Luteirhabdus pelagi gen. nov., sp. nov., a novel member of the family Flavobacteriaceae, isolated from the West Pacific Ocean

A Gram-stain-negative, aerobic, and yellow-pigmented bacterium, designated A3-108T, was isolated from seawater of the West Pacific Ocean. Cells were non-motile and rod-shaped, with carotenoid-type pigments. Strain A3-108T grew at pH 6.0–8.5 (optimum 6.5) and 15–40 °C (optimum 28 °C), in the presence of 0.5–10% (w/v) NaCl (optimum 1.0%). It possessed the ability to produce H2S. Based on the 16S rRNA gene analysis, strain A3-108T exhibited highest similarity with Aureisphaera salina A6D-50T (90.6%). Phylogenetic analysis shown that strain A3-108T affiliated with members of the family Flavobacteriaceae and represented an independent lineage. The principal fatty acids were iso-C15:0, iso-C17:0 3-OH, iso-C15:1 G, and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The sole isoprenoid quinone was MK-6. The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified aminolipid and one unidentified lipid. The ANIb, in silico DDH and AAI values among the genomes of strain A3-108T and three reference strains were 67.3–71.1%, 18.7–22.1%, and 58.8–71.4%, respectively. The G + C content was 41.0%. Distinctness of the phylogenetic position as well as differentiating chemotaxonomic and other phenotypic traits revealed that strain A3-108T represented a novel genus and species of the family Flavobacteriaceae, for which the name Luteirhabdus pelagi gen. nov., sp. nov. is proposed (type strain, A3-108T = CGMCC 1.18821T = KCTC 82563T). Supplementary Information The online version contains supplementary material available at 10.1007/s00203-021-02557-3.


Introduction
The family Flavobacteriaceae, belonging to the class Flavobacteriia, the phylum Bacteroidetes, was first proposed by Jooste (1985), validly published by Reichenbach (1992), then emended by Bernardet et al. (1996Bernardet et al. ( , 2002 and García-López et al. (2019). Prior to 2021, the family Flavobacteriaceae comprised 150 genera with validly published names. This study focuses on the description of a novel genus and species with the type strain A3-108 T was isolated from seawater collected at the seamount area in the West Pacific Ocean.
Seamounts are defined as the huge uplifts located below sea level, and exceed 1000 m in height, and are unique environments widely distributed on the deep-ocean subseafloor (Yesson et al. 2011). The West Pacific Ocean has the most concentrated area of global seamount systems (Qin and Yin 2011). Seamounts are important habitats for marine organisms (Clark et al. 2010). In the upper water column, primary productivity is influenced by topographically induced turbulent mixing in the seamounts' ecosystem (Boehlert and Genin 1987;Polzin et al. 1997), which has a strong impact on physical/chemical parameters and organism communities (Mashayek et al. 2017;Muck et al. 2014). Currently, there are few studies on microbial communities in seamount environments.

Samples and isolation
During the investigation of bacterial diversity, a seawater sample from the seamount area in the West Pacific Ocean (at a depth of 300 m, 23.2° N, 162.3° E), was collected by a rosette sampler connected with CTD system (SBE911 plus; Sea-Bird Electronics, Inc. USA) in 2018. Aboard the ship, the seawater sample was subjected to the culture process immediately. Approximately 100 µL seawater samples were diluted using serial dilution technique and added to different media. The strain A3-108 T was isolated aerobically on natural seawater agar (1 L filtered natural seawater supplemented with 0.5 g peptone (BD Difco), 0.1 g yeast extract (BD Difco), 20 g agar (BD Difco), pH 7.2-7.4) and purified by repeated restreaking. The purity was confirmed by the uniformity of cell morphology. Unless otherwise stated, strain A3-108 T was routinely cultured in marine broth 2216 (MB, BD Difco) or on marine agar 2216 (MA, BD Difco) at 30 °C and maintained at − 80 °C with 30% (v/v) glycerol. Galbibacter mesophilus CGMCC 1.15663 T and Marixanthomonas ophiurae JCM 14121 T , were obtained from the CGMCC (China General Microbiological Culture Collection Center) and JCM (Japan Collection of Microorganisms), respectively. An additional reference strain Marinirhabdus gelatinilytica NH83 T was obtained from our laboratory (Wu et al. 2016).

16S rRNA gene and genome sequence determination
High-quality genomic DNA was extracted by Nucleic Acid Purification kit (Dongsheng Biotech). The 16S rRNA gene was amplified by the universal primers 27F/1492R (27F: 5'-AGA GTT TGA TCC TGG CTC AG-3'; 1492R: 5'-GGY TAC CTT GTT ACG ACT T-3'). The PCR thermal cycling conditions were as follows: 30 cycles of 98 °C for 10 s, 55 °C for 10 s, and 72 °C for 30 s. The PCR products were purified and sequenced by Sanger sequencing to obtain the almost complete 16S rRNA gene sequence.
The genomic DNA of strain A3-108 T and Galbibacter mesophilus CGMCC 1.15663 T were sequenced by Solexa paired-end sequencing technology with the Illumina NovaSeq 6000 PE150 platform (Novogene Co. Ltd, Tianjing). One paired-end library was constructed with insert size of 350 bp. The sequencing generated approx. 1 Gb clean data (approx. 500-fold genome coverage). De novo assembly of the reads was carried out using SOAPdenovo (version 2.0.1) (Luo et al. 2012). The completeness of genome sequences was addressed using the bioinformatics tool CheckM (http:// ecoge nomics. github. io/ CheckM/) (Parks et al. 2015). The complete 16S rRNA gene was annotated via the RNAmmer 1.2 Server (Lagesen et al. 2007) and compared with gene sequences obtained from PCR to ensure its authenticity.

Phylogenetic status and DNA relatedness
The 16S rRNA gene sequence was compared with the corresponding sequences of closely related organisms via online EzBioCloud service (https:// www. ezbio cloud. net) (Yoon et al. 2017). Based on 16S rRNA gene similarity, 23 species were selected and aligned for phylogenetic analysis by CLUSTALW software (Thomson et al. 1994). Phylogenetic trees were constructed using MEGA 7.0 program package (Kumar et al. 2016) using the methods of neighbor-joining (Saitou and Nei 1987), maximum-parsimony (Fitch 1971) and maximum-likelihood (Felsenstein 1981). Evolutionary distances of the neighborjoining method were calculated according to the Kimura-2-parameter algorithm model (Kimura 1980). A phylogenomic tree was constructed based on singlecopy orthologous clusters (OCs) of strain A3-108 T and its related taxa of the family Flavobacteriaceae. The related genome sequences were obtained from the NCBI Gen-Bank database and annotated using the Prokka server (Seemann 2014). Orthologous clusters (OCs) were selected by Proteinortho (version 5.16b) (Lechner et al. 2014). Singlecopy OCs were filtered by an in-house shell script. Protein sequences were aligned using MAFFT (version 7) (Katoh and Standley 2013). Aligned sequences were refined via trimAL (version 1.4.1) (Capella-Gutiérrez et al. 2009) and concatenated by an in-house shell script. The best substitution model was estimated by IQ-Tree software (version 1.6.1) (Nguyen et al. 2015) and the model LG + F + R4 was selected. The maximum-likelihood phylogenomic tree was reconstructed through IQ-Tree software and visualized applying MEGA 7.0 software (Kumar et al. 2016).

Phenotypic characteristics
Cell morphology, ultrastructure, size, and the presence of flagellum were observed by transmission electron micrographs (JEM-1230, JEOL). Gram reaction was determined by the Gram-Stain method (Brown and Hopps 1973). Motility was examined by stab culture with semi-solid medium, using MB supplemented with 0.5% (w/v) agar (Wolfe and Berg 1989). The temperature range for growth was investigated by incubating in MB at 4,15,20,28,30,37,40,45, and 50 °C. The pH range for growth was determined in MB with different pH (pH 5.0-10.5, in 0.5 pH unit intervals) using appropriate biological buffers at 50 mM concentration (MES for pH 5.0-6.0, PIPES for pH 6.5-7.0, Tricine for pH 7.5-8.5, CAPSO for pH 9.0-10.0 and CAPS for pH 10.5). The optimal conditions with NaCl for growth were measured using NaCl-free MB (prepared according to the MB formula, but without NaCl) with different NaCl concentrations (0, 0.5, 1.0, 3.0, 5.0, 7.5, 10.0, 15.0, 20.0, and 25%, w/v). Cell densities were monitored by measuring with a UV/Visible Spectrophotometer at 600 nm (Ultrospec 6300 pro; Amersham Biosciences). Anaerobic growth was tested by the Anaero-Pack (Mitsubishi) adding sodium nitrate (10 mM), sodium sulfate (10 mM), and sodium thiosulfate (10 mM) as potential electron acceptors on the MA. The growth curve of strain A3-108 T was determined by incubation in MB with the optimal growth condition (30 °C, 180 rpm), and cell densities were measured every 2 h incubation via measuring OD 600 in a UV/Visible Spectrophotometer (Genesys 50; Thermofisher Scientific). The doubling time and the specific growth rate were calculated by formulas: doubling time (t d /h) = ln2/k; specific growth rate (μ/ h −1 ) = 1/t d ; k represents relative growth rate (slope of the curve) (Monod 1949).
Flexirubin-type pigments were detected by a bathochromic shift test (Fautz and Reichenbach 1980). Carotenoidtype pigments were detected by pigment absorption spectrum analysis as described by Hildebrand et al. (1994). Pigments were extracted with acetone/methanol (7:2, v/v) and performed by a Beckman DU 800 Spectrophotometer (detection wavelength from 300 to 800 nm).

Chemotaxonomic analysis
The cellular fatty acids of the strain A3-108 T and three reference strains were determined under identical conditions in parallel. Approximately 20 mg of cells were harvested by the quadrant streak method on MA plates at 30 °C for 3 days (quadrant 3 exhibiting confluent growth). Fatty acids were extracted by saponification, methylation, and extraction as described previously (Sasser 1990). The cellular fatty acids were analyzed by 6890 gas chromatograph according to Microbial Identification System (MIDI).

Genomic analysis
The draft genome sequence was annotated using the RAST server online (https:// rast. nmpdr. org/ rast. cgi) (Aziz et al. 2008), and annotation information including predicted coding sequences (CDSs), proteins and RNAs were obtained. Metabolic pathways were predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) online annotation server (Kanehisa et al. 2016).
The phylogenetic trees manifested that strain A3-108 T fall into the family Flavobacteriaceae and formed a separated branch apart from other genera of the family with high bootstrap values (Fig. 1). Phylogenetic analysis indicated that the strain A3-108 T represented an independent lineage in family Flavobacteriaceae. Furthermore, the maximum-likelihood phylogenomic tree based on single-copy orthologous clusters (OCs) demonstrated that strain A3-108 T affiliated with the family Flavobacteriaceae and clustered with Marixanthomonas ophiurae KMM 3046 T (Fig. 2).

Genomic features and DNA-DNA relatedness
Based on the bioinformatic tool CheckM, the genome completeness of strain A3-108 T was 99.2%, with 0.27% Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony algorithms. Bar, 0.05 substitutions per nucleotide position 1 3 contamination. The genome sequence estimated to be ≥ 95% completeness, with ≤ 5% contamination, was considered to be an excellent reference genome for deeper analyses (Pruesse et al. 2007). The final genome of strain A3-108 T comprised a total size of 3.40 Mb with 99 contigs, and G + C content was 41.0%. The assembled scaffolds annotated by RAST online, harbored a total of 3250 coding genes, 37 tRNAs and 5 rRNAs. The general genomic features of strain A3-108 T and reference strains are shown in Supplementary Table S1. The ANIb and in silico DDH among the genome of strain A3-108 T and the reference strains were 67.3-71.1% and 18.7-22.1%, respectively (Supplementary Table S2). The ANI values were far below the species threshold of 94-96% (Richter and Rosselló-Móra 2009) and the genus demarcation boundary of 90% (Barco et al. 2020). The in silico DDH values were below the threshold value 70% that corresponded to the species boundary (Wayne et al. 1987). In addition, the AAI values were 58.8-71.4% among the genome of strain A3-108 T and the reference strains (Supplementary Table S2), which were below the species cutoff 95-96% (Konstantinidis and Tiedje 2005) and the threshold of 60-80% to distinguish genera from each other (Luo et al. 2014). The OrthoANI values between strain A3-108 T and the reference strains were 67.7-71.5% (Supplementary Table S2). The ANI, in silico DDH and AAI values indicted a low taxonomic relatedness between strain A3-108 T and the reference strains of the family Flavobacteriaceae.

Phenotypic features
Strain A3-108 T was Gram-stain-negative, aerobic, nonmotile, and rod-shaped with 0.5-0.8 μm in width and 1.6-3.9 μm in length ( Supplementary Fig. S1). No flagellum was observed. Colonies were yellow, circular, convex, opaque, smooth, and 1-2 mm in diameter after 3 days of incubation at 30 °C on MA. The growth range of pH, temperature and NaCl concentrations were pH 6.0-8.5,  Fig. S2). Strain A3-108 T was positive for oxidase, arginine dihydrolase, nitrite reduction, and H 2 S production. Carotenoid-type pigments were present but flexirubin-type pigments were not ( Supplementary Fig. S3). Additional phenotypic properties are given in the species description, Tables 1, 3 and Supplementary Table S3.
Chemotaxonomic analysis supported the result of the phylogenetic analysis. The sole respiratory quinone detected in strain A3-108 T was consistent with members of the family Flavobacteriaceae (Bernardet 2015). The components iso-C 17:0 3-OH and iso-C 15:0 were major fatty acids in strain A3-108 T and the reference strains ( Table 2). The presence of phosphatidylethanolamine (PE) was conserved in strain A3-108 T and the related genera (Table 3).
The chemotaxonomic results also showed some differences clearly in fatty acid compositions and polar lipid profiles between strain A3-108 T and the reference strains. The component iso-C 15:1 G was presented as major fatty acid in the strain A3-108 T (15.7%), while it was presented as moderate fatty acid in Marinirhabdus gelatinilytica NH83 T , Galbibacter mesophilus CGMCC 1.15663 T and Marixanthomonas ophiurae JCM 14121 T (3.2-7.8%). In addition, the component anteiso-C 15:1 A was only detected in the strain A3-108 T . In addition, the fatty acid of strain A3-108 T were different from the reference strains in the compositions and proportions (Table 2). With respect to polar lipid profiles, the component of the unidentified aminophospholipid (APL) was presented as major polar lipid in the strain A3-108 T , while it was not presented in Galbibacter mesophilus CGMCC 1.15663 T . Besides, the unidentified aminolipid (AL), one of the major polar lipids, was not presented in the related strain Marixanthomonas ophiurae JCM 14121 T . In addition, moderate polar lipids, including an unidentified glycolipid (GL) and two aminoglycolipids (AGL1-2), were presented in the strain A3-108 T , while they were not detected in the reference strains. Besides, lysophosphatidylethanolamine (LPE) and sphingolipid (SL) were only detected in Marixanthomonas ophiurae JCM 14121 T (Supplementary Fig. S4 and Wu et al. 2016;Romanenko et al. 2007;Hameed et al. 2014).
The type strain A3-108 T (CGMCC 1.18821 T = KCTC 82563 T ) is isolated from the seawater, collected from the West Pacific Ocean (at depth of 300 m, 23.2°N, 162.3°E). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain A3-108 T is MW244395 and the GenBank accession number for the whole genome sequence is JAECMS000000000.