Mobile genetic elements contribute to prokaryotic evolution by moving DNA within and between genomes. Mesorhizobium spp. are soil bacteria that host large (up to 800 kb) integrated mobile genetic elements called Integrative and Conjugative Elements (ICEs). These ICEs carry genes essential for establishment of a nitrogen-fixing symbiosis with legumes and frequently transfer to native Mesorhizobium spp. in Australian soils, converting them into legume symbionts. Complex regulation of the quorum sensing and gene transfer systems occurs through a series of feedback loops governed by a transcriptionally active hormone receptor TraR and a second transcriptional activator FseA. FseA activates transcription of the ICE excision-stimulating protein (RdfS). The transfer activation proteins are repressed by the dual-target antiactivator protein QseM, which binds either TraR or FseA to prevent ICE transfer. Here we present the NMR structure of QseM, which represents the monomeric archetype of a novel family of helix-turn-helix (HTH) domain proteins that have evolved to become antagonistic paralogs of the FseA family. Unlike the dimeric DNA-binding FseA protein, QseM has an overall negative surface charge and lacks an ability to bind DNA. In vivo studies using structure-based mutagenesis revealed QseM acts by binding an N-terminal helix within FseA that would normally maintain critical contacts between FseA N and C-terminal domains. We also present the crystal structure of RdfS, which reveals it to be a winged-helix-turn-helix protein that forms superhelical head-to-tail oligomers in crystals and exists as tetramers in solution. Surface plasmon resonance analysis was used to characterise RdfS DNA-binding specificity and how it relates to RdfS oligomerisation.