Segregational and structural instability of recombinant plasmid carrying genes for naphthalene degrading pathway

Abstract

The stability of recombinant plasmid carrying genes for naphthalene mineralization was determined. A strain of Pseudomonas putida capable of mineralizing naphthalene (Nap+) via salicylate (Sal+) was isolated, and all regulatory and structural genes for the whole pathway were found to be encoded on a 25 kb EcoRI fragment of an approximately 83 kb plasmid present in this strain. The 25 kb EcoRI fragment was cloned into a tetracycline- resistant (Tc(R)) cloning vector pLAFR3 and the recombinant plasmid, pRKJ3 (Nap+, Sal+, Tc(R)), thus obtained was transferred into the plasmid-free strain Pseudomonas putida KT2442 in order to test the stability of the plasmid. Plasmid pRKJ3 was found to be segregationally and/or structurally unstable, depending on the growth conditions. Two types of novel derivative strains having the phenotypes Nap-, Sal+, Tc(R) and Nap-, Sal-, Tc(R) with specific deletions of approximately 2 kb and 18 kb, respectively, were obtained.