Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4

Abstract

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3 end of a tRNA Ser gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA Ser gene. In the other bacterial species where this tRNA gene is less or not conserved, secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5 end of the core in the strand exchange reaction.The possibilities for genetic manipulation of the lactobacilli, a group of organisms of considerable economic importance, are limited by the lack of genetic tools such as efficient gene transfer systems and vectors for stable maintenance of genes.Integrative vectors are well adapted for chromosomal integration of cloned DNA sequences. Bacteriophage site-specific integration elements have been used to develop integrative vectors for multiple species such as Escherichia coli (3), Mycobacterium spp. (26, 45), Staphylococcus aureus (25), Arthrobacter aureus (28), Lactococcus lactis (12, 46), and Lactobacillus gasseri (37). Wide-spectrum integrative vectors for actinomycetes have also been constructed by using the integrative function of Streptomyces plasmid pSAM2 (1,31,32,43).We previously characterized the genetic elements required for the integration of the temperate phage mv4 into the Lactobacillus delbrueckii subsp. bulgaricus chromosome (15). This integration occurs by specific recombination between the phage attachment site attP and the bacterial attachment site attB. These two sites have a 17-nucleotide (nt)-long identical sequence defined as the core, within which the crossover takes place during the recombination event. This mechanism follows the Campbell model (8) and is catalyzed by the phage-encoded recombinase belonging to the integrase family. Analysis of the attB site revealed that mv4 DNA integrates into a tRNA gene (tRNA Ser ), a phenomenon common to many bacterial species (4,6,7,16,19,21,26,32,33,38,47). A nonreplicative vector, pMC1, bearing the phage attP site ...

Site-Specific Integrative Elements of Rhizobiophage 16-3 Can Integrate into Proline tRNA (CGG) Genes in Different Bacterial Genera

“…Considering the conservative nature of tRNAs, one can expect that the target sites for a particular integrative system could occur in several bacterial species at various levels of relatedness. This view was confirmed by well-documented examples: the site-specific recombination systems of Ms6, mv4, VWB, pSAM2, pSE101, and pSLP1 were shown to function in closely related bacteria (3,5,19,39,51,52). Moreover A2 provides an example of integration in both gram-negative and gram-positive species (2).…”

Prophage Genomics

“…A similar observation was made for prophages 8232.5 and 315.5. Such low specificity of the phage integrase with respect to conserved nucleotides in the core sequence was also observed in other phages from LAB (S. thermophilus phage Sfi21 [33] and Lactobacillus phage mv4 [5]). It is therefore unlikely that the competition for integration sites is a limiting factor in the establishment of polylysogenic cells.…”

“…L. delbrueckii prophage mv4, for which closely related virulent phages were also described (142,208), became the focus for research into the site specificity of the integration system (4,5). Lactobacillus gasseri phage A2, in comparison, is the bestcharacterized LAB phage with respect to its DNA-packaging mechanism (75).…”

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).…”