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FtsK-dependent dimer resolution on multiple chromosomes in the pathogen Vibrio cholerae.

Val ME, Kennedy SP, El Karoui M, Bonné L, Chevalier F, Barre FX - PLoS Genet. (2008)

Bottom Line: However, the process is FtsK-independent.In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division.Chromosome II dimer resolution thus stands as a bona fide chromosomal process.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Centre de Génétique Moléculaire, UPR 2167, Gif-sur-Yvette, France.

ABSTRACT
Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.

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V. cholerae FtsK Orienting Polar Sequences.A. Growth competition of E. coli cells encoding FtsK hybrids. N: cells carrying a complete deletion of the C-terminal domain and linker region of FtsKEc; NLCEc: cells carrying full length FtsKEc; NLCVc and NLCHi: cells in which the C-terminal domain FtsKEc was replaced by the one of FtsKVc and FtsKHi, respectively. f: frequency of cells that the parental N strain fails to produce at each generation compared to the FtsK hybrids. B. 5′-GGGCAGGG-3′ inhibits recombination activation by FtsKVc. Plasmid recombination at E. coli dif by XerCDEc was induced with 0.5% arabinose. Ec[NRE]: FtsK50CEc[NRE]; Vc[NRE]: FtsKVc[NRE]. KOPS-0: substrate without GGGCAGGG sequences; KOPS-2: substrate with triple overlapping GGGCAGGG sequences in the non-permissive orientation on both sides of the two dif sites. C. Scheme of the two V. cholerae chromosomes showing the distributions of the GGGCAGGG and GGGNAGGG motifs. Upper bars: motifs found in the leading strand; Lower bars: motifs found in the lagging strand. Number, frequency, skew and skew significance (p-skew) are indicated for each motif. Recently acquired genomic regions are indicated (superintegron, CTX and TLC prophages and the Vibrio Pathogenicity Island VPI).
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pgen-1000201-g006: V. cholerae FtsK Orienting Polar Sequences.A. Growth competition of E. coli cells encoding FtsK hybrids. N: cells carrying a complete deletion of the C-terminal domain and linker region of FtsKEc; NLCEc: cells carrying full length FtsKEc; NLCVc and NLCHi: cells in which the C-terminal domain FtsKEc was replaced by the one of FtsKVc and FtsKHi, respectively. f: frequency of cells that the parental N strain fails to produce at each generation compared to the FtsK hybrids. B. 5′-GGGCAGGG-3′ inhibits recombination activation by FtsKVc. Plasmid recombination at E. coli dif by XerCDEc was induced with 0.5% arabinose. Ec[NRE]: FtsK50CEc[NRE]; Vc[NRE]: FtsKVc[NRE]. KOPS-0: substrate without GGGCAGGG sequences; KOPS-2: substrate with triple overlapping GGGCAGGG sequences in the non-permissive orientation on both sides of the two dif sites. C. Scheme of the two V. cholerae chromosomes showing the distributions of the GGGCAGGG and GGGNAGGG motifs. Upper bars: motifs found in the leading strand; Lower bars: motifs found in the lagging strand. Number, frequency, skew and skew significance (p-skew) are indicated for each motif. Recently acquired genomic regions are indicated (superintegron, CTX and TLC prophages and the Vibrio Pathogenicity Island VPI).

Mentions: We next investigated if FtsKVc could serve to bring together the CDR sites carried by dimers of chromosome I or by dimers of chromosome II. Several key residues implicated in KOPS recognition have been identified in the γ domain of FtsKEc (Figure 5A; N1296; R1300; E1303; [35]). The conservation of these residues in FtsKVc suggested that it could recognize the same motifs (Figure 5A; N926; R930; E933). If this was indeed the case, replacing the C-terminal domain of FtsKEc with the one of FtsKVc should completely rescue CDR in E. coli cells since FtsKVc fully activates recombination by XerCDEc at difEc (Figure 5B, XerCDEc, difEc, FtsKVc). Indeed, the fitness of such cells equaled the fitness of wild-type E. coli cells in growth competition experiments (Figure 6A, NLCVc and NLCEc), in contrast to cells only expressing the N-terminal domain of FtsKEc or a fusion with the C-terminal domain of H. influenzae FtsK (Figure 6A, N and NLCHi).


FtsK-dependent dimer resolution on multiple chromosomes in the pathogen Vibrio cholerae.

Val ME, Kennedy SP, El Karoui M, Bonné L, Chevalier F, Barre FX - PLoS Genet. (2008)

V. cholerae FtsK Orienting Polar Sequences.A. Growth competition of E. coli cells encoding FtsK hybrids. N: cells carrying a complete deletion of the C-terminal domain and linker region of FtsKEc; NLCEc: cells carrying full length FtsKEc; NLCVc and NLCHi: cells in which the C-terminal domain FtsKEc was replaced by the one of FtsKVc and FtsKHi, respectively. f: frequency of cells that the parental N strain fails to produce at each generation compared to the FtsK hybrids. B. 5′-GGGCAGGG-3′ inhibits recombination activation by FtsKVc. Plasmid recombination at E. coli dif by XerCDEc was induced with 0.5% arabinose. Ec[NRE]: FtsK50CEc[NRE]; Vc[NRE]: FtsKVc[NRE]. KOPS-0: substrate without GGGCAGGG sequences; KOPS-2: substrate with triple overlapping GGGCAGGG sequences in the non-permissive orientation on both sides of the two dif sites. C. Scheme of the two V. cholerae chromosomes showing the distributions of the GGGCAGGG and GGGNAGGG motifs. Upper bars: motifs found in the leading strand; Lower bars: motifs found in the lagging strand. Number, frequency, skew and skew significance (p-skew) are indicated for each motif. Recently acquired genomic regions are indicated (superintegron, CTX and TLC prophages and the Vibrio Pathogenicity Island VPI).
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pgen-1000201-g006: V. cholerae FtsK Orienting Polar Sequences.A. Growth competition of E. coli cells encoding FtsK hybrids. N: cells carrying a complete deletion of the C-terminal domain and linker region of FtsKEc; NLCEc: cells carrying full length FtsKEc; NLCVc and NLCHi: cells in which the C-terminal domain FtsKEc was replaced by the one of FtsKVc and FtsKHi, respectively. f: frequency of cells that the parental N strain fails to produce at each generation compared to the FtsK hybrids. B. 5′-GGGCAGGG-3′ inhibits recombination activation by FtsKVc. Plasmid recombination at E. coli dif by XerCDEc was induced with 0.5% arabinose. Ec[NRE]: FtsK50CEc[NRE]; Vc[NRE]: FtsKVc[NRE]. KOPS-0: substrate without GGGCAGGG sequences; KOPS-2: substrate with triple overlapping GGGCAGGG sequences in the non-permissive orientation on both sides of the two dif sites. C. Scheme of the two V. cholerae chromosomes showing the distributions of the GGGCAGGG and GGGNAGGG motifs. Upper bars: motifs found in the leading strand; Lower bars: motifs found in the lagging strand. Number, frequency, skew and skew significance (p-skew) are indicated for each motif. Recently acquired genomic regions are indicated (superintegron, CTX and TLC prophages and the Vibrio Pathogenicity Island VPI).
Mentions: We next investigated if FtsKVc could serve to bring together the CDR sites carried by dimers of chromosome I or by dimers of chromosome II. Several key residues implicated in KOPS recognition have been identified in the γ domain of FtsKEc (Figure 5A; N1296; R1300; E1303; [35]). The conservation of these residues in FtsKVc suggested that it could recognize the same motifs (Figure 5A; N926; R930; E933). If this was indeed the case, replacing the C-terminal domain of FtsKEc with the one of FtsKVc should completely rescue CDR in E. coli cells since FtsKVc fully activates recombination by XerCDEc at difEc (Figure 5B, XerCDEc, difEc, FtsKVc). Indeed, the fitness of such cells equaled the fitness of wild-type E. coli cells in growth competition experiments (Figure 6A, NLCVc and NLCEc), in contrast to cells only expressing the N-terminal domain of FtsKEc or a fusion with the C-terminal domain of H. influenzae FtsK (Figure 6A, N and NLCHi).

Bottom Line: However, the process is FtsK-independent.In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division.Chromosome II dimer resolution thus stands as a bona fide chromosomal process.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Centre de Génétique Moléculaire, UPR 2167, Gif-sur-Yvette, France.

ABSTRACT
Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.

Show MeSH
Related in: MedlinePlus