The broad-host-range plasmid pTF-FC2 requires a primase-like protein for autonomous replication in Escherichia coli

Abstract

A 3202-bp fragment of plasmid pTF-FC2, cloned into PUC19, had previously been identified as the minimum region required for replication in either Pseudomonas aeruginosa or Escherichia coli polA- mutants. During the course of experiments to construct broad-host-range cloning vectors based on the pTF-FC2 replicon, it was found that the 3202-bp fragment had an absolute requirement for some function of the pUC19 vector. This requirement was eliminated in the presence of co-resident pTF-FC2 derivatives. An additional 1239-bp fragment from pTF-FC2, immediately adjacent to the 3202-bp fragment, was identified which restored the ability of the pTF-FC2 replicon to replicate autonomously. Sequence analysis of the region revealed a single open reading frame encoding a 40-kDa polypeptide, which was synthesised in an in vitro transcription/translation system. A comparison of the amino acid sequence of this protein with sequence data banks revealed limited homology with the RepB' primase of the IncQ plasmid, RSF1010. An M13 delta lac 110 replication-deficient phage system was used to demonstrate that the 40-kDa protein did function as a primase with respect to replication at the origin of replication (vegetative) of pTF-FC2.