Diverse dif module content and configurations in the r3-T5 group of Rep_3/OrfX plasmids from Acinetobacter species...

The dif module mobile element system is found naturally in Acinetobacter plasmids with a backbone that encodes a Rep_3 replication initiation function and an OrfX open reading frame. Here, the characteristics of plasmid pMSHR_A204 (10,025 bp) from the r3-T5 subgroup of this family and related plasmids were examined. pMSHR_A204 consists of a 3.27 kbp backbone C module and five accessory dif modules (two C and three D modules), including the tet39 tetracycline resistance module and a large fusion module carrying higBA2 toxin-antitoxin genes. Rearrangements of the dif module content, both inversions and deletions involving pdif sites with identical or nearly identical spacers, were detected experimentally. Sequences of 35 unique r3-T5 plasmids retrieved from GenBank and originating from three different Acinetobacter species and 27 countries were annotated and compared. The backbone C module was highly conserved but was associated with a variety of accessory dif modules. Most plasmids carried a dif module containing toxin-antitoxin genes. Eleven plasmids were cointegrates with a second backbone from another r3 type. Most non-cointegrate r3-T5 plasmids carried odd numbers of dif modules (e.g., 2 D modules and 1 C module), but several included a fusion (CD) module and two or four dif modules. Varied configurations caused by reactions between pdif sites with identical or nearly identical spacers were detected within groups of plasmids with the same overall dif module content. One pair differed by a single dif module. These rearrangements were consistent with models for movement of dif modules between plasmids, including via a cointegrate intermediate.

The plasmids that are associated with Acinetobacter isolates appear to be restricted to this genus. However, the unique properties of the many and various plasmid types found in Acinetobacter species have received little attention relative to the plasmids of other gram-negative pathogens. In addition, the novel dif module mobile element system, also found uniquely in Acinetobacter species, was recently shown to be naturally associated with a specific type of Acinetobacter plasmid. To investigate the dynamics of dif module movement, here, the properties and configurations of one group of plasmids with identical or closely related backbones that carry different dif modules or the same modules in different configurations have been examined. This will increase understanding of the global distribution and diversity of plasmids that carry dif modules, as well as provide insight into the way dif modules relocate.