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A defined terminal region of the E. coli chromosome shows late segregation and high FtsK activity.

Deghorain M, Pagès C, Meile JC, Stouf M, Capiaux H, Mercier R, Lesterlin C, Hallet B, Cornet F - PLoS ONE (2011)

Bottom Line: 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.Our results show that a restricted terminal region of the chromosome is specifically dedicated to the last steps of chromosome segregation and to their coupling with cell division by FtsK.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Microbiologie et de Génétique Moléculaire, CNRS, Toulouse, France.

ABSTRACT

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 KOPS-recognition 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 steps of chromosome segregation and to their coupling with cell division by FtsK.

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Related in: MedlinePlus

Measuring FtsK Activity.(A) The dif-lacI dif cassette is shown with the dif sites as black and white squares. It was inserted at chosen loci of a Δ(xerC)Δ(dif)Δ(lacI) strain. Transformation with the XerC-producing plasmid pFC241 allows recombination, giving rise to blue colonies on indicator medium (right). (B) Map of the chromosome with the insertion loci used for insertion of the dif-lacI dif or parS-Kn cassettes. The replication origin (closed circle) and replication terminators (black flags) are indicated. Coordinates are in Kb.
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pone-0022164-g001: Measuring FtsK Activity.(A) The dif-lacI dif cassette is shown with the dif sites as black and white squares. It was inserted at chosen loci of a Δ(xerC)Δ(dif)Δ(lacI) strain. Transformation with the XerC-producing plasmid pFC241 allows recombination, giving rise to blue colonies on indicator medium (right). (B) Map of the chromosome with the insertion loci used for insertion of the dif-lacI dif or parS-Kn cassettes. The replication origin (closed circle) and replication terminators (black flags) are indicated. Coordinates are in Kb.

Mentions: Recombination between dif sites requires a direct interaction between FtsKγ and XerD [32], [33]. We reasoned that XerCD/dif recombination could be used to measure the relative frequencies at which FtsK interacts with different chromosome loci. To this end, we constructed a dif-lacI-dif cassette and inserted it in the chromosome of a strain deleted for dif, lacI and xerC (Figure 1A; Materials and methods). Recombination between dif sites was induced by transformation with the XerC-producing plasmid pFC241, which provoked the loss of lacI and derepression of the lacZ gene thus allowing the measurement of recombination frequencies from the formation of blue colonies on indicator plates (Figure 1A).


A defined terminal region of the E. coli chromosome shows late segregation and high FtsK activity.

Deghorain M, Pagès C, Meile JC, Stouf M, Capiaux H, Mercier R, Lesterlin C, Hallet B, Cornet F - PLoS ONE (2011)

Measuring FtsK Activity.(A) The dif-lacI dif cassette is shown with the dif sites as black and white squares. It was inserted at chosen loci of a Δ(xerC)Δ(dif)Δ(lacI) strain. Transformation with the XerC-producing plasmid pFC241 allows recombination, giving rise to blue colonies on indicator medium (right). (B) Map of the chromosome with the insertion loci used for insertion of the dif-lacI dif or parS-Kn cassettes. The replication origin (closed circle) and replication terminators (black flags) are indicated. Coordinates are in Kb.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140498&req=5

pone-0022164-g001: Measuring FtsK Activity.(A) The dif-lacI dif cassette is shown with the dif sites as black and white squares. It was inserted at chosen loci of a Δ(xerC)Δ(dif)Δ(lacI) strain. Transformation with the XerC-producing plasmid pFC241 allows recombination, giving rise to blue colonies on indicator medium (right). (B) Map of the chromosome with the insertion loci used for insertion of the dif-lacI dif or parS-Kn cassettes. The replication origin (closed circle) and replication terminators (black flags) are indicated. Coordinates are in Kb.
Mentions: Recombination between dif sites requires a direct interaction between FtsKγ and XerD [32], [33]. We reasoned that XerCD/dif recombination could be used to measure the relative frequencies at which FtsK interacts with different chromosome loci. To this end, we constructed a dif-lacI-dif cassette and inserted it in the chromosome of a strain deleted for dif, lacI and xerC (Figure 1A; Materials and methods). Recombination between dif sites was induced by transformation with the XerC-producing plasmid pFC241, which provoked the loss of lacI and derepression of the lacZ gene thus allowing the measurement of recombination frequencies from the formation of blue colonies on indicator plates (Figure 1A).

Bottom Line: 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.Our results show that a restricted terminal region of the chromosome is specifically dedicated to the last steps of chromosome segregation and to their coupling with cell division by FtsK.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Microbiologie et de Génétique Moléculaire, CNRS, Toulouse, France.

ABSTRACT

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 KOPS-recognition 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 steps of chromosome segregation and to their coupling with cell division by FtsK.

Show MeSH
Related in: MedlinePlus