Molecular mechanism of Phage-encoded activation of Xer recombination – PhenX

A major difference between bacteria and eukaryotes is intrinsic to the structure of their chromosomes: they are linear in eukaryotes and circular in bacteria. DNA circularity can result in the formation of chromosome dimers, which physically impede the segregation of genetic information at cell division. However, bacteria have evolved a highly conserved chromosomally encoded recombination (Xer) machinery to resolve chromosome dimers by the addition of crossovers at a specific unique site of their circular chromosomes, dif.
Numerous mobile elements exploit Xer to integrate into the dif site of one of their host chromosomes. Intriguingly, Integrative Mobile Elements exploiting Xer (IMEX) are often associated to pathogenicity. A salient example is given by the evolutionary history of the agent of the cholera, Vibrio cholerae. V. cholerae is found in briny waters all over the world. However, most strains are not pathogenic or only cause local outbreaks of gastroenteritis. The diarrhoea that is responsible for the epidemic propagation and high death rate associated with cholera is due to a toxin that is encoded in the genome of an IMEX, the cholera toxin phage (CTXf). Interactions between CTXf and several other IMEX participate in the constant and rapid emergence of new cholera epidemic strains. Foremost among those is the toxin-linked cryptic satellite phage, TLCf, whose integration seems to be a prerequisite for CTXf integration.
The Xer machinery belongs to the family of tyrosine recombinases. It is very similar to Cre, the resolvase of phage P1, whose action mechanism has been thoroughly characterised at the atomic resolution. However, several features of the Xer machinery differentiate it from Cre and most other tyrosine recombinases. In particular, it is under the control of a large integral membrane cell division protein, FtsK. IMEX escape this control. We recently identified a Xer activation factor in the genome of TLCf, XafT (unpublished results). XafT is a small cytoplasmic protein with no sequence or structural similarities to FtsK. It contains a domain of unknown function that is encoded in many other IMEX. We have been able to reconstitute a full Xer recombination reaction in vitro using purified XafT.
The goal of this project is to obtain a detailed atomic-scale understanding of the Xer recombination process and of its control. To this end, we will take advantage of the simple limited component Xer reaction promoted by XafT to characterise in vitro the structural changes that occur in the recombination synapse upon activation. Although the research proposed is essentially academic, several aspects of it may have a direct impact in medical and/or environmental fields since it could lead to the discovery and /or rational design of compounds that inhibit Xer activity. In particular, such compounds could be used to help prevent infection by pathogenic Vibrios and/or toxigenic conversion of non-pathogenic Vibrios by TLCf and CTXf.
The PhenX project is multidisciplinary and associates four teams with complementary expertise. Team 1 is composed of geneticists, molecular and cell biologists with a strong experience on V. cholerae. Its members have already contributed to the molecular characterization of several Xer recombination pathways, including the FtsK- and XafT-pathways. Team 2 will bring its great experience on the structural characterization and modelling of nucleoprotein complexes. Team 3 is one of the world leaders on multiplex Förster resonance energy transfer, which will serve to monitor the changes induced by XafT on Xer recombination synapses. Team 4 is composed of chemists that maintain a bank of 5000 compounds that we will screen for their activity on Xer recombination in vivo. They will also bring their expertise for the rational design of putative Xer inhibitors and/or activators based on the structural data we will have obtained.