Two OB-fold proteins from a Gram-positive conjugative element engage in relaxosome assembly and DNA processing.

Integrative and conjugative elements (ICEs) are widespread genomic islands that propagate by bacterial conjugation. ICEs often spread antibiotic resistance genes and other adaptive genes conferring evolutionary advantages to their hosts. At the initiation of conjugation, the ICE DNA is processed by the relaxase, a transesterase that recognises its origin-of-transfer. ICESt3/Tn916/ICEBs1 superfamily of ICEs is widespread in Gram-positive bacteria, and it encodes uncanonical relaxases classified within the unique MOBT family. This study elucidates the roles of OrfL and OrfM, two OB-fold proteins from ICESt3, in its conjugative transfer. We demonstrated that OrfL and OrfM form a complex and function as relaxosomal auxiliary proteins with RelSt3, the ICESt3 MOBT relaxase. Through conjugation assays, we established that they are both essential for ICESt3 conjugation. The NMR 3D structure of OrfM revealed an OB-fold protein with several flexible regions. Our results also suggest a networking role for OrfL as it was shown by BACTH to interact with both the relaxase and with the cellular PcrA helicase involved in rolling-circle replication of mobile genetic elements in Firmicutes. Our data provide a description of essential participation of OB-fold proteins in a relaxosome, contrasting with most of other known relaxosomes in both Gram-negative and Gram-positive bacteria, which primarily involve ribbon-helix-helix proteins.