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

Meile JC, Mercier R, Stouf M, Pages C, Bouet JY, Cornet F - BMC Microbiol. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université de Toulouse, Université Paul Sabatier, Laboratoire de Microbiologie et Génétique Moléculaires, F-31000 Toulouse, France.

ABSTRACT

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.

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Positioning of chromosome loci in living cells. (A) A scheme of the E. coli chromosome with relevant features indicated. The replication origin (ori) and the two inner replication terminators (TerA and TerC) defining the zone of replication termination are shown. The grey arrows indicate the sense of replication. The loci used for insertion of the parS site are shown in red. Coordinates are in kb. (B) Micrographs of cells harbouring the YFP-ParB foci at the ori locus. From top left to bottom right: phase contrast; membrane staining (FM 4-64); DNA staining (DAPI); YFP-ParB foci; overlay phase/DNA/YFP-ParB; overlay membrane/DNA/ParB. (C) Linescan analysis of fluorescence signals along cell length (L, top panel) and cell diameter (W, middle panel). Linescans of fluorescence intensities (Y-axis, in Gray Level units) for the cell membrane (red); DNA (blue) and YFP-ParB (green) are shown along the two cell axes (X-axis in μm). Red arrowheads indicate the cell boundaries and green arrowheads show the positions of YFP-ParB foci. The bottom panel shows micrographs of the cell scanned in the panels above with the two linescans used (from left to right: phase contrast; YFP-ParB; DNA; membrane; overlay YFP-ParB/DNA/membrane). Scale bars are 2 μm.
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Figure 1: Positioning of chromosome loci in living cells. (A) A scheme of the E. coli chromosome with relevant features indicated. The replication origin (ori) and the two inner replication terminators (TerA and TerC) defining the zone of replication termination are shown. The grey arrows indicate the sense of replication. The loci used for insertion of the parS site are shown in red. Coordinates are in kb. (B) Micrographs of cells harbouring the YFP-ParB foci at the ori locus. From top left to bottom right: phase contrast; membrane staining (FM 4-64); DNA staining (DAPI); YFP-ParB foci; overlay phase/DNA/YFP-ParB; overlay membrane/DNA/ParB. (C) Linescan analysis of fluorescence signals along cell length (L, top panel) and cell diameter (W, middle panel). Linescans of fluorescence intensities (Y-axis, in Gray Level units) for the cell membrane (red); DNA (blue) and YFP-ParB (green) are shown along the two cell axes (X-axis in μm). Red arrowheads indicate the cell boundaries and green arrowheads show the positions of YFP-ParB foci. The bottom panel shows micrographs of the cell scanned in the panels above with the two linescans used (from left to right: phase contrast; YFP-ParB; DNA; membrane; overlay YFP-ParB/DNA/membrane). Scale bars are 2 μm.

Mentions: To label chromosomal loci such that their position could be determined, we used insertions of the parS site from the bacteriophage P1 and production of the YFP-Δ30ParB fusion protein (Methods) [19,20]). The parS site was first inserted at four different loci located at 3909 kb (ori), 316 kb (right, inside the right MD), 738 kb (NS-right) and 1568 kb (ter) on the E. coli chromosome map (Figure 1A). The resulting strains showed equivalent growth rates and normal cell shape whether or not they produced the YFP-Δ30ParB protein (doubling times in synthetic medium of 45 min. at 42°C and 70 min at 30°C).


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

Meile JC, Mercier R, Stouf M, Pages C, Bouet JY, Cornet F - BMC Microbiol. (2011)

Positioning of chromosome loci in living cells. (A) A scheme of the E. coli chromosome with relevant features indicated. The replication origin (ori) and the two inner replication terminators (TerA and TerC) defining the zone of replication termination are shown. The grey arrows indicate the sense of replication. The loci used for insertion of the parS site are shown in red. Coordinates are in kb. (B) Micrographs of cells harbouring the YFP-ParB foci at the ori locus. From top left to bottom right: phase contrast; membrane staining (FM 4-64); DNA staining (DAPI); YFP-ParB foci; overlay phase/DNA/YFP-ParB; overlay membrane/DNA/ParB. (C) Linescan analysis of fluorescence signals along cell length (L, top panel) and cell diameter (W, middle panel). Linescans of fluorescence intensities (Y-axis, in Gray Level units) for the cell membrane (red); DNA (blue) and YFP-ParB (green) are shown along the two cell axes (X-axis in μm). Red arrowheads indicate the cell boundaries and green arrowheads show the positions of YFP-ParB foci. The bottom panel shows micrographs of the cell scanned in the panels above with the two linescans used (from left to right: phase contrast; YFP-ParB; DNA; membrane; overlay YFP-ParB/DNA/membrane). Scale bars are 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Positioning of chromosome loci in living cells. (A) A scheme of the E. coli chromosome with relevant features indicated. The replication origin (ori) and the two inner replication terminators (TerA and TerC) defining the zone of replication termination are shown. The grey arrows indicate the sense of replication. The loci used for insertion of the parS site are shown in red. Coordinates are in kb. (B) Micrographs of cells harbouring the YFP-ParB foci at the ori locus. From top left to bottom right: phase contrast; membrane staining (FM 4-64); DNA staining (DAPI); YFP-ParB foci; overlay phase/DNA/YFP-ParB; overlay membrane/DNA/ParB. (C) Linescan analysis of fluorescence signals along cell length (L, top panel) and cell diameter (W, middle panel). Linescans of fluorescence intensities (Y-axis, in Gray Level units) for the cell membrane (red); DNA (blue) and YFP-ParB (green) are shown along the two cell axes (X-axis in μm). Red arrowheads indicate the cell boundaries and green arrowheads show the positions of YFP-ParB foci. The bottom panel shows micrographs of the cell scanned in the panels above with the two linescans used (from left to right: phase contrast; YFP-ParB; DNA; membrane; overlay YFP-ParB/DNA/membrane). Scale bars are 2 μm.
Mentions: To label chromosomal loci such that their position could be determined, we used insertions of the parS site from the bacteriophage P1 and production of the YFP-Δ30ParB fusion protein (Methods) [19,20]). The parS site was first inserted at four different loci located at 3909 kb (ori), 316 kb (right, inside the right MD), 738 kb (NS-right) and 1568 kb (ter) on the E. coli chromosome map (Figure 1A). The resulting strains showed equivalent growth rates and normal cell shape whether or not they produced the YFP-Δ30ParB protein (doubling times in synthetic medium of 45 min. at 42°C and 70 min at 30°C).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université de Toulouse, Université Paul Sabatier, Laboratoire de Microbiologie et Génétique Moléculaires, F-31000 Toulouse, France.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus