Cloning of pAhX22, a small cryptic plasmid from Aeromonas hydrophila, and construction of a pAhX22-derived shuttle vector

In this study, a cryptic plasmid from Aeromonas hydrophila (pAhX22) was cloned and characterized. pAhX22 was 2523 bp long, had a GC content of 59.9%, and contained two putative open reading frames (ORFs). orf1 and orf2 encoded putative proteins of 458 amino acids and 88 amino acids, respectively; these putative proteins might be involved in plasmid replication. An Escherichia coli–A. hydrophila shuttle vector, pAEsv-1 (4587 bp, Kan^R), was constructed using in-fusion cloning, combining pAhX22 with the kanamycin-resistance gene and the origin of replication from E. coli expression vector pET-28a. The transformation efficiency of pAEsv-1 in A. hydrophila strains ranged from 2.2 × 10⁶ to 1.0 × 10⁷ CFU/μg DNA, while transformation efficiency in E. coli DH5α was about 1.6 × 10⁶ CFU/μg DNA. pAEsv-1 was segregationally and structurally stable in A. hydrophila in the absence of selective pressure. A green fluorescent protein gene (gfp) from pHT315-gfp was successfully cloned and expressed in A. hydrophila strain X2 using pAEsv-1, and 82.3% ± 2.5% of cells maintained the recombinant plasmid after one week in liquid culture without kanamycin. These results suggested that pAEsv-1 might potentially be used as a stable cloning vector for A. hydrophila, which might facilitate genetic studies of A. hydrophila.