Rep_3 plasmids found in Acinetobacter species that include orfX also harbour dif modules

Plasmids found in Acinetobacter species are not found in other Gram-negative species. There are many distinct plasmid types and the majority encode a Rep_3 family RepA replication initiation protein. Among these, a number were known to carry dif modules. Here, the representative plasmid for each of the 78 reported R3 types was examined to identity features of the plasmid backbone that are associated with the carriage of dif modules. A conserved open reading frame designated orfX (IPR047783) was found downstream of repA in 35 of them, and the backbones of those 35 plasmids were bounded by recombination sites recognised by XerC and XerD, known as pdif sites. These plasmid backbones are all equivalent to a C-type dif module as the pdif sites are in the orientation D/C at one end and C/D at the other end, i.e. the XerC binding sites are internal. Hence, to provide the XerD binding sites and generate a complete plasmid at least one D-type dif module is needed. Phylogenies of the RepA and OrfX proteins revealed that plasmids with closely-related RepA proteins are not always associated with closely-related OrfX proteins and vice-versa indicating extensive backbone recombination. Folded structures of diverse OrfX proteins predicted using AlphaFold 3 revealed an N-terminal HTH domain followed by a long α-helix that is predicted to promote dimerization and a disordered C-terminus. Given the correlation between the presence of orfX and one or more dif modules, the possibility that OrfX is involved in dif module movement deserves to be investigated.