HIGH-EFFICIENCY GENE INACTIVATION AND REPLACEMENT SYSTEM FOR GRAM-POSITIVE BACTERIA

A system for high-efficiency single- and double-crossover homologous i ntegration in gram-positive bacteria has been developed, with Lactococ cus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a p BR322 replicon for propagation in Escherichia coli at 37-degrees-C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 wer e used to show that the single-crossover integration frequency was log arithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we s howed that homologous integration can occur along the entire chromosom e. We made use of the reported stimulatory effect of rolling-circle re plication on intramolecular recombination to develop a protocol for ge ne replacement. Cultures were first maintained at 37-degrees-C to sele ct for a bacterial population enriched for plasmid integrants; activat ion of the integrated rolling-circle plasmid by a temperature shift to 28-degrees-C resulted in efficient plasmid excision by homologous rec ombination and replacement of a chromosomal gene by the plasmid-carrie d modified copy. More than 50% of cells underwent replacement recombin ation when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection wa s applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an ex tremely high efficiency by making use of the thermosensitive rolling-c ircle nature of the delivery vector. This procedure is applicable to n umerous gram-positive bacteria.