Identification and characterization of a new replication region in the Neisseria gonorrhoeae beta-lactamase plasmid pFA3

Abstract

The 7.1-kilobase-pair (kbp) plasmid pFA3 specifies TEM beta-lactamase production in Neisseria gonorrhoeae. We studied the minimal region required for replication of this plasmid in Escherichia coli by constructing a set of nested deletions of the 3.4-kbp PstI-HindIII fragment. The smallest fragment capable of maintenance in E. coli when ligated to a streptomycin-spectinomycin resistance cassette was 2.0 kbp in size and was different from another autonomously replicating fragment of pFA3 reported by K. H. Yeung and J. Dillon (Plasmid 20:232-240, 1988). The fragment contained single BamHI and XbaI sites and specified a 39-K protein. Fragments subcloned from the minimal region or constructed by deletion from the 3' or 5' ends were not capable of autonomous replication. Mutants constructed by end filling and religating DNA cleaved at the BamHI or XbaI sites were not capable of autonomous replication and no longer produced the 39K protein. These results suggest that replication is dependent on the 39K protein. DNA sequence analysis of the region showed an A-T-rich region followed by four 22-bp direct repeats followed by an open reading frame encoding a 39K basic protein.