Abstract
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.
ACKNOWLEDGMENTS
This work was supported by the College of Veterinary Medicine, Mississippi State University, and by USDA ARS CRIS project 31000-012-00D.
Footnotes
Citation Kumru S, Tekedar HC, Waldbieser GC, Karsi A, Lawrence ML. 2016. Genome sequence of the fish pathogen Flavobacterium columnare genomovar II strain 94-081. Genome Announc 4(3):e00430-16. doi:10.1128/genomeA.00430-16.
REFERENCES
- 1.Bernardet JF, Segers P, Vancanneyt M, Berthe F, Kersters K, Vandamme P. 1996. Cutting a Gordian Knot: emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis Norn. Nov. (basonym, Cytophaga aquatilis Strohl and Tait 1978). Int J Syst Bacteriol 46:128–148. doi: 10.1099/00207713-46-1-128. [DOI] [Google Scholar]
- 2.Bernardet JF, Grimont PAD. 1989. Deoxyribonucleic acid relatedness and phenotypic characterization of Flexibacter columnaris sp. nov., nom. rev., Flexibacter psychrophilus sp. nov., nom. rev., and Flexibacter maritimus Wakabayashi, Hikida, and Masumura 1986. Int J Syst Bacteriol 39:346–354. [Google Scholar]
- 3.Davis HS. 1922. A new bacterial disease of fresh-water fishes. Bull US Bur Fish 38:261–280. [Google Scholar]
- 4.Arias CR, Welker TL, Shoemaker CA, Abernathy JW, Klesius PH. 2004. Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish. J Appl Microbiol 97:421–428. doi: 10.1111/j.1365-2672.2004.02314.x. [DOI] [PubMed] [Google Scholar]
- 5.Shoemaker CA, Olivares-Fuster O, Arias CR, Klesius PH. 2008. Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus). Vet Microbiol 127:353–359. doi: 10.1016/j.vetmic.2007.09.003. [DOI] [PubMed] [Google Scholar]
- 6.Triyanto, Wakabayashi H. 1999. Genotypic diversity of strains of Flavobacterium columnare from diseased fishes. Fish Pathol 34:65–71. doi: 10.3147/jsfp.34.65. [DOI] [Google Scholar]
- 7.Darwish AM, Ismaiel AA. 2005. Genetic diversity of Flavobacterium columnare examined by restriction fragment length polymorphism and sequencing of the 16S ribosomal RNA gene and the 16S-23S rDNA spacer. Mol Cell Probes 19:267–274. doi: 10.1016/j.mcp.2005.04.003. [DOI] [PubMed] [Google Scholar]
- 8.Tekedar HC, Karsi A, Gillaspy AF, Dyer DW, Benton NR, Zaitshik J, Vamenta S, Banes MM, Gülsoy N, Aboko-Cole M, Waldbieser GC, Lawrence ML. 2012. Genome sequence of the fish pathogen Flavobacterium columnare ATCC 49512. J Bacteriol 194:2763–2764. doi: 10.1128/JB.00281-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Soto E, Mauel MJ, Karsi A, Lawrence ML. 2008. Genetic and virulence characterization of Flavobacterium columnare from channel catfish (Ictalurus punctatus). J Appl Microbiol 104:1302–1310. doi: 10.1111/j.1365-2672.2007.03632.x. [DOI] [PubMed] [Google Scholar]
- 10.Berlin K, Koren S, Chin CS, Drake JP, Landolin JM, Phillippy AM. 2015. Assembling large genomes with single-molecule sequencing and locality-sensitive hashing. Nat Biotechnol 33:623–630. doi: 10.1038/nbt.3238. [DOI] [PubMed] [Google Scholar]
- 11.Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R. 2014. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST) Nucleic Acids Res 42:D206–D214. doi: 10.1093/nar/gkt1226. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Angiuoli SV, Gussman A, Klimke W, Cochrane G, Field D, Garrity G, Kodira CD, Kyrpides N, Madupu R, Markowitz V, Tatusova T, Thomson N, White O. 2008. Toward an online repository of Standard Operating Procedures (SOPs) for (Meta)genomic annotation. J Integr Bio 12:137–141. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. 2007. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91. doi: 10.1099/ijs.0.64483-0. [DOI] [PubMed] [Google Scholar]