GENETIC-ANALYSIS OF THE MINIMAL REPLICON OF PLASMID PIP417 AND COMPARISON WITH THE OTHER ENCODING 5-NITROIMIDAZOLE RESISTANCE PLASMIDS FROMBACTEROIDES SPP

The nucleotide sequence of the DNA replication origin region of a Bact eroides vulgatus plasmid, pIP417, encoding 5-nitroimidazole resistance has been determined. This region of 1934 bp presents some characteris tics similar to those of other replication protein-dependent origins. It contains a large open reading frame which could encode a basic Rep protein (RepA) of 36.8 kDa. Upstream of this ORF exist an AT-rich regi on, three direct repeats (iterons) of 21 bp, multiple DnaA binding sit es, and sites for the integration host factor (SHF). Moreover, the ami no acid sequence of the pIP417 RepA protein shows similarities with th ose of other Rep proteins encoded by plasmids of gram-negative bacteri a: pRO1600 from Pseudomonas aeruginosa; pPS10 from Pseudomonas syringa e; pFA3 from Neisseria gonorrhoeae; and two cryptic plasmids from Camp ylobacter hyointestinalis and Butyrivibrio fibrisolvens. Although RepA can be expressed in an Escherichia coli in vitro transcription-transl ation assay, vectors containing the pIP417 replication origin did not replicate in E. coli The homology of the pIP417 replication region wit h the corresponding regions of other Bacteroides spp. plasmids was als o studied by Southern blot hybridization. The results indicated that t he repA gene of plasmid pIP417 is homologous to that of plasmid pIP421 , but not of plasmid pIP419. The replication region of plasmid pIP421 was sequenced and showed about 80% identity at the nucleotide level wi th that of pIP417. A small (3634-bp) cloning vector (pFK12) of entirel y defined nucleotide sequence was constructed for Bacteroides spp. (C) 1995 Academic Press, Inc.