Comparative Biology of Two Natural Variants of the IncQ-2 Family Plasmids,pRAS3.1 and pRAS3.2

Plasmids pRAS3.1 and pRAS3.2 are two closely related, natural variants of the IncQ-2 plasmid family that have identical plasmid backbones except for two differences. Plasmid pRAS3.1 has five 6-bp repeat sequences in the promoter region of the mobB gene and four 22-bp iterons in its oriV region, whereas pRAS3.2 has only four 6-bp repeats and three 22-bp iterons. Plasmid pRAS3.1 was found to have a higher copy number than pRAS3.2, and we show that the extra 6-bp repeat results in an increase in mobB and downstream mobA/repB expression. Placement of repB (primase) behind an arabinose-inducible promoter in trans resulted in an increase in repB expression and an approximately twofold increase in the copy number of plasmids with identical numbers of 22-bp iterons. The pRAS3 plasmids were shown to have a previously unrecognized toxin-antitoxin plasmid stability module within their replicons. The ability of the pRAS3 plasmids to mobilize the oriT regions of two other plasmids of the IncQ-2 family, pTF-FC2 and pTC-F14, suggested that the mobilization proteins pRAS3 are relaxed and can mobilize oriT regions with substantially different sequences. Plasmids pRAS3.1 and pRAS3.2 were highly incompatible with plasmids pTF-FC2 and pTC-F14, and this incompatibility was removed on inactivation of an open reading frame situated downstream of the mobCDE mobilization genes rather than being due to the 22-bp oriV-associated iterons. We propose that the pRAS3 plasmids represent a third, γ incompatibility group within the IncQ-2 family plasmids.Plasmids of the IncQ family are small (<20 kb), have a broad host range, and are highly promiscuous due to their ability to be mobilized very efficiently by self-transmissible plasmids such as the IncP plasmids. They have been divided into two families, IncQ-1 and IncQ-2, based on the amino acid sequence relatedness of their RepA (helicase), RepB (primase), and RepC (DNA-binding) replication proteins and because the mobilization proteins of the two families are unrelated, consisting of three or five genes, respectively (31). IncQ-1 group plasmids include RSF1010 and the near-identical R1162, pDN1, pIE1107, pIE1115, and pIE1130, while IncQ-2 plasmids include pTF-FC2, pTC-F14, and pRAS3.IncQ-2 plasmids pRAS3.1 and pRAS3.2 were isolated in Norway from the fish pathogens Aeromonas salmonicida subsp. salmonicida and atypical A. salmonicida, respectively, while investigating plasmids that conferred resistance to tetracycline (21). The two plasmids encode identical replication and mobilization proteins, with the most important differences in the plasmid backbone being that pRAS3.1 has four 22-bp iterons in its oriV region and five 6-bp repeat sequences upstream of its mobB gene, whereas pRAS3.2 has only three iterons and four 6-bp repeat sequences. No biological studies were carried out in the initial report of the pRAS3 plasmids. As a contribution to our studies on the evolution of IncQ plasmids, our longer-term aim is to address the question of why two natural versions of the plasmid exist. Here we report on the major differences in the biology of the two plasmids. In addition, we discovered the presence of repC and mobB genes that were not detected when the sequence of pRAS3 plasmids was previously reported. We also discovered a putative toxin-antitoxin (TA) postsegregational system different from that found in other members of the IncQ plasmids and tested it for functionality.The IncQ-1 plasmids are subdivided into incompatibility groups α, β, and γ, (31), whereas the IncQ-2 plasmids are subdivided into two incompatibility groups, α and β (14). In this work we also report on the incompatibility between the pRAS3 plasmids and other members of the IncQ-2 plasmid family as well as the IncQ-1 family plasmids. Furthermore, we compare the functional relatedness of the pRAS3 mobilization system with that of previously studied IncQ-2 plasmids.