Description and Whole-Genome Sequencing of Mariniflexile litorale sp. nov., Isolated from the Shallow Sediments of the Sea of Japan

A Gram-negative, aerobic, rod-shaped, non-motile, yellow-pigmented bacterium, KMM 9835T, was isolated from the sediment sample obtained from the Amur Bay of the Sea of Japan seashore, Russia. Phylogenetic analyses based on the 16S rRNA gene and whole genome sequences positioned the novel strain KMM 9835T in the genus Mariniflexile as a separate line sharing the highest 16S rRNA gene sequence similarities of 96.6% and 96.2% with Mariniflexile soesokkakense RSSK-9T and Mariniflexile fucanivorans SW5T, respectively, and similarity values of <96% to other recognized Mariniflexile species. The average nucleotide identity and digital DNA–DNA hybridization values between strain KMM 9835T and M. soesokkakense KCTC 32427T, Mariniflexile gromovii KCTC 12570T, M. fucanivorans DSM 18792T, and M. maritimum M5A1MT were 83.0%, 82.5%, 83.4%, and 78.3% and 30.7%, 29.6%, 29.5%, and 24.4%, respectively. The genomic DNA GC content of strain KMM 9835T was 32.5 mol%. The dominant menaquinone was MK-6, and the major fatty acids were iso-C15:0, iso-C15:1ω10c, and C15:0. The polar lipids of strain KMM 9835T consisted of phosphatidylethanolamine, two unidentified aminolipids, an unidentified phospholipid, and six unidentified lipids. A pan-genome analysis showed that the KMM 9835T genome encoded 753 singletons. The annotated singletons were more often related to transport protein systems (SusC), transcriptional regulators (AraC, LytTR, LacI), and enzymes (glycosylases). The KMM 9835T genome was highly enriched in CAZyme-encoding genes, the proportion of which reached 7.3%. Moreover, the KMM 9835T genome was characterized by a high abundance of CAZyme gene families (GH43, GH28, PL1, PL10, CE8, and CE12), indicating its potential to catabolize pectin. This may represent part of an adaptation strategy facilitating microbial consumption of plant polymeric substrates in aquatic environments near shorelines and freshwater sources. Based on the combination of phylogenetic and phenotypic characterization, the marine sediment strain KMM 9835T (=KCTC 92792T) represents a novel species of the genus Mariniflexile, for which the name Mariniflexile litorale sp. nov. is proposed.


Chemotaxonomic Analyses
For the lipid analyses of bacterial strain KMM 9835 T , M. soesokkakense KCTC 32427 T and M. maritimum KCTC 72895 T were cultivated on MA 2216 at 28 • C. Lipids were ex-tracted according to the method of Folch et al. [17].Two-dimensional thin layer chromatography of polar lipids was performed on Silica gel 60 F254 (10 × 10 cm, Merck, Darmstadt, Germany), applying chloroform-methanol-water (65:25:4, v/v) for the first direction, chloroform-methanol-acetic acid-water (80:12:15:4, v/v) for the second one [18], and spraying with specific reagents [19].Fatty acid methyl esters (FAMEs) were prepared following the procedure of the Microbial Identification System (MIDI) [20].A chromatograph (Shimadzu, Kyoto, Japan) with a flame ionization detector equipped with a SPB-5 capillary column (30 m × 0.25 mm × 0.25 mkm) was used.Identification of FAMEs was carried out by comparing the equivalent chain length values and retention times of the samples to those of the standards (Standard bacterial acid methyl ester mix 47080-U, Supelco, Bellefonte, PA, USA).In addition, FAMEs were investigated using a GC-MS Shimadzu QP2020 (Shimadzu, Kyoto, Japan) with a column Shimadzu SH-Rtx-5MS (30 m × 0.25 mm × 0.25 mkm) and a temperature program from 160 • C to 320 • C at a rate of 2 • C/min).To determine the position of double bonds and methyl groups, fatty acids were analyzed as 4,4-dimethyloxazoline derivatives [21] using GC-MS with the SH-Rtx-5MS column at a temperature from 180 • C to 320 • C, 2 • C/min.Menaquinones were examined by HPLC, as described by Hirashi et al. (1996) [22].A Shimadzu LC-30 chromatograph with a photodiode array detector (SPD-M30A) was used.Absorption spectra of lipid extracts redissolved in methanol at a concentration of 0.2 mg/mL were obtained on a Shimadzu UV-2600 spectrophotometer.The presence of flexirubin pigments was determined as described by Fautz and Reichenbach [23].

16S rRNA Gene Sequence and Phylogenetic Analysis
Genomic DNA of strain KMM 9835 T was extracted using the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions.The 16S rRNA gene was PCR-amplified using 27F (5 ′ -AGAGTTTGATCMTGGCTCAG-3 ′ ) and 1492R (5 ′ -TACGGTTACCTTGTTACGACTT-3 ′ ) primers with 5 min of denaturation (96 • C) followed by 25 cycles of 30 s of denaturation (95 • C), 30 s of annealing (55 • C), and 1 min 20 s of elongation (72 • C), finalizing with 5 min of elongation (72 • C).The obtained amplicons of standard bacterial acid were sequenced and compared with those of their closest relatives using the EzBioCloud service [24].Phylogenies were performed on the GGDC web server (http://ggdc.dsmz.de/,accessed on 16 May 2024) [25] using the DSMZ pipeline [26] applied to a single gene.Maximum likelihood (ML) and maximum parsimony (MP) trees were inferred from the alignment with RAxML [27] and TNT [28], respectively.The neighbor-joining (NJ) tree was reconstructed with MEGA version 11 [29] using the Kimura two-parameter model of nucleotide substitutions.The robustness of phylogenetic trees was estimated by the bootstrap analysis of 1000 replicates.

Whole-Genome Sequencing, Phylogenomic, and Comparative Analyses
Genomic DNAs were extracted from strains KMM 9835 T and M. soesokkakense KCTC 32427 T by the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany).The DNA quality was estimated by agarose gel electrophoresis, and the DNA quantity was measured on the Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Singapore).The DNA libraries were prepared with Nextera DNA Flex kits (Illumina, San Diego, CA, USA) and were sequenced on an Illumina MiSeq instrument using paired-end runs with a 250-bp read length.The nanopore library was prepared for KMM 9835 T using EXP-NBD104 and SQK-LSK109 kits (Oxford Nanopore Technologies, Oxford, UK), according to the Native barcoding genomic DNA protocol.The reads were trimmed using Trimmomatic version 0.39 [30] and their quality assessed using FastQC version 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 21 August 2021 and 30 November 2023).Filtered reads of the M. soesokkakense RSSK-9 T genome were assembled into contigs with SPAdes version 3. 15.3 [31], and genome metrics were calculated with QUAST version 5.0.2 [32].Dorado version 0.4.3 (Oxford Nanopore Technologies, Oxford, UK) was used with default parameters to quality filter Nanopore reads and filter out sequences 1000 bp in length.
Comparisons of the Average Nucleotide Identity (ANI), Average Amino Acid Identity (AAI), and digital DNA-DNA hybridization (dDDH) values of the strains KMM 9835 T and KCTC 32427 T with their closest neighbors were performed with the online servers ANI/AAI-Matrix [36] and TYGS platform [37], respectively.The phylogenomic analysis was performed using PhyloPhlAn software version 3.0.1 based on a set of 400 conserved bacterial protein sequences using the RAxML program under the PROTCATLG model with bootstrapping of 100 replicates (flag −b +−100) [38].
On the 16S rRNA phylogenetic trees, the position of strain KMM 9835 T was uncertain due to low bootstrap support when M. maritimum M5A1M T was included in the analysis (Figure 1).M5A1M T exclusion and TRM1-10 addition resulted in KMM 9835 T clustering with Mariniflexile spp.strains (except for TYO-10 T ) under strong bootstrap support (Figure S1).There are currently seven species of the genus Mariniflexile with validly published names, but only three genomes of type strains M. gromovii, M. fucanivorans, and M. maritimum are available.In this study, the genome sequence of the fourth type strain, M. soesokkakense KCTC 32427 T , was obtained.The genomes of three type strains (DSM 18792 T , KCTC 12570 T , and M5A1M T ) were retrieved from NCBI (Table 1).The phylogenomic tree based on concatenated sequences extracted from the genomes of Mariniflexile species and related taxa showed that strain KMM 9835 T formed a distinct line within the genus Mariniflexile (Figure 2).There are currently seven species of the genus Mariniflexile with validly published names, but only three genomes of type strains M. gromovii, M. fucanivorans, and M. maritimum are available.In this study, the genome sequence of the fourth type strain, M. soesokkakense KCTC 32427 T , was obtained.The genomes of three type strains (DSM 18792 T , KCTC 12570 T , and M5A1M T ) were retrieved from NCBI (Table 1).The phylogenomic tree based on concatenated sequences extracted from the genomes of Mariniflexile species and related taxa showed that strain KMM 9835 T formed a distinct line within the genus Mariniflexile (Figure 2).The ANI/AAI values between the genomes of strain 9835 T and the type strains of M. soesokkakense KCTC 32427 T , M. gromovii KCTC 12570 T , M. fucanivorans DSM 18792 T , and M. maritimum M5A1M T were 83.0%/83.5%,82.5%/81.7%,83.4%/80.7%,and 78.3%/77.6%,respectively, which were lower than the 95−96% threshold value accepted for species delineation [55].The dDDH values (formula d4) between strain KMM 9957 T and the four relatives, ranging from 24.4% (M.maritimum M5A1M T ) to 30.7% (M.soesokkakense KCTC 32427 T ), were below the 70% threshold value accepted for species delineation [56,57].These overall genomic relatedness indices (OGRIs) and phylogenomic position suggest that KMM 9835 T represents a novel species in the genus Mariniflexile.

Genomic Characteristics and Pan-Genome Analysis of the Mariniflexile Genus
The complete genome of strain KMM 9835 T was de novo assembled into one chromosome with an estimated size of 4,521,428 bp and an overall G+C content of 62.1%.The two genome-extracted 16S rRNA gene sequences were 100% identical to the PCR-amplified one (OQ300347).The genome contains 3752 protein coding sequences, 36 tRNAs, and 6 rRNA genes (two 16S-23S-5S operons).The indices recommended to evaluate the quality of the genomic data [58,59] are shown in Table 1.The observed characteristics satisfy the proposed minimal standards for the taxonomy of prokaryotes and indicate high genome The ANI/AAI values between the genomes of strain 9835 T and the type strains of M. soesokkakense KCTC 32427 T , M. gromovii KCTC 12570 T , M. fucanivorans DSM 18792 T , and M. maritimum M5A1M T were 83.0%/83.5%,82.5%/81.7%,83.4%/80.7%,and 78.3%/77.6%,respectively, which were lower than the 95−96% threshold value accepted for species delineation [55].The dDDH values (formula d4) between strain KMM 9957 T and the four relatives, ranging from 24.4% (M.maritimum M5A1M T ) to 30.7% (M.soesokkakense KCTC 32427 T ), were below the 70% threshold value accepted for species delineation [56,57].These overall genomic relatedness indices (OGRIs) and phylogenomic position suggest that KMM 9835 T represents a novel species in the genus Mariniflexile.

Genomic Characteristics and Pan-Genome Analysis of the Mariniflexile Genus
The complete genome of strain KMM 9835 T was de novo assembled into one chromosome with an estimated size of 4,521,428 bp and an overall G+C content of 62.1%.The two genome-extracted 16S rRNA gene sequences were 100% identical to the PCR-amplified one (OQ300347).The genome contains 3752 protein coding sequences, 36 tRNAs, and 6 rRNA genes (two 16S-23S-5S operons).The indices recommended to evaluate the quality of the genomic data [58,59] are shown in Table 1.The observed characteristics satisfy the proposed minimal standards for the taxonomy of prokaryotes and indicate high genome quality.In total, seven Mariniflexile strains were taken for comparative genome analysis, two of which have been sequenced in this study (KMM 9835 T and KCTC 32427 T ).In addition to the genomes of three type strains (DSM 18792 T , KCTC 12570 T , and M5A1M T ), two high-quality genomes of Mariniflexile sp.strains (AS56 and TRM1-10) were retrieved from NCBI.Their basic genome indices are listed in Table 1.The ML phylogenomic tree, including two last genomes, clearly showed that strains AS56 and TRM1-10 might present two novel species of the genus Mariniflexile (Figure S2).The genome sequences contain from 3157 (M.maritimum M5A1M T ) to 4104 (M.fucanivorans DSM 18792 T ) genes, from 36 (KMM 9835 T and M. soesokkakense KCTC 32427 T ) to 41 (TRM1-10) tRNAs, and from one up to two rrn operon copies (KMM 9835 T and TRM1-10).
The first gene (dnaA) in the genome sequence was automatically assigned as the origin of replication; however, its position did not align with a GC skew plot.To identify the origin (oriC) and terminus (ter) of replication, the Ori-Finder 2022 server [45] was utilized.Regions adjacent to the mnmG and leuB genes were predicted as oriC and ter, respectively.These regions aligned with the GC skew plot and had the highest Ori-Finder scores.The chromosomal level of genomic assemblies obtained for strains KMM 9835 T and TRM1-10 made it possible to estimate the exact numbers of rrn operons (Table 1); both rrn operons of KMM 9835 T are located on the leading strands (Figure 3).The 13 retron-type RNA-directed DNA polymerase (EC 2.7.7.49) genes and one CRISPR-Cas region, among them seven full-length, were found in the genome strain KMM 9835 T (Figure 3).Three loci had truncated ORFs.
The KMM 9835 T chromosome map was built and visualized using Proksee [42] (Figure 3).Genome annotations were carried out using the RAST tool kit [40] and Prokka [41].The first gene (dnaA) in the genome sequence was automatically assigned as the origin of replication; however, its position did not align with a GC skew plot.To identify the origin (oriC) and terminus (ter) of replication, the Ori-Finder 2022 server [45] was utilized.Regions adjacent to the mnmG and leuB genes were predicted as oriC and ter, respectively.These regions aligned with the GC skew plot and had the highest Ori-Finder scores.The chromosomal level of genomic assemblies obtained for strains KMM 9835 T and TRM1-10 made it possible to estimate the exact numbers of rrn operons (Table 1); both rrn operons of KMM 9835 T are located on the leading strands (Figure 3).The 13 retron-type RNAdirected DNA polymerase (EC 2.7.7.49) genes and one CRISPR-Cas region, among them seven full-length, were found in the genome strain KMM 9835 T (Figure 3).Three loci had truncated ORFs.To determine genus-related features, a pan-genome analysis of Mariniflexile species (Table 1) was performed using orthologous clustering and metabolic pathway reconstruction with the anvi'o platform [50].The Mariniflexile pan-genome (Figure 4) comprised a total of 9163 gene clusters (distance: Euclidean; linkage: Ward) with 25,933 gene calls.The core genome included 2202 core gene clusters covering 15,113 genes, of which 1625 were single-copy genes (SCGs).The accessory shell and cloud clusters were composed of 913 (3317 genes) and 1074 (2320 genes) clusters, respectively.A unique part of the pangenome included 4974 gene clusters (5183 genes) of singletons.The largest and smallest numbers of singletons were observed in the genomes of Mariniflexile sp.TRM1-10 (1083 clusters) and M. soesokkakense KCTC 32427 T (377 clusters).The annotated singletons in the KMM 9835 T genome were more prevalent and often related to transport protein systems (SusC), transcriptional regulators (AraC, LytTR, and LacI), and enzymes (Glycosy-lases).According to the genome size modeling, the Mariniflexile pan-genome is open with a γ value of 0.58 (Figure S3).

CAZymes Repertoires and Predicted PULs Analysis
The dbCAN annotation analysis revealed that strains within the Mariniflexile genus possess a highly diverse repertoire of CAZymes and PULs, which may allow them to utilize a wide range of polysaccharides.The proportion of CAZyme-encoding genes in their genomes varied from 5.3% to 7.9%, with the maximum amount predicted in strains TRM1-10 and KMM 9835 T (Figure 5a).In the KMM 9835 T genome, CAZymeencoding genes reached 7.3%, comprising 164 glycoside hydrolases (GHs) classified into 52 families, 65 glycosyltransferases (GTs) into 10 families, 29 polysaccharide lyases (PLs) into 9 families, 19 carbohydrate esterases (CEs) into 9 families, and 2 auxiliary activities (AAs) into 2 families (Figure 5a).
The highest number of GHs found in KMM 9835 T was related to the GH43 family (19 predicted encoding genes) containing arabinases and xylosidases (Figure 5b).That indicates the potential of the novel strain KMM 9835 T to cleave arabinose moieties from xylans and pectins [60].The greatest number of GH43-encoding genes among those of Mariniflexile spp.were also predicted in Mariniflexile sp.TRM1-10, isolated from the rhizosphere of tomato (Figure 5b).It can be assumed that strain TRM1-10 is able to utilize pectin, which is a polysaccharide primarily characteristic of land plants.The other abundant GHs of strain KMM 9835 T were found to belong to GH2 beta-galactosydases (16 genes) catalyzing the degradation of different oligosaccharides.Members of the GH28 and GH92 families were also widely distributed within the KMM 9835 T genome.The GH28 family was represented by 11 putative polygalacturonases that may cleave the backbone glycosidic linkages of pectin using a hydrolytic reaction [61].The GH92 family contains α-mannosidases responsible for N-glycan cleavage that are common in many flavobacterial species [62].All of these GHs were found within PULs (Figure 3), with the majority of GH92 members concentrated in PUL3, which is predicted to hydrolyze mucin-rich substrates.Most annotated polysaccharide lyases were classified as PL1 and PL10 families, possessing pectin and pectate lytic activities.Among carbohydrate esterases, the CE8 and CE12 families recognized for facilitating the degradation of pectin by deacetylation and demethylation [63] were the most abundant.The Mariniflexile sp.TRM1-10 and M. soesokkakense KCTC 32427 T genomes shared similarities with the KMM 9835 T distribution of CAZyme gene families (GH43, GH28, PL1, PL10, CE8, and CE12), which are responsible for pectin degradation.This may represent part of their adaptation strategy for facilitating microbial consumption of plant polymeric substrates in aquatic environments near shorelines and freshwater sources.The highest number of GHs found in KMM 9835 T was related to the GH43 family (19 predicted encoding genes) containing arabinases and xylosidases (Figure 5b).That indicates the potential of the novel strain KMM 9835 T to cleave arabinose moieties from xylans and pectins [60].The greatest number of GH43-encoding genes among those of Mariniflexile spp.were also predicted in Mariniflexile sp.TRM1-10, isolated from the rhizosphere of tomato (Figure 5b).It can be assumed that strain TRM1-10 is able to utilize pectin, which is a polysaccharide primarily characteristic of land plants.The other abundant GHs of strain KMM 9835 T were found to belong to GH2 beta-galactosydases (16 genes) catalyzing the degradation of different oligosaccharides.Members of the GH28 and GH92 families were also widely distributed within the KMM 9835 T genome.The GH28 family was represented by 11 putative polygalacturonases that may cleave the backbone glycosidic linkages of pectin using a hydrolytic reaction [61].The GH92 family contains α-mannosidases responsible for N-glycan cleavage that are common in many flavobacterial species [62].All of these GHs were found within PULs (Figure 3), with the majority of GH92 members concentrated in PUL3, which is predicted to hydrolyze mucin-rich substrates.Most annotated polysaccharide lyases were classified as PL1 and PL10 families, possessing pectin and pectate lytic activities.Among carbohydrate esterases, the CE8 and CE12 families recognized for facilitating the degradation of pectin by deacetylation and demethylation [63] were the most abundant.The Mariniflexile sp.TRM1-10 and M. soesokkakense KCTC 32427 T genomes shared similarities with the KMM 9835 T distribution of CAZyme gene families (GH43, GH28, PL1, PL10, CE8, and CE12), which are responsible for pectin degradation.This may represent part of their adaptation strategy for facilitating microbial consumption The second most frequent enzyme family in the KMM 9835 T CAZome is GTs (65 encoding genes) (Figure 5a).GT2 and GT4 accounted for the highest proportion in the GT families, followed by GT51 in KMM 9835 T and the related strain genomes (Figure 5b).GT2 and GT4 have been shown to perform the synthesis of αand β-glycans and glycoconjugates [64], while GT51 enzymes, known as peptidoglycan glycosyltransferase, take part in the synthesis of murein in both Gram-positive and Gram-negative bacteria [65].
Signal peptide prediction in the KMM 9835 T CAZome provided by dbCAN [48] revealed that about half of the total number of GHs, PLs, and CEs genes have signal peptide sequences targeting their products to the periplasmic space.In addition, some secretory CAZymes were predicted to contain CTDs and can be transported by the unique Bacteroidota T9SS [11,66], supporting their extracellular role in polysaccharide metabolism.
Based on biochemical characteristics (Table 2), Mariniflexile spp., except for M. gromovii KMM 6038 T , showed the ability to hydrolyze starch [1,4,8,10].Deep genomic analysis revealed that the genomes of novel bacteria KMM 9835 T , M. soesokkakense KCTC 32427 T , M. maritimum KCTC 72895 T , and M. fucanivorans DSM 18792 T contain genes encoding for the starch utilization system (Sus) [67], which explains the difference in the substrate utilization abilities of all the above-mentioned bacteria.However, these genes were not found in the M. gromovii KMM 6038 T genome, which explains the difference in their substrate utilization abilities.In the KMM 9835 T genome, the sus locus includes six genes, encoding outer membrane SusCD (E/F)G and periplasmic SusAB proteins.SusG is an α-amylase with CBM48, GH13_10, and C-terminal T9SS domains, which allows for hydrolysis of α-1-4 glucosidic linkages at the cell surface.Interestingly, SusC, SusD, hybrid SusE/F, and two neighboring hypothetical proteins shared 75-90% similarity with those from Tenacibaculum adriaticum DSM 18961 T , Gelidibacter salicanalis PAMC21136 T , and Algibacter agarivorans JCM 18285 T .Moreover, this region in the KMM 9835 T genome was detected as a HGT region by the AlienHunter tool in Proksee [42].Neopullulanase SusA (GH13_46) and α-glucosidase SusB (GH97) may be responsible for the degradation of oligosaccharides to maltose.Thus, a full starch utilization system was found in all the Mariniflexile genomes except for M. gromovii KMM 6038T, which is consistent with the data from the biochemical tests.
Finally, we hypothesize that the environment can influence the gene composition of Mariniflexile species, providing them with an adaptive potential to degrade natural polysaccharides specific to the particular ecological niche.The most obvious difference between Mariniflexile strains isolated from the seashore or plants (KMM 9835 T , Mariniflexile sp.TRM1-10, and M. soesokkakense KCTC 32427 T ) and from sea water or marine organisms (M.fucanivorans DSM 18792 T , M. gromovii KCTC 12570 T , and Mariniflexile sp.AS56) is that they have similar CAZomes, despite their distant positions on the phylogenomic tree (Figure S2); KMM 9835 T is phylogenetically close to strain AS56 and most distant from the strains TRM1-10 and M. soesokkakense KCTC 32427 T (Figure 5b).

Morphological, Physiological, and Biochemical Characteristics
Strain KMM 9835 T was found to be Gram-negative, aerobic, non-motile bacteria.Colonies were yellow-pigmented shiny ones with regular edges of 2−3 mm in diameter on MA 2216.Electron microscopy observation revealed rod-shaped cells, 1.5−2.0µm long and 0.7−0.9µm in diameter, and extracellular material production was observed (Figure 6).Phenotypic characteristics of strain KMM 9862 T are given in Tables 2 and 3, Figure S4, and in the species description.The bacterium KMM 9835 T was able to grow in the narrow salinity range of 0-5% NaCl and at a temperature of 5-36 • C but was not able to utilize carbohydrates sources in the API 20E and API 20NE tests.It should be noted that the novel strain grew slowly without NaCl addition (0% NaCl) and weakly with 5% NaCl.The genomic DNA G+C content of strain KMM 9835 T was 32.5 mol% (Table 2).

Conclusions
The phylogenetic relationships observed on the basis of 16S rRNA gene and whole genome sequences and genetic distinctness as revealed by ANI and dDDH analyses were supported by phenotypic differences of the novel isolate KMM 9835 T in its growth temperature and salinity ranges, enzyme activity, and substrate hydrolysis.Differential phenotypic characteristics are indicated in Table 2. Based on the combined phylogenetic evidence and phenotypic characteristics, it is proposed to classify marine sediment strain KMM 9835 T as a novel species, Mariniflexile litorale sp.nov.
The dominant menaquinone is MK-6, and the major fatty acids are iso-C15:0, iso-C15:1 ω10c, and C15:0.The polar lipids comprise phosphatidylethanolamine, two unidentified aminolipids, an unidentified phospholipid, and six unidentified lipids.The DNA GC content of 32.5% is calculated from the genome sequence.
The DDBJ/GenBank accession numbers for the 16S rRNA gene and genome sequences of strain KMM 9835 T are OQ300347 and JASCRQ010000000 (GCF_031128465.2),respectively.
The type strain of the species is strain KMM 9835 T (=KCTC 92792 T ), isolated from the sediment sample collected from the Amur Bay of the Sea of Japan seashore, Russia.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms12071413/s1, Figure S1: NJ/ML/MP tree based on 16S rRNA gene sequences available from the GenBank database showing relationships between the novel strain KMM 9835 T (in bold), Mariniflexile species, and related taxa of the family Flavobacteriaceae.The NJ tree was reconstructed using the Kimura two-parameter model, and the ML tree was inferred under the GTR+GAMMA model.The branches are scaled in terms of the expected number of substitutions per site.The numbers above the branches represent bootstrap values with 1000 replicates larger than 60% (NJ/ML/MP).The bar indicates 0.02 accumulated substitutions per nucleotide position;

Data Availability Statement:
The type strain of the species is strain KMM 9835 T (=KCTC 92792 T ), isolated from the sediment sample collected from the Amur Bay of the Sea of Japan seashore, Russia.The DDBJ/GenBank accession numbers for the 16S rRNA gene and genome sequences of strain KMM 9835 T are OQ300347 and JASCRQ010000000, respectively.The DDBJ/GenBank accession number for the genome sequence of strain Mariniflexile soesokkakense KCTC 32427 T is JAZHYP000000000.

Conflicts of Interest:
The authors declare no conflicts of interest.

Figure 1 .
Figure 1.NJ/ML/MP tree based on 16S rRNA gene sequences available from the GenBank database showing relationships between the novel strain KMM 9835 T (in bold), Mariniflexile species, and related taxa of the family Flavobacteriaceae.The NJ tree was reconstructed using the Kimura two-parameter model.The ML tree was inferred under the GTR + GAMMA model.The branches are scaled in terms of the expected number of substitutions per site.The numbers above the branches represent bootstrap values with 1000 replicates larger than 60% (NJ/ML/MP).The bar indicates 0.02 accumulated substitutions per nucleotide position.

Figure 1 .
Figure 1.NJ/ML/MP tree based on 16S rRNA gene sequences available from the GenBank database showing relationships between the novel strain KMM 9835 T (in bold), Mariniflexile species, and related taxa of the family Flavobacteriaceae.The NJ tree was reconstructed using the Kimura twoparameter model.The ML tree was inferred under the GTR + GAMMA model.The branches are scaled in terms of the expected number of substitutions per site.The numbers above the branches represent bootstrap values with 1000 replicates larger than 60% (NJ/ML/MP).The bar indicates 0.02 accumulated substitutions per nucleotide position.

Figure 2 .
Figure 2. ML tree based on concatenated sequences of 341 translated proteins showing the phylogenetic position of strain KMM 9835 T among Mariniflexile species and related taxa.The tree was inferred under the PROTCATLG evolutionary model using 100 replicates for bootstrapping.Bar: 0.20 substitutions per amino acid position.

Figure 2 .
Figure 2. ML tree based on concatenated sequences of 341 translated proteins showing the phylogenetic position of strain KMM 9835 T among Mariniflexile species and related taxa.The tree was inferred under the PROTCATLG evolutionary model using 100 replicates for bootstrapping.Bar: 0.20 substitutions per amino acid position.

Figure 3 .
Figure 3. Chromosome map of strain KMM 9835 T created using the Proksee server[42].The scale is shown in megabases (Mbp) on the inside circle.Starting with the inner rings, the first two circles represent GC content (in black) and GC skew (G−C)/(G+C) (in violet blue and light green).The next two dark red circles show reverse and forward strand CDSs.Moving outward, the dark green circle shows PULs designated as CGCs, annotated by the dbCAN server[49].The outermost circle shows the CRISPR-Cas region (in black).The figure also shows retron-type RNA-directed DNA polymerase (EC 2.7.7.49) (designated as retron 1-13 with black labels), rrn operons (blue labels), oriC (leuB_2 and dnaA), and ter (mnmG) (red labels).

Figure 3 .
Figure 3. Chromosome map of strain KMM 9835 T created using the Proksee server[42].The scale is shown in megabases (Mbp) on the inside circle.Starting with the inner rings, the first two circles represent GC content (in black) and GC skew (G−C)/(G+C) (in violet blue and light green).The next two dark red circles show reverse and forward strand CDSs.Moving outward, the dark green circle shows PULs designated as CGCs, annotated by the dbCAN server[49].The outermost circle shows the CRISPR-Cas region (in black).The figure also shows retron-type RNA-directed DNA polymerase (EC 2.7.7.49) (designated as retron 1-13 with black labels), rrn operons (blue labels), oriC (leuB_2 and dnaA), and ter (mnmG) (red labels).

Figure 4 .
Figure 4.The pan-genome of seven strains of Mariniflexile spp.generated with anvi'o [50].Circle bars represent the presence/absence of 9163 pan-genomic clusters in each genome.Gene clusters are organized as core (green), shell (yellow), cloud (red), and singleton (purple) gene clusters using Euclidian distance and Ward ordination.The heatmap in the upper right corner shows pairwise values of average nucleotide identity (ANI) in percentages.The bars under the heatmap show, relative to each genome, the number of gene clusters (0-3881), number of singleton gene clusters (0-1010), GC-content (0-0.37778), and total length (0-4,858,325).The strain KMM 9835 T is colored red.

Figure 4 .
Figure 4.The pan-genome of seven strains of Mariniflexile spp.generated with anvi'o [50].Circle bars represent the presence/absence of 9163 pan-genomic clusters in each genome.Gene clusters are organized as core (green), shell (yellow), cloud (red), and singleton (purple) gene clusters using Euclidian distance and Ward ordination.The heatmap in the upper right corner shows pairwise values of average nucleotide identity (ANI) in percentages.The bars under the heatmap show, relative to each genome, the number of gene clusters (0-3881), number of singleton gene clusters (0-1010), GC-content (0-0.37778), and total length (0-4,858,325).The strain KMM 9835 T is colored red.Other information included in the figure comprises the maximum number of paralogs, combined homogeneity index, single-copy gene clusters (SCG clusters), and KOfam and KEGG modules (green and light green circles).

Table 2 .
Differential characteristics of strain 9835 T and the type strains of the most closely related Mariniflexile species.

Table 3 .
Cellular fatty acid composition (%) of strain KMM 9835 T and type strains of related Mariniflexile species.

Table 3 .
Cellular fatty acid composition (%) of strain KMM 9835 T and type strains of related Mariniflexile species.