Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum

Abstract

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3 end of a tRNA Ser gene. Phage mv4 DNA integration into the tRNA Ser gene preserved an intact tRNA Ser gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA Ser gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present.Gram-positive Lactobacillus strains are extensively used as preservatives in food products and as lactic acid producers in dairy fermentations, but development of bacteriophages is the main cause of fermentation failures. Taxonomic studies on phages from Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis led to the determination of two genetic groups (21, 32). The phage representative of the most widespread group, bacteriophage mv4, has been well characterized (13). Phage mv4 is a temperate phage which infects and lysogenizes L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains. The 36-kb genome of the phage has been mapped physically, and its DNA is circularly permuted (20). Several genes have been characterized, such as those encoding structural proteins (52) or genes involved in cell lysis (6). The phage attachment site (attP) has previously been located on the mv4 genome, and several attachment sites on the chromosome of independently isolated lysogens have been identified (20).Most temperate bacteriophages integrate their DNA into the host chromosome by a site-specific recombination process following the Campbell model (10). This mechanism involves two specific attachment sites, one on the bacterial chromosome (attB) and the other one on the phage genome (attP). The recombination process is catalyzed by a phage-encoded integrase. There are many well-characterized examples of sitespecific recombination in gram-negative bacteriophages, and the best-studied system is that of bacteriophage (for a review, see reference 22). The integration system of phages of grampositive bacteria is less well documented, but data on sitespecific recombination are available for phages L54a, 11, and 13 of Staphylococcus aureus (14,25,55,56), for mycobacteriophage L5 (26), and for phages of...

MM1, a Temperate Bacteriophage of the Type 23F Spanish/USA Multiresistant Epidemic Clone of Streptococcus pneumoniae : Structural Analysis of the Site-Specific Integration System

“…There are well-characterized examples of site-specific recombination in gram-negative bacteria, especially that of bacteriophage (30). Although the integration system of phages of gram-positive bacteria is less well documented, data are available for several phages of S. aureus (11,31,59), for bacteriophage T12 of Streptococcus pyogenes (36), for the actinophage RP3 (18), and for several lactic acid bacterial phages (8,14,42,57). We have now identified the attP-containing phage DNA, the bacterial attachment site attB, and the host-phage junctions attL and attR of the MM1 prophage.…”

Genome Evolution at the Genus Level: Comparison of Three Complete Genomes of Hyperthermophilic Archaea

“…This supports the existence of a common mode of DNA integration within tRNA genes between the Crenarchaea and the Euryarchaea. In Eubacteria, some tRNA genes can constitute the integration site of plasmids and phages (Reiter et al 1989;Dupont et al 1995) and may also be the target site of recombination of pathogenicity islands (Hou 1999). Thus, the involvement of tRNA genes in sitespecific recombination appears as a widespread mechanism able to promote integration of various autonomous genetic elements.…”

Bacteriophage mv4 Site-Specific Recombination: the Central Role of the P2 mv4 Int-Binding Site

“…The site-specific integration system of the Lactobacillus delbrueckii mv4 bacteriophage has been characterized (5,6). The mv4 integrase has no absolute requirement for accessory factors (7).…”