Computational protocol: Complete Genome Sequence of Nonagglutinating Lactococcus garvieae Strain 122061 Isolated from Yellowtail in Japan

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Protocol publication

[…] Lactococcus garvieae is a Gram-positive, α-hemolytic, nonmotile, and non-spore-forming ovoid coccus that forms short chains and white colonies. In aquaculture, yellowtail infected with L. garvieae show high mortality (). A protective vaccine containing formalin-inactivated cells, which can be injected or orally administered, was developed for the genus Seriola, dramatically decreasing the damage caused by L. garvieae infection (, ). In Japan, a slide agglutination test with rabbit antiserum is routinely used to identify L. garvieae isolated from fish ().Since 2012, L. garvieae, which does not agglutinate with regular antiserum, has been isolated from vaccinated yellowtail (Seriola quinqueradiata). The isolates were identified as nonagglutinating L. garvieae (). The biochemical properties and morphological characteristics of nonagglutinating L. garvieae were the same as those of typical L. garvieae (). However, nonagglutinating L. garvieae differed from typical L. garvieae in terms of the susceptibility to lytic phage and antigenicity of autoclave-extracted cellular antigens (). Interestingly, the protective effect of a commercially available vaccine against nonagglutinating L. garvieae was found to be limited (). Thus, nonagglutinating L. garvieae is one of the most dangerous pathogens of fish farms in Japan. To comprehensively analyze the genome of nonagglutinating L. garvieae, we determined the complete genome sequence of nonagglutinating L. garvieae strain 122061.L. garvieae strain 122061 was isolated from vaccinated yellowtail in 2012 and grown in Todd Hewitt Broth (Difco, Sparks, MD, USA) at 25°C for 24 h. Genomic DNA was extracted using the Maxwell Cell DNA purification kit (Promega, Madison, WI, USA) and used to construct SMRTbell sequencing libraries (Pacific Biosciences, Menlo Park, CA, USA). The whole-genome sequence was determined using PacBio RS II (Pacific Biosciences). De novo assembly using Hierarchical Genome Assembly Process 3 (PacBio DevNet; Pacific Biosciences) generated two circular contigs composed of a chromosome and a plasmid. Functional annotation was performed using Prokka (http://www.vicbioinformatics.com/software.prokka.shtml). The chromosomal genome was 1,985,694 bp with a G+C content of 38.2%. The plasmid was 14,414 bp with a G+C content of 34.7%. Prokka predicted a total of 2,036 genes including 1,961 coding genes, 16 rRNA genes, and 59 tRNA genes in the chromosomal genome of nonagglutinating L. garvieae strain 122061. The plasmid was predicted to contain 16 coding genes. The average nucleotide identity (ANI) between the typical L. garvieae strain lg2 (AP090129) and nonagglutinating L. garvieae strain 122061 was estimated at 90.7% by the ANI calculator (http://enve-omics.ce.gatech.edu/ani/index). Here, we report the complete genome sequence of nonagglutinating L. garvieae for, to our knowledge, the first time. This genome sequence can be used to comparatively analyze the genomes of different L. garvieae strains. […]

Pipeline specifications

Software tools Prokka, ANI Calculator
Applications WGS analysis, Nucleotide sequence alignment
Organisms Lactococcus garvieae