BackgroundThe population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE).Methodology/Principal FindingsThe MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size.Conclusion/SignificanceThe gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between “environmental” strains, the main contributors to the genetic diversity within the subspecies, and “domesticated” strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the “domesticated” strains essentially arose through substantial genomic flux within the dispensable genome.
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...
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