Molecular characterization of functional modules of plasmid pWKS1 of Paracoccus pantotrophus DSM 11072.

The complete nucleotide sequence of the small, cryptic plasmid pWKS1 (2697 bp) of Paracoccus pantotrophus DSM 11072 was determined. The G+C content of the sequence of this plasmid was 62 mol%. Analysis revealed that over 80% of the plasmid genome was covered by two ORFs, ORF1 and ORF2, which were capable of encoding putative peptides of 44.1 and 37.8 kDa, respectively. Mutational analysis showed that ORF2 was crucial for plasmid replication. The translational product of ORF2 shared local homologies with replication proteins of several theta-replicating lactococcal plasmids, as well as with the Rep proteins of plasmids residing in Gram-negative hosts. An A+T-rich region, located upstream of the rep gene and containing three tandemly repeated 21 bp long iteron-like sequences, served as the origin of replication (oriV). ORF1 encoded a putative mobilization protein with similarities to mobilization proteins (Mob) from the broad-host-range plasmid pBBR1 and plasmids of Gram-positive bacteria. A plasmid bearing the MOB module of pWKS1 (the mob gene and the oriT sequence) could be mobilized for transfer (by IncP RP4 transfer apparatus) at low frequency between different strains of Escherichia coli. MOB modules of pWKS1 and pBBR1 were functionally complementary to each other. Hybridization analysis revealed that only plasmid pSOV1 (6.5 kb), among all of the paracoccal plasmids identified so far, carries sequences related to pWKS1. Plasmid pWKS1 could replicate in 10 species of Paracoccus and in Agrobacterium tumefaciens, Rhizobium leguminosarum and Rhodobacter sphaeroides, but it could not replicate in E. coli.