knowing that MukBEF forms foci within cells, when assayed by immunocytochemistry (den Blaauwen et al., 2001), or with a fluorescent fusion protein (Ohsumi et al., 2001), we analysed their position and dynamics with respect to chromosomal loci. For simultaneous visualization, the chromosomal mukB gene was replaced by the functional mukB-gfp4 fusion gene, and FROS in cells grown at 30°C was used to track ori1 (3908 kb), oriL (3713 kb), oriR (4139 kb), R2 (366 kb) or ter2 (1801 kb) loci (Fig. 3A).


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)

Colocalization of MukB with the origin region.A. Colocalization is indicated as a number (%) on black lines linking MukB and each given loci positioned on the circular map of the E. coli chromosome (ori1, R2, ter2 loci from OS18 and OS29, OS69, OS19 cells respectively). Colocalization (%) of ter2 with ori1 in IL05 cells is indicated on the purple line.B. Snapshot analysis of MukB-GFP and ori1 positioning. Images of representative wild-type cells (OS18; see histogram for legend). Histogram representing the proportion of the different MukB patterns for each cell type (A–D, Fig. 2). Green bars correspond to MukB/ori1 colocalization, and patterns 1, 2 and 3 correspond to one, two or three MukB foci colocalizing with one ori1 focus. When cells contains two ori1 foci, each ori1 focus can colocalize either with one or two MukB, generating pattern 1/1, 1/2 or 2/2. Grey bars and pattern 0 correspond to the absence of colocalization of one ori1 focus with MukB, including cells containing one ori1 focus or two ori1 foci (patterns 0/0, 0/1 and 0/2 are included in pattern 0). Pattern ‘a’ corresponds to cells with an extra MukB focus distant from the ori1/MukB colocalizing foci.C. Time-lapse tracking simultaneously MukB and ori1 (without or with ori1 duplication, left and right respectively; OS18 cells). The number of MukB foci per cluster (bottom panel) and the time intervals between images (below panels) are indicated.D. Representation of the cell-cycle dynamics of ori1 and MukB deduced from snapshot and time-lapse analysis.
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Related In: Results  -  Collection

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fig03: Colocalization of MukB with the origin region.A. Colocalization is indicated as a number (%) on black lines linking MukB and each given loci positioned on the circular map of the E. coli chromosome (ori1, R2, ter2 loci from OS18 and OS29, OS69, OS19 cells respectively). Colocalization (%) of ter2 with ori1 in IL05 cells is indicated on the purple line.B. Snapshot analysis of MukB-GFP and ori1 positioning. Images of representative wild-type cells (OS18; see histogram for legend). Histogram representing the proportion of the different MukB patterns for each cell type (A–D, Fig. 2). Green bars correspond to MukB/ori1 colocalization, and patterns 1, 2 and 3 correspond to one, two or three MukB foci colocalizing with one ori1 focus. When cells contains two ori1 foci, each ori1 focus can colocalize either with one or two MukB, generating pattern 1/1, 1/2 or 2/2. Grey bars and pattern 0 correspond to the absence of colocalization of one ori1 focus with MukB, including cells containing one ori1 focus or two ori1 foci (patterns 0/0, 0/1 and 0/2 are included in pattern 0). Pattern ‘a’ corresponds to cells with an extra MukB focus distant from the ori1/MukB colocalizing foci.C. Time-lapse tracking simultaneously MukB and ori1 (without or with ori1 duplication, left and right respectively; OS18 cells). The number of MukB foci per cluster (bottom panel) and the time intervals between images (below panels) are indicated.D. Representation of the cell-cycle dynamics of ori1 and MukB deduced from snapshot and time-lapse analysis.
Mentions: Knowing that MukBEF forms foci within cells, when assayed by immunocytochemistry (den Blaauwen et al., 2001), or with a fluorescent fusion protein (Ohsumi et al., 2001), we analysed their position and dynamics with respect to chromosomal loci. For simultaneous visualization, the chromosomal mukB gene was replaced by the functional mukB-gfp4 fusion gene, and FROS in cells grown at 30°C was used to track ori1 (3908 kb), oriL (3713 kb), oriR (4139 kb), R2 (366 kb) or ter2 (1801 kb) loci (Fig. 3A).

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