From the Cover: 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.In all organisms, the processing of chromosome ends or termini relies on specific activities for replication and segregation. In eukaryotes, telomeres are often targeted by mobile genetic elements, which may even substitute for telomeric functions (1). Circular chromosomes found in prokaryotes have no telomeres but harbor chromosome dimer resolution sites, called dif sites, on which dedicated Xer recombinases (XerC and XerD in most cases) act (2, 3). Besides their role in chromosome maintenance, dif sites are targeted by numerous mobile genetic elements, referred to as integrating mobile element exploiting Xer (IMEX) (4). How IMEXs integrate into dif without inactivating its cellular function and how they are stably maintained in their integrated state has remained unclear despite study over the past decade (4–7). Here we answer these questions by studying the gonococcal genomic island (GGI), an IMEX stably integrated into the dif site of pathogenic Neisseria species that encodes crucial functions for gene exchange and virulence (8, 9).In Escherichia coli, chromosome dimers form by homologous recombination during replication and are resolved by site-specific recombination between sister dif sites catalyzed by the XerC and XerD recombinases (Fig. 1) (3). The 28-bp dif site carries binding sites for each recombinase, separated by a 6-bp central region at the border of which strand exchanges are catalyzed. After assembly of the recombination complex (synapse), one pair of strands is exchanged by the XerD monomers, leading to a branched DNA intermediate (Holliday junction, HJ) subsequently resolved by XerC. Dimer resolution is integrated into the general processing of the terminal region of the chromosome (ter region) during cell division (10). FtsK, a DNA translocase associated with the division apparatus, segregates this region at the onset of cell division (10, 11). The translocation motor, FtsKαβ, is located in the C terminal of FtsK (12). Translocation is oriented toward the dif site located at the center of the ter region via a direct interaction between the extreme C-terminal subdomain of FtsK, FtsKγ, and the KOPS DNA motifs (13). Upon reaching the XerCD/dif complex, FtsK stops translocating and activates recombination via direct interaction with XerD (14, 15) (Fig. 1). The mechanisms of translocation arrest and of recombination activation are poorly understood but they both involve FtsKγ. However, these activities appear to be distinct from each other because FtsKγ can activate recombination in vivo and in vitro when isolated from the FtsKαβ motor or fused to XerC or XerD (16).Open in a separate windowFig. 1.The XerCD/dif recombination. (A) Chromosome dimer formation by homologous recombination (HR) during replication and resolution by site-specific recombination between the two dif sites. The dif site is represented as green and purple boxes for the XerC-binding and the XerD-binding sites, respectively. ori (black circle), some KOPS motifs (arrows), and the ter domain (thick line) are represented. The mechanism of XerCD/dif recombination is represented in the box. XerC (green circles) and XerD (purple circles) bind two distant dif sites to create a synapse. Hexamers of the FtsK C-terminal domain [FtsKC: FtsKαβ: (diamonds) + FtsKγ: (triangle) contacting XerD] translocate toward dif and contact XerD. This activates XerD (Y indicates the active recombinases), which catalyzes the first-strand exchange. This process leads to the formation of an HJ intermediate within which XerC is active and catalyzes the second-strand exchange (3). (B) Integration and excision of the GGI (dotted line) by XerCD catalysis. KOPS, dif_Ng, and dif_GGI sites are represented as in A. An alignment of dif_Ng, dif_GGI and consensus dif sequence (27, 28) is shown on the left. Substituted positions in dif_GGI are represented as lowercase characters and highlighted by stars.In numerous bacteria, the XerCD/dif system is hijacked by IMEXs, which integrate their host genome into dif sites by using XerCD-mediated catalysis (4). In all of the reported cases, integration of IMEXs recreates a bona fide dif site, thereby not interfering with chromosome dimer resolution, which would lead to their counter-selection. The best-described examples are Vibrio cholerae IMEXs, which carry crucial virulence determinants (5–7, 17). These IMEXs have developed different strategies to integrate and to remain stably integrated, although the mechanisms ensuring their stable maintenance are not fully understood. Neisseria species contain an unusually long IMEX called the gonococcal genomic island (GGI) (8). In Neisseria gonorrheae, the GGI is 57 kb long and encodes a type IV secretion system that exports the chromosomal DNA of its host, rendering it available to neighboring cells for gene exchange by genetic transformation (8, 18). The GGI carries a dif site, dif_GGI, consisting of a XerC-binding site, a central region homologous to the Neisseria dif site, dif_Ng, and a divergent XerD-binding site (Fig. 1B). Comparison of N. gonorrheae strains harboring or lacking the GGI, together with functional data, indicates that the GGI integrates by XerCD-dependent recombination (9). The nonreplicative excised circular form of the GGI can be detected and the GGI can also be lost, showing that excision occurs, although at low frequencies (9). Although the GGI was identified over a decade ago, it has remained unclear how DNA flanked by two Xer recombination sites is stably maintained at a chromosomal locus processed by FtsK during each cell cycle. In this study, we have combined in vitro and in vivo approaches to show that dif_GGI is indeed an active Xer recombination site at which the Neisseria Xer recombinases catalyze recombination when activated by FtsKγ. However, we find that recombination between dif_Ng and dif_GGI is inhibited by translocating FtsK. Inhibition is a result of the absence of translocation arrest at XerCD_Ng/dif_GGI complexes that most likely precludes recombination activation, an absence that causes the complex to dismantle. We conclude that, depending on the sequence of the recombination site, Xer recombination complexes have the intrinsic capacity to be activated or inhibited by FtsK.