Preparation of Bacterial Plasmid DNA

Methods for isolation of small plasmids (usually cloning vehicles) from genetically characterized strains of Enterobacteriaceae (Escherichia coli and Salmonella) are well established (1 –3 ). This chapter seeks to complement them by describing reliable basic methods for detecting and isolating larger, native, plasmids from less well-characterized bacteria. The methods presented here were developed for isolating plasmids from the Gram-negative bacterium Legionella pneumophila . Like the enteric bacteria, L. pneumophila belongs to the gamma-division of the Proteobacteria (4 ), but differs markedly from them in many respects. Its DNA is AT rich (GC = 38 mole%) (5 ), and its cell wall is less susceptible to lysozyme (6 ), owing to its distinctly different structure. The protocols presented herein are similar to those used for isolating plasmids from bacteria belonging to a wide range of very different genera, e.g., Mycobacterium (7 ), Rhizobium (8 ,9 ), and may be used as the basis of techniques for plasmid isolation from yet different species.