IncN plasmid replicon. A deletion and subcloning analysis.

A DNA segment of approximately 2000 base-pairs bounded by restriction enzyme sites for PvuII and containing the minimal replicon of an N group plasmid was characterized. A natural derivative of this miniplasmid was found to have undergone a deletion within one of two tandem iteron families, the group I iterons. Further analysis showed that all plasmid-determined functions essential for stable maintenance in Escherichia coli were localized to a contiguous region of DNA of 1019 nucleotides that excludes entirely these iterons. However, the loss of these iterons led to an increase in plasmid copy number. This indicates that members of the group I iteron-family have a role in determining plasmid copy number perhaps by titrating a plasmid-specified trans-acting product. The 2000 base-pair segment contains six open reading frames of 40 or more amino acid residues. The essential segment contains a 368 nucleotide region that must be present in cis and within which there are three "GATC" sequences and a putative Escherichia coli DnaA protein-binding sequence (dnaA box). An interesting feature is that the cis-acting region is present entirely within a presumptive rep gene. The essential segment contains four open reading frames, only one of which has an Escherichia coli canonical ribosome-binding site. The 2000 base-pair miniplasmid has two separable regions determining N group plasmid incompatibility.