Muricauda okinawensis sp. Nov. and Muricauda yonaguniensis sp. Nov., Two Marine Bacteria Isolated from the Sediment Core near Hydrothermal Fields of Southern Okinawa Trough

Two strains, 81s02T and 334s03T, were isolated from the sediment core near the hydrothermal field of southern Okinawa Trough. The cells of both strains were observed to be rod-shaped, non-gliding, Gram-staining negative, yellow-pigmented, facultatively anaerobic, catalase and oxidase positive, and showing optimum growth at 30 °C and pH 7.5. The strains 81s02T and 334s03T were able to tolerate up to 10% and 9% (w/v) NaCl concentration, respectively. Based on phylogenomic analysis, the average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) values between the two strains and the nearest phylogenetic neighbors of the genus Muricauda were in range of 78.0–86.3% and 21.5–33.9%, respectively. The strains 81s02T and 334s03T shared 98.1% 16S rRNA gene sequence similarity to each other but were identified as two distinct species based on 81.4–81.5% ANIb, 85.5–85.6% ANIm and 25.4% dDDH values calculated using whole genome sequences. The strains 81s02T and 334s03T shared the highest 16S rRNA gene sequence similarity to M. lutimaris SMK-108T (98.7%) and M. aurea BC31-1-A7T (98.8%), respectively. The major fatty acid of strains 81s02T and 334s03T were identified similarly as iso-C15:0, iso-C17:0 3-OH and iso-C15:1 G, and the major polar lipids of the both strains consisted of phosphatidylethanolamine and two unidentified lipids. The strains contained MK-6 as their predominant menaquinone. The genomic G+C contents of strains 81s02T and 334s03T were determined to be 41.6 and 41.9 mol%, respectively. Based on the phylogenetic and phenotypic characteristics, both strains are considered to represent two novel species of the genus Muricauda, and the names Muricauda okinawensis sp. nov. and Muricauda yonaguniensis sp. nov. are proposed for strains 81s02T (=KCTC 92889T = MCCC 1K08502T) and 334s03T (=KCTC 92890T = MCCC 1K08503T).


Sampling, Isolation and Maintenance
The samples were recovered by a gravity corer from southern OT during the HOBAB4 cruise of the R/V Kexue at Station S2 in 2016. Sediment core HOBAB4-S2 (24 • 52 49.91 N; 122 • 37 19.70 E; water depth, 1505 m) was collected from a rifted basin between the Yonaguni Knoll IV and Tangyin hydrothermal field. Marine sediment dilutions (up to 10 −2 , 0.1 g sediment in 9.9 mL artificial sea water) were spread-plated on marine agar 2216 (MA, pH 7.2; BD Difco, New York, NY, USA) [39] and incubated at 25 • C under aerobic condition. Two isolates, designated as 81s02 and 334s03, were obtained after being incubated for 7 days from the samples of 81 cm and 334 cm below surface, respectively. The strains were stored at −80 • C in 20% (v/v) glycerol. The type strains Muricauda ruestringensis B1 T (=DSM 13258 T ), Muricauda aurea BC31-1-A7 T (=MCCC M23246 T ), Muricauda aquimarina SW-63 T (=JCM 11811 T ) and Muricauda lutimaris SMK-108 T (=KCTC 22173 T ), purchased from the Marine Culture Collection of China (MCCC), were used as reference strains for comparative purposes. Unless otherwise described, the strains were cultivated on marine agar 2216 (MA; Difco) at pH 7.5 and 30 • C.

16S rRNA Gene Sequence and Phylogeny
Genomic DNA was extracted using a genomic DNA extraction kit (TIANGEN Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. The 16S rRNA gene was amplified by the universal primers 27F and 1492R, as previously described [40]. The PCR product was purified and ligated into the PMD19-T vector (TaKaRa, Kusatsu, Japan) and cloned according to the manufacturer's instructions. Sequencing was performed by BGI (Qingdao, China). A BLAST search (https://www.ncbi.nlm.nih.gov, accessed on 10 April 2023) and the EzTaxon-e server (http://www.ezbiocloud.net, accessed on 10 April 2023) were used to calculate the pairwise sequence similarity based on the almost complete 16S rRNA gene sequences [41]. The multiple alignments of sequences for the new strains (81s02 T and 334s03 T ) and other type strains of the most closely related species were performed using CLUSTAL X [42]. A phylogenetic analysis was subsequently performed and the phylogenetic trees were constructed with MEGA version X [43] using the neighborjoining (NJ) [44], maximum parsimony (MP) [45] and maximum likelihood (ML) [46] algorithms. The bootstrap analyses were based on 1000 re-samplings and the complete deletion option was used for the analysis [47], and the distances were calculated according to the two-parameter model of Kimura [48].

Chemotaxonomic Characterization
The cell biomass of strains for chemotaxonomic analysis was collected and freeze-dried after incubation in MB at 30 • C for 48 h. The respiratory isoprenoid quinones of strains 81s02 T and 334s03 T were extracted and analyzed by HPLC as described [66][67][68]. The polar lipids of novel isolates were extracted and detected using 2D thin-layer chromatography (TLC). Total lipid material was examined using molybdatophosphoric acid and specific functional groups were investigated using spray reagents specific for each, according to Tindall et al. [69]. The fatty acids were determined for the strains 81s02 T and 334s03 T , as well as for M. aquimarina SW-63 T , M. aurea BC31-1-A7 T , M. lutimaris SMK-108 T and M. ruestringensis B1 T , according to the standard protocol of the Microbial Identification System (MIDI, Sherlock Version 6.3). Fatty acids were methylated and analyzed using an Agilent 6890 N gas chromatography instrument (Santa Clara, CA, USA) and identified using the RTSBA6 database of the microbial identification system [70].

Phylogenetic and Genome Analysis
The almost full-length 16S rRNA gene sequences of strains 81s02 T (1488 bp, OQ547168) and 334s03 T (1488 bp, OQ547169) were determined and confirmed with the draft genome sequence. The strains shared 98.1% 16S rRNA gene sequence similarity to each other. Sequence similarity values calculated for the strains 81s02 T and 334s03 T indicated the greatest degree of similarity to M. lutimaris SMK-108 T (98.7%) and M. aurea BC31-1-A7 T (98.8%), respectively. Moreover, strain 81s02 T exhibited 16S rRNA gene sequence similarities of 98.7, 98. The draft genome sequence of strain 81s02 T (JARFVA000000000) resulted in 14 contigs and yielded a genome of 4,030,812 bp in length after assembly. Contigs varied in length from 1908 to 1,216,753 bp, and the N50 value was 852,588. The draft genome sequence of strain 334s03 T (JARFVB000000000) resulted in 44 contigs and yielded a genome of 4,292,830 bp in length after assembly. Contigs varied in length from 1164 to 968,529 bp, and the N50 value was 323,675. The sequencing depths of coverage were 347× and 294× for strains 81s02 T and 334s03 T , respectively. The genomic DNA G+C contents of strains 81s02 T and 334s03 T were 41.6 and 41.9 mol%, respectively, which were determined from the genome sequence. ANIb and ANIm values between new strains (81s02 T and 334s03 T ) and reference strains ranged from 78.0% to 86.3%, which were significantly lower than the threshold value (95-96%) for the delineation of genomic species [71]. The dDDH values based on the draft genomes between new strains and reference strains ranged from 21.5% to 33.9%, which were far below cut-off values (70%) for species differentiation [57] (Table S1). Moreover, the ANIb, ANIm and dDDH values between strain 81s02 T and 334s03 T were 81.4-81.5, 85.5-85.6 and 25.4%, respectively. The overall topological structures of the phylogenetic and phylogenomic trees (Figures 1 and 2) clearly showed that strains 81s02 T and 334s03 T fell within the clade comprising species of the genus Muricauda. The phylogenetic trees using MP and ML algorithms also showed essentially the similar topology (Figures S1 and S2). threshold value (95-96%) for the delineation of genomic species [71]. The dDDH value based on the draft genomes between new strains and reference strains ranged from 21.5% to 33.9%, which were far below cut-off values (70%) for species differentiation [57] (Tabl S1). Moreover, the ANIb, ANIm and dDDH values between strain 81s02 T and 334s03 were 81.4-81.5, 85.5-85.6 and 25.4%, respectively. The overall topological structures of th phylogenetic and phylogenomic trees (Figures 1 and 2) clearly showed that strains 81s02 and 334s03 T fell within the clade comprising species of the genus Muricauda. The phylo genetic trees using MP and ML algorithms also showed essentially the similar topology ( Figures S1 and S2).
Hydrolysis of: Enzymic activities:  * Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed feature 2 contained C 14:0 3-OH and/or iso-C 16:1 I; Summed feature 3 contained C 16:1 ω7c and/or C 16:1 ω6c; Summed feature 9 contained C 16:0 10-methyl and/or iso-C 17:1 ω9c.

Genome Attributes and Comparative Genome Analysis
The draft genome of strain 81s02 T contained 3662 ORFs and 42 tRNAs, while strain 334s03 T contained 3864 ORFs and 38 tRNAs. Pathway analyses on the KEGG website suggested that all eleven strains in this study within genus Muricauda had a complete glycolysis pathway (Embden-Meyerhof pathway, EMP) although the genes encoding some reaction steps were diverse (Figure 4). A complete pentose phosphate pathway (PPP) was also found in all eleven genomes, and it was more conserved compared to the EMP, where all genes encoding the PPP found in all strains were the same, aside from ripA, which was only found in strain M. oceani 501str8 T . On the other hand, all strains were lacking the gene edd, encoding phosphogluconate dehydratase, indicating that the Entner-Doudoroff pathway might not present in Muricauda strains. Several Muricauda species contained the genes nasC and nirA, such as M. aurea, M. chongwuensis, M. brasiliensi, M. oceani and M. ruestringensis, while the other species only contained nirA but not nasC. Commonly, genes nasC and nirA were recognized to reduce nitrate to nitrite and reduce nitrite to ammonia, respectively [76]. Therefore, Muricauda species had a diverse nitrate reduction capability. Moreover, a complete assimilatory sulfate reduction pathway was found in M. ruestringensis B1 T through the existence of genes cysC, cysD, cysN, cysJ and cysI. It was indicated that M. ruestringensis (the type species of genus Muricauda) had the capability of reducing sulfate to sulfite or hydrogen sulfide. However, other members in this study were lacking certain genes, showing an uncomplete sulfate reduction pathway. In addition, KEGG annotation showed that rhamnose containing glycans biosynthesis protein, polysaccharide biosynthesis/export protein, and lipopolysaccharide (dTDP-L-rhamnose) biosynthesis/assembly protein-related genes were found in all eleven Muricauda strains, and it has been suggested that it helps Muricauda survive in marine environments and assists them to endure extremes of temperature, salinity and nutrient availability. Metabolic features related to functional categories from RAST showed that genes associated with "virulence, disease and defense" existed in the genomes of Muricauda strains, which might be important for Muricauda to resist the toxic compounds in the environments ( Figure S4). A large number of genes were involved in the class of "stress response", which might provide these species the ability to adapt to special environments stresses, such as pressure, oxygen concentration, temperature, pH and salinity in marine ecosystem.
only found in strain M. oceani 501str8 T . On the other hand, all strains were lacking the gene edd, encoding phosphogluconate dehydratase, indicating that the Entner-Doudoroff pathway might not present in Muricauda strains. Several Muricauda species contained the genes nasC and nirA, such as M. aurea, M. chongwuensis, M. brasiliensi, M. oceani and M. ruestringensis, while the other species only contained nirA but not nasC. Commonly, genes nasC and nirA were recognized to reduce nitrate to nitrite and reduce nitrite to ammonia, respectively [76]. Therefore, Muricauda species had a diverse nitrate reduction capability. Moreover, a complete assimilatory sulfate reduction pathway was found in M. ruestringensis B1 T through the existence of genes cysC, cysD, cysN, cysJ and cysI. It was indicated that M. ruestringensis (the type species of genus Muricauda) had the capability of reducing sulfate to sulfite or hydrogen sulfide. However, other members in this study were lacking certain genes, showing an uncomplete sulfate reduction pathway. In addition, KEGG annotation showed that rhamnose containing glycans biosynthesis protein, polysaccharide biosynthesis/export protein, and lipopolysaccharide (dTDP-L-rhamnose) biosynthesis/assembly protein-related genes were found in all eleven Muricauda strains, and it has been suggested that it helps Muricauda survive in marine environments and assists them to endure extremes of temperature, salinity and nutrient availability. Metabolic features related to functional categories from RAST showed that genes associated with "virulence, disease and defense" existed in the genomes of Muricauda strains, which might be important for Muricauda to resist the toxic compounds in the environments (Figure S4). A large number of genes were involved in the class of "stress response", which might provide these species the ability to adapt to special environments stresses, such as pressure, oxygen concentration, temperature, pH and salinity in marine ecosystem. Carbohydrate-active enzymes (CAZymes) are involved in many metabolic pathways and in the biosynthesis and degradation of various biomolecules, such as bacterial exopolysaccharides, starch, cellulose and lignin [77]. Thus, genes putatively coding for carbon metabolism were analyzed among different species within Muricauda, including Carbohydrate-active enzymes (CAZymes) are involved in many metabolic pathways and in the biosynthesis and degradation of various biomolecules, such as bacterial exopolysaccharides, starch, cellulose and lignin [77]. Thus, genes putatively coding for carbon metabolism were analyzed among different species within Muricauda, including CAZymes, redox enzymes with auxiliary activities (AAs) and those with carbohydratebinding modules (CBMs) ( Figure S5). CAZyme families were classified into four major groups: glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs) and carbohydrate esterases (CEs). Strains 81s02 T and 334s03 T harbored similar CAZymeencoding genes compared to other species of Muricauda. These genes were mainly present in the groups of GHs and GTs. A number of GH3, GH13, GH109, GT2 and GT4 were identified in all genomes; however, GH43 was not observed in the genome of strain 81s02 T , which was present in other ten strains. Moreover, PL7, PL9 and CBM62 were only observed in the genome of strain M. aurea BC31-1-A7 T , while PL8 was only observed in the genome of strain M. chongwuensis HICW T , suggesting a different process for carbon metabolism in these strains. Therefore, a more expanded investigation for the carbon utilization of the genus Muricauda is required to gain insights into a complete process for the carbon metabolism in the future.
The type strain is 81s02 T (=KCTC 92889 T = MCCC 1K08502 T ) and was isolated from the sediment core near the hydrothermal fields of the southern Okinawa Trough. The GenBank accession numbers for the 16S rRNA gene and the draft whole genome data of the strain 81s02 T are OQ547168 and JARFVA000000000, respectively.
The type strain is 334s03 T (=KCTC 92890 T = MCCC 1K08503 T ) and was isolated from the sediment core near the hydrothermal fields of the southern Okinawa Trough. The GenBank accession numbers for the 16S rRNA gene and the draft whole genome data of strain 334s03 T are OQ547169 and JARFVB000000000, respectively.

Supplementary Materials:
The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/microorganisms11061580/s1, Figure S1: Phylogenetic tree, based on the 16S rRNA gene sequences using the maximum likelihood algorithm showing the position of strains 81s02 T and 334s03 T . GenBank accession numbers used are given in the parentheses. Bootstrap values higher than 50% are indicated at branch nodes. Formosa maritima 1494 T was used as an outgroup. Bar, 0.02 substitutions per nucleotide position; Figure S2: Phylogenetic tree, based on the 16S rRNA gene sequences using the maximum parsimony algorithm showing the position of strains 81s02 T and 334s03 T . GenBank accession numbers used are given in the parentheses. Bootstrap values higher than 50% are indicated at branch nodes; Figure S3: Two-dimensional thin-layer chromatogram of polar lipids; Figure S4: Metabolic features related to functional categories of 11 Muricauda strains. The encoding gene involved in the categories of "Photosynthesis", "Iron acquisition and metabolism" and "Motility and Chemotaxis" was not annotated; Figure S5: Genes putatively coding for carbon metabolism among different Muricauda species based on the CAZy database. Table S1: ANIb, ANIm and dDDH values between pairs of type strains of Muricauda species.
Author Contributions: Strain 8102 T and 334s03 T were isolated by X.D. and W.C. Material preparation, data collection and analysis were performed by W.C., X.D. and M.J. The samples was collected and provided by Z.Z. The original draft was written by W.C. The writing, review and editing for the manuscript was performed by M.J., Z.Z. and F.C. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The datasets used during the current study are available from the corresponding author on reasonable request.