Genome analysis of Lactococcus garvieae

Lactococcus garvieae is a pathogen that causes septicemia in fish and serious damage to fish aquaculture worldwide (Vendrell et al., 2006). This pathogen has also been found in domestic animals, in humans associated with different tissue infections (Li et al., 2008) and in various food matrices, as artisanal cheeses, meat, vegetables and cereals, sometimes as a major component (Fortina et al., 2003; Ferrario et al., 2012). Despite its widespread distribution and emerging clinical significance, little is known about the genetic content of this microorganism. Recently, a molecular polyphasic approach comprising PCR-rybotyping, REP and RAPD-PCR analyses and MLRT revealed high variability, with the separation of L. garvieae population in two independent genomic lineages, not entirely coherent with the strains niche of isolation (Ferrario et al., 2012). In order to improve our knowledge about the genetic heterogeneity within the species, we performed the genome sequencing of two strains, representative of the two main genomic lineages previously obtained, using a whole-genome shotgun strategy with an Illumina Genome Analyzer Hiseq 1000. These strains revealed similar genome size (2,014,328 and 2,087,705 bases) a similar number of predicted genes (about 2,000 CDSs and 42 tRNAs) and similar G+C content (38.3%). The newly sequenced genomes and the genomes of five L. garvieae strains already available in databases, were used for subsequent analyses. The seven strains belong to different Sequence Types (ST), as determined from recent Multi Locus Sequence Type (MLST) studies and, likely, represent the genetic diversity of the L. garvieae species. The information derived from the comparison of the seven genomes suggested that this species can be described by its “pan-genome”, which includes a core genome containing genes present in all strains and a “dispensable” genome. The latter is composed of genes absent from one or more strains and genes that are unique to each strain, such as genes involved in sucrose and lactose fermentation. It is interesting to note that gene clusters involved in exopolysaccharide/capsule biosynthesis, correlated to pathogenicity in fish, were part of dispensable genome, and were only present in some strains, among those isolated from fish. Aside from the capsule gene clusters, we identified other possible virulence genes, as candidate genes encoding haemolysins and cell surface adhesins. For these potential virulence factors we carried out Real-time quantitative PCR experiments to evaluate their differential expression in conditions simulating environmental colonization and infection sites. Many shared genes fall into the class of hypothetical proteins and protein of unknown function, suggesting that many aspects of basic L. garvieae biology still need to be explored.