Discovering genetic determinants for cell-to-cell adhesion in two prevalent conjugative lactococcal plasmids

Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of the putative adhesins of the pNP40 and pUC11B conjugation systems, encoded by traAd and trsAd, respectively. Despite their significant sequence divergence, TraAd and TrsAd represent the most conserved component between the pNP40- and the pUC11B-encoded conjugation systems and share similar peptidoglycan-hydrolase domains. Protein structure prediction using AlphaFold2 highlighted the structural similarities between their predicted domains, as well as the potential homo-dimeric state of both proteins. Expression of the putative surface adhesins resulted in a cell clumping phenotype not only among cells expressing these surface adhesins but also between adhesin-expressing and non-producing cells. Furthermore, mutant derivatives of plasmids pNP40 or pUC11B carrying a mutation in traAd or trsAd, respectively, were shown to act as efficient donors provided the corresponding recipient expresses either traAd or trsAd, thus demonstrating in trans reciprocal complementarity of these proteins in conjugation systems.