Use of the integration elements encoded by the temperate lactococcal bacteriophage TP901-1

Lone Brøndsted, Karin Hammer

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    Previously we 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 bacteriophage TP901-1 genome into the chromosome of Lactococcus lactis subsp. cremoris.In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate 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 plasmid into the attB site on the L. lactis subsp. cremoris chromosome. Furthermore, the first detailed deletion analysis of an attP region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of the attP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of L. lactis subsp. cremoris when the orf1 gene is donated in trans. The functional 56-bp attP region of TP901-1 is substantially smaller than minimal attP regions identified for other phages. Based on the deletion analysis, several 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 lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in L. lactis was constructed. Two promoter-reporter integration vectors containing the reporter gene gusA or lacLM, encoding beta-glucuronidase or beta-galactosidase, respectively, were constructed. Immediately upstream of both genes are found translational stop codons in all three
    Original languageEnglish
    JournalJournal of Bacteriology
    Issue number2
    Pages (from-to)752-758
    Publication statusPublished - 1999

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