Cloning vectors derived from the Pseudomonas plasmid pVS1.

The Pseudomonas plasmid pVS1, which has about seven copies, was reduced to a minimal replicon and used to construct stable gene-cloning vehicles. The host for all cloning experiments was P. aeruginosa strain PAO. Two nonmobilizable plasmids, pME260 and pME290, and one RP1-mobilizable plasmid, pME285, were constructed. The vectors pME260 (6.3 kb) and pME290 (6.8 kb) carry the Tn801 bla gene specifying carbenicillin (Cb) resistance, a good selective marker in Pseudomonas, and the Tn903 aph gene encoding kanamycin (Km) resistance, with useful restriction sites for insertional inactivation. The Mob+ vector pME285 (10.6 kb) carries the aph gene and the Tn501-derived merRTCA genes coding for mercuric ion resistance, another good selective marker in Pseudomonas. The hypothetical merD gene, which may follow the merA gene in Tn501 but is absent from pME285, appeared to be dispensable for mercuric ion resistance in P. aeruginosa. The Mob- vector pME290 could be introduced by transformation and maintained in strains of P. aeruginosa, P. fluorescens, P. putida, P. acidovorans, P. stutzeri, P. mendocina, P. cepacia, and P. syringae. The plasmid was compatible with IncP-1 and IncP-4 replicons.