Speaker
Description
Antibiotic resistance causes over one million deaths annually worldwide and poses an increasing threat to healthcare systems. Its rapid spread is largely driven by horizontal gene transfer (HGT) through bacterial conjugation, a process that disseminates resistance and virulence genes. In Bacillus subtilis, gene expression from conjugative plasmid pLS20 is regulated by a two-factor response regulator composed by the anti-repressor Rap and the repressor Rco. Our group discovered that this regulatory system shares marked structural similarity with the main tumor suppressor p53 family related proteins.
We present the first structure of the Rap/Rco, obtained at BL-13 XALOC (ALBA synchrotron, Spain), together with CryoEM data obtained at Glacios 200kV in JEMCA (ALBA synchrotron, Spain) and at EBIC (Diamond light source, UK). Our structures describe the mechanism for Rap anti-repression and Rco release upon Rap tetramerization. Biophysical characterization confirms that Rco repressor keeps a p53-like architecture both in its repressive form and when bound to Rap anti-repressor. AI-guided tool Foldseek further expands the structural similarities to other p53 related proteins. Finally, in vivo complementation assays show that Rco rescues the function of the Caenorhabditis elegans p53 homolog.
These results define the molecular determinants underlying the Rap-mediated control of bacterial conjugation and antibiotic resistance, and further highlight the structural similarities of this regulatory strategy across species. This work takes advantage of ALBA capabilities as a facility for integrative structural biology. In the future, this project will be benefitted by the new instruments that will be developed in parallel to the ALBA-II project, like 300kV cryoET and BioSAXS beamline CALIMA, to bridge the resolution gap between atomic models and cellular events related to conjugation control and antibiotic resistance.