Isolation and physical characterization of zymomonas plasmids

Abstract

Isolation of plasmids from two species, Zymomonas mobilis ATCC 10988 and Zymomonas anaerobia NCIB 8227 and Zymomonas strains, ZM2 and ZM6, was attempted. The purpose was to use the plasmids as vectors for cloning genes in the host organisms. The isolated plasmids were partially characterized to figure their-sizes and sites of restriction endonucleases on them. It was found that Zymomonas mobilis ATCC 10988 contained five different plasmids with the size range between $2\times10^6$ and $25\times10^6$ dalton. From Zymomonas anaerobia NCIB 8227 two types of plasmids sized around $18\times10^6$ and $2\times10^6$ dalton respectively were isolated. The larger plasmid was designated as pZA1 and the smaller one as pZA2 for the further studies. The two strains of Zymomonas mobilis, ZM2 and ZM6, however, were found to harbor only one type of plasmid which sized about $17\times10^6$ dalton. The identity of plasmids isolated from the two different strains, ZM2 and ZM6, was confirmed by the electrophoresis pattern of restriction enzyme digestion. The two plasmids, pZA1 and pZA2, were subjected to electron microscopy. By measuring their sizes through the pictures of electron microscopy, $16.9\times10^6$ and $2\times10^6$ dalton, respectively, were obtained. These values were well correlated with the sizes obtained through the electrophoresis. The responses of these two plasmids, pZA1 and pZA2 against 10 different restriction endonucleases, HindIII, EcoRI, PvuII, HaeII, HpaI, SalI, XhoI BamHI, PstI and KpnI were checked. As expected, the larger plasmid, pZA1, showed sites for all the 10 enzymes, however, the 2 megadalton plasmid, pZA2, responded to only four enzymes, HindIII, EcoRI, PvuII and HaeII. A rough restriction endonuclease map of pZA1 for HpaI and XhoI was constructed. The possibility of levan forming genes`` location on plasmids pZA1 or pZA2 was tested using spontaneously occurred strain and allyl alcohol resistant strain, but the result was negative.