Isolation and characterization of a ColE1-like plasmid from Enterobacter agglomerans with a novel variant of rom gene.

Complete nucleotide sequence of a plasmid isolated from Enterobacter agglomerans has been determined. The plasmid, called pPIGDM1, consists of 2495 base pairs. The analysis of its nucleotide sequence suggested that pPIGDM1 may be a ColE1-like replicon. We confirmed this hypothesis by constructing a pPIGDM1-derived plasmid harboring the cat gene (pBW4), which could be introduced into Escherichia coli cells, and demonstrating that pBW4 cannot replicate in the absence of the polA function and that its copy number is significantly decreased in the pcnB mutant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-derived plasmids can be amplified both by chloramphenicol method and in isoleucine-starved relA mutants but not in relA+ bacteria. Inactivation of the putative rom gene by insertion of an amplicillin-resistance gene resulted in significant increase in pPIGDM1-derived plasmid copy number in E. coli-despite the fact that amino acid sequence of the putative RNA 1 modulator (Rom) protein is only 55.7% identical to the ColE1 analog. The pPIGDM1-derived rom-like coding sequence is also homologous to the rom-like gene present in the Proteus vulgaris plasmid pPvul. We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compatible with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF13-derived) in E. coli, one may consider pPIGDM1 as a progenitor of new cloning vehicles compatible with most (if not all) of currently used plasmid vectors. Moreover, this plasmid may serve as a source of the new rom-like gene coding for a protein useful in investigation of RNA-protein interactions. A role for the pPIGDM1 plasmid in the host strain is not known.