A Cryptic Plasmid from Stutzerimonas stutzeri DIA-8 Provides a Basis for Shuttle Vector Development in Escherichia coli and Pseudomonas putida

We characterized the cryptic plasmid pM27181 (15,416 bp) isolated from the Stutzerimonas stutzeri strain DIA-8. Sequencing and bioinformatic analysis allowed identification of 20 open reading frames, including genes involved in replication (rep), mobilization (relaxase/mobilization protein), segregation (parA, parG), and site-specific recombination (XerC-like protein), as well as the putative Csr/Rsm regulatory module. Deletion analysis and random ligation yielded three minireplicons: pEBNT-1-K, pEBNT-2, and pEBNT-3. Each contains a replication module comprising a rep gene and an iteron-containing region. All three variants replicated autonomously in both Escherichia coli and Pseudomonas putida. Loss of one of the two iterons in variant pEBNT-3 relative to pEBNT-2 has a host-dependent effect. In P. putida, this deletion increased plasmid copy number, possibly due to attenuation of negative feedback, but reduced segregational stability. In E. coli cells the deletion decreased copy number without a corresponding decrease in segregational stability. pEBNT-1-K retained mobilization activity, whereas no mobilization was detected for pEBNT-2 and pEBNT-3 under the conditions tested, suggesting a reduced risk of horizontal dissemination. These findings suggest that replication control is host-dependent and may involve interaction among the Rep protein, iteron repeats, and intracellular factors. The minireplicons provide a basis for developing genetic tools for pseudomonads and a model for studying plasmid replication in different bacterial hosts.