Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1

Abstract The stability ( stb ) locus of IncFII plasmid NR1 was mapped to a 1700 base-pair Nae I- Taq I restriction fragment. A series of unstable plasmids that contained insertion, deletion, and point mutations that inactivated the stability function was isolated. The unstable point mutants examined were all stabilized (complemented) in trans by a copy of the wild-type stb locus, suggesting that the mutations had inactivated diffusible gene products. The nucleotide sequence of the stb locus contained two tandem open reading frames, designated stbA and stbB , that encoded essential trans -acting protein products with predicted sizes of 36,000 M r and 13,000 M r , respectively. A third open reading frame, stbC , that could encode a peptide of 8000 M r , was contained within stbB in the complementary DNA strand. Plasmid-encoded proteins of 36,000 M r and 13,000 M r were identified in minicell experiments as the products of stbA and stbB , respectively. Unstable deletion mutants that retained the promoter proximal region of the stb locus upstream from stbA but had deleted both stbA and stbB were stabilized in trans by plasmids that could supply StbA and StbB. In contrast, deletion mutants that had. lost the stbAB promoter region were not complemented in trans , indicating that this region contained an essential cis -acting site (or sites). Unlike some other loci that mediate stable plasmid inheritance, cloned copies of the wild-type stb locus of NR1 did not exert strong incompatibility (i.e. trans destabilization) against other stb + derivatives of plasmid NR1 present in the same cell.