ABSTRACT
We report the draft genome sequences of Cetobacterium somerae 2G Large and two Cetobacterium isolates, 2A and 8H, which may represent novel species. The isolates were recovered from the intestines of catfish, and the genomes will assist in research to understand their potential use as probiotics in aquaculture.
ANNOUNCEMENT
The genus Cetobacterium is comprised of two described species, C. ceti (1) and C. somerae (2). Our research, among that of others, has shown a high prevalence of Cetobacterium in the gastrointestinal tracts of fish (3–6). This finding has led us to explore the role of these bacteria in the physiology of the fish gastrointestinal tract and their potential use as probiotics in aquaculture. The draft genomes reported here will assist in this research.
Pond-raised channel catfish (Ictalurus punctatus) were collected at the E. W. Shell Fisheries Center in Auburn, AL, and euthanized with 300 mg liter−1 neutral buffered Tricaine-S (Western Chemical) according to approved Auburn University IACUC protocol 2016–2946. The intestines were aseptically removed and transferred to an anaerobic chamber (Shel Lab) with 5% hydrogen, 5% carbon dioxide, and 90% nitrogen. The intestinal contents were removed, diluted 1:1 (vol/vol) with Gifu anaerobic broth (GAM; HiMedia Laboratories) containing 0.05% l-cysteine (GAM-L), and serially diluted, and then dilutions were plated on GAM agar and brucella blood agar with hemin and vitamin K (Hardy Diagnostics). The plates were incubated at 25°C for 48 h under anaerobic conditions. Colonies with typical characteristics of Cetobacterium spp. (gray, smooth, and circular) were subcultured to GAM agar. Single isolated colonies were expanded in GAM-L broth anaerobically at 25°C and cryopreserved at −80°C in 20% glycerol. Isolates were identified as Cetobacterium using partial 16S rRNA sequencing, and three were chosen for genome sequencing (2G Large, 2A, and 8H) based on phylogenetic analysis of the 16S rRNA gene sequences (data not shown).
Genomic DNA (gDNA) was extracted using a Qiagen DNeasy blood and tissue kit (Qiagen) and sent to MR DNA (Molecular Research LP, Shallowater, TX) for sequencing using a Pacific Biosciences (PacBio) Sequel sequencer. The gDNA was sheared using Covaris g-TUBEs (Covaris, Inc.), and a multiplex microbial library was prepared using a SMRTbell Express template prep kit 2.0 (PacBio). The three DNA samples were pooled in equimolar ratios prior to the final library purification, and the average fragment size was determined to be 9,160 bp. The SMRTbell library pool was then sequenced using a 10-h movie time on the PacBio Sequel, and default parameters were used for all software unless otherwise noted. De novo assembly of each genome was accomplished using the SMRT Analysis Hierarchical Genome Assembly Process (HGAP) and Falcon pipelines and then polished with Arrow (SMRT Link version 7.0). The single-pass reads were mapped against the seed reads, which averaged 15.6 kb in length across the three genomes. The average percentage of bases successfully realigned to each draft assembly was 95.5%, with a mean concordance of 88.8%. The assembled genome sequences were submitted to NCBI for annotation using the Prokaryotic Genome Annotation Pipeline (PGAP; version 4.12). The assembly and annotation metrics are shown in Table 1.
Assembly and annotation metrics
Average nucleotide identities (ANI) between isolates 2G Large, 2A, and 8H and the genomes of C. somerae ATCC BAA-474T (NCBI accession no. AXZF00000000) and C. ceti ATCC 700028T (NCBI accession no. FUWX00000000) were calculated using the OrthoANIu algorithm at EZBioCloud (7). Genome distances revealed that isolate 2G Large had high genomic similarity to C. somerae (98.57%). The ANI values obtained for comparisons of all other genomes were less than 80%, well below the 95 to 96% threshold used to distinguish between members of closely related species (8, 9), suggesting that isolates 2A and 8H represent two novel species in the genus Cetobacterium.
Data availability.The draft genome sequences and raw read sequences for the three strains have been deposited in GenBank and SRA under the accession numbers listed in Table 1.
ACKNOWLEDGMENTS
Funding for this research was provided through a Non-Assistance Cooperative Agreement between Auburn University and the United States Department of Agriculture–Agricultural Research Service (project no. 6010-32000-027-04-S; Examining the interrelationships between virulent Aeromonas hydrophila and the microflora of farmed catfish). The 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.
FOOTNOTES
- Received 27 August 2020.
- Accepted 7 October 2020.
- Published 29 October 2020.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
