Identification of cis- and trans-acting elements in pHW126, a representative of a novel group of rolling circle plasmids

pHW126, pIGRK, pIGMS31 and pRAO1 are the only known members of a novel and as yet uncharacterised family of rolling circle plasmids. pHW126 contains only two open reading frames, of which one shows homology to pMV158-family mobilisation proteins. Here we provide evidence that the second open reading frame encodes a replication protein (Rep). Mutation or deletion of this gene resulted in replication deficient constructs, but providing functional Rep from a compatible vector rescued these constructs, indicating that Rep acts in trans. An approximately 300 bp cis-acting region representing the origin of replication was identified upstream of the rep gene. The origin was identified to be composed of three parts: an accessory region, a conserved stretch and four perfect tandem repeats. The two latter elements were essential for replication. Constructs with a deletion of the accessory region could still replicate, but their loss rate was high, indicating that the accessory region is necessary for plasmid maintenance under non-selective conditions. Interestingly, pHW126 could replicate in all Enterobacteriaceae tested while Agrobacterium tumefaciens and Pseudomonas syringae were inappropriate hosts. Thus, pHW126 seems to have a rather limited host range.