Use of the Integration Elements Encoded by the Temperate Lactococcal Bacteriophage TP901-1 To Obtain Chromosomal Single-Copy Transcriptional Fusions in Lactococcus lactis

Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of theorf1 gene (presumably encoding a promoter), and theattP region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome ofLactococcus lactis subsp.cremoris (B. Christiansen, L. Brøndsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164–5173, 1996). In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when theorf1 gene is separated from theattP region, the Orf1 protein is able to promote site-specific integration of anattP -carrying plasmid into theattB site on theL. lactis subsp.cremoris chromosome. Furthermore, the first detailed deletion analysis of anattP region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of theattP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome ofL. lactis subsp.cremoris when theorf1 gene is donated intrans . The functional 56-bpattP region of TP901-1 is substantially smaller than minimalattP regions identified for other phages. Based on the deletion analysis, several repeats located within theattP region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (attP andorf1 ) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions inL. lactis was constructed. Two promoter-reporter integration vectors containing the reporter genegusA orlacLM , encoding β-glucuronidase or β-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 suitable for cloning of the promoter of interest. By transformation ofL. lactis subsp.cremoris MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment siteattB used by bacteriophage TP901-1.