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

Abstract

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 (B. Christiansen, L. Brondsted, 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 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 reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest. By transformation of L. 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 site attB used by bacteriophage TP901-1.

FIG. 1

Sequence of the attP region of the temperate bacteriophage TP901-1. The bold sequence shows the coding region for the integrase of TP901-1 (Orf1). The core region is boxed, and the identical region is underlined. Direct and inverted repeats are indicated with black arrows above the sequence. Repeats identified by Christiansen et al. (8) are R1, R2, R3, R4, R5, and R6, whereas the recently identified inverted repeat is P1. The numbers and small arrows under the sequence indicate the borders of the different attP fragments. The largest attP fragment contains all bases shown, e.g., 1 to 333. Base 1 corresponds to base 2499 in the sequence deposited in GenBank.

FIG. 2

Deletion analysis of the attP region of the temperate bacteriophage TP901-1. For each plasmid, the nucleotides of the cloned attP fragment are indicated by numbers, which correspond to the numbers in Fig. 1. The cloned attP fragment is indicated by a black line, and the ability of each fragment to promote site-specific integration of the plasmid is indicated (yes or no). The large black arrow indicates the 3′ end of the orf1 gene, the black box indicates the core region, and small arrows indicate direct and inverted repeats.

FIG. 3

(A) Structures of the promoter-reporter integration vectors pLB85 and pLB86. The direction of transcription is indicated by arrows. The black box indicates the 207-bp attP region of TP901-1. (B) Results of site-specific integration of the promoter-reporter vectors in the chromosome of L. lactis subsp. cremoris. The multiple cloning sites (mcs) are indicated by horizontal lines, and the direction of transcription is indicated by arrows. The black box indicates the attP part of attL and attR of TP901-1, and the thin black arrow indicates the orientation of transcription of chromosomal genes located within the attB region (8).