Molecular interplay between Integrative Mobile Elements exploiting Xer drives the evolution of cholera pandemics

XerC and XerD are ubiquitous bacterial recombinases dedicated to chromosome dimer resolution. They add a crossover at a specific chromosomal site, dif, when activated by the FtsK cell division protein. Several integrative mobile elements exploiting Xer (IMEXs) have been reported. IMEXs hijack XerCD to integrate at dif in an FtsK-independent manner. The most notable IMEX is the CTX{Phi} bacteriophage of Vibrio cholerae, which encodes the Cholera toxin. V. cholerae isolates often contain several sequentially integrated IMEX, forming arrays. In the current cholera pandemic lineage, CTX{Phi} integrated in the dif site of the primary V. cholerae chromosome, difI, after the integration of another IMEX, TLC{Phi} (Toxin-Linked Cryptic). The strict order of TLC{Phi} and CTX{Phi} integration has been explained via a dif site-correction hypothesis, where the difI site of the environmental ancestor of the current pandemic isolates was non-functional and was corrected by the integration of TLC{Phi}. Here, we traced the inheritance of difI variants and IMEXs in environmental and pandemic isolates. We show that IMEX arrays undergo rearrangements not congruent with the core genome phylogeny and that TLC{Phi} integration preceded CTX{Phi} integration in pandemic lineages. We demonstrate that the difI sites of environmental and pandemic isolates are equally functional for dimer resolution and CTX{Phi} integration, and show that TLC{Phi} and another IMEX, VGJ{Phi}, encode a Xer activation factor that facilitates CTX{Phi} integration, suggesting an alternate explanation for the integration order of IMEX in environmental and pandemic isolates. Significance statementCholera is an increasing threat due to climate change. Vibrio cholerae pandemic potential is intricately linked to its infection by a lysogenic phage, the CTX{Phi}, which encodes the Cholera Toxin. CTX{Phi} uses the Xer recombination of its host for genomic integration. We established that CTX{Phi} is integrated downstream of the TLC{Phi} phage satellite in pandemic lineages and that it is frequently integrated downstream of the VGJ{Phi} phage in environmental strains. Our results show that TLC{Phi} and VGJ{Phi} encode for distinct Xer activator proteins (Xafs) that facilitate CTX{Phi} integration, providing a mechanistic explanation for the observed integration order. This novel molecular interplay is crucial to understanding the development of pandemic V. cholerae, with a view to preventing future expansions.