Complete genome sequence of Chryseobacterium sp. strain KCF3-3, isolated from the body surface of channel catfish, Ictalurus punctatus

Members of the genus Chryseobacterium are rod-shaped, non-spore-forming, nonmotile Gram-negative bacteria that are found in diverse environments (1). Here, we report the complete genome sequence of Chryseobacterium sp. KCF3-3 to enhance our understanding of its ecological diversity. We isolated the strain from the body surface of channel catfish, Ictalurus punctatus in 2019 (Table 1). Mucus samples were collected from four I. punctatus specimens and cultured on Reasoner’s 2A agar plates at 25°C for 24 h to obtain the isolates. After three rounds of purification, isolates were identified by molecular phylogenetic analysis based on the 16S rRNA gene sequences. The primers used for amplification were 27 F-W (5′-AGRGTTTGATCMTGGCTCAG-3′) and 1,492 R-W (5′-GGYTACCTTGTTACGACTT-3′). The neighbor-joining methods were used to analyze the genetic relationship (2). Consequently, the strain KCF3-3, which is classified within the genus Chryseobacterium, was obtained.

The genomic DNA of strain KCF3-3 was extracted using NucleoBond HMW DNA (Macherey-Nagel) and further purified using DNA Clean Beads (MGI Tech Co., Ltd.) according to the manufacturer’s protocol. The concentration of the DNA solution was measured using the QuantiFluor dsDNA system on a Quantus Fluorometer (Promega). The DNA was fragmented to approximately 10–20 kbp using g-TUBE (Covaris) by centrifuging three times at 2,100 × g. Fragmented lengths were confirmed by measuring with a 5200 Fragment Analyzer (Agilent Technologies). The DNA library was prepared using the SMRTbell Express Template Prep Kit 2.0 according to the Procedure and Checklist instructions (Part number 101–730-400 ver. 06). Polymerase complexes were prepared by Revio Polymerase kit (PacBio), and sequencing was performed using Revio (PacBio) by Bioengineering Lab. Co., Ltd. in 2024. SMRT Link (ver. 13.0.0.207600) was used to remove overhang adaptors, and consensus sequence reads with an average quality value of less than 20 per read were removed. Filtlong (ver. 0.2.1) was used to eliminate reads shorter than 1,000 bases and yielded 18,579 reads with an N₅₀ value of 14,599 bp. De novo assembly was performed using Flye (ver. 2.9.2-b1786) (3, 4), and Bandage (ver. 0.8.1) (5) and CheckM2 (ver. 1.0.1) (6) were used for quality assessment of the assembled genome. Flye detects overlapping contig ends as circular candidates and automatically circularizes them (3). Genome annotation was performed using the Prokka software (ver. 1.14.6) (7).

The chromosome size of the strain KCF3-3 was 5,623,437 bp, and the contig was a single circularized genome. The genome coverage was 44.0×, and the GC content was 36.0%. The genome contains 4,939 coding sequences. Prokka predicted 105 RNA genes (87 tRNA genes and 18 rRNA genes). These results and other genomic information are shown in Table 1. Clusters of Orthologous Groups (COG) categories were estimated using DFAST (https://dfast.ddbj.nig.ac.jp) (8, 9), and GC contents and GCskew were calculated using GCcalc (https://github.com/WenchaoLin/GCcalc). These results were visualized using Circos (ver. 0.69) (10) (Fig. 1). All software used default parameters, unless otherwise specified.

This work was partially supported by the JST-Mirai Program of the Japan Science and Technology Agency (JST) Grant No. JPMJMI18CF and the Academic Research Grant of the Reiwa Environmental Foundation (No. JP0000008) for M.K. The authors express their gratitude to Dr. Motonori Kudou, Dr. Naoshige Izumikawa, and Dr. Hiroyuki Hamada for their collaboration in the sampling of channel catfish in Lake Kasumigaura. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics.