Elucidation of the molecular Interactions controlling Xer recombination – ELIXER

The ELIXER project aims to elucidate the molecular mechanisms underlying the Xer site-specific recombination system of bacteria. Xer recombination is a critical process that resolves chromosome dimers formed during homologous recombination, ensuring faithful segregation of bacterial chromosomes. In addition, the Xer system mediates the integration of many mobile elements into the genome of their host. With the exception of a few species, the Xer system is composed of two tyrosine recombinases, XerC and XerD, which are under the control of species-specific activators. XerC and XerD can perform recombination via two pathways, depending on whether the reaction is initiated by XerD or XerC. The ‘XerD-first’ pathway is the canonical pathway which followed for chromosome dimer resolution. The alternative ‘XerC-first pathway’ is the pathway used by most mobile elements. The project focuses on the Vibrio cholerae Xer system, in which the canonical XerD-first is exploited by a phage implicated in the evolution toward pathogenicity and the alternative XerC-first pathway is exploited by a phage that carries the cholera toxin genes. This duality makes it an ideal model to study the dynamics and regulation of tyrosine recombinase (YR)–mediated strand exchange.
ELIXER will combine structural biology, biochemistry, and genetics to characterize the full catalytic cycle of Xer recombination. Cryo-EM and X-ray crystallography will be used to determine the structures of key intermediate states, including pre-cleavage, post-cleavage, and Holliday junction (HJ) complexes. The role of FtsK? and XafT—two essential accessory proteins—in activating XerD will be investigated, with particular attention to their influence on synaptic complex formation and the control of catalytic directionality.
The ELIXER consortium brings together three complementary partners with strong expertise in recombination, structural biology, and access to major infrastructures. Partner 1 (I2BC) leads the project and focuses on in vivo and in vitro assays; Partner 2 (I2BC) contributes structural and biochemical expertise. Partner 3 (Synchrotron-Soleil) brings advanced expertise in Cryo-EM and crystallography data acquisition and analysis.
The project is structured into two main work packages. WP1 focuses on the structural characterization of XerCD-DNA complexes, while WP2 addresses biochemical and genetic validation of protein–DNA and protein–protein interactions inferred from structural data. Novel strategies for assembling asymmetric recombination complexes using DNA-bending proteins (e.g., IHF) or chemical-inducible dimerization domains will be employed to isolate structurally informative intermediates. Deep sequencing and mutagenesis approaches will be used to dissect the sequence determinants of recombinase binding and activity, as well as to probe species-specific activation mechanisms.
By resolving the conformational states of the XerCD recombination cycle, ELIXER will answer longstanding questions regarding the regulation of recombination order, the role of accessory factors, and the structural basis of species specificity. These insights will not only advance our fundamental understanding of YR-mediated recombination but also pave the way for the rational design of new molecules capable of selectively inhibiting Xer recombinase activity. Such inhibitors could serve as novel antimicrobial agents or prevent the integration of pathogenic mobile elements like CTXF in commensal microbiota.