Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites

Abstract

The temperate bacteriophage +adh integrates its genome into the chromosomal DNA ofLactobaciUus gasseri ADH by a site-specific recombination process. Southern hybridization analysis of BclI-digested genomic DNA from six relysogenized derivatives of the prophage-cured strain NCK102 displayed phage-chromosomal junction fragments identical to those of the lysogenic parent. The +adh attachment site sequence, attP, was located within a 365-bp EcoRI-HindIII fragment of phage 4adh. This fragment was cloned and sequenced. DNA sequence analysis revealed striking features common to the attachment sites of other site-specific recombination systems: five direct repeats of the sequence TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived from the sequence of the aftP-containing fragment enabled us to amplify predicted junction fragment sequences and thus to identify attL, attR, and attB. The core region was defined as the 16-bp sequence TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific insertion of phage +adh were located in a 4.5-kb BcII fragment. This fragment was cloned in plasmid pSA34 to generate the insertional vector pTRK182. Plasmid pTRK182 was introduced into L. gassedi NCK102 by electroporation. Hybridization analysis showed that a single copy of pTRK182 had integrated at the attB site of the NCK102 erythromycin-resistant transformants. This is the first site-specific recombination system described in lactobacilli, as well as the first attP-based site-specific integration vector constructed for L. gassed ADH.Programmed DNA rearrangements mediated by site-specific recombination are described in several biological phenomena, including, among others, integration and excision of temperate bacteriophages (1,4,17,19,21,42) and Streptomyces spp. plasmids (5, 6, 23, 25, 37) into chromosomal DNA, DNA inversion controlling gene expression (12,36), control of plasmid amplification of the 2,um plasmid in Saccharomyces cerevisiae (9), dimer resolution and stable propagation of phage P1 (2), and resolution of cointegrates structures formed by transposons . The hallmarks of all of these site-specific recombination systems are that DNA exchange occurs between two specialized, short DNA sequences and that general recombination function(s) are not required.Integrative recombination of phage lambda provides the classical example of site-specific integration and excision of a temperate bacteriophage (42). Extensive studies of this system have allowed the characterization of its components in great detail and definition of the mechanism and regulatory circuits involved in the recombination process. This information provides a model which has facilitated the characterization of other genetic elements encoding sitespecific recombination systems.Lysogeny within the gram-positive, rod-shaped lactobacilli has been extensively reported (10,31 Lactobacillus gassedi ADH, has a hexagonal head and a noncontractile tail, and its genome is a linear doublestranded DNA molecule of 43 kb (27). Genetic and physical ...

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