Deefgea salmonis sp. nov., isolated from gills of rainbow trout (Oncorhynchus mykiss)

A Gram-stain-negative, milky white, aerobic, rod-shaped bacterium named strain H3-26T was isolated from gills of Oncorhynchus mykiss in Lhasa, Tibet Autonomous Region, PR China. Strain H3-26T grew at 4–30 °C and pH 5.0–11.0 (optimum, 25 °C and pH 7.0) with 0–1% (w/v) NaCl (optimum, 0%). The 16S rRNA gene sequence of strain H3-26T showed the highest similarity to Deefgea rivuli WB 3.4-79T (98.42%), followed by Deefgea chitinilytica Nsw-4T (96.91%). Phylogenetic analysis based on 16S rRNA genes indicated that strain H3-26T was a new member of the genus Deefgea. The digital DNA–DNA hybridization and average nucleotide identity values between the genome sequence of strain H3-26T and Deefgea spp. were 21.2–21.9% and 76.3–77.4%, respectively. The genomic DNA G+C content of strain H3-26T was 48.74%. The predominant fatty acids were C12:0, C14:0, C16:0 and C16:1 ω7c. Based on phenotypic, phylogenetic, and genotypic data, strain H3-26T is considered to represent a novel species of the genus Deefgea, for which the name Deefgea salmonis sp. nov. is proposed. The type strain is H3-26T (= JCM 35050T = CICC 25103T).


Introduction
The genus Deefgea, a member of the family Neisseriaceae, which were mainly isolated from water and fish. Deefgea spp. may be related to fish's diseases and are a component of intestinal microflora in fish (Jeon et al. 2017;Shtykova et al. 2018;Terova et al. 2021). Up until now, only two validly species (Deefgea rivuli WB 3.4-79 T and Deefgea chitinilytica Nsw-4 T ) and one draft genome sequence (Deefgea sp. CFH1-16) were published (Stackebrandt et al. 2007;Chen et al. 2010;Han 2021). The physiological and biochemical characteristics, phylogenetic and genotypic data of genus Deefgea were lack of systematic understanding (Jung and Jung-Schroers. 2011). During the investigation of pathogenic microorganism of rainbow trout in Lhasa, a milky white bacterium, named strain H3-26, was isolated from the gills of Oncorhynchus mykiss. Genomic, phylogenetic and phenotypic data obtained from strain H3-26 support the definition of a new Deefgea species, for which the name Deefgea salmonis sp. nov. is proposed.

Isolation and cultivation of strain H3-26 T
In June 2020, a study of pathogenic microorganism of farmed rainbow trout in Lhasa led to the isolation of a strain of Deefgea.

Phylogenetic analysis based on 16S rRNA gene
Genomic DNA of strain H3-26 T was extracted using Min-iBEST Bacterial Genomic DNA Extraction Kit Version 2.0 (TaKaRa Biotechnology Co., Tokyo, Japan). Amplification of the 16S rRNA gene was performed using the extracted highly purified genomic DNA as a template under the following conditions: 95 °C for 10 min, followed by 94 °C for 45 s, 56 °C for 45 s, and 72 °C for 90 s for 30 cycles with a final 10 min extension at 72 °C, the PCR products were detected by agarose gel electrophoresis and then sent to GENEWIZ.lnc for sequencing. Primers used for amplification and sequencing of 16S rRNA were 27F/1492R (Lane 1991). The 16S rRNA gene was aligned in EzBioCloud (Yoon et al. 2017a, b). Maximum-likelihood, neighbor-joining and maximum-evolution trees were constructed using MEGA7.0 software with bootstrap values of 1000 replicates (Felsenstein 1985;Kumar et al. 2016).

Genome sequencing and comparative genomic analysis
Deefgea chitinilytica LMG 24817 T was obtained from Laboratory of Microbiology, Ghent University (LMG) for genome sequencing. The genomic DNA of strain H3-26 T and Deefgea chitinilytica LMG 24817 T was sequenced with BGISEQ-500 platform in China Center of Industrial Culture Collection. The genomic sequence information of H3-26 T and Deefgea chitinilytica Nsw-4 T had been submitted to the National Centre for Biotechnology Information (NCBI) database under the accession number JAJAWG000000000 and WOFE00000000. Draft genome assemblies were prepared from the ONT reads using Apades v3.11.0, gene prediction using Glimmer 3.02 software. Based upon the close relationship with the test strain in phylogenetic analyses, the draft genome sequences of Deefgea rivuli WB 3.4-79 T (JHVM00000000) were obtained from NCBI database. The digital DNA-DNA hybridization (dDDH) values and confidence intervals were calculated using the recommended settings of Genome-to-Genome Distance Calculator (Meier-Kolthof et al. 2013). The average nucleotide identity (ANI) was determined between strains H3-26 T and closely related strains of the genus Deefgea using OrthANIu (Yoon et al. 2017a, b). The wholegenome evolution trees were constructed using Type (Strain) Genome Server (Meier-Kolthoff et al. 2022).

Phenotypic characterization
The phenotypic characteristics of H3-26 T were tested on R2A agar in parallel after incubation for 24 h at 25 °C. Cell morphology of strain H3-26 T cultured at 25 °C for 24 h was observed by both light microscopy (CX31, Olympus) and scanning electron microscopy (Hitachi FE-SEM SU8010). The temperature for optimal growth was tested at 4-45 °C (4, 10, 15, 20, 25, 30, 37, 40 and 45 °C). The pH range for growth was determined by measuring the OD 600 of the culture grown in R2A broth, which was adjusted prior to sterilization to various pH values (pH 3.0-12.0 with an interval of 1.0 units) using appropriate biological buffers (Chung et al. 1995). The salt tolerance was determined with various NaCl concentrations (0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10%, w/v). Gram-straining reaction was carried out according to Claus (1992). Oxidase activity was tested by oxidase test strips with 1% (w/v) tetra-methyl-p-phenylenediamine. Catalase activity was determined by bubble production after mixing a loopful of cells with 3% (v/v) H 2 O 2 . Hydrolyses of starch and Tween 80 were tested on R2A agar with starch (1%, w/v) and Tween 80 (1%, v/v), respectively. Cell motility was tested by the hanging drop method with 0.2% agar. Anaerobic growth was checked using the Oxoid AnaeroGen system. Other tests to determine the biochemical characteristics were carried out using API 50CH, API 20E, API 20NE, and API ZYM strips according to the manufacturer's instructions (BioMérieux).

Determination of fatty acid profiles
After incubation on R2A at 25 °C for 48 h, cells were collected for fatty acids test. Fatty acids were saponified, methylated and extracted according to the standard protocol of the Sherlock Microbial Identification System (MIDI), analyzed via gas chromatography and identified using the Sherlock Aerobic Bacterial Database (RTSBA 6.2B) (Miller 1982).

Phylogenetic analysis
Compared to the sequences deposited in EzBioCloud, the 16S rRNA gene sequence of strain H3-26 T shared highest similarity with Deefgea rivuli WB 3.4-79 T (98.42%), followed by Deefgea chitinilytica Nsw-4 T (96.91%). Phylogenetic analysis of H3-26 T based on 16S rRNA genes confirmed its placement within the Deefgea genus, to form a separate branch of evolution with a very high bootstrap support (98-100%). A neighbor-joining tree derived from full 16S rRNA alignments is shown in Fig. 1, similar results were obtained using maximum-likelihood and maximumevolution methods (Figs. S1, S2).

Genomic characteristics and comparative genomics analysis
The draft genome of strain H3-26 T contained 26 contigs with an N 50 value of 387,129 bp and an N 90 value of 69,269 bp. The genome size of strain H3-26 T is 3.26 Mb. A total of 2995 genes were predicted in the draft genome of strain H3-26 T . The genomic DNA G+C content of H3-26 T is 48.74 mol%, which is similarity with Deefgea rivuli WB 3.4-79 T (48.50%) and Deefgea chitinilytica Nsw-4 T (48.29%). The whole-genome evolution tree of H3-26 T and 19 related bacteria shown that Deefgea salmonis H3-26 T , Deefgea chitinilytica Nsw-4 T and Deefgea rivuli WB 3.4-79 T formed a stable evolutionary branch (Fig. 2). Furthermore, the dDDH (d4) and ANI values between H3-26 T and other related strains were 18.1-22.1% and 68.3-77.4%, which were lower than the threshold values of 70% and 95-96% for species discrimination. The homologous genes analyses of strain H3-26 T , Deefgea chitinilytica Nsw-4 T and Deefgea rivuli WB 3.4-79 T are shown in a Venn diagram (Fig. S3). 2432, 2604 and 2656 genes were identified in the genomes of strain H3-26 T , Deefgea chitinilytica Nsw-4 T and Deefgea rivuli WB 3.4-79 T , respectively, with 2221 genes shared in all of them. In the genomes of strain H3-26 T , Deefgea chitinilytica Nsw-4 T and Deefgea rivuli WB 3.4-79 T , 10, 16 and 15 genes were identified as unique genes with no detectable homologous in each other. Both 16S rRNA gene and whole genome in the phylogenetic trees demonstrated that strain H3-26 T had the closest phylogenetic relationship with members of the genus of Deefgea.
The type strain H3-26 T (= JCM 35050 T = CICC 25103 T ) was isolated from gills of rainbow trout in Lhasa, Tibet Autonomous Region, PR China.
The GenBank accession numbers of 16S rRNA gene sequences and whole genome sequence of strain H3-26 T are OK077561 and JAJAWG000000000, respectively.
Author contributions YT and HP conceived the project. MC, CZ, JZ, LT and WW performed the experiments. MC and HP analyzed the data, and MC, HP and YT drafted and revised the manuscript. All authors have read and approved the final version of the manuscript.
Funding Natural Science Foundation of Tibet (XZ2019ZRG-80(Z)) and the "Double First-Class" construction project of Hunan Agricultural University (SYL201802002) funded this work.
Data availability All data have been made fully available to the public.

Declarations
Conflict of interest All the authors declared that they have no conflict of interest.