Characterization of pEC156, a ColE1-type plasmid from Escherichia coli E1585-68 that carries genes of the EcoVIII restriction-modification system

The complete 4312-bp sequence of the pEC156 plasmid from Escherichia coli E 1585-68, which carries genes encoding the EcaVITI restriction-modification (R-Nr) system, an isosch mer of HindIII from Haemophilus influenzae, has be en determined. Two clustered and convergently oriented open reading frames, large enough to encode genes of the EcoVIII R-M system, were found. The tr anscriptional start points were mapped by the primer extension method. The relative molecular masses of the EcoVIII endonuclease and EcoVIII methyltra nsferase deduced from the nucleotide sequence are 35,554 and 33,910, respec tively. Nucleotide sequence analysis of pEC156 suggests that this plasmid i s a ColE1-type replicon. It consists of an origin of replication and two un translated genes encoding RNA I and RNA II, both involved in the regulation of plasmid DNA replication. The replication region also contains the gene encoding a 64-aa Rom-like protein. Inactivation of the putative rom gene by insertion of a kanamycin-resistance cassette resulted in 4.5-fold increase in pEC156-derived plasmid copy number in E. coli cells. All of these eleme nts (RNA I, RNA II, and rom) reveal a high level of similarity to ColE1 hom ologs. The replication of all ColE1-type plasmids is dependent on the activ ity of E. coli DNA polymerase 1. It was shown that a pEC156 derivative (pIB 8) carrying an antibiotic resistance gene indeed failed to replicate in an E. coli polA12(ts) mutant at 43 degreesC, and its copy number was reduced i n the E. coli pcnB80 mutant. These results prove that pEC156 is a ColE1-typ e replicon. (C) 2001 Academic Press.