Dimer Resolution Site Research Articles

A Fork Trap in the Chromosomal Termination Area Is Highly Conserved across All Escherichia coli Phylogenetic Groups.

Termination of DNA replication, the final stage of genome duplication, is surprisingly complex, and failures to bring DNA synthesis to an accurate conclusion can impact genome stability and cell viability. In Escherichia coli, termination takes place in a specialised termination area opposite the origin. A ‘replication fork trap’ is formed by unidirectional fork barriers via the binding of Tus protein to genomic ter sites. Such a fork trap system is found in some bacterial species, but it appears not to be a general feature of bacterial chromosomes. The biochemical properties of fork trap systems have been extensively characterised, but little is known about their precise physiological roles. In this study, we compare locations and distributions of ter terminator sites in E. coli genomes across all phylogenetic groups, including Shigella. Our analysis shows that all ter sites are highly conserved in E. coli, with slightly more variability in the Shigella genomes. Our sequence analysis of ter sites and Tus proteins shows that the fork trap is likely to be active in all strains investigated. In addition, our analysis shows that the dif chromosome dimer resolution site is consistently located between the innermost ter sites, even if rearrangements have changed the location of the innermost termination area. Our data further support the idea that the replication fork trap has an important physiological role that provides an evolutionary advantage.

Characterization of the Chromosome Dimer Resolution Site in Caulobacter crescentus.

Chromosome dimers occur in bacterial cells as a result of the recombinational repair of DNA. In most bacteria, chromosome dimers are resolved by XerCD site-specific recombination at the dif (deletion-induced filamentation) site located in the terminus region of the chromosome. Caulobacter crescentus, a Gram-negative oligotrophic bacterium, also possesses Xer recombinases, called CcXerC and CcXerD, which have been shown to interact with the Escherichia colidif site in vitro Previous studies on Caulobacter have suggested the presence of a dif site (referred to in this paper as dif1CC ), but no in vitro data have shown any association with this site and the CcXer proteins. Using recursive hidden Markov modeling, another group has proposed a second dif site, which we call dif2CC , which shows more similarity to the dif consensus sequence. Here, by using a combination of in vitro experiments, we compare the affinities and the cleavage abilities of CcXerCD recombinases for both dif sites. Our results show that dif2CC displays a higher affinity for CcXerC and CcXerD and is bound cooperatively by these proteins, which is not the case for the original dif1CC site. Furthermore, dif2CC nicked substrates are more efficiently cleaved by CcXerCD, and deletion of the site results in about 5 to 10% of cells showing an altered cellular morphology.IMPORTANCE Bacteria utilize site-specific recombination for a variety of purposes, including the control of gene expression, acquisition of genetic elements, and the resolution of dimeric chromosomes. Failure to resolve dimeric chromosomes can lead to cell division defects in a percentage of the cell population. The work presented here shows the existence of a chromosomal resolution system in C. crescentus Defects in this resolution system result in the formation of chains of cells. Further understanding of how these cells remain linked together will help in the understanding of how chromosome segregation and cell division are linked in C. crescentus.

FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex

In bacteria, the FtsK/Xer/dif (chromosome dimer resolution site) system is essential for faithful vertical genetic transmission, ensuring the resolution of chromosome dimers during their segregation to daughter cells. This system is also targeted by mobile genetic elements that integrate into chromosomal dif sites. A central question is thus how Xer/dif recombination is tuned to both act in chromosome segregation and stably maintain mobile elements. To explore this question, we focused on pathogenic Neisseria species harboring a genomic island in their dif sites. We show that the FtsK DNA translocase acts differentially at the recombination sites flanking the genomic island. It stops at one Xer/dif complex, activating recombination, but it does not stop on the other site, thus dismantling it. FtsK translocation thus permits cis discrimination between an endogenous and an imported Xer/dif recombination complex.

Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome.

Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.

Effect of LexA on Chromosomal Integration of CTXϕ in Vibrio cholerae.

The genesis of toxigenic Vibrio cholerae involves acquisition of CTXϕ, a single-stranded DNA (ssDNA) filamentous phage that encodes cholera toxin (CT). The phage exploits host-encoded tyrosine recombinases (XerC and XerD) for chromosomal integration and lysogenic conversion. The replicative genome of CTXϕ produces ssDNA by rolling-circle replication, which may be used either for virion production or for integration into host chromosome. Fine-tuning of different ssDNA binding protein (Ssb) levels in the host cell is crucial for cellular functioning and important for CTXϕ integration. In this study, we mutated the master regulator gene of SOS induction, lexA, of V. cholerae because of its known role in controlling levels of Ssb proteins in other bacteria. CTXϕ integration decreased in cells with a ΔlexA mutation and increased in cells with an SOS-noninducing mutation, lexA (Ind(-)). We also observed that overexpression of host-encoded Ssb (VC0397) decreased integration of CTXϕ. We propose that LexA helps CTXϕ integration, possibly by fine-tuning levels of host- and phage-encoded Ssbs. Cholera toxin is the principal virulence factor responsible for the acute diarrheal disease cholera. CT is encoded in the genome of a lysogenic filamentous phage, CTXϕ. Vibrio cholerae has a bipartite genome and harbors single or multiple copies of CTXϕ prophage in one or both chromosomes. Two host-encoded tyrosine recombinases (XerC and XerD) recognize the folded ssDNA genome of CTXϕ and catalyze its integration at the dimer resolution site of either one or both chromosomes. Fine-tuning of ssDNA binding proteins in host cells is crucial for CTXϕ integration. We engineered the V. cholerae genome and created several reporter strains carrying ΔlexA or lexA (Ind(-)) alleles. Using the reporter strains, the importance of LexA control of Ssb expression in the integration efficiency of CTXϕ was demonstrated.

Mechanistic insights into filamentous phage integration in Vibrio cholerae.

Vibrio cholerae, the etiological agent of acute diarrhoeal disease cholera, harbors large numbers of lysogenic filamentous phages, contribute significantly to the host pathogenesis and provide fitness factors to the pathogen that help the bacterium to survive in natural environment. Most of the vibriophage genomes are not equipped with integrase and thus exploit two host-encoded tyrosine recombinases, XerC and XerD, for lysogenic conversion. Integration is site-specific and it occurs at dimer resolution site (dif) of either one or both chromosomes of V. cholerae. Each dif sequence contains two recombinase-binding sequences flanking a central region. The integration follows a sequential strand exchanges between dif and attP sites within a DNA-protein complex consisting of one pair of each recombinase and two DNA fragments. During entire process of recombination, both the DNA components and recombinases of the synaptic complex keep transiently interconnected. Within the context of synaptic complex, both of the actuated enzymes mediate cleavage of phosphodiester bonds. First cleavage generates a phosphotyrosyl-linked recombinase-DNA complex at the recombinase binding sequence and free 5′-hydroxyl end at the first base of the central region. Following the cleavage, the exposed bases with 5′-hydroxyl ends of the central region of dif and attP sites melt from their complementary strands and react with the recombinase-DNA phosphotyrosyl linkage of their recombining partner. Subsequent ligation between dif and attP strands requires complementary base pair interactions at the site of phosphodiester bond formation. Integration mechanism is mostly influenced by the compatibility of dif and attP sequences. dif sites are highly conserved across bacterial phyla. Different phage genomes have different attP sequences; therefore they rely on different mechanisms for integration. Here, I review our current understanding of integration mechanisms used by the vibriophages.

Differential management of the replication terminus regions of the two Vibrio cholerae chromosomes during cell division.

The replication terminus region (Ter) of the unique chromosome of most bacteria locates at mid-cell at the time of cell division. In several species, this localization participates in the necessary coordination between chromosome segregation and cell division, notably for the selection of the division site, the licensing of the division machinery assembly and the correct alignment of chromosome dimer resolution sites. The genome of Vibrio cholerae, the agent of the deadly human disease cholera, is divided into two chromosomes, chrI and chrII. Previous fluorescent microscopy observations suggested that although the Ter regions of chrI and chrII replicate at the same time, chrII sister termini separated before cell division whereas chrI sister termini were maintained together at mid-cell, which raised questions on the management of the two chromosomes during cell division. Here, we simultaneously visualized the location of the dimer resolution locus of each of the two chromosomes. Our results confirm the late and early separation of chrI and chrII Ter sisters, respectively. They further suggest that the MatP/matS macrodomain organization system specifically delays chrI Ter sister separation. However, TerI loci remain in the vicinity of the cell centre in the absence of MatP and a genetic assay specifically designed to monitor the relative frequency of sister chromatid contacts during constriction suggest that they keep colliding together until the very end of cell division. In contrast, we found that even though it is not able to impede the separation of chrII Ter sisters before septation, the MatP/matS macrodomain organization system restricts their movement within the cell and permits their frequent interaction during septum constriction.

A novel, broad-range, CTXΦ-derived stable integrative expression vector for functional studies.

CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ.

FtsK actively segregates sister chromosomes in Escherichia coli

Bacteria use the replication origin-to-terminus polarity of their circular chromosomes to control DNA transactions during the cell cycle. Segregation starts by active migration of the region of origin followed by progressive movement of the rest of the chromosomes. The last steps of segregation have been studied extensively in the case of dimeric sister chromosomes and when chromosome organization is impaired by mutations. In these special cases, the divisome-associated DNA translocase FtsK is required. FtsK pumps chromosomes toward the dif chromosome dimer resolution site using polarity of the FtsK-orienting polar sequence (KOPS) DNA motifs. Assays based on monitoring dif recombination have suggested that FtsK acts only in these special cases and does not act on monomeric chromosomes. Using a two-color system to visualize pairs of chromosome loci in living cells, we show that the spatial resolution of sister loci is accurately ordered from the point of origin to the dif site. Furthermore, ordered segregation in a region ∼200 kb long surrounding dif depended on the oriented translocation activity of FtsK but not on the formation of dimers or their resolution. FtsK-mediated segregation required the MatP protein, which delays segregation of the dif-surrounding region until cell division. We conclude that FtsK segregates the terminus region of sister chromosomes whether they are monomeric or dimeric and does so in an accurate and ordered manner. Our data are consistent with a model in which FtsK acts to release the MatP-mediated cohesion and/or interaction with the division apparatus of the terminus region in a KOPS-oriented manner.

Validation of Bacterial Replication Termination Models Using Simulation of Genomic Mutations

In bacterial circular chromosomes and most plasmids, the replication is known to be terminated when either of the following occurs: the forks progressing in opposite directions meet at the distal end of the chromosome or the replication forks become trapped by Tus proteins bound to Ter sites. Most bacterial genomes have various polarities in their genomic structures. The most notable feature is polar genomic compositional asymmetry of the bases G and C in the leading and lagging strands, called GC skew. This asymmetry is caused by replication-associated mutation bias, and this “footprint" of the replication machinery suggests that, in contrast to the two known mechanisms, replication termination occurs near the chromosome dimer resolution site dif. To understand this difference between the known replication machinery and genomic compositional bias, we undertook a simulation study of genomic mutations, and we report here how different replication termination models contribute to the generation of replication-related genomic compositional asymmetry. Contrary to naive expectations, our results show that a single finite termination site at dif or at the GC skew shift point is not sufficient to reconstruct the genomic compositional bias as observed in published sequences. The results also show that the known replication mechanisms are sufficient to explain the position of the GC skew shift point.

VGJɸ integration and excision mechanisms contribute to the genetic diversity of Vibrio cholerae epidemic strains

Most strains of Vibrio cholerae are not pathogenic or cause only local outbreaks of gastroenteritis. Acquisition of the capacity to produce the cholera toxin results from a lysogenic conversion event due to a filamentous bacteriophage, CTX. Two V. cholerae tyrosine recombinases that normally serve to resolve chromosome dimers, XerC and XerD, promote CTX integration by directly recombining the ssDNA genome of the phage with the dimer resolution site of either or both V. cholerae chromosomes. This smart mechanism renders the process irreversible. Many other filamentous vibriophages seem to attach to chromosome dimer resolution sites and participate in the rapid and continuous evolution of toxigenic V. cholerae strains. We analyzed the molecular mechanism of integration of VGJ, a representative of the largest family of these phages. We found that XerC and XerD promote the integration of VGJ into a specific chromosome dimer resolution site, and that the dsDNA replicative form of the phage is recombined. We show that XerC and XerD can promote excision of the integrated prophage, and that this participates in the production of new extrachromosomal copies of the phage genome. We further show how hybrid molecules harboring the concatenated genomes of CTX and VGJ can be produced efficiently. Finally, we discuss how the integration and excision mechanisms of VGJ can explain the origin of recent epidemic V. cholerae strains.

Comprehensive prediction of chromosome dimer resolution sites in bacterial genomes

BackgroundDuring the replication process of bacteria with circular chromosomes, an odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. However, many bacteria harbor a conserved dimer resolution machinery consisting of one or two tyrosine recombinases, XerC and XerD, and their 28-bp target site, dif.ResultsTo study the evolution of the dif/XerCD system and its relationship with replication termination, we report the comprehensive prediction of dif sequences in silico using a phylogenetic prediction approach based on iterated hidden Markov modeling. Using this method, dif sites were identified in 641 organisms among 16 phyla, with a 97.64% identification rate for single-chromosome strains. The dif sequence positions were shown to be strongly correlated with the GC skew shift-point that is induced by replicational mutation/selection pressures, but the difference in the positions of the predicted dif sites and the GC skew shift-points did not correlate with the degree of replicational mutation/selection pressures.ConclusionsThe sequence of dif sites is widely conserved among many bacterial phyla, and they can be computationally identified using our method. The lack of correlation between dif position and the degree of GC skew suggests that replication termination does not occur strictly at dif sites.

Molecular keys of the tropism of integration of the cholera toxin phage

Cholera toxin is encoded in the genome of CTXvarphi, a lysogenic filamentous phage of Vibrio cholerae. CTXvarphi variants contribute to the genetic diversity of cholera epidemic strains. It has been shown that the El Tor variant of CTXvarphi hijacks XerC and XerD, two host-encoded tyrosine recombinases that normally function to resolve chromosome dimers, to integrate at dif1, the dimer resolution site of the larger of the two V. cholerae chromosomes. However, the exact mechanism of integration of CTXvarphi and the rules governing its integration remained puzzling, with phage variants integrated at either or both dimer resolution sites of the two V. cholerae chromosomes. We designed a genetic system to determine experimentally the tropism of integration of CTXvarphi and thus define rules of compatibility between phage variants and dimer resolution sites. We then showed in vitro how these rules are explained by the direct integration of the single-stranded phage genome into the double-stranded bacterial genome. Finally, we showed how the evolution of phage attachment and chromosome dimer resolution sites contributes to the generation of genetic diversity among cholera epidemic strains.

FtsK-Dependent Dimer Resolution on Multiple Chromosomes in the Pathogen Vibrio cholerae

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.

Analysis of the Terminus Region of the Caulobacter crescentus Chromosome and Identification of the dif Site

The terminus region of the Caulobacter crescentus chromosome and the dif chromosome dimer resolution site were characterized. The Caulobacter genome contains skewed sequences that abruptly switch strands at dif and may have roles in chromosome maintenance and segregation. Absence of dif or the XerCD recombinase results in a chromosome segregation defect. The Caulobacter terminus region is unusual, since it contains many essential or highly expressed genes.

Prophage lambda induces terminal recombination in Escherichia coli by inhibiting chromosome dimer resolution. An orientation-dependent cis-effect lending support to bipolarization of the terminus.

A prophage lambda inserted by homologous recombination near dif, the chromosome dimer resolution site of Escherichia coli, is excised at a frequency that depends on its orientation with respect to dif. In wild-type cells, terminal hyper- (TH) recombination is prophage specific and undetectable by a test involving deletion of chromosomal segments between repeats identical to those used for prophage insertion. TH recombination is, however, detected in both excision and deletion assays when Deltadif, xerC, or ftsK mutations inhibit dimer resolution: lack of specialized resolution apparently results in recombinogenic lesions near dif. We also observed that the presence near dif of the prophage, in the orientation causing TH recombination, inhibits dif resolution activity. By its recombinogenic effect, this inhibition explains the enhanced prophage excision in wild-type cells. The primary effect of the prophage is probably an alteration of the dimer resolution regional control, which requires that dif is flanked by suitably oriented (polarized) stretches of DNA. Our model postulates that the prophage inserted near dif in the deleterious orientation disturbs chromosome polarization on the side of the site where it is integrated, because lambda DNA, like the chromosome, is polarized by sequence elements. Candidate sequences are oligomers that display skewed distributions on each oriC-dif chromosome arm and on lambda DNA.

Recombinase binding specificity at the chromosome dimer resolution site dif of Escherichia coli

Xer site-specific recombination functions in Escherichia coli chromosome segregation and cell division apparently by resolving chromosome dimers, which arise through homologous recombination, to monomers. Xer recombination requires two closely related site-specific recombinases, XerC and XerD, which bind cooperatively to the recombination site dif and catalyse separate pairs of strand exchanges. The dif site is an imperfect palindrome whose left and right halves are bound by XerC and XerD, respectively. By using variant dif sites in which the symmetry between the XerC and XerD binding sites was increased incrementally, the determinants in the dif site that specifically direct binding of XerC and XerD to their cognate sites were elucidated. The primary specificity nucleotides in the XerC and XerD binding sites were identified and their relative contributions to specificity assessed. The biological affects of these mutations on site-specific recombination, chromosome segregation and cell division were examined. The specificity determinants are confined to the non-palindromic outer ends of the binding sites. Replacement of the wild-type dif site with mutated dif sites at the normal location in the replication terminus region of the chromosome revealed that the sequence of the dif site can be altered substantially while retaining apparently normal chromosome segregation activity.

Accessory proteins impose site selectivity during ColE1 dimer resolution.

The cer-Xer dimer resolution system of plasmid ColE1 is highly selective, acting only at sites on the same molecule and in direct repeat. Recombination requires the XerCD recombinase and accessory proteins ArgR and PepA. The Escherichia coli chromosome dimer resolution site dif and the type II hybrid site use the same recombinase but are independent of ArgR and PepA and show no site selectivity. This has led to the proposal that ArgR and PepA are responsible for the imposition of constraint. We describe here the characterization of a novel class of "conditionally constrained' multimer resolution sites whose properties support this hypothesis. In the presence of ArgR and PepA, plasmids containing conditionally constrained sites are monomeric, but in their absence, extensive multimerisation is seen. A mutant ArgR derivative (ArgR110), which is defective in cer-mediated dimer resolution, remains able to prevent plasmid multimerisation by a conditionally constrained site. This implies that the accessory factors block recombination in trans rather than ensuring rapid multimer resolution. When the distance between the ArgR and XerCD binding sites in a conditionally constrained site was altered by a non-integral number of helical turns, the site became unconstrained. Constraint was restored by the insertion of a full helical turn.

Involvement of ArgR and PepA in the pairing of ColE1 dimer resolution sites

Dimer formation and associated copy number depression is an important cause of multicopy plasmid instability. Natural multicopy plasmids employ site-specific recombination to convert dimers to monomers, thus maximizing the number of independently segregating molecules at cell division. Resolution of dimers of Escherichia coli plasmid ColE1 requires the plasmid cer site and at least four chromosome-encoded proteins: the XerC and XerD recombinases, and accessory factors ArgR and PepA. It has been suggested that ArgR has a role in the initial pairing of recombination sites and we describe here an attempt to detect this process in vivo. Our approach exploits a previous observation that a cer-like site known as the type II hybrid supports inter-molecular recombination and causes extensive multimerization of plasmids. We report that type-II-mediated multimerization can be repressed by a cer site in cis or in trans and propose that this is due to a physical interaction between the sites. If this hypothesis is correct, suppression of multimer formation provides an assay of site pairing. Our results demonstrate that the putative pairing interaction is independent of the topological relationship of the sites and that both PepA and ArgR are involved. Although most recombination-deficient mutant derivatives of ArgR are unable to pair recombination sites, we have found two (ArgR110 and ArgR115) which retain pairing activity. The validity of the pairing hypothesis is discussed in the light of alternative explanations for our data.

Derivatives of ColE1 cer show altered topological specificity in site-specific recombination.

ColE1 contains a 250-bp sequence (cer) which is required in cis for the conversion of plasmid dimers to monomers. Recombination between cer and parB (a dimer resolution site from plasmid CloDF13) occurs in vivo at low frequency. The properties of the resulting hybrid sites have been studied. The type I hybrid closely resembles wild-type cer. It supports intramolecular recombination and requires the products of the chromosomal xerA, xerB and xerC genes together with the 250-bp site. In contrast, the type II hybrid (although differing from type I by only 2 bp) functions independently of the topological relationship of the participating sites, supporting both inter- and intramolecular recombination. Furthermore, recombination between type II sites is independent of the products of the xerA and xerB genes and requires a site of less than 50 bp.