Use of the integration elements encoded by the temperate lactococcal bacteriophage TP901-1 to obtain chromosomal single-copy transcriptional fusions in Lactococcus lactis

Previously me showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the orf1 gene (presumably encoding a promoter), and the attP region are necessary and also sufficient for integration of the bacte riophage TP901-1 genome into the chromosome of Lactococcus lactis subsp. cr emoris (B. Christiansen, L. Brondsted, F. K. Vogensen, and K. Hammer, J. Ba cteriol, 178:5164-5173, 1996), In this work, a further analysis of the phag e-encoded elements involved in integration was performed, Here we demonstra te that even when the orf1 gene is separated from the attP region, the Orf1 protein is able to promote site-specific integration of an attP-carrying p lasmid into the attB site on the L. lactis subsp. cremoris chromosome. Furt hermore, the first detailed deletion analysis of an attP region of a phage infecting lactic acid bacteria was carried out. We show that a fragment con taining 56 bp of the attP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome oft. lactis subsp. cremoris when the orf1 gene is donated in trans, The fun ctional 56-bp attP region of TP901-1 is substantially smaller than minimal attP regions identified for other phages, Based on the deletion analysis, s everal repeats located within the attP region seem to be necessary for site -specific integration of the temperate bacteriophage TP901-1, By use of the integrative elements (attP and orf1) expressed by the temperate lactococca l bacteriophage TP901-1, a system for obtaining stable chromosomal single-c opy transcriptional fusions in L. lactis was constructed. Two promoter-repo rter integration vectors containing the reporter gene gusA or lacLM, encodi ng beta-glucuronidase or beta-galactosidase, respectively, were constructed . Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suit able for cloning of the promoter of interest. By transformation of L. lacti s subsp. cremoris MG1363 containing the integrase gene on a replicating pla smid, the promoter-reporter integration vectors integrated with a high freq uency site specifically into the chromosomal attachment site attB used by b acteriophage TP901-1.