Genome Sequence of the Fish Pathogen Flavobacterium columnare Genomovar II Strain 94-081.

Flavobacterium columnare causes columnaris disease in fresh and brackish water worldwide. F. columnare strain 94-081 was isolated from a diseased channel catfish in 1994; its genome sequence is the first completed genomovar II sequence.

GENOME ANNOUNCEMENT

Flavobacterium columnare is a long Gram-negative rod in the family Flavobacteriaceae within the Bacteroidetes phylum (1, 2). Several Flavobacterium species cause disease in fish, and F. columnare is the causative agent of columnaris disease in fresh and brackish water fish (3). F. columnare strains are grouped into three major genomovars (4–7), and within the species F. columnare, strains vary in their colony morphology and virulence in fish. Our group reported the complete genome sequence of F. columnare strain ATCC 49512, which is a representative strain from genomovar I (accession no. CP003222.2) (8). Here, we report the first complete genome sequence of a representative strain from F. columnare genomovar II, strain 94-081 (5). Strain 94-081 is virulent in healthy channel catfish (Ictalurus punctatus), which is an important aquaculture species in the United States (9).

To obtain the F. columnare strain 94-081 genome, a sequencing run was conducted on a PacBio RS II (25× coverage), and de novo assembly was conducted using the PBcR pipeline within Whole Genome Assembler version 8.2 beta (10). A mate-pair sequencing run on a MiSeq (Illumina) (5.8 million reads × 75 bp) was conducted, and these reads were mapped onto the PacBio assembly to correct sequence errors, resulting in three contigs >10 kb. Assembly analysis and primer design were conducted using Sequencher 5.3 (Gene Codes Corporation) and CLC Workbench 6.5.1 (CLC bio), and a circular chromosome sequence was obtained by long-range PCR and Sanger sequencing by primer walking.

The final chromosome had 3,321,600 bp, with 30.8% G+C content. For annotation, NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) and Rapid Annotations using Subsystems Technology (RAST) were used (11, 12). PGAAP annotation showed that the circular F. columnare 94-081 genome had 2,779 coding genes, four rRNA operons, and 74 tRNAs. In comparison, the F. columnare genomovar I strain ATCC 49512 has 3,162,462 bp, with 31.5% G+C content, 2,632 coding genes, five rRNA operons, and 74 tRNAs.

Function-based comparison of F. columnare strain 94-081 and F. columnare ATCC 49512 by RAST showed that F. columnare strain 94-081 has 34 unique protein-coding genes, including cysteine synthase, teichoic acid export ATP-binding protein TagH, retron-type RNA-directed DNA polymerase, and metal-dependent hydrolases of the β-lactamase superfamily III. F. columnare strain 94-081 has 412 unique protein-coding genes (82% are hypothetical); some of these include phage antirepressor protein, type II restriction enzyme BsuBI, and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein Cas2.

The two-way average nucleotide identity (ANI) between the strain 94-081 and ATCC 49512 genomes was 90.71%, which suggests the two strains being classified in different species (13).

Nucleotide sequence accession number.

The completed F. columnare strain 94-081 genome was submitted to GenBank under the accession no. CP013992.