Biology subjects

Barre, F.-X.

Publications and source records attributed to Barre, F.-X..

3 recordsLinked to original sources

bioRxiv2026

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.

microbiology↗

bioRxiv2020

L-arabinose induces the formation of viable non-proliferating Vibrio cholerae spheroplasts

A general survival strategy of many life forms faced with harmful growth conditions is to enter into a non-proliferating state until conditions suitable for growth are restored. In bacteria, this survival strategy is associated with antimicrobial tolerance, chronic infections and environmental dispersion. In particular, the agent of the deadly human disease cholera, Vibrio cholerae, undergoes a morphological transition from a rod-shaped proliferative form to a spherical non-proliferating form after exposure to cold or cell wall targeting antibiotics. Growth is resumed when the adverse conditions have ceased. Here, we show that a component of the hemicellulose and pectin of terrestrial plants, L-arabinose, triggers the formation of non-proliferating V. cholerae spherical cells, which are able to return to growth when L-arabinose is removed from the growth medium. We found that the cell wall of L-arabinose treated V. cholerae cells has a peptidoglycan composition similar to the cell wall of spheroplasts and that they revert to a wild-type morphology through the formation of branched cells like L-forms. Unlike L-forms, however, they are osmo-resistant. Through a random Tn genetic screen for mutants insensitive to L-arabinose, we identified genes involved in the uptake and catabolism of galactose and in glycolysis. We hypothesize that L-arabinose is enzymatically processed by the galactose catabolism and glycolysis pathways until it is transformed in a product that cannot be further recognized by V. cholerae enzymes. Accumulation of this enzymatic by-product triggers the formation of viable non-dividing cell wall deficient spherical cells.

microbiology↗

bioRxiv2017

Enhancing Multiplex Genome Editing by Natural Transformation (MuGENT) via inactivation of ssDNA exonucleases

Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ~50% without selection in cis. Consequently, editing constructs are generated in a single PCR reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.

microbiology↗