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 a gglomerans,rs has been determined. The plasmid, called pPIGDM1, consis ts of 2495 base pairs. The analysis of its nucleotide sequence suggest ed that pPIGDM1 may be a ColE1-like replicon, We confirmed this hypoth esis by constructing a pPIGDM1-derived plasmid harboring the cat gene (pBW4), which could be introduced into Escherichia coli cells, and dem onstrating that pBW4 cannot replicate in the absence of the polA funct ion and that its copy number is significantly decreased in the pcnB mu tant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-der ived plasmids can be amplified both by chloramphenicol method and in i soleucine-starved relA mutants but not in relA(+) bacteria. Inactivati on of the putative rom gene by insertion of an ampicillin-resistance g ene resulted in significant increase in pPIGDM1-derived plasmid copy n umber in E. coli despite the fact that amino acid sequence of the puta tive RNA I modulator (Rom) protein is only 55.7% identical to the ColE 1 analog. The pPIGDM1-derived rom-like coding sequence is also homolog ous to the rom-like gene present in the Proteus vulgaris plasmid pPvu1 . We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compati ble with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF1 3-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 n ew rom-like gene coding for a protein useful in investigation of RNA-p rotein interactions. A role for the pPIGDM1 plasmid in the host strain is not known. (C) 1997 Academic Press.