ICEs have been largely overlooked as vectors of AR and they still remain poorly characterised. Most of the molecular understanding derives from the ICEs belonging to Gram-positive bacteria, and very few proteins implicated in ICE conjugation have been biochemical characterized. In ICECOUP, to the best of our knowledge, it is the first time that a T4CP belonging to an ICE from a Gram-negative bacterium has been purified and its ATPase enzymatic activity has been demonstrated. The characterisation of MobBICE is relevant for understanding Gram-negative bacteria horizontal gene transfer mechanisms, taking into account that its encoding gene, mobBICE, is present (more than 90% identity covering the whole sequence) in at least 2000 strains of Klebsiella pneumoniae, 200 of E. coli and other Enterobacteriae, according to genomic analysis.
Preliminary results of this project revealed that there is an interaction between the oriT of ICEEc1 and one of the conjugative plasmids present in the E. coli strain ECOR31. Nevertheless, no interaction was detected between the oriT, the relaxase and the T4CP of ICEEc1 and the equivalent elements of other MGE, such as the mobilisable plasmid CloDF13 or the conjugative plasmid R388. Defining the boundaries, characteristics and restrictions of these dynamic interactions is essential and, once more, underscores the necessity of experimental evidence besides genomic predictions to address complicated biological problems such as AR dissemination. A thorough characterization is also crucial for the identification and use of possible conjugation inhibitors. This project was also pioneer, as far as we know, in the use of a molecular component of an ICE as a target to control mobilization.
Overall, these results require further research to draw consistent conclusions. Nevertheless, ICECOUP has laid the foundation of a novel research line that will be continued within the host laboratory. In conclusion, this project has provided novel insights into the conjugation process of an ICE that in the long run can be used to design novel strategies to combat AR, such as the use of conjugation inhibitors targeting the T4CPs.
