Snuella sedimenti sp. nov., isolated from marine sediment

A Gram stain-negative, aerobic, motile by gliding, rod-shaped bacterial strain CAU 1569T was isolated from marine sediment on Shido Island in Incheon. It grew at 20–37 °C (optimum, 30 °C), pH 6.0–9.0 (optimum, 7.0), 2–6% NaCl (w/v) (optimum, 2%). Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain CAU 1569T formed a distinct lineage with only the type strain of Snuella. Strain CAU 1569T showed high similarity to S. lapsa KACC 14152T (95.8%), Mariniflexile gromovii KMM KCTC 12570T, Aestuariibaculum marinum KCTC 52521T (95.4%), A. suncheonense KACC 16186T (94.6%) and Yeosuana aromativorans KCCM 42019T (94.4%). The genome contained 57 contigs, 3,437 protein-coding gene, 3 rRNAs (5, 16, and 23S), 43 tRNAs, and with a 35.7 mol% G + C content. The DDH value between strain CAU 1569T and S. lapsa KACC 14152T was 39.4 ± 0.6%. The only isoprenoid quinone was menaquinone 6 (MK-6). The major fatty acids were iso-C15:0, C15:1-iso G, and C17:0 iso 3-OH. Strain CAU 1569T contained diphosphatidylglycerol, aminoglycolipid, unidentified aminolipid, and three unidentified lipids. Based on phylogenetic, genomic, physiologic, and chemotaxonomic characterizations, strain CAU 1569T represents a novel Snuella species, which the name Snuella sedimenti sp. nov. is proposed. The type of strain is CAU 1569T (= KCTC 82409T = MCCC 1K05670T).


Introduction
The family Flavobacteriaceae was described by Reichenbach (1991) and emended by Bernardet et al. (1996Bernardet et al. ( , 2002. At the time of writing, the family contains 150 genera with the validly published and correct name (https:// lpsn. dsmz. de/ family/ flavo bacte riace ae). Members of the family Flavobacteriaceae are isolated from various environments, especially in marine such as tidal flat sediment (Kim et al. 2008), mud (Nedashkovskaya et al. 2004), and deep-sea seamount (Wang et al. 2020), coastal surface seawater (Bhumika et al. 2013), surface of algae, sponges and corals (Gavriilidou et al. 2020).The genus Snuella, a member of the family Flavobacteriaceae, was first proposed by Yi and Chun (2011) and comprises only one species, Snuella lapsa, which was isolated from a tidal flat sediment. In the course of investigating novel bacteria, strain CAU 1569 T was isolated from a marine sediment of Shido Island sample collected in the Republic of Korea, was identified as a novel bacterium based on its unique taxonomic position using a polyphasic approach, including the determination of phenotypic, genomic, and chemotaxonomic characteristics.

Bacterial isolation and ecology
A sediment sample was collected from Shido Island, Incheon (37° 32′ 11.0" N 126° 25′09.0" E) in South Korea. A yellow colony was isolated from 100-µl aliquots of serial

16S rRNA gene sequence and phylogeny
Genomic DNA of strain CAU 1569 T was extracted using a genomic DNA extraction kit (iNtRON, Seongnam, Korea) according to the manufacturer's instructions. The amplification of 16S rRNA gene was performed as described previously (Nam et al. 2004). Identification and calculation of pairwise identity of 16S rRNA gene sequence similarity between strain CAU 1569 T and the most closely related strains were performed from the EzBioCloud server (http:// www. ezbio cloud. net/ eztax on) (Kim et al. 2012) and Gen-Bank database. Multiple alignment and phylogenetic analysis were carried out MEGA7 program (Kumar et al. 2016). Phylogenetic trees were constructed using algorithms, including neighbour-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981), and maximum-parsimony (MP) (Fitch 1971) approaches for distance analysis. The topology of the phylogenetic tree was estimated by bootstrap values with 1000 replicates (Felsenstein 1985). The DNA-DNA pairing between strain CAU 1569 T and the closest strain S. lapsa KACC 14152 T was performed using the fluorometric microplate method (Ezaki et al. 1989).

Physiological characterization
For morphological analysis of strain CAU 1569 T , cells were grown on MA for 2 days at 30 °C. Cell morphology was observed under a DM 1000 light microscope (Leica, Wetzlar, Germany) and the JEM 1010 transmission electron microscope (JEOL, Tokyo, Japan) using cells growing at the exponential phase. Motility of strain CAU 1569 T was determined by a semisolid agar tube (Wolfe and Berg 1989) and gliding motility was performed using hanging-drop method as described by Bernardet et al. (2002). Gram staining was carried out using a Gram staining kit (bioMérieux, Craponne, France). Strain CAU 1569 T was inoculated at various temperatures (4, 10, 20, 25, 30, 37, and 45 °C) in MA, pH (4.5-12.0 at 0.5 pH unit intervals; adjusted with 1 M HCl or 1 M NaOH), and NaCl concentrations in NaCl-free MB supplemented with 0 and 15% (1% increments, w/v). Catalase and oxidase activity were examined using bubble production in 3% (v/v) H 2 O 2 solution and oxidase reagent (bioMérieux), respectively. Hydrolysis of casein and starch were determined as described previously (Smibert and Krieg 1994). Identification of gram-negative bacteria and carbon sources assimilation were evaluated using API 20E, API 20NE and API 50CH kits (bioMérieux), respectively, and the results were recorded after 48 h of incubation at 30 °C. Enzyme activity was examined using API ZYM kit (bioMérieux) and the results were analyzed after 24 h of incubation at 37 °C.

Chemotaxonomic characterization
Isoprenoid quinones were extracted and analyzed by reverse-phase high-performance liquid chromatography (HPLC) as previously described (Komagata and Suzuki 1987). For analysis of fatty acids, strain CAU 1569 T and the five reference strains were cultured on MA at 30 °C and cell mass was harvested after 3 days (exponential growth phase, optical density at 600 nm = 0.8). Fatty acids of strain CAU 1569 T were saponified, methylated and extracted using standard Microbial Identification System (MIDI, version 6.2B) methods. Fatty acids were analyzed by gas chromatography (Hewlett Packard 6890) and identified using the TSBA6 database of the Microbial Identification System (Sasser 2006). Polar lipids were extracted from cell mass harvested at exponential growth phase as described by Minnikin et al. (1984) and separated by two-dimensional TLC as described by Embley and Wait (1994). The polar lipids were identified using specific spraying reagents as described Kim et al. (Kim et al. 2015).

Phylogenetic and genome characterization
The nearly complete the 16S rRNA gene sequence of CAU 1569 T (1,511 bp) was investigated for similarity among sequences of related bacterial strains in the GenBank databases (access May 2021) and submitted to the GenBank/ EMBL/DDBJ under the accession number MN544292. The 16S rRNA gene sequences analysis revealed that strain CAU 1569 T showed the highest pairwise similarity to S. lapsa KACC 14152 T (95.8%), followed by M. gromovii KCTC 12570 T (95.5%) and A.marinum KCTC 52521 T (95.4%). The neighbour-joining phylogenetic tree indicated that strain CAU 1569 T clustered in a single branch of S. lapsa KACC 14152 T (Fig. 1). The DDH value between strain CAU 1569 T and S. lapsa KACC 14152 T was 39.4 ± 0.6%, which is clearly < 70% value was recommended by Wayne et al. (1987) for species differentiation. The draft genome of strain CAU 1569 T was composed 57 contigs (average length 76,651 bp) and a total genome size of 4.4 Mb. DNA G + C content of CAU 1569 T was 35.7 mol%, this value is lower than that of closely related members of the genus Snuella (Table 1). The K-mer coverage was 272.8, and the N50 value was 147,344 bp. The genome of strain CAU 1569 T was composed contained 3,437 protein-coding genes with 3,462 coding sequences, 3 rRNAs (5, 16, and 23S), and 43 tRNAs. The overall genome-related index (OGRI) was not calculated according to minimal standards for using genome data (Chun et al. 2018), which is the threshold value of 16S sequence similarity (< 98.5%) of a novel species. The subsystem genome features of strain CAU 1569 T with RAST server are summarized in Supplementary Fig. 1. Genome annotations (> 7%) of strain CAU 1569 T were included "Amino Acids and Derivatives" (214 genes), "Carbohydrates" (122 genes), "Respiration" (26 genes), and "Cofactors, Vitamins, Prosthetic Groups, Pigments" (148 genes).

Physiological characterization
Strain CAU 1569 T was motile by gliding and rod shaped (0.6-0.8 µm in width and 1.8-2.5 µm in length). Cells have no flagella (Supplementary Fig. 2). Gliding motility, which does not require flagella or pili, is a common characterization among the family Flavobacteriaceae (Gavriilidou et al. 2020). The characteristics between strain CAU 1569 T and closely related reference strains of the genus Snuella were summarized in Table 1. Strain CAU 1569 T differed from closely related species, S. lapsa KACC 14152 T , based on negative reactions for oxidase production, arginine dihydrolase, acid production from D-ribose and positive reactions for cystine arylamidase and α-glucosidase enzyme activity. Strain CAU 1569 T differed from M. gromovii KCTC 12570 T based on negative reaction for α-chymotrypsin and positive reaction for α-glucosidase. In addition, strain CAU 1569 T differed from A. marinum KCTC 52521 T , A. suncheonense KACC 16186 T based on negative reaction at α-galactosidase. Compared with Y. aromativorans KCCM 42019 T , strain CAU 1569 T showed differences at the negative reaction for β-galactosidase and arginine dihydrolase. Based on the comparison of phenotypic characteristics, strain CAU 1569 T could be distinguished from recognized species of the genus Snuella. The list of all negative reactions of strain CAU 1569 T from API test strips are shown in Supplementary Table 1.

Chemotaxonomic characterization
The only isoprenoid quinone detected from strain CAU 1569 T was MK-6, which is a typical feature of the genus Snuella (Yi and Chun, 2011) as well as that of other members of the family (Bernardet et al. 1992). The major fatty acids (> 10% of the total fatty acids) of strain CAU 1569 T were iso-C 15:0 (22.3%) , C 17:0 iso 3-OH (15.7%) and C 15:1-iso G (12.0%). The fatty acids profile of strain CAU 1569 T was similar to those of reference strains, especially iso-C 15:0, C 15:1-iso G, and C 17:0 iso 3-OH were major fatty acids ( Table 2). The major polar lipids of strain CAU 1569 T were diphosphatidylglycerol and aminoglycolipid, and minor components were unidentified aminolipid, and three unidentified lipids (Supplementary Fig. 3). The major polar lipids of strain CAU 1569 T were similar with those of S. lapsa KACC 14152 T in that diphosphatidylglycerol and aminoglycolipid are major components.
T h e t y p e st r a i n i s CAU 1 5 6 9 T ( = KC T C 82409 T = MCCC 1K05670 T ), which was isolated from marine sediment from Shido Island, Incheon, South Korea.  Nedashkovskaya et al. (2006)  Cystine arylamidase