M. BILIĆ AND V. DELIĆ . 1997. A multiple-copy plasmid pPZG500 (3·8 kb) was isolated from a phytopathogenic bacterium Erwinia citreus ATCC 31623. This is the smallest plasmid so far isolated from the genus Erwinia. The plasmid was partially characterized by a set of restriction enzymes and the unique restriction sites were mapped for HindIII, EcoRI, EcoRV and XBaI, while three sites were found for BglII. Nineteen other enzymes did not cut pPZG500. By deletion analyses minimal regions required for replication (ori) and segregational stability (par) were localized on 1·4 kb EcoRV/BglII and 0·7 kb Bgl/II/EcoRI fragments, respectively. The erythromycin resistance marker (Em r ) was cloned into pPZG500 and two plasmid derivatives, pPZG502 and pPZG503, were constructed expressing erythromycin resistance as a good selective marker for recombinant selection in Erw. citreus and Escherichia coli. The segregational stability of both constructed plasmids during 90 generations in E. coli JM109 and Erw. citreus C-4 showed that plasmid pPZG503 lacking the presumptive par region was lost from the population at a higher rate. The results of this study demonstrate that plasmid pPZG500 and derivatives are suitable prerequisites for the construction of useful cloning vector(s) in the genus Erwinia.
INTRODUCTIONdevelopment of a suitable plasmid vector system for gene cloning in the genus Erwinia has been lacking and has relied The genus Erwinia comprises a number of extensively studied on artificial plasmids, mainly derivatives of pBR322, pUC8 species of bacteria. Most of them are usually associated with and others (Anderson et al. 1985;Grindley et al. 1988; plants as pathogens, saprophytes or as members of the epiHugovieux-Cotte-Pattat and Robert-Baudouy 1992; Xia et phytic flora causing 'soft-rot' and 'fireblight' diseases on comal. 1992;Suzuki et al. 1993). As recombinant DNA techmercially important plants (reviewed by Starr 1981).nology is dependent on a good vector system for the particular Recombinant DNA technology has been applied to some genus of bacteria, it was of interest to find and characterize Erwinia species making them economically useful in biocryptic plasmid(s) as a basis for construction of a possible technological processes (reviewed by Delić et al. 1989; natural and stable cloning vector. Robert-Baudouy 1991).Attention was concentrated on Erwinia citreus ATCC Many cryptic plasmids were isolated from different 31623, a fruit pathogen of mandarin oranges (Sonoyama et Erwinia strains ranging from 4·2-340 kb (Coplin et al. 1981;, and on the genetically modified organism able to Falkenstein et al. 1989;Steinberger et al. 1990;Fu et al. convert glucose directly to 2-keto-L-gulonate, a chemical 1995), but none of them were used for vector construction.precursor for vitamin C (Anderson et al. 1985; Grindley et The main reason was that most of them were uncharacterized al. 1988). The aim of this study was to characterize and at the molecular level. In spite of the relative importance, the construct versatile useful clo...