ABSTRACT
Flavobacterium covae is one of four Flavobacterium spp. that cause columnaris disease in teleost fish. Here, we report the draft genomes of two isolates, LSU-066-04 and LV-359-01, and their predicted virulence factors.
ANNOUNCEMENT
The fish pathogen Flavobacterium columnare was previously organized into four genetic groups, but recent phylogenetic analysis has shown that F. columnare encompasses four separate species, all of which are etiologic agents of columnaris disease (1–3). In 2004, the bacterial isolate LSU-066-04 was cultured from a largemouth bass showing symptoms of columnaris disease from the Louisiana Fish and Game Hatchery in Forest Hill, Louisiana (4). The isolate LV-359-01 was cultured from a channel catfish (Ictalurus punctatus) production farm experiencing a columnaris outbreak in Lake Village, Arkansas in 2001. Both isolates were initially described as F. columnare genomovar II using methods from Arias et al. and were shown to be virulent in channel catfish disease models (4, 5).
From cryogenic stocks, cultures of both isolates were grown overnight in 10 mL of modified Shieh medium at 28°C with shaking (6). Genomic DNA was isolated using an E.Z.N.A. bacterial DNA isolation kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s protocol. Genomic DNA was quantified using AccuGreen fluorescence on the Qubit 3.0 fluorometer using the double-stranded DNA (dsDNA) high sensitivity program (Biotium, Fremont, CA, USA). Paired-end reads of 250 bp were generated using Illumina Nextera with indexed fragment libraries on an Illumina NovaSeq 6000 sequencer (Novogene, Durham, NC, USA). End trimming, error correction, and contig assembly were conducted using CLC Genomics Workbench 20.0 (Qiagen, Aarhus, Denmark). Contig sequences of >1 kb were uploaded to KBase, and CheckM was used to assess genome purity. Genome annotation was conducted using Rapid Annotation using Subsystem Technology (RAST) v. 2.0 (7, 8). Using the published genomes, average nucleotide identities (ANI) and DNA-DNA hybridization estimate comparisons for each type strain of F. columnare (accession number PCMX01000000), F. covae (accession number CP067379), F. davisii (accession number CP067378), and F. oreochromis (accession number CP067377) were calculated using OrthoANI algorithm in the Orthologous Average Nucleotide Identity Tool (OAT) and Genome-to-Genome Distance Calculator (GGDC) 3.0 (Leibniz Institute DSMZ, Braunschweig, Germany) (9, 10). LSU-066-04 had a genome size of 3.07 Mb with 100 contigs with an N50 of 75,071, G+C% content of 30.4%, 2,724 coding sequences, and 64 total RNAs. LV-359-01 had a genome size of 3.14 Mb with 95 contigs with an N50 of 70,015, G+C% content of 30.5%, 2,832 coding sequences, and 62 total RNAs. Both isolates had ANI values of >99% to Flavobacterium covae AL-02-36Tand DNA-DNA hybridization estimates of >95%. The ANI and GGDC values to F. columnare ATCC 23463T were 92 to 93% and 4 to 11%, indicating that both strains belong to the species F. covae (1, 11, 12).
To predict the presence of virulence factors in F. covae LSU-066-04 and F. covae LV-359-01, a tBLASTx analysis of each genome was conducted with previously described virulence factors associated with columnaris disease. Interestingly, both F. covae isolates were predicted to carry all of the genes previously determined to have a role in columnaris disease, with high percent similarities to each gene (Table 1). These predicted virulence factors included genes associated with T9SS, denitrification, and anaerobic growth. It is likely that there are many other virulence factors expressed by these F. covae strains, but their functional roles in virulence have not yet been determined. The genome sequences for these two virulent strains LSU-066-04 and LV-359-01 will provide a resource to explore the specific factors that contribute to F. covae virulence and contrast this with virulence factors expressed by the other Flavobacterium spp. that cause columnaris disease.
TABLE 1.
Virulence factors predicted to be produced by F. covae LSU-066-04 and F. covae LV-359-01 based on previous studies; percent similarities are to F. covae AL-02-36T
| Gene | Reference | Strain(s) used | Flavobacterium species | LSU-066-04 percent similarity; E value; query cover | LV-359-01 percent similarity; E value; query cover |
|---|---|---|---|---|---|
| porV | 13, 14 | C#2 and MS-FC-4 | F. covae and F. columnare | 99.29%; 0.0; 100% | 99.29%; 0.0; 100% |
| gldN | 13, 14 | C#2, IA-S-4, and MS-FC-4 | F. covae, F. columnare, and F. columnare | 98.67%; 0.0; 100% | 98.67%; 0.0; 100% |
| gldK | 13 | C#2 and IA-S-4 | F. covae and F. columnare | 99.78%; 0.0; 100% | 100.00%; 0.0; 100% |
| gldL | 13 | C#2 and IA-S-4 | F. covae and F. columnare | 100.00%; 4e−116; 100% | 100.00%; 4e−116; 100% |
| gldM | 13 | C#2 and IA-S-4 | F. covae and F. columnare | 99.50%; 0.0; 100% | 99.50%; 0.0; 100% |
| sprA | 13 | C#2 and IA-S-4 | F. covae and F. columnare | 99.36%; 0.0; 100% | 99.84%; 0.0; 100% |
| sprE | 13 | C#2 and IA-S-4 | F. covae and F. columnare | 97.44%; 0.0; 100% | 97.18%; 0.0; 100% |
| sprT | 13 | C#2 and IA-S-4 | F. covae and F. columnare | 99.15%; 6e−163; 100% | 99.15%; 6e−163; 100% |
| nirS | 15 | 94-801 | F. covae | 100.00%; 0.0; 100% | 100.00%; 0.0; 100% |
| cylA | 15 | MS-FC-4 | F. columnare | 100.00%; 0.0; 100% | 100.00%; 0.0; 100% |
| C6N29_11540 | 14 | MS-FC-4 | F. columnare | 100.00%; 0.0; 100% | 100.00%; 0.0; 100% |
| C6N29_11545 | 14 | MS-FC-4 | F. columnare | 99.44%; 0.0; 100% | 99.37%; 0.0; 100% |
Data availability.
This Whole Genome Shotgun project for both LSU-066-04 and LV-359-01 has been deposited at GenBank under the accession numbers JALDSR000000000 and JALDSS000000000, respectively. The versions described in this paper are versions JALDSR010000000 and JALDSS010000000, respectively. LSU-066-04 is under the BioSample accession number SAMN26856626, and LV-359-01 is under the BioSample accession number SAMN26856627. Both strains are under BioProject accession number PRJNA818358 with the SRA accession numbers SRX15461022 and SRX1461023.
ACKNOWLEDGMENTS
The USDA is an equal opportunity provider and employer. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture. This work was supported through USDA-ARS Research Project #6010-32000-027-014-D and Non-Assistance Cooperative Agreement #6010-32000-027-000-S.
Contributor Information
Mark R. Liles, Email: lilesma@auburn.edu.
Frank J. Stewart, Montana State University
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Associated Data
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Data Availability Statement
This Whole Genome Shotgun project for both LSU-066-04 and LV-359-01 has been deposited at GenBank under the accession numbers JALDSR000000000 and JALDSS000000000, respectively. The versions described in this paper are versions JALDSR010000000 and JALDSS010000000, respectively. LSU-066-04 is under the BioSample accession number SAMN26856626, and LV-359-01 is under the BioSample accession number SAMN26856627. Both strains are under BioProject accession number PRJNA818358 with the SRA accession numbers SRX15461022 and SRX1461023.