Development of an expression strategy using a lytic phage to trigger explosive plasmid amplification and gene expression.

Abstract

A novel plasmid-based expression strategy, exploiting two features of lytic bacteriophages, was developed in Lactococcus lactis. Components of this system include a phage origin of replication and phage expression signals, which were induced to high efficiency upon phage infection of the host. Phage-specific expression signals were cloned from phi 31 in a promoter-screening strategy using the lacZ gene from Streptococcus thermophilus. One clone exhibited a significant induction in beta-galactosidase production and concomitant increase in lacZ mRNA during the phi 31 infection cycle of the host. Molecular characterization of the cloned insert revealed 888 bp positioned near the phi 31 cos site. Primer extension analysis showed that transcription was induced approximately 20 min following phi 31 infection at four points, apparently organized in two sets of tandem promoters on the cloned phage insert. One of these middle phage promoters also showed a basal level of activity prior to phage infection. The phi 31 promoter lacZ cassette was cloned into a low-copy-number vector plasmid containing the phi 31 origin of replication (ori31) and the resulting low-copy-number plasmid exhibited negligible beta-galactosidase production in L. lactis. However, > 2,000 units were detected following a deliberate infection with phi 31. A control expression plasmid without ori31 could only be induced to 85 units. The combination of these phage-inducible expression signals together with ori31 functioned synergistically to drive rapid and high efficiency expression of a heterologous gene in L. lactis.