USE OF CONTINUOUS-CULTURE FOR THE SELECTION OF PLASMIDS WITH IMPROVED SEGREGATIONAL STABILITY

In this report a method that enables the selection of stable plasmid variants and the isolation of DNA sequences that improve plasmid maintenance is described. The method is based on the principle that in populations of cells carrying derivatives of a plasmid that differ only in the level of segregational stability, when grown in a chemostat under conditions with selective pressure on the plasmid, cells that carry more stable plasmid variants will be enriched. We developed the system for Lactococcus lactis using segregationally unstable derivatives of the gram-positive theta plasmid pAM beta 1 as selection vectors. The results showed that the method is suitable for the enrichment, and subsequent purification, of three classes of plasmids with improved maintenance properties. The first class involved mutations in the pAM beta 1-derived selection plasmid. These mutations resulted in increased copy numbers, thereby rendering the plasmid segregationally more stable. The other two classes were based on the insertion of additional, stability promoting, sequences in the selection plasmids. We showed that these sequences can constitute either replication functions derived from another plasmid or functions directly involved in plasmid maintenance. The site-specific resolution function of pAM beta 1 was used as an example of the latter functions. We anticipate that the method described should have general applicability for the development of stable host-vector systems in bacteria. (C) 1995 Academic Press, Inc.