FtsK translocation on DNA stops at XerCD-dif

Escherichia coli FtsK is a powerful, fast, double-stranded DNA translocase, which can strip proteins from DNA. FtsK acts in the late stages of chromosome segregation by facilitating sister chromosome unlinking at the division septum. KOPS-guided DNA translocation directs FtsK towards dif, located within the replication terminus region, ter, where FtsK activates XerCD site-specific recombination. Here we show that FtsK translocation stops specifically at XerCD-dif, thereby preventing removal of XerCD from dif and allowing activation of chromosome unlinking by recombination. Stoppage of translocation at XerCD-dif is accompanied by a reduction in FtsK ATPase and is not associated with FtsK dissociation from DNA. Specific stoppage at recombinase-DNA complexes does not require the FtsKγ regulatory subdomain, which interacts with XerD, and is not dependent on either recombinase-mediated DNA cleavage activity, or the formation of synaptic complexes.     

A dual role for the FtsK protein in Escherichia coli chromosome segregation

FtsK is a multifunctional protein that acts in Escherichia coli cell division and chromosome segregation. Its C-terminal domain is required for XerCD-mediated recombination between dif sites that resolve chromosome dimers formed by recombination between sister chromosomes. We report the construction and analysis of a set of strains carrying different Xer recombination sites in place of dif, some of which recombine in an FtsK-independent manner. The results show that FtsK-independent Xer recombination does not support chromosome dimer resolution. Furthermore, resolution of dimers by the Cre/loxP system also requires FtsK. These findings reveal a second role for FtsK during chromosome dimer resolution in addition to XerCD activation. We propose that FtsK acts to position the dif regions, thus allowing a productive synapse between dif sites.     

Are two better than one? Analysis of an FtsK/Xer recombination system that uses a single recombinase

Bacteria harbouring circular chromosomes have a Xer site-specific recombination system that resolves chromosome dimers at division. In Escherichia coli, the activity of the XerCD/dif system is controlled and coupled with cell division by the FtsK DNA translocase. Most Xer systems, as XerCD/dif, include two different recombinases. However, some, as the Lactococcus lactis XerS/dif_SL system, include only one recombinase. We investigated the functional effects of this difference by studying the XerS/dif_SL system. XerS bound and recombined dif_SL sites in vitro, both activities displaying asymmetric characteristics. Resolution of chromosome dimers by XerS/dif_SL required translocation by division septum-borne FtsK. The translocase domain of L. lactis FtsK supported recombination by XerCD/dif, just as E. coli FtsK supports recombination by XerS/dif_SL. Thus, the FtsK-dependent coupling of chromosome segregation with cell division extends to non-rod-shaped bacteria and outside the phylum Proteobacteria. Both the XerCD/dif and XerS/dif_SL recombination systems require the control activities of the FtsKγ subdomain. However, FtsKγ activates recombination through different mechanisms in these two Xer systems. We show that FtsKγ alone activates XerCD/dif recombination. In contrast, both FtsKγ and the translocation motor are required to activate XerS/dif_SL recombination. These findings have implications for the mechanisms by which FtsK activates recombination.     

Asymmetric DNA requirements in Xer recombination activation by FtsK

In bacteria with circular chromosomes, homologous recombination events can lead to the formation of chromosome dimers. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover by two tyrosine recombinases, XerC and XerD, at a specific site on the chromosome, dif. Recombination depends on a direct contact between XerD and a cell division protein, FtsK, which functions as a hexameric double stranded DNA translocase. Here, we have investigated how the structure and composition of DNA interferes with Xer recombination activation by FtsK. XerC and XerD each cleave a specific strand on dif, the top and bottom strand, respectively. We found that the integrity and nature of eight bottom-strand nucleotides and three top-strand nucleotides immediately adjacent to the XerD-binding site of dif are crucial for recombination. These nucleotides are probably not implicated in FtsK translocation since FtsK could translocate on single stranded DNA in both the 5′–3′ and 3′–5′ orientation along a few nucleotides. We propose that they are required to stabilize FtsK in the vicinity of dif for recombination to occur because the FtsK–XerD interaction is too transient or too weak in itself to allow for XerD catalysis.     

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

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.     

The unconventional Xer recombination machinery of Streptococci/Lactococci

Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (dif_SL) associated with a dedicated tyrosine recombinase (XerS). In contrast to the E. coli Xer system, only a single recombinase is required to recombine dif_SL, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when dif_SL sites are located on chromosome dimers. Moreover, the XerS/dif_SL recombination requires the streptococcal protein FtsK_SL, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs.     

The Xer activation factor of TLCΦ expands the possibilities for Xer recombination

The chromosome dimer resolution machinery of bacteria is generally composed of two tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific site, dif. The reaction depends on a cell division protein, FtsK, which activates XerD by protein-protein interactions. The toxin-linked cryptic satellite phage (TLCΦ) of Vibrio cholerae, which participates in the emergence of cholera epidemic strains, carries a dif-like attachment site (attP). TLCΦ exploits the Xer machinery to integrate into the dif site of its host chromosomes. The TLCΦ integration reaction escapes the control of FtsK because TLCΦ encodes for its own XerD-activation factor, XafT. Additionally, TLCΦ attP is a poor substrate for XerD binding, in apparent contradiction with the high integration efficiency of the phage. Here, we present a sequencing-based methodology to analyse the integration and excision efficiency of thousands of synthetic mini-TLCΦ plasmids with differing attP sites in vivo. This methodology is applicable to the fine-grained analyses of DNA transactions on a wider scale. In addition, we compared the efficiency with which XafT and the XerD-activation domain of FtsK drive recombination reactions in vitro. Our results suggest that XafT not only activates XerD-catalysis but also helps form and/or stabilize synaptic complexes between imperfect Xer recombination sites.     

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.     

Asymmetric activation of Xer site-specific recombination by FtsK

Chromosome dimers, which frequently form in Escherichia coli, are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific site on the chromosome, dif, together with the cell division protein FtsK. The C-terminal domain of FtsK (FtsK(C)) is a DNA translocase implicated in helping synapsis of the dif sites and in locally promoting XerD strand exchanges after synapse formation. Here we show that FtsK(C) ATPase activity is directly involved in the local activation of Xer recombination at dif, by using an intermolecular recombination assay that prevents significant DNA translocation, and we confirm that FtsK acts before Holliday junction formation. We show that activation only occurs with a DNA segment adjacent to the XerD-binding site of dif. Only one such DNA extension is required. Taken together, our data suggest that FtsK needs to contact the XerD recombinase to switch its activity on using ATP hydrolysis.     

Sister Chromatid Exchange Frequencies in Escherichia coli Analyzed by Recombination at the dif Resolvase Site

Sister chromatid exchange (SCE) in Escherichia coli results in the formation of circular dimer chromosomes, which are converted back to monomers by a compensating exchange at the dif resolvase site. Recombination at dif is site specific and can be monitored by utilizing a density label assay that we recently described. To characterize factors affecting SCE frequency, we analyzed dimer resolution at the dif site in a variety of genetic backgrounds and conditions. Recombination at dif was increased by known hyperrecombinogenic mutations such as polA, dut, and uvrD. It was also increased by a fur mutation, which increased oxidative DNA damage. Recombination at dif was eliminated by a recA mutation, reflecting the role of RecA in SCE and virtually all homologous recombination in E. coli. Interestingly, recombination at dif was reduced to approximately half of the wild-type levels by single mutations in either recB or recF, and it was virtually eliminated when both mutations were present. This result demonstrates the importance of both RecBCD and RecF to chromosomal recombination events in wild-type cells.     

The Xer/dif site-specific recombination system of Campylobacter jejuni

Chromosome dimers, which form during the bacterial life cycle, represent a problem that must be solved by the bacterial cell machinery so that chromosome segregation can occur effectively. The Xer/dif site-specific recombination system, utilized by most bacteria, resolves chromosome dimers into monomers using two tyrosine recombinases, XerC and XerD, to perform the recombination reaction at the dif site which consists of 28–30 bp. However, single Xer recombinase systems have been recently discovered in several bacterial species. In Streptococci and Lactococci a single recombinase, XerS, is capable of completing the monomerisation reaction by acting at an atypical dif site called dif _SL (31 bp). It was recently shown that a subgroup of ε-proteobacteria including Campylobacter spp. and Helicobacter spp. had a phylogenetically distinct Xer/dif recombination system with only one recombinase (XerH) and an atypical dif motif (difH). In order to biochemically characterize this system in greater detail, Campylobacter jejuni XerH was purified and its DNA-binding activity was characterized. The protein showed specific binding to the complete difH site and to both halves separately. It was also shown to form covalent complexes with difH suicide substrates. In addition, XerH was able to catalyse recombination between two difH sites located on a plasmid in Escherichia coli in vivo. This indicates that this XerH protein performs a similar function as the related XerS protein, but shows significantly different binding characteristics.     

Engineered synaptic tools reveal localized cAMP signaling in synapse assembly

The physiological mechanisms driving synapse formation are elusive. Although numerous signals are known to regulate synapses, it remains unclear which signaling mechanisms organize initial synapse assembly. Here, we describe new tools, referred to as “SynTAMs” for synaptic targeting molecules, that enable localized perturbations of cAMP signaling in developing postsynaptic specializations. We show that locally restricted suppression of postsynaptic cAMP levels or of cAMP-dependent protein-kinase activity severely impairs excitatory synapse formation without affecting neuronal maturation, dendritic arborization, or inhibitory synapse formation. In vivo, suppression of postsynaptic cAMP signaling in CA1 neurons prevented formation of both Schaffer-collateral and entorhinal-CA1/temporoammonic-path synapses, suggesting a general principle. Retrograde trans-synaptic rabies virus tracing revealed that postsynaptic cAMP signaling is required for continuous replacement of synapses throughout life. Given that postsynaptic latrophilin adhesion-GPCRs drive synapse formation and produce cAMP, we suggest that spatially restricted postsynaptic cAMP signals organize assembly of postsynaptic specializations during synapse formation.     

Synapsis of Tn3 recombination sites: unpaired sites destabilize synapses by a partner exchange mechanism

Catalysis of site-specific recombination is preceded by the formation of a synapse comprising two DNA sites and multiple subunits of the recombinase, together with other "accessory" proteins in some cases. We investigated the stability of synapses of Tn3 resolvase-bound res recombination sites, in plasmids containing either two or three res sites. Although synapses are long-lived in plasmids with just two res sites, persisting for tens of minutes, a synapse of any two sites is relatively short-lived in plasmids with three res sites. The three alternative pairwise synapses that can be formed in three-res plasmids re-assort rapidly relative to the rate of recombination. We propose a "partner exchange" mechanism for this re-assortment, involving direct attack on a synapse by an unpaired res site. This mechanism reconciles studies on selective synapsis in multi-res substrates, which imply rapid interchange of synaptic pairings, with studies indicating that synapses of two Tn3res sites are stable.     

An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes

A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.     

Mobilization of pdif modules in Acinetobacter: A novel mechanism for antibiotic resistance gene shuffling?

XerCD-dif site-specific recombination is a well characterized system, found in most bacteria and archaea. Its role is resolution of chromosomal dimers that arise from homologous recombination. Xer-mediated recombination is also used by several plasmids for multimer resolution to enhance stability and by some phage for integration into the chromosome. In the past decade, it has been hypothesized that an alternate and novel function exists for this system in the dissemination of genetic elements, notably antibiotic resistance genes, in Acinetobacter species. Currently the mechanism underlying this apparent genetic mobility is unknown. Multidrug resistant Acinetobacter baumannii is an increasingly problematic pathogen that can cause recurring infections. Sequencing of numerous plasmids from clinical isolates of A. baumannii revealed the presence of possible mobile modules: genes were found flanked by pairs of Xer recombination sites, called plasmid-dif (pdif) sites. These modules have been identified in multiple otherwise unrelated plasmids and in different genetic contexts suggesting they are mobile elements. In most cases, the pairs of sites flanking a gene (or genes) are in inverted repeat, but there can be multiple modules per plasmid providing pairs of recombination sites that can be used for inversion or fusion/deletion reactions; as many as 16 pdif sites have been seen in a single plasmid. Similar modules including genes for surviving environmental toxins have also been found in strains of Acinetobacter Iwoffi isolated from permafrost cores; this suggests that these mobile modules are an ancient adaptation and not a novel response to antibiotic pressure. These modules bear all the hallmarks of mobile genetic elements, yet, their movement has never been directly observed to date. This review gives an overview of the current state of this novel research field.     

Unlinking chromosome catenanes in vivo by site-specific recombination

A challenge for chromosome segregation in all domains of life is the formation of catenated progeny chromosomes, which arise during replication as a consequence of the interwound strands of the DNA double helix. Topoisomerases play a key role in DNA unlinking both during and at the completion of replication. Here we report that chromosome unlinking can instead be accomplished by multiple rounds of site-specific recombination. We show that step-wise, site-specific recombination by XerCD-dif or Cre-loxP can unlink bacterial chromosomes in vivo, in reactions that require KOPS-guided DNA translocation by FtsK. Furthermore, we show that overexpression of a cytoplasmic FtsK derivative is sufficient to allow chromosome unlinking by XerCD-dif recombination when either subunit of TopoIV is inactivated. We conclude that FtsK acts in vivo to simplify chromosomal topology as Xer recombination interconverts monomeric and dimeric chromosomes.     

Species specificity in the activation of Xer recombination at dif by FtsK

In Escherichia coli, chromosome dimers are resolved to monomers by the addition of a single cross-over at a specific locus on the chromosome, dif. Recombination is performed by two tyrosine recombinases, XerC and XerD, and requires the action of an additional protein, FtsK. We show that Haemophilus influenzae FtsK activates recombination by H. influenzae XerCD at H. influenzae dif. However, it cannot activate recombination by E. coli XerCD. Reciprocally, E. coli FtsK cannot activate recombination by the H. influenzae recombinases at H. influenzae dif. We took advantage of this species specificity to gain further insight into the mechanism of activation of Xer recombination at dif by FtsK. We mapped the region of FtsK implicated in species specificity to the extreme 140-amino-acid C-terminal residues of the protein. Our results suggest that FtsK interacts directly with XerCD in order to activate recombination at dif.     

New players tip the scales in the balance between excitatory and inhibitory synapses

Synaptogenesis is a highly controlled process, involving a vast array of players which include cell adhesion molecules, scaffolding and signaling proteins, neurotransmitter receptors and proteins associated with the synaptic vesicle machinery. These molecules cooperate in an intricate manner on both the pre- and postsynaptic sides to orchestrate the precise assembly of neuronal contacts. This is an amazing feat considering that a single neuron receives tens of thousands of synaptic inputs but virtually no mismatch between pre- and postsynaptic components occur in vivo. One crucial aspect of synapse formation is whether a nascent synapse will develop into an excitatory or inhibitory contact. The tight control of a balance between the types of synapses formed regulates the overall neuronal excitability, and is thus critical for normal brain function and plasticity. However, little is known about how this balance is achieved. This review discusses recent findings which provide clues to how neurons may control excitatory and inhibitory synapse formation, with focus on the involvement of the neuroligin family and PSD-95 in this process.