Genetic analysis of the minimal replicon of plasmid pIP417 and comparison with the other encoding 5-nitroimidazole resistance plasmids from Bacteroides spp

Abstract

The nucleotide sequence of the DNA replication origin region of a Bacteroides vulgatus plasmid, pIP417, encoding 5-nitroimidazole resistance has been determined. This region of 1934 bp presents some characteristics 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 region, three direct repeats (iterons) of 21 bp, multiple DnaA binding sites, and sites, and sites for the integration host factor (IHF). Moreover, the amino acid sequence of the pIP417 RepA protein shows similarities with those of other Rep proteins encoded by plasmids of gram-negative bacteria: pRO1600 from Pseudomonas aeruginosa; pPS10 from Pseudomonas syringae; pFA3 from Neisseria gonorrhoeae; and two cryptic plasmids from Campylobacter hyointestinalis and Butyrivibrio fibrisolvens. Although RepA can be expressed in an Escherichia coli in vitro transcription-translation assay, vectors containing the pIP417 replication origin did not replicate in E. coli. The homology of the pIP417 replication region with the corresponding regions of other Bacteroides spp, plasmids was also studied by Southern blot hybridization. The results indicated that the 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 with that of pIP417. A small (3634-bp) cloning vector (pFK12) of entirely defined nucleotide sequence was constructed for Bacteroides spp.