PBuzz: A cryptic rolling-circle plasmid from a commensal Escherichia coli has two inversely oriented oriTs and is mobilised by a B/O plasmid

Abstract

Ampicillin, streptomycin and sulphamethoxazole resistant commensal E. coli 838-3B contains five plasmids that range in size from >90 kb to <2 kb. The resistance genes blaTEM (ampicillin), strA (streptomycin) and sul2 (sulphamethoxazole) transferred along with a B/O plasmid named p838B-R. However, three plasmids smaller than 7 kb were also found in transconjugants, suggesting that they could be mobilised by the B/O plasmid. The complete sequences of p838B-R and pBuzz, a small plasmid mobilised by p838B-R with 70% efficiency, were determined. p838B-R is 94,803 bp and contains an 8400 bp resistance island that includes the three antibiotic resistance genes. The p838B-R backbone contains a complete conjugative transfer region, including an oriT site upstream of nikAB that resembles the experimentally-defined oriT of R64. The 1982 bp pBuzz contains a rep gene and sites associated with replication that resemble those of pC194/pUB110 family rolling-circle plasmids. It also contains two, inversely oriented copies of an 84 bp sequence that differs from the oriT region in p838B-R at just 6 positions. These oriT-like sites likely explain the ability of pBuzz to co-transfer with the B/O plasmid using the NikB relaxase and NikA accessory protein encoded by p838B-R, i.e. pBuzz utilises relaxase-in trans mobilisation. Several rolling-circle plasmids related to pBuzz were found in the GenBank non-redundant nucleotide database. They contain diverse potential oriTs, including sequences similar to known oriTs found in conjugative plasmids of I-complex (I1, B/O, K, Z and I2), L or M types.