Halocynthiibacter laminarini gen. nov., sp. nov. and Halocynthiibacter xylanolyticus sp. nov., marine anaerobic laminarin and xylan degraders in the phylum Bacteroidota

Halocynthiibacter laminarini gen. nov., sp. nov. and Halocynthiibacter xylanolyticus sp. nov., marine anaerobic laminarin and xylan degraders in the phylum Bacteroidota | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Halocynthiibacter laminarini gen. nov., sp. nov. and Halocynthiibacter xylanolyticus sp. nov., marine anaerobic laminarin and xylan degraders in the phylum Bacteroidota Tra T.H. Nguyen, Tien Q. Vuong, Ho Le Han, Song-Gun Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4632626/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The bacterial group of the phylum Bacteroidota greatly contributes to the global carbon cycle in marine ecosystems through its specialized ability to degrade marine polysaccharides. In this study, it is proposed that two novel facultative anaerobic strains, DS1-an-13321 T and DS1-an-2312 T , which were isolated from a sea squirt, represent a novel genus, Halocynthiibacter , with two novel species in the family Prolixibacteraceae. The 16S rRNA sequence similarities of these two strains were 91.26% and 91.37%, respectively, against Puteibacter caeruleilacunae JC036 T , which is the closest recognized neighbor. The complete genomes of strains DS1-an-13321 T and DS1-an-2312 T each consisted of a single circular chromosome with a size of 4.47 and 5.19 Mb, respectively. The average amino acid identity and the percentage of conserved proteins against the type species of the genera in the family Prolixibacteraceae ranged from 48.33–52.35% and 28.34–37.37%, respectively, which are lower than the threshold for genus demarcation. Strains DS1-an-13321 T and DS1-an-2312 T could grow on galactose, glucose, maltose, lactose, sucrose, laminarin, and starch, and only DS1-an-2312 T could grow on xylose and xylan under fermentation conditions. These strains produced acetic acid and propionic acid as the major fermentation products. Genome mining of the genomes of the two strains revealed 27 and 34 polysaccharide utilization loci, which included 155 and 249 carbohydrate-active enzymes (CAZymes), covering 57 and 65 CAZymes families, respectively. The laminarin-degrading enzymes in both strains were cell-associated, and showed exo-hydrolytic activity releasing glucose as a major product. The xylan-degrading enzymes of strain DS1-an-2312 T was also cell-associated, and had endo-hydrolytic activities, releasing xylotriose and xylotetraose as major products. The evidence from phenotypic, biochemical, chemotaxonomic, and genomic characteristics supported the proposal of a novel genus with two novel species in the family Prolixibacteraceae , for which the names Halocynthiibacter laminarini gen. nov., sp. nov. and Halocynthiibacter xylanolyticus sp. nov. are proposed. The type strain of Halocynthiibacter laminarini is DS1-an-13321 T (= KCTC 25031 T = DSM 115329 T ) and the type strain of Halocynthiibacter xylanolyticus is DS1-an-2312 T (= KCTC 25032 T = DSM 115328 T ). Biological sciences/Biotechnology Biological sciences/Microbiology Polyphasic taxonomy KEGG CAZyme polysaccharide degradation Prolixibacteraceae PUL Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Polysaccharides constitute a significant proportion of the carbon inventory in marine organic matter and form a major structural component of both macro- and micro-algae [ 1 ]. Roughly half of this organic matter is released to the marine environment as dissolved and particulate organic matter (DOM, and POM) [ 2 ]. This is further degraded by the activities of marine heterotrophic bacteria, which contribute to carbon cycling in marine ecosystems by producing smaller molecular weight compounds and minerals [ 1 , 3 ]. Among marine heterotrophic bacteria, Bacteroidota is one of the most abundant groups, after Cyanobacteria and Proteobacteria [ 4 ]. These bacteria engage in different strategies to contribute to regulate the carbon cycle. Indeed, Cyanobacteria are known as a primary producer because of their photoautotrophic ability in marine ecosystems [ 4 , 5 ], whereas abundant members of marine Alphaproteobacteria are known to preferentially utilize simple sugars and are found in the water column [ 4 , 6 ]. In contrast, Bacteroidota are known as polysaccharide degraders [ 7 ]. Many studies have revealed the ability of Bacteroidota to degrade marine polysaccharides from dissolved or particulate organic matters, and the cell wall component of macro- and micro-algal cells [ 4 , 7 ]. This highlights the crucial role of this process in sequestering carbon within the marine environment, thereby significantly contributing to the carbon cycle in marine ecosystems [ 4 , 8 ]. To access this rich source of polysaccharides, Bacteroidota have evolved a unique apparatus for their polysaccharide degradation strategy [ 9 ]. This apparatus was first reported in Bacteroides thetaiotaomicron in the human gut microbiota [ 9 , 10 ]. It consists of SusD, an outer membrane-bound protein responsible for capturing polysaccharides [ 9 ], and outer membrane-bound carbohydrate-active enzymes (CAZymes) that partly degrade target polysaccharides into oligosaccharides [ 9 , 11 ]. Subsequently, these oligosaccharides are imported into the cell periplasm via a membrane-bound SusC transporter [ 9 ]. Other CAZymes then further degrade these oligosaccharides to monosaccharides, which are transported into the cytoplasm through dedicated transporters [ 7 , 9 ]. This entire process is regulated by a regulator that senses the products of polysaccharide degradation. These SusC, SusD, CAZymes, transporters, and regulators are encoded by genes that are clustered together in a region of the genome and are known as polysaccharide utilization loci (PUL) [ 7 ]. Bacteroidota was first described as a novel phylum by Krieg et al. in 2010, in the second edition of Bergey’s Manual of Systematic Bacteriology [ 12 ]. The name Bacteroidota was revised (from Bacteroidetes ) by Oren and Garrity in 2021 [ 13 ]. At the time of writing, the phylum Bacteroidota consists of six classes ( https://lpsn.dsmz.de/phylum/bacteroidota ): Bacteroidia Krieg 2012, Cytophagia Nakagawa 2012, Flavobacteriia Bernardet 2012, Sphingobacteriia Kampfer 2012, Chitinophagia Munoz et al. 2017, and Saprospiria Hahnke et al. 2018. Among these, three classes, Bacteroidia, Cytophagia , and Flavobacteriia , are the largest, and a huge number of studies on the polysaccharide degradation capability of the member bacteria have been reported. To date, members of the class Flavobacteriia are found to be abundant in the macroalgal phycosphere microbiome and in microalgal blooms and are well documented for macro- and microalgal polysaccharide degradation [ 14 , 15 ]. Several studies have meanwhile demonstrated the ability of members of the class Cytophagia to degrade polysaccharides [ 16 , 17 ] and predicted potential polysaccharide degradation [ 18 ], although there are fewer available literature sources of marine Cytophagia bacteria degrading polysaccharides [ 19 ]. On the other hand, members of the class Bacteroidia are mostly anaerobes and have been well studied for polysaccharide degradation in the mammalian gut microbiota [ 20 ]. Several studies on human gut Bacteroidia bacteria degrading marine polysaccharide under anaerobic conditions also have been reported [ 21 ]. However, few studies on marine Bacteroidia that anaerobically degrade polysaccharides can be found in the literature [ 22 ]. Xylan is the most abundant form of hemicellulose found in nature and is produced in marine and terrestrial environments [ 23 ]. Xylan from terrestrial plants contains a β-1,4-linkage D-xylopyranosyl backbone and units of acetate, ɑ-L-arabinofuranose, 4-O-methyl-glucuronic acid, or ferulic acid [ 23 ] whereas xylan from marine sources is homoxylan, which is found only in algae [ 24 ]. The backbone of marine xylan mainly consists of a 1,3-glycoside bond and a mixture of 1,3 and 1,4 glycoside bonds [ 24 ]. The molecular structure of xylan protects the cell walls of plants and algae from abiotic or biotic stresses, and it is very difficult to degrade xylan biologically. Biodegradation of xylan results in the production of xylose and xylooligosaccharides, which have potential roles in biotechnological applications such as biofuel, biomedicine, and food supplements [ 25 – 28 ]. Initially, xylan is degraded randomly by endoxylanases GH5, GH10, GH11, or GH30 [ 29 ] to release unbranched xylooligosaccharides, which are then degraded into xylose by β-xylosidase GH39, or GH43 [ 30 ]. Several studies have focused on the aerobic degradation of xylan by marine bacteria [ 24 , 31 , 32 ]. However, to our knowledge, research on the anaerobic degradation of xylan by marine Bacteroidia bacteria has been limited. Laminarin serves as the principal energy reserve glycan identified in brown algae [ 33 ] and some microalgae [ 34 ], with an estimated annual production of approximately 12–18 gigatons globally [ 35 ]. It is constructed by combining β-(1→3)-linked glucose-based linear chains and a lower ratio of β-(1→6)-linked side chains from glucose moieties [ 35 ]. Laminarin degradation results in the production of glucose and laminarin oligosaccharides, which can potentially be used in biotechnological applications in the biofuel, cosmetics, biomedical, and food industries [ 36 ]. To biologically degrade laminarin, endo-acting β-1,3-glucanases (glycoside hydrolase family 17, GH17) specifically break down the β-(1→3)-linkage in the main chain, while exo-acting β-1,6-glucosidases (GH30) specifically hydrolyze the β-(1→6)-linkage at the side chain of laminarin. Additionally, exo-acting β-1,3-glucosidases (GH3) specifically hydrolyze the β-(1→3)-linkage of oligosaccharides into glucoses [ 35 ]. The ability of laminarin degradation is widely distributed in diverse marine heterotrophic bacteria. However, there has been limited research on the anaerobic degradation of laminarin by marine Bacteroidia bacteria. The family Prolixibacteraceae of the order Bacteroidales , class Bacteroidia , phylum Bacteroidota was first proposed by Huang et al. 2014. At the time of writing, as described on the List of Prokaryotic Names with Standing in Nomenclature (LPSN, https://www.bacterio.net/ ), the family accommodates 12 valid genera, including Aquipluma Watanabe et al. 2020, Draconibacterium Du et al 2014, Gaoshiqia Yu et al. 2023, Mangrovibacterium Huang et al. 2014, Maribellus Zhou et al. 2019, Mariniphaga Iino et al. 2014, Meniscus Irgens 1977, Prolixibacter Holmes et al. 2007, Puteibacter Sun et al. 2020, Roseimarinus Wu et al. 2015, Sunxiuqinia Qu et al. 2011, and Tangfeifania Liu et al. 2014. They have been isolated from various habitats, particularly hypolimnion water [ 37 ], river sediment [ 38 ], mangrove sediment [ 39 ], marine sediment [ 40 ], seawater [ 22 ], and crude oil [ 41 ]. Most of the members of the family Prolixibacteraceae are Gram-staining negative, rod-shaped, non-spore-producing bacteria. They have no motility and their oxygen requirement ranges from aerobic to facultative anaerobic conditions. The predominant quinone component is menaquinone 7 (MK-7) [ 42 ]. Several isolation methods have been applied to isolate members of the family Prolixibacteraceae , including enrichment [ 22 ], a dilution technique [ 38 ], and nitrogen-free medium cultivation [ 39 ]. In this study, we used another strategy to isolate novel bacteria by using a low nutrient isolation medium, which was prepared from 60% strength seawater (at the collection site) with 1.5% agar (BD) and a piece of filter paper placed on the surface of the agar as the sample carrier [ 19 ]. We isolated two novel bacteria from a sea squirt under anaerobic conditions. In this study, we identified and characterized two novel anaerobic isolates, DS1-an-13321 T (strictly anaerobic) and DS1-an-2312 T (facultative anaerobic), through genetic, morphological, biochemical, and chemotaxonomic analyses. These isolates were proposed as novel genus with two novel species within the family Prolixibacteraceae. Genome mining revealed that these isolates contain hundreds of CAZyme genes spanning 65 CAZyme families. Their glycoside hydrolases were the most abundant among CAZymes, and exhibited up to double frequency of GHs per genome compared to the average value in marine Bacteroidota. Additionally, DS1-an-13321 T was found to utilize laminarin as a sole carbon source under anaerobic conditions, while DS1-an-2312 T could utilize both laminarin and xylan. The final hydrolytic product of laminarin degradation was glucose, whereas xylan degradation primarily yielded xylotriose and xylotetraose. This study is the first to combine genome mining with in vitro experiments to analyze xylan and laminarin degradation in anaerobic marine Bacteroidia , and the results enhance our understanding of the polysaccharide degradation strategies of anaerobic marine Bacteroidia and their role in marine carbon cycling. MATERIALS AND METHODS Ecology, isolation, and cultivation of isolates The source of isolation was a sea squirt collected by a fisherman at a depth of 18 m beneath the surface of seawater in the East Sea, Republic of Korea (38º38’48.5” N, 129º44’29,2” E). The sample was then stored in an ice-pack container and delivered to the laboratory on the same day. For isolation, a low nutrient solid medium prepared by sixty percent strength seawater (collected from the sampling site) solidified with 1.5% agar (BD) was used. After autoclaving the medium, 50 mg/L cycloheximide (Aldrich Sigma) was added to the agar medium [ 19 ]. All the steps for bacterial isolation were performed in an anaerobic chamber (Coy Lab Products, USA; N 2 : H 2 : CO 2 = 94.5%:1.5%:5%). The plates were stored in an anaerobic chamber overnight to remove oxygen. A slice taken from the mouth of the squirt was placed on the top of a piece of filter paper (Whatman No.2) on the isolation plates. The plates were placed in an anaerobic jar with a bag of BD GasPak EZ anaerobe container system (BD) and incubated at 23 ℃. After 4–5 days of incubation, the colony surrounding the filter paper was transferred and streaked onto fresh marine agar plates (BD marine agar 2216) under anaerobic conditions. After several transfers, single colonies of the two strains were obtained. The pure cultures of the two strains were preserved in 20% glycerol at -80 ℃, and lyophilized in ampoules stored at 4 ℃. Phylogenetic analysis based on the 16S rRNA gene sequence For a phylogenetic analysis, the 16S rRNA gene sequences of the two isolates were determined. The 16S rRNA sequences of the two strains were sequenced using four universal primers: 27F (5’-AGAGTTTGATCCTGGCTCAG-3’), 518F (5’-CCAGCAGCCGCGGTAATAC-3’), 805R (5’-GACTACCAGGGTATCTAATC-3’), and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) [ 19 ], via Sanger sequencing. The complete sequences were manually assembled using Vector NTI software (Invitrogen). Next, pairwise sequence alignment was performed on EzBioCloud ( https://www.ezbiocloud.net/ ). The related sequences downloaded from EzBioCloud were aligned using ClustalW in BioEdit software (version 7.2.5). The trimmed sequences were then used to make phylogenetic trees in MEGA11 using three algorithms: neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) [ 19 ]. The estimated matrix on MEGA11 based on the alignment fasta file showed that the optimal model for the MP tree was the general time reversible (GTR) model, and the rates and patterns were Gamma distributed with Invariant sites (G + I). Moreover, the Kimura two-parameter model was used for the NJ algorithm, and tree-bisection-reconnection (TBR) was used for the ML algorithm. The bootstrap resampling method with 1000 replicates was used to evaluate the phylogenetic tree. The sequence of strain Agarivorans albus (accession number AB681878) was obtained from NCBI as the outgroup, and the sequence of Marinilabilia salmonicolor NCIMB 2216 T (accession number D12672) was retrieved from NCBI as the type strain of the closest family, Marinilabiliaceae. Pairwise alignment of 16S rRNA sequences revealed that the strains DS1-an-2312 T and DS1-an-13321 T had the highest similarity to Puteibacter caeruleilacunae JC036 T , with similarity values of 91.57% and 91.48%, respectively. Therefore, the isolated strains were considered to belong to a novel genus in the family Prolixibacteraceae or to a novel family in the order Bacteroidales. To delineate the two strains in terms of phylogeny, the type species of each genus in the family Prolixibacteraceae and the type strains of the second closest family, Marinilabiliaceae , were included in a phylogenetic tree. The resulting phylogenetic tree, reconstructed based on three algorithms, revealed that the two novel isolates belonged to a cluster within the family Prolixibacteraceae and formed a separate cluster from other genera in this family (Fig. 2 . ) . Based on the phylogenetic position of the two isolates on the phylogenetic tree and their top hit similarity, three strains, Puteibacter caeruleilacunae KCTC 25263 T , Prolixibacter bellariivorans KCTC 25261 T , and Sunxiuqinia elliptica KCTC 32215 T , were selected as the reference strains. Physiological characterization The morphology of colonies of the two novel isolates was observed on MA plates after three days of cultivation under anaerobic conditions. Gram staining was performed according to the standard protocol, and the prepared slices were observed under a light microscope (Nikon Eclipse 80i). Scanning electron microscopy (SEM, JEOL JSM 7600F) was used to observe the cell morphology [ 19 ]. To determine their temperature range, the two strains were cultured in marine broth in test tubes purged with nitrogen gas and then incubated over a temperature range from 10–30 ℃ for three days and one week at 4 ℃ and 35 ℃. To determine the pH range, marine broth in test tubes purged with nitrogen gas was used to determine the growth of the two novel isolates using the following buffer systems at 50 mM: 5.5-6.0 (MES), 6.5-7 (PIPES), 7.5-8.0 (HEPES), 8.5 (Bis-Tris), and 9.0–10.0 (CHES) [ 43 ]. In a NaCl tolerance experiment, Zobell broth [ 43 ] in artificial seawater [ 44 ] was supplemented with NaCl concentrations of 0, 0.5, and 1–10% (with intervals of 1.0%, w/v). To determine the oxygen requirements, the two isolates were cultivated on MA plates in anaerobic jars under anaerobic conditions provided by a bag of BD GasPak EZ anaerobe container system (BD) and under microaerophilic conditions provided by a bag of BD GasPak EZ CO 2 container system. The plates were inoculated for three days at 28 ℃. The biochemical characteristics of the two novel isolates and their reference strains were determined on marine agar as the basal media at 28 ℃ for three days under anaerobic conditions unless otherwise specified. The hydrolysis of starch was tested by supplying 0.2% starch to MA plates and detecting a clear zone after staining with iodine solution. The hydrolysis of cellulose was assessed on carboxymethyl cellulose agar plates [ 19 ] using artificial seawater [ 44 ] with 2% NaCl instead of distilled water, and a clear zone was detected after embedding in Congo Red and washing with 1% (w/v) NaCl solution. The hydrolysis of Tween 20 and Tween 80 was performed by adding 0.1% (v/v) Tweens 20 and 80 to MA plates, respectively. Catalase activity was determined by dropping a 3% H 2 O 2 solution onto the surface of cells [ 19 ]. Oxidase activity was tested by reacting cells with an oxidase reagent (bioMerieux). All five strains were cultured on DNase agar (Difco) using artificial seawater with 2% NaCl instead of distilled water to test DNase activity. Chitin (1%, w/v) was added to MA plates, and chitin hydrolysis activity was detected by the appearance of a clear zone [ 19 ]. Gelatinase activity was evaluated on nutrient gelatin (Remel Gelatin medium) in which distilled water was replaced with artificial seawater [ 44 ] supplemented with 2% (w/v) NaCl for one week at 25 ℃, and a positive result was recognized by liquidation of the medium [ 45 ]. To determine anaerobic metabolism, the growth of the two novel isolates on a variety of carbon sources was assessed. An inoculum of the strains was prepared in 5 mL of marine broth (MB, Difco) supplemented with 20 mM HEPES (Sigma) and adjusted to pH 7.0 using 2N NaOH. The strains were cultivated on basal media (BS) supplemented with carbon substrates (0.2%, w/v). The composition of the BS was as follows: 23 g NaCl, 1.3 g KCl, 1 g MgCl 2 .6H 2 O, 0.1 g CaCl 2 .2H 2 O, 0.5 g NH 4 Cl, 0.2 g KH 2 PO 4 , 3.82 g Na 2 SO 4 , 0.08 g FeCl 2 .4H 2 O, 1 mL trace elements (KCTC Media No. 918, https://kctc.kribb.re.kr/access/search/viewMedia?sn=918 ), 1 mL vitamin solution [ 46 ], 0.1 g yeast extract, 2.5 g NaHCO 3 , 4.76 g HEPES, and 1 L deionized H 2 O, adjusted to pH 7.0 using 2N NaOH. The following carbon substrates were tested: fructose, galactose, glucose, xylose, maltose, lactose, sucrose, and starch. BS without substrates was used as the control. For fermentation conditions, Na 2 SO 4 was omitted from the composition of the basal medium. For sulfate reduction conditions, additional carbon sources (10 mM), which served as an electron donor, were evaluated, including acetate, benzoate, formate, fumarate, hexanoate, octanoate, lactate, propionate, and pyruvate [ 47 ], and sulfate (27 mM) was tested as an electron acceptor. For an additional anaerobic respiration test, nitrate (10 mM), nitrite (10 mM), sulfite (10 mM), and thiosulfate (10 mM) were added to BS to replace sulfate (27 mM) as electron acceptors. The growth of the strains was monitored under a microscope (Nikon Eclipse 80i) and compared with that of the control. The concentration of sulfate in the culture broth was measured [ 48 ]. The utilization of mono-, di-, and polysaccharides was quantified by measuring the production of reducing sugars during growth using a DNS assay [ 49 ]. Fermentation products were determined after five days of cultivation using high-performance liquid chromatography (HPLC), as described previously [ 50 ]. The chromatograph of the 5-day culture was compared with that of the 0-hour culture. Different and high peaks were identified by aligning the retention times with those of candidate standard compounds. All experiments were performed in duplicate. Chemotaxonomic characterization The fatty acid profiles of strains DS1-an-2312 T and DS1-an-13321 T , as well as their reference strains, were determined after they were grown on MA plates for three days. Cells were harvested for a fatty acid analysis following the procedure outlined in the MIDI protocol (version 6.2). Subsequently, the extracted fatty acid methyl esters were injected into a gas chromatograph system, and their fatty acid components were identified based on the RTSBA 6.0 database. To determine the quinone type, freeze-dried DS1-an-2312 T and DS1-an-13321 T cells were mixed with chloroform-methanol (2:1, v/v) and shaken overnight. The extract was collected through paper filtration, concentrated via evaporation, and recovered by adding acetone. The acetone suspension was then applied to a thin-layer chromatography (TLC) plate (Kieselgel 60F 254 , 20 × 20 cm, Merck) and separated using a combination of petroleum ether-diethyl ether (9:1, v/v). The quinone band on the TLC plate was visualized with UV light, and the samples were harvested and recovered in 100% acetone. The quinone extracts were further analyzed via reversed-phase chromatography using a mobile phase of methanol-isopropanol (7:5, v/v) and a wavelength of 270 nm to detect the quinone components [ 45 ]. The polar lipids of strains DS1-an-2312 T and DS1-an-13321 T were extracted from their freeze-dried cells following the detailed methods of Komagata and Suzuki [ 51 ]. The extracted lipids were then applied onto a silica gel TLC plate and developed on the plate in two dimensions. The first dimension involved a combination of chloroform-methanol-water (65:25:4, v/v/v), and the second a combination of chloroform-methanol-acetic acid-water (80:15:12:4, v/v/v/v) [ 45 ]. To identify the polar lipid profiles of the two novel isolates, TLC plates were sprayed with individually appropriate reagents: molybdatophosphoric acid for total lipids, ninhydrin for lipids with amino groups, molybdenum blue for lipids with phosphate groups, and ɑ-naphthol in a sulfuric acid solution for lipids with sugar groups. Genome analysis For genomic analysis, genomic DNA was extracted based on the method of Vengadesh et al. [ 52 ] with several modifications. Briefly, the isolated strains cultured anaerobically for two days in marine broth were harvested. The cell mass was mixed with proteinase K (20 mg/mL, 20 mM Tris-HCl, pH 8.0) in cetyltrimethylammonium bromide (CTAB) solution. The mixtures were incubated at 37 ℃ for one hour. Next, one volume of phenol-chloroform-isoamyl alcohol (PCI, 25:24:1, v/v/v) was added to the mixture, followed by centrifugation at 11 000 × g. The aqueous phase was collected and transferred to a new tube, and the nucleic acids were precipitated by adding a 0.6 volume of isopropyl alcohol and a one-tenth volume of 3 M sodium acetate. The tubes were inverted to ensure a consistent mixture and then incubated at 4 ℃. After 1–2 hours of incubation, the tubes were centrifuged at 16 000 × g for 20 minutes at 4 ℃. Next, the suspension was removed and replaced twice with 100 µL of 70% ethanol (cooled at -20 ℃), twice. The pellet was dried at room temperature and re-dissolved in DNase-free water. RNA was then removed from the solution by treatment with RNase A at 40 ℃ for one hour. The DNA component subsequently was separated by one volume of PCI and precipitated with alcohol as aforementioned. The DNA was re-dissolved in DNase-free water and used for genome sequencing. The whole-genome sequences of both novel strains were obtained by a combination of two sequencing methods, short-read Illumina sequencing (Macrogen, Inc., Seoul, Republic of Korea) and Nanopore sequencing. For Illumina sequencing, the short-length DNA of each strain was used to construct a library based on the protocol of the TruSeq DNA PCR-Free Sample Preparation Guide, part #15036187 Rev. D. For nanopore sequencing, high-molecular-weight DNA was used to prepare a library for nanopore sequencing according to the SQK-LSK109 protocol (version GDE_9063_v109_revN_14Aug2019). The genomes of both strains were de novo assembleed by Canu (version 2) [ 53 ] based on a combination of raw data from Nanopore and Illumina sequencing. Medaka ( version 1.3.2, https://github.com/nanoporetech/medaka ) was used as a polishing tool for assembly by counting the occurrences of each nucleotide at each position on the assembled sequence to predict the true base at that position. The quality of the assembled genome and annotation completeness were assessed by BUSCO ( https://busco.ezlab.org/ ) [ 54 ]. CheckM (version 1.1.3) was used to estimate the contamination and completeness of the genome [ 55 ]. The genome was annotated on Prokka (version 1.12) [ 56 ]. The average nucleotide identity (ANI) tool on EzBioCloud ( https://www.ezbiocloud.net/tools/ani ) [ 57 ] and the genome-to-genome distance calculator (version 2.1) on DSMZ ( https://ggdc.dsmz.de/ggdc.php# ) [ 58 ] were used for digital DNA-DNA hybridization to distinguish the novel isolates from their closest valid taxa. The whole-genome sequences of the reference strains, including Puteibacter caeruleilacunae JC036 T (GCA_005217565), Prolixibacter bellariivorans JCM 13498 T (GCF_000621705), Sunxiuqinia elliptica CGMCC 1.9156 T (GCF_900113005), Maribellus luteus XSD2 T (GCA_003576475), Dracinibacterium orientale FH5 T (GCA_000626635), Aquipluma introreducens MeG22 T (AP018694), Mangrovibacterium diazotrophicum DSM 27148 T (GCF_003610535), Mariniphaga anaerophila DSM 26910 T (GCF_900129025), Tangfeifania diversioriginum DSM 27063 T (GCF_900141875), and Gaoshiqinia sediminis A06 T (GCF_025907915.1), were retrieved from NCBI. The average amino acid identity (AAI) was calculated using the AAI calculator from the Kostas laboratory ( http://enve-omics.ce.gatech.edu/aai/ ) [ 59 ]. Pairwise comparisons for the percentage of conserved proteins (POCP) were calculated based on the method provided by Qin et al. [ 60 ]. The amino acid FASTA sequences of the reference strains were retrieved from NCBI. Functional genes within each genome were also annotated using KEGG and deciphered to pathways using KEGG Decoder [ 61 ] and KEGG-Expander ( https://github.com/bjtully/BioData/tree/masterEGGDecoder ). Other databases were used for annotation including the Clusters of Orthologous Genes (COGs) [ 62 ] and Gene Ontology (GO) [ 63 ] databases. Carbohydrate-active enzymes were identified through the CAZy database ( http://www.cazy.org/ ) [ 64 ] and the dbCAN server ( https://bcb.unl.edu/dbCAN2/blast.php ) [ 65 ]. Biosynthesis gene clusters (BGCs) and metabolic gene clusters (MGCs) were predicted by antiSMASH 7.1.0 [ 66 ] and gutSMASH [ 67 ], respectively. Prophages were predicted in the genomes using PHASTER [ 68 ]. CRISPR-Cas in the genomes was predicted using CRISPRCasFinder [ 69 ]. The subcellular location of proteins in prokaryotes was predicted via PSORTb (version 3.0.3) [ 70 ]. The predicted model of xylan degradation and xylose utilization was drawn by using Affinity Designer (version 1.10.6.1665). Polysaccharide-degrading ability To test polysaccharide utilization, two novel strains were anaerobically grown on BS media supplemented with individual polysaccharides (0.1–0.2%, w/v), including alginate, cellulose, chitin, κ-, λ-, and ι-carrageenan, fucoidan, laminarin, starch, and xylan. Ten microliters of each culture was observed under a light microscope (Nikon Eclipse 80i) every two days to monitor growth. By comparing the cell numbers each day and with those of the control (no carbon source), the growth of both strains was recorded. The production of reducing sugars in the supernatant was detected by reacting the cell-free supernatant with 3,5-dinitrosalicylic acid (DNS) [ 49 ]. Growth of DS1-an-13321 T and DS1-an-2312 T on laminarin and xylan The ability of strains DS1-an-13321 T and DS1-an-2312 T to grow on laminarin and the ability of strain DS1-an-2312 T to grow on xylan were further studied. During the growth of strains DS1-an-13321 T and DS1-an-2312 T on laminarin and xylan, black particles and clumped cells were produced in the culture broth, and the density of the cells in culture could not be measured by a spectrophotometer. The growth of DS1-an-13321 T and DS1-an-2312 T on the corresponding substrates was therefore monitored under a light microscope (Nikon Eclipse 80i). We counted the cells in culture every 6 h under a microscope to track the growth of both strains on laminarin and xylan to make growth curves. Briefly, every 6 h, 100 µL of broth culture was harvested and diluted tenfold. Subsequently, 10 µL aliquots of diluted solution were dropped onto glass slides, stained with safranine (BD), and cells were counted under 400× magnification by a microscope (Nikon Eclipse 80i). Five frames were randomly selected for statistical analysis. Subsequently, the cells in each frame were counted, and the average cell number and standard deviation were calculated considering the dilution factors. Location of laminarin and xylan-degrading enzymes For laminarin-degrading enzymes, cells were harvested by centrifugation from the 4-day cultures of strains DS1-an-13321 T and DS1-an-2312 T on laminarin. The cell pellet was washed and resuspended in phosphate buffer solution (PBS, pH 7.2). Subsequently, the cell suspension was incubated with laminarin (final concentration of 0.1%, w/v) for 6 h at 30 ºC. The supernatant was stirred with 50% (w/v) (NH 4 ) 2 SO 4 (final concentration) to precipitate the extracellular protein. Overnight dialysis was applied to remove the salt. The resulting solution was incubated with laminarin (at a final concentration of 0.1%, w/v) for 6 h at 30 ºC. The enzyme reaction was assessed by detection of reducing sugars by a DNS assay [ 49 ]. The change of reducing sugars from the beginning to the end of the enzyme reaction was determined. The procedure used to determine the location of the active xylan-degrading enzymes was the same as that used for laminarin-degrading enzymes. However, in the enzyme reaction, the final concentration of xylan was 0.2% (w/v). Degradation of laminarin and xylan by whole cell enzymes Four-day old strains DS1-an-13321 T and DS1-an-2312 T cells grown on laminarin (0.1%, w/v) were harvested. The cells were harvested by centrifugation, washed three times, and resuspended in PBS. The cell suspension was then used as a crude enzyme of cell-associated laminarin-degrading enzymes to test for enzyme activity. In the case of DS1-an-2312 T grown on xylan (0.2%, w/v), 4-day old DS1-an-2312 T cells grown on xylan were harvested by centrifugation. The cell pellet and remaining insoluble xylan were washed three times to remove remaining soluble sugars and resuspended in PBS. The cell suspensions were used as crude enzymes, and enzyme activity was directly tested. The DNS assay was applied to detect the production of reducing sugars during the hydrolysis of laminarin and xylan. The products of the hydrolysis reaction were further analyzed in the next step. The hydrolysis products of the crude enzymes were assessed by thin layer chromatography (TLC) according to the method of Lee et al. [ 71 ]. In brief, the crude enzymes from strains DS1-an-13321 T and DS1-an-2312 T were incubated with each substrate (1 mg/mL, final concentration) of glucose (G), laminaribiose (L2), laminaritriose (L3), laminaritetraose (L4), laminaripentaose (L5), laminarihexaose (L6), and laminarin (Ln) at 30 ºC for 24 h. The reaction of the crude enzymes without substrates and the reaction of each substrate without the crude enzymes were included as controls. The enzyme-reaction tubes were centrifuged at 8000 rpm for 2 min at room temperature to harvest the degradation products of the reaction. The supernatant was applied to a TLC plate without concentration. The TLC plate was then developed in a chloroform-acetic acid-water (6:7:1, v/v/v) solvent system [ 71 ]. The TLC plates were visualized by spraying a mixture of ethanol-sulfuric acid (95:5, v/v), followed by drying in an oven at 150 ºC for 5 to 10 min. For hydrolysis products from xylan degradation of DS1-an-2312 T , the cell pellet was washed three times with PBS to remove the remaining soluble sugar from the culture. Subsequently, to assess the mode of action of the DS1-an-2312 T xylan-degrading enzyme, the reducing sugars present at the initial crude enzyme solution and the reducing sugars that were produced during the enzyme reaction were compared. The limitation of this test is the remaining insoluble xylan in the cell pellet; thus, during enzyme incubation with the whole cell, this remaining xylan that had not yet been consumed was further degraded, and the resulting reducing sugars was released into the suspension. Thus, the presence of hydrolytic products during enzyme reaction of crude enzyme was detected via TLC in the same manner as in the analysis of the laminarin-hydrolytic products. The oligosaccharides of xylan and laminarin were purchased from Megazyme, and xylan and laminarin were purchased from Sigma. Results and Discussion Isolation, cultivation, and identification Two strains were isolated from a sea squirt (Fig. 1 a) collected at a depth of 18 m of the East Sea, Republic of Korea. A portion of the feeding area of the sea squirt (Fig. 1 b) was cut and placed on the surface of filter paper on a low nutrient agar medium that was prepared by sixty percent seawater collected from the same sampling location. Pure cultures of the two novel strains were obtained by transferring colonies exhibiting gliding traits around the filter paper. The strains were transferred several times onto fresh marine agar (MA) plates until no contaminants were detected. The morphologies of the pure cultures of strains DS1-an-13321 T and DS1-an-2312 T are presented in Figs. 1 c and d , respectively. Strain DS1-an-13321 T formed nearly round colonies with a tortilla color in the center and a cream color at the periphery, while strain DS1-an-2312 T exhibited irregular colonies with a round tortilla color in the center and broad swarming at the outer edge of the colonies. The cells of both strains were long rods with a length exceeding 20 µm during the log-phase of growth and a width ranging from 0.25–0.5 µm (Figs. 1 e, f, and Table 1 ). Interestingly, both strains showed a transition from a long rod to a spherical shape at the end of the stationary phase ( Fig. S1 ). Strains DS1-an-13321 T and DS1-an-2312 T were maintained on MA plates at 23 ºC and 28 ºC, respectively. For long-term preservation, two novel isolates were preserved by using a liquid-dry method. Table 1 Comparative physiological characteristics of two novel strains, DS1-an-13321 T and DS1-an-2312 T , and their representative reference strains in the family Prolixibacteraceae . Strains: 1, DS1-an-13321 T ; 2, DS1-an-2312 T ; 3, Puteibacter caeruleilacunae JC036 T [ 43 ]; 4, Prolixibacter bellariivorans JCM 13498 T [ 73 ]; 5, Sunxiuqinia elliptica CGMCC 1.9156 T [ 74 ]. * Data obtained from [ 43 ]. Characteristic 1 2 3 4 5 Colony Nearly round shape colony and bright brown color Irregular shape, bright brown color Creamy white* White* Slightly pink* Cell diameter (µm) Long-rod shape, long-shape: 0.25–0.5 x ~ 20, Spherical shape: 0.48–0.95 Long-rod shape long-shape: 0.25–0.5 x ~ 20, Spherical shape: 0.48–0.95 Rod shape, 1-3x0.3-0.5 Filamentous-rod shape, 0.33 x 10.5–12.5 Rod shape, 0.4 x 0.8–0.9 Temperature range (℃) 15–30 10–32 4–37 4–42 15–42 Optimum growth temperature (℃) 20–30 20–30 28 22 30 pH range 6.0-8.5 (6.5–8.5) 6.0–8.0 (7.0-7.5) 5.0–9.0 (7.0) 5.0–9.0 (7.0) 5.0–9.0 (7.0–8.0) NaCl (%) range(optimum) 2–4 (2–3) 1–4 (2–3) 2–6 (3) 0.5-8 (2) 0.5–10 (3) Oxygen requirement Microaerophilic Microaerophilic Facultative anaerobic Facultative anaerobes Strictly aerobic H2S production + + + - + Hydrolysis Gelatin + + + - - Starch + + - + - DNase + + - - ND Utilization Fructose - - + - + Galactose + + + + - Lactose + + ND + - Sucrose + + + + ND Xylose - + ND + + Source of isolation Sea squirt (-18 m) Sea squirt (-18 m) Seawater at a depth of 90 m at Yongle blue Hole in the South China sea. Surface of an electricity-harvesting electrode incubated in marine sediments. Sediment of a seashore pond for sea cucumber culture in Jimo, Qingdao, on the east coast of China. The 16S rRNA gene sequences of the pure cultures were determined. The analysis of the sequences on the EzBioCloud server ( https://www.ezbiocloud.net/ ) revealed that the highest 16S rRNA gene sequence similarities to Puteibacter caeruleilacunae JC036 T for strains DS1-an-13321 T and DS1-an-2312 T was 91.26% and 91.37%, respectively. Moreover, 16S rRNA similarities of the two novel isolates with type strains of all existing genera in the family Prolixibacteraceae , which is the parent taxon of Puteibacter caeruleilacunae , were calculated ( Table S1 ). This analysis demonstrated that the 16S rRNA similarity between the two isolates and existing members of the family Prolixibacteraceae fell within the range of 86.70 to 91.37%, while a similarity of 98.75% was observed between the two isolates. Based on these results, we hypothesized that the two novel isolates represent a novel genus and two novel species. Therefore, 16S rRNA-based phylogenetic tree, genome-based phylogenetic tree, genomic indices, and polyphasic taxonomic study were performed to evaluate this hypothesis. 16S rRNA-based and genome-based phylogeny A phylogenetic tree based on 16S rRNA gene sequences by combining three algorithms, ML, NJ, and MP, showed the taxonomic position of the two novel isolates within the families Prolixibacteraceae and Marinilabiliaceae in the phylum Bacteroidota (Fig. 2 ). Interestingly, strain DS1-an-13321 T formed a single cluster with strain DS1-an-2312 T , but the two strains were separated from all other representatives in the family Prolixibacteraceae , even Puteibacter caeruleilacunae JC036 T (approximately 91% of 16S rRNA similarity). Based on 92 core genes from their genomes, a genome-based phylogenetic tree was constructed under the UBCG pipeline using the ML algorithm to clarify the taxonomic position of the two isolates within the families Prolixibacteraceae and Marinilabiliaceae (Fig. 3 ). The two novel isolates clustered with their closest relative, Puteibacter caerulelacunae JC036 T , with a high bootstrap value of 92%. However, this cluster was separated from other representatives in the families Prolixibacteraceae and Marinilabiliaceae. Considering the results from both the 16S-based and genome-based phylogenetic trees, the two novel strains could be classified as a novel genus within the family Prolixibacteraceae. To evaluate the taxonomic proposal of the two novel isolates, the genomic indices of the two isolates against the type species in all genera of the family Prolixibacteraceae , including the ANI, dDDH, AAI, and POCP values, were calculated (Table 2 ). The ANI and dDDH values between strain DS1-an-13321 T and strain DS1-an-2312 T were 73.99% and 20.9%, respectively. These values were under the cutoff values of the ANI (95%) [ 57 ] and dDDH (70%) [ 58 ] for species demarcation. In addition, the ANI and dDDH values between the two isolates and other representatives in the family Prolixibacteraceae ranged from 66.17–67.76% and 24.0-37.4%, respectively. All these values were much lower than the cutoff values of the ANI (95%) and dDDH (70%). Hence, the two novel isolates were deemed candidates for two novel species classified in the family Prolixibacteraceae. For a comparison of the amino acid sequences, the AAI and POCP values between the two novel isolates and representatives in the family Prolixibacteraceae ranged from 48.33–52.35% and 29.01–37.37%, respectively. Both of these ranges fell under the cutoff values for genus demarcation of AAI (60%) [ 72 ] and POCP (50%) [ 60 ] but strain DS1-an-13321 T shared 71.34% and 73.55% for the AAI and POCP values, respectively, with strain DS1-an-2312 T . Therefore, the genomic indices provide strong evidence for the proposal of strains DS1-an-13321 T and DS1-an-2312 T as candidates for two novel species in a novel genus in the family Prolixibacteraceae. Table 2 ANI values calculated using the EzBioCloud service, digital DNA-DNA hybridization values calculated with Genome-to-Genome Distance Calculator 3.0, AAI values calculated using the AAI calculator (Kostas lab), POCP values calculated among novel strains and their reference strains: 1, DS1-an-13321 T (CP081303); 2, DS1-an-2312 T (CP082230); 3, Puteibacter caeruleilacunae JC036 T (SJCO00000000); 4, Prolixibacter bellariivorans JCM 13498 T (JHXO00000000); 5, Sunxiuqinia elliptica CGMCC 1.9156 T (FONW00000000); 6, Maribellus luteus XSD2 T (QWGR00000000); 7, Draconibacterium orientale FH5 T (CP007451); 8, Aquipluma nitroreducens MeG22 T (AP018694); 9, Mangrovibacterium diazotrophicum DSM 27148 T (RAPN00000000); 10, Mariniphaga anaerophila DSM 26910 T (FQUM00000000); 11, Tangfeifania diversioriginum DSM 27063 T (FQZE00000000); 12, Gaoshiqia sediminis A06 T (JAPAAF000000000). Strain 1 2 3 4 5 6 7 8 9 10 11 12 ANI value (%) 1 100 73.99 67.48 67.76 66.92 66.69 67.04 67.31 67.12 66.68 66.31 66.42 2 73.99 100 67.56 67.34 66.75 66.47 66.77 66.83 66.93 66.17 66.32 66.84 DNA-DNA hybridization (%) 1 100.0 20.9 24.1 37.4 27.6 24.7 25.5 26.6 30.0 29.8 31.6 24.0 2 20.9 100.0 24.9 36.2 24.5 29.8 24.9 26.6 24.5 28.4 28.9 31.9 AAI values (%) 1 100.00 71.34 51.17 52.35 50.12 48.83 48.99 48.72 49.02 49.19 48.84 49.81 2 71.34 100.00 51.05 51.24 49.69 48.75 48.86 48.33 49.05 48.78 48.5 49.95 POCP values (%) 1 100.00 73.55 34.56 37.37 35.47 30.50 32.85 29.01 31.26 32.49 31.25 33.69 2 73.55 100.00 35.48 36.57 35.02 30.67 32.45 28.34 30.88 31.92 30.52 32.96 Physiological characterization The two novel strains showed differences with other representatives in the family Prolixibacteraceae. Both strains DS1-an-13321 T and DS1-an-2312 T could hydrolyze starch and DNA, while the closest strain Puteibacter caeruleilacunae JC036 T [ 43 ] cannot hydrolyze starch or DNA, Prolixibacter bellariivorans JCM 13498 T [ 73 ] cannot hydrolyze DNA, and Sunxiuqinia elliptica CGMCC 1.9156 T [ 74 ] cannot hydrolyze starch (Table 1 ). Only strain DS1-an-13321 T is strictly anaerobic, while most others are facultative anaerobic, and only strain Sunxiuqinia elliptica CGMCC 1.9156 T is strictly aerobic [ 74 ]. Interestingly, even though DS1-an-13321 T and DS1-an02312 T belong to one genus, only strain DS1-an-2312 T could utilize xylose (Table 1 ). The detailed characteristics of the two novel isolates and representatives in the family Prolixibacteraceae are presented in Table 1 . Chemotaxonomic characterization The fatty acid profiles of the two novel isolates and their reference strains are shown in Table 3 . The two novel isolates were distinguished from each other and from their closest valid species by the different major fatty acid components and their proportions. The major fatty acid components (> 10% of the total fatty acids) of strain DS1-an-13321 T were iso -C 15:0 (28.08%), anteiso -C 15:0 (21.27%), and iso -C 15:0 3OH (13.58%). In addition, the predominant fatty acid components (> 10% of the total fatty acids) of strain DS1-an2312 T were anteiso -C 15:0 (20.08%), iso -C 15:0 (16.51%), iso -C 17:0 3OH (12.83%), and iso -C 15:0 3OH (10.4%). The primary components (> 10% of the total fatty acids) of their three reference strains included iso -C 15:0 , anteiso -C 15:0 , and iso -C 17:0 3OH. The primary isoprenoid quinone of both novel strains was menaquinone-7 (MK-7), which is common to other species in the family Prolixibacteraceae . The polar lipids of strain DS1-an-13321 T included phosphatidylethanolamine (PE), phosphatidylserine (PS), two amino-lipids (AL1-2), an aminophospholipid (APL), a phospholipid (PL), and two unidentified lipids (L1-2) ( Fig. S1 . a ). The polar lipid components of strain DS1-an-2312 T consisted of phosphatidylethanolamine (PE), three aminophospholipids (APL1-3), and two unidentified lipids (L1-2) ( Fig. S1 . b ). Table 3 Comparison of the cellular fatty acid compositions (%) of two novel strains, DS1-an-13321 T and DS1-an-2312 T , and their closest relatives in the family Prolixibacteraceae. Strains: 1, DS1-an-13321 T ; 2, DS1-an-2312 T ; 3, Puteibacter caeruleilacunae JC036 T ; 4, Prolixibacter bellariivorans JCM 13498 T ; 5, Sunxiuqinia elliptica CGMCC 1.9156 T . The percentages of the fatty acids are presented as numbers. -, not detected. Values 10% are in bold. Summed features 1: C 15:1 iso H/C 13:0 3OH; Summed features 2: C 14:0 3OH/C 16:1 iso I; Summed features 3: C 16:1 ω7c/C 16:1 ω6c; Summed features 4: C 17:1 iso I/anteiso B; Summed features 8: C 18:1 ω7c; Summed features 9: C 16:0 10-methyl. Fatty acid 1 2 3 4 5 Straight-chain saturated C 14:0 TR - 2.02 TR - C 16:0 1.12 1.04 5.04 3.64 1.78 C 17:0 - TR TR TR 1.49 C 18:0 TR TR 1.64 TR 0.55 Straight-chain unsaturated C 15:1 ω6c - - TR TR 1.35 C 16:1 ω5c - - TR TR 1.09 C 17:1 ω6c TR - - TR 3.43 C 18:1 ω9c TR TR 2.27 TR 0.8 Branched iso-C 12:0 TR 1.04 - - - iso-C 13:0 2.32 2.27 TR TR 0.82 iso-C 14:0 1.41 2.84 TR TR 2.6 iso-C 15:0 28.08 16.51 38.09 27.75 14.67 iso-C 16:0 1.1 1.31 TR TR 3.94 iso-C 16:1 H - - - - 0.98 iso-C 16:0 H - - - - - iso-C 17:0 3.12 1.03 TR 1.16 3.28 anteiso-C 13:0 1.44 TR - TR 0.49 anteiso-C 15:0 21.27 20.08 11.63 25.3 8.27 anteiso-C 17:0 TR TR - TR 1.41 Hydroxylated C 14:0 2-OH TR 1.14 - - - C 15:0 2-OH 1.02 TR TR TR 1.4 C 15:0 3-OH 5.52 6.68 2.5 3.45 - C 16:0 3-OH TR TR 3.72 1.24 - C 17:0 2-OH 1 2.5 1.16 4.6 6.3 C 17:0 3-OH TR TR - - 0.86 C 18:0 3-OH - - - TR - iso-C 14:0 3-OH TR 1.27 - - 0.26 iso-C 15:0 3-OH 13.58 10.4 6.75 8.03 4.29 iso-C 16:0 3-OH 1.2 3.64 1.83 2.39 3.39 iso-C 17:0 3-OH 8.4 12.83 8.75 11.69 18.59 Summed features 1 - - 1.49 - 0.97 2 - 1.72 - - - 3 - - 1.12 TR 1.95 4 - - - - 6.77 8 - - 1.49 - 0.48 9 1.71 4.51 - 1.59 5.52 Anaerobic metabolism The type of anaerobic metabolism of strains DS1-an-13321 T and DS1-an-2312 T was assessed. Under fermentation conditions, both strains were able to utilize galactose, glucose, lactose, maltose, sucrose, and starch. Strain DS1-an-2312 T was also able to utilize xylose, whereas DS1-an-13321 T was not able to utilize xylose. The presence of sulfate did not change the carbon utilization pattern of either strain, as also observed under fermentation conditions, and a decrease of sulfate was not observed (data not shown). Additionally, strain DS1-an-13321 T could weakly grow on pyruvate, but grew actively on glucose. Strain DS1-an-2312 T could grow on lactate or pyruvate. The growth of strains DS1-an-13321 T and DS1-an-2312 T on glucose was also observed in the presence of nitrate and thiosulfate, but no reduction of nitrate or thiosulfate was observed. Interestingly, only strain DS1-an-2312 T could grow in the presence of sulfite. However, according to the API 20NE test, neither DS1-an-13321 T nor DS1-an-2312 T could perform nitrate/nitrite reduction under anaerobic conditions. Moreover, in the presence of sulfite, the growth of DS1-an-13321 T was suppressed, and in the presence of nitrite, the growth of both strains was suppressed. These results indicated that strains DS1-an-13321 T and DS1-an-2312 T have fermentation metabolism even in the presence of a high concentration of sulfate (27 mM). This high concentration of sulfate in the basal medium mimicked the sulfate concentration in natural sea water [ 75 ]. The fermentation products of strains DS1-an-13321 T and DS1-an-2312 T from the above experiments were analyzed through HPLC [ 50 ]. By comparing the retention times of standard SCFAs under the same analysis conditions, the major fermentation products of strains DS1-an-13321 T and DS1-an-2312 T on galactose, glucose, maltose, lactose, sucrose, and starch and those of strain DS1-an-2312 T on xylose were acetic acid and propionic acid. Polysaccharide utilization For polysaccharide utilization, strains DS1-an-13321 T and DS1-an-2312 T were tested for growth on alginate, cellulose, chitin, κ-, λ-, and ι-carrageenan, fucoidan, laminarin, starch, and xylan in basal media. Their growth was observed under a microscope. Strain DS1-an-13321 T was found to utilize laminarin and starch as the sole carbon source, while strain DS1-an-2312 T could utilize xylan in addition to laminarin and starch (Table 4 ). However, a DNS assay could not detect any reducing sugars from the culture broth. This observation might be related to the “selfish” lifestyle of polysaccharide degraders in the phylum Bacteroidota [ 7 , 76 ]. Table 4 Growth of strains DS1-an-13321 T and DS1-an-2312 T on organic substrates under anaerobic conditions. Substrate DS1-an-13321 T DS1-an-2312 T Monosaccharide Fructose - - Galactose + + Glucose + + Xylose - + Disaccharide Lactose + + Maltose + + Sucrose + + Polysaccharide Alginate - - Cellulose - - Chitin - - κ-Carrageenan - - λ-Carrageenan - - ι-Carrageenan - - Fucoidan - - Laminarin + + Starch + + Xylan - + Several studies have reported that members of the family Prolixibacteraceae can utilize polysaccharides. For instance, Mangrovibacterium lignilyticum is enriched in media containing lignin [ 77 ], Tangfeifania diversioriginum is capable of hydrolyzing starch, and Sunxiuqinia indica [ 78 ] and Gaoshiqia sediminis [ 79 ] contain 176 genes and 297 genes related to CAZymes, respectively. However, there has not yet been a study based on a combination of in silico analysis and in vitro experiments to assess polysaccharide utilization among members of the family Prolixibacteraceae. Interestingly, both novel isolates could utilize laminarin, which was not previously reported for members of the family Prolixibacteraceae , and strain DS1-an-2312 T could degrade xylan. Therefore, in this study, we further investigated laminarin and xylan utilization by the isolates through in vitro examination and in silico genomic mining. Growth of DS1-an-13321 T and DS1-an-2312 T on laminarin and xylan Based on the cell counting method, with counts taken every six hours for growth on laminarin and every 24 hours for growth on xylan, the growth curves of DS1-an-13321 T and DS1-an-2312 T on laminarin and xylan are shown in Fig. 4 . The growth of strain DS1-an-13321 T on laminarin reached the late log phase on day 3, while the late log phase of strain DS1-an-2312 T occurred on day 4. The growth of strain DS1-an-2312 T on xylan reached the late log phase on day 3. Based on these results, cultures at their late log phase were harvested for further enzyme activity studies. Laminarin degradation was detected in enzyme reaction of cell-associated proteins of strains DS1-an-13321 T and DS1-an-2312 T with laminarin, but it was not found in the supernatant of their broth cultures. This indicated that laminarin-degrading enzymes were cell-associated proteins. Likewise, the xylan-degrading enzymes of strain DS1-an-2312 T were also associated with cells. To trace the mode of action of the laminarin- and xylan-degrading enzymes of DS1-an-13321 T and DS1-an-2312 T , cell-associated enzymes were harvested from each strain and the degradation products were determined by a TLC analysis. Based on the RF values of the enzyme reactant and standard compounds, the major hydrolytic product on the laminarin or laminarin oligosaccharides of both strains was identified as glucose (Fig. 5 , and Figs. S3a, b ). These results indicated that the laminarin-degrading enzymes of both strains exhibited exo-hydrolytic activities. For the xylan-degrading enzyme, the hydrolytic products by the crude enzyme of strain DS1-an-2312 T on xylan were xylosetriose and xylotetraose (Fig. 6 ), indicating that the cell-associated xylan-degrading enzyme of DS1-an-2312 T exhibited endo-hydrolytic enzyme activities. Genome analysis The whole genomes of DS1-an-13321 T and DS1-an-2312 T , determined by a combination of Nanopore and Illumina platforms, were obtained with high completeness (BUSCO values: 94.3% and 93.5%, respectively). Both the DS1-an-13321 T and DS1-an-2312 T genomes comprised a single circular chromosome with sizes of 4,465,088 bp and 5,187,288 bp, respectively, and had G + C content 35.9% and 36.5%, respectively ( Table S2) . The whole-genome sequence of strain DS1-an-13321 T contained 3,545 predicted genes including 3,341 coding genes and eight pseudogenes. Among these, there were 158 tRNAs, five noncoding RNAs, and 41 rRNA genes (15 5S rRNAs, 13 16S rRNAs, and 13 23S rRNAs) ( Table S2 ). On the other hand, genome analysis of strain DS1-an-2312 T revealed that the strain harbored 3,807 predicted genes, consisting of 3,634 coding genes and six pseudogenes. Among these, there were 128 tRNAs, five noncoding RNAs, and 40 rRNA genes (14 5S rRNAs, 13 16S rRNAs, and 13 23S rRNAs) ( Table S2 ). AntiSMASH and gutSMASH revealed that the genome of strain DS1-an-13321 T encodes one BGC belonging to the nonribosomal peptide synthetase family and nine MGCs, while the genome of strain DS1-an-2312 T encodes one BGC belonging to the linear azoline-containing peptides family and nine MGCs. In strain DS1-an-13321 T , the three COGs with the greatest number of genes were related to cell wall/membrane/envelope biogenesis, the mobilome (prophases, transposons), translation, ribosomal structure, and biogenesis. Moreover, in strain DS1-an-2312 T , the three COGs with the greatest number of genes were related to cell wall/membrane/envelope biogenesis, inorganic ion transport and metabolism, and carbohydrate transport and metabolism ( Fig. S4 ). Genome mining revealed that the genomes of strains DS1-an-13321 T and DS1-an-2312 T contain a high number of genes encoding carbohydrate-active enzymes (CAZymes). The CAZy database ( http://www.cazy.org/ ), which contains information about carbohydrate-active enzymes, including glycoside hydrolase (GH, cleavage of glycosidic bonds), glycosyl transferase (GT, construction of glycosidic bonds), polysaccharide lyase (PL, nonhydrolytic hydrolysis of glycosidic bonds), carbohydrate esterase (CE, cleavage of carbohydrate esters), auxiliary activity (AA, a redox enzyme that works in conjunction with other CAZymes), and carbohydrate-binding modules (CBM, adhesion to carbohydrates), was used to assess the detailed composition of CAZymes in the genomes of the two isolates, and the results are presented in Table S3 . Strain DS1-an-13321 T encoded a total of 155 CAZymes consisting of 84 GHs, 32 GTs, 13PLs, seven CEs and 19 CBMs, while strain DS1-an-2312 T encoded a total of 249 CAZymes, an amount 1.6 times greater than that of DS1-an-13321 T , consisting of 128 GHs, 37 GTs, 27 PLs, 35 CEs, and 22 CBMs. The number of GH genes per genome in strain DS1-an-13321 T was 18.81 (GHs/Mb), while in strain DS1-an-2312 T it was 24.46 (GHs/Mb); both of these values are significantly greater than the average value of 12 GHs/Mb in the genomes of other members of marine bacteria of the class Bacteroidia [ 80 ]. The genomes of DS1-an-13321 T and DS1-an-2312 T contained 27 and 34 PULs, respectively, which are approximately one-third lower than the average number of PULs in human gut Bacteroides and similar to the number of PULs found in the genus Prevotella (average 23 PULs/genome) [ 20 , 81 ]. The genomes of strains DS1-an-13321 T and DS1-an-2312 T contained genes predicted to be involved in laminarin degradation. The genome of strain DS1-an-13321 T harbored genes encoding for four GH3, one GH16, and one GH30, while strain DS1-an-2312 T harbored genes encoding for seven GH3, two GH16, and one GH30 ( Table S4) , which has been reported to contribute to the degradation of laminarin in other marine bacteria [ 14 , 82 , 83 ]. Unlike other laminarin degraders of Gramella spp. [ 82 ], Formosa spp. [ 83 ] (class Flavobacteriia ) or Bacteroides spp. [ 84 , 85 ] (class Bacteroidia ), neither strain DS1-an-13321 T nor DS1-an-2312 T harbored genes encoding GH3 and GH16, which in general collocate with each other and collocate with SusC/SusD (a signature for the PUL structure). Instead, GH3 and GH16 of strains DS1-an-13321 T and DS1-an-2312 T were located separately, and each gene was collocated with SusC/SusD in the genomes. For strain DS1-an13321 T , we found that PUL8 contained a tandem of SusD/TBDR, an unknown protein, and two copies of GH3. Additionally, these two GH3s were predicted to be located in the periplasmic space (PSORTb scores of 9.44 and 9.76). Furthermore, strain DS1-an-13321 T harbored an unidentified PUL (21_un_PUL), and the gene cluster contained SusD (K4L44_09375), SusC (K4L44_09380), TonB-dependent receptor (TBDR) (K4L44_09385), two unknown proteins (K4L44_09390, K4L44_09405), superoxide dismutase, Ni (K4L44_09395), GH3 (K4L44_09400), IS4 family transposase (K4L44_09410), and GH30 (K4L44_09415) ( Fig. S5a ). Within this gene cluster, we detected GH3 (cleavage β-1,3-glucan) and GH30 (cleavage β-1,6-glucan), which PSORTb could predict at multiple locations on the cell. In contrast, strain DS1-an-2312 T consisted of three PULs (PUL9, PUL29, and an unidentified PUL (12_un_PUL)), which contained GH3 without the presence of GH16 and GH30. We also found that the GH10 and GH5 genes in PUL9 and PUL29, respectively, were predicted to degrade the xylan main chain, indicating that these two PULs may play a role in xylan degradation rather than laminarin degradation. Additionally, in the 12_un_PUL ( Fig. S5b ), we detected several genes of SusC/SusD and GH3. The GH3 was predicted to locate in the periplasmic space with PSORTb score 9.44. To identify the active gene cluster responsible for laminarin degradation, further transcriptomic analysis is required. In this study, the novel strain DS1-an-2312 T was identified as an anaerobic bacterium capable of utilizing xylan as a sole carbon source. Whole-genome analysis of strain DS1-an-2312 T revealed the presence of CAZymes involved in the effective degradation of the natural polymer xylan ( Table S4 , and S6 ). We found that strain DS1-an-2312 T harbored genes encoding two GH5, one GH10, one GH30, and three GH141 enzymes ( Table S4) . Notably, using PULDB, all four potential xylan utilization loci were identified, PUL8, PUL9, PUL25, and PUL29 ( Fig. S5c ). A detailed analysis of these PUL indicated that PUL9 and PUL29 would have greater potential for xylan degradation in strain DS1-an-2312 T ( Table S5 ). Specifically, PUL9 contained SusC and SusD, which are responsible for capturing polysaccharides and delivering oligosaccharides into the cytoplasm [ 9 , 11 ]. It also contained a GH10 enzyme, which exhibited the highest amino acid similarity of 26% (covering 80% of the sequence) to endo-1,4-β-xylanase (UniProt accession code G4MLU0) and 24.4% (covering 81% of the sequence) to a reported GH10 module glycoside hydrolase of Caldicellulosiruptor danielii (PDB accession code 6D5C_A). Additionally, the PUL9 of strain DS1-an-2312 T contained a GH3 enzyme, which exhibited the highest similarity (43.5%, covering 83% of the sequence) to β-xylosidase of Formosa agariphila (UniProt accession code T2KMH0). Similarly, PUL29 contained the SusC, SusD, and a multidomain protein consisting of one GH5 subfamily 46 domain and two CBM6 modules. The multidomain protein exhibited 32.8% similarity to endoglucanase C of Acetivibrio thermocellus (UniProt accession code A3DJ77). Moreover, these two enzymes, GH5 and GH10, were not found in the genome of strain DS1-an-13321 T , which cannot utilize xylose and xylan as a sole carbon source under anaerobic conditions (Table 4 ). Additionally, through genome analysis, physiological characterization, and TLC analysis, it was inferred that strain DS1-an-2312 T exhibited strong xylan degradation capabilities (Tables 1 , 4 , Figs. S5 and 6 ). In the xylan degradation process, endo-1,4-β-xylanase and β-xylosidase enzymes degrade xylan to xylooligosaccharides (Fig. 7 ). The genome of strain DS1-an-2312 T encoded both endo-1,4-β-xylanase (GH10) and β-xylosidase (GH3) enzymes ( Table S5 ). Additionally, arabinofuranosidase GH30, a multisubstrate-specific family enzyme, acts as an endo-1,4-β-xylanase and degrades xylooligosaccharides. Subsequently, xylooligosaccharides are transported into the cell membrane. Bacterial strains typically use active transport mechanisms, with some routes utilizing high and low affinity transporters. Only the genome of strain DS1-an-2312 T , not DS1-an-13321 T , encoded the xylose transporter (XylE), which is a low-affinity transporter associated with xylooligosaccharide transportation via a proton motive force [ 86 , 87 ], and xylose isomerase (XylA), which facilitates the reversible conversion of D-xylose into D-xylulose [ 88 ]. The whole-genome sequence of DS1-an-2312 T was analyzed and the xylose metabolic pathway of the strains was modelled (Fig. 7 ). It was hypothesized that the metabolic pathway of the novel species involves xylose isomerase, as indicated by the presence of genes such as xylose isomerase (K5X82_00205) in its genome. In the isomerase pathway, the xylose transporter XylE (K5X82_00210) is responsible for the uptake of xylooligosaccharides. Xylooligosaccharides are degraded into D-xylose at the periplasm under the function of GH3 (K5X82_03105) (Fig. 7 ). This model hypothesis was supported by a TLC experiment (Fig. 6 ), where no detectable D-xylose was detected in the enzyme-reaction supernatant after removing the whole-cell-associated proteins of strain DS1-an-2312 T . The xylose isomerase xylA (K5X82_00205) enzyme converts D-xylose to D-xylulose, which is phosphorylated to D-xylulose-5-phosphate by the xylulokinase enzyme [ 87 , 89 , 90 ]. The phosphoketolase enzyme further degrades D-xylulose-5-phosphate (a 5-carbon compound) into acetyl phosphate (a 2-carbon compound) and glyceraldehyde-3-phosphate (a 3-carbon compound). Some anaerobic bacteria, such as Clostridium sp, and lactic acid bacteria, can cleave xylulose-5-P by phosphoketolase into these compounds [ 86 , 90 – 93 ]. Taken together, the results of this study not only support two novel strains that represent a novel genus with two novel species in the family Prolixibacteraceae , class Bacteroidia , phylum Bacteroidota but also expand our understanding of the strategies employed by marine Bacteroidia bacteria to access and degrade polysaccharides anaerobically. By mimicking natural nutrient conditions for isolation, pure cultures of the type strains of the two novel species were obtained. Both strains were capable of fermenting glucose, galactose, maltose, lactose, sucrose, and starch, with only DS1-an-2312 T exhibiting the ability to utilize xylose. The major fermentation products of strains DS1-an-13321 T and DS1-an-2312 T were acetic acid and propionic acid. Genome mining revealed that both novel species contained rich sources of CAZymes. In vitro experiments demonstrated that both novel species could degrade laminarin and starch, with only DS1-an-2312 T capable of utilizing xylan under anaerobic conditions. Both strains possessed cell-associated laminarin-degrading enzymes, exhibiting exo-hydrolytic enzyme activity and producing glucose as the major final product. In addition, strain DS1-an-2312 T possessed a cell-associated xylan-degrading enzyme with endo-hydrolytic enzyme activity, producing xylotriose and xylotetraose as the major final products. These results highlight the potential biotechnological applications of the two novel species and their strategies for adaptation under anoxic conditions in marine ecosystems through fermentation and polysaccharide degradation. For further study of the molecular mechanism of laminarin and xylan degradation in both novel species, future work will involve transcriptomic and proteomic analyses. Description of Halocynthiibacter gen. nov. Halocynthiibacter gen. nov. (Ha.lo.cyn.thi.i.bac'ter. N.L. fem. n. Halocynthia , an animal genus, N.L. masc. n. bacter , a rod; N.L. masc. n. Halocynthiibacter , a rod from Halocynthia ). Cells are Gram-stain-negative, anaerobic, rod-shaped, and oxidase- and catalase-negative. Prominent fatty acid components are iso- C 15:0 , anteiso- C 15:0 , iso- C 15:0 3-OH, and iso -C 17:0 3-OH. The major respiratory quinone type is menaquinone-7 (MK-7). Glucose, galactose, maltose, lactose, sucrose, and starch are fermented to produce a mixture of acid as major products. The genus Halocynthiibacter belongs to the family Prolixibacteraceae , phylum Bacteroidota. The type species is Halocynthiibacter xylanolyticus. Description of Halocynthiibacter laminarini sp. nov. Halocynthiibacter laminarini sp. nov. (la.mi.na.ri'ni. N.L. gen. n. laminarini , of laminarin, referring to its ability to hydrolyze laminarin). Cells are Gram-strain-negative, mesophilic, neutrophilic, strictly anaerobic, long rod-shaped at the log phase, and spherical at the end of the stationary phase of growth. They are oxidase- and catalase-negative. Round and bright brown colonies appeared on the surface of MB agar plates. Growth occurs at 15–30 ℃ (optimum, 20–30 ℃), at pH 6.0-8.5 (optimum, 6.5–8.5), and with 2–4% NaCl (optimum, 2–3%). H 2 S is produced. Positive for hydrolysis of gelatin, DNA, laminarin, and starch. Galactose, glucose, maltose, lactose, sucrose, and starch are fermented to produce acetic acid and propionic acid as the major products. The major fatty acid components are iso -C 15:0 , anteiso -C 15:0 , iso -C 15:0 3-OH, and iso -C 17:0 3-OH. Menaquinone 7 (MK-7) is the major quinone. The polar lipid profile comprises phosphatidylethanolamine (PE), two unidentified amino-lipids (AL1-2), one unidentified aminophospholipid (APL), one unidentified phospholipid (PL), one identified lipid (L), and one phosphatidylserine (PS). The type strain DS1-an-13321 T (= KCTC 25031 T = DSM 115329 T ) was isolated from a sea squirt at a depth of 18 m under the surface of seawater. The genome contains one circular chromosome that is 4.47 Mb long. The G + C content is 35.9%, as calculated from whole-genome sequencing. Description of Halocynthiibacter xylanolyticus sp. nov. Halocynthiibacter xylanolyticus sp. nov. (xy.la.ni.ly'ti.cus. N.L. neut. n. xylanum , xylan; Gr. masc. adj. lytikos , dissolving; N.L. masc. adj. xylanilyticus , xylan-dissolving). Cells are gram-stain-negative, mesophilic, neutrophilic, long rod-shaped at the log phase and spherical at the end of the stationary phase of growth. They are oxidase- and catalase- negative. Irregularly shaped and bright brown colonies appeared on the surface of MB agar plates. Growth occurs at 10–32 ℃ (optimum, 20–30 ℃), at pH 6.0–8.0 (optimum, 7.0-7.5), and with 1–4% NaCl (optimum, 2–3%). H 2 S is produced. Positive for hydrolysis of gelatin, DNA, laminarin, starch, and xylan. Galactose, glucose, xylose, maltose, lactose, sucrose, and starch are fermented to produce acetic acid and propionic acid as the major products. The major fatty acid components are iso -C 15:0 , anteiso -C 15:0 , iso -C 15:0 3-OH, and iso -C 17:0 3-OH. Menaquinone 7 (MK-7) is the major quinone. The polar lipid profile comprises phosphatidylethanolamine (PE) and three unidentified aminophospholipids (APL1-3). The type strain DS1-an-2312 T (= KCTC 25032 T = DSM 115328 T ) was isolated from a sea squirt at a depth 18 m under the surface of sea water. The genome contains one circular chromosome that is 5.19 Mb long. The G + C content is 36.52%, as calculated from whole-genome sequencing. Declarations Conflicts of interest The authors declare no competing interests. Author Contribution T.T.H.N. performed experiments of isolation, identification, and characterization of the bacterial strains including anaerobic metabolism, polysaccharide degradation, and genome analysis. T.T.H.N also wrote the manuscript. T.Q.V. analyzed genomes, constructed a UBGC genome tree, determined POCP indices, and drew the xylan degradation pathway. H.L.H carried out the genome analysis for xylose utilization and proposed the schematic model of xylose utilization, and wrote the paragraphs regarding xylose utilization. S-G.K. supervised the experiments and finalized the manuscript. All authors reviewed the manuscript. Acknowledgments The authors thank Prof. Dr. Bernhard Schink from the University of Konstanz (Germany) and Prof. Dr. Aharon Oren from Edmond J. Safra Campus, The Hebrew University of Jerusalem (Israel), for their help with the nomenclature of the new genus and two novel species names. The authors also thank Mrs. Mi-Kyung Eom and Dr. Li Zhun at KCTC for maintaining the two novel isolates and for providing the SEM images, respectively. This research was supported by The Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5232423) and a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2021M3H9A1030164). Data Availability The DNA sequences generated from the study are available at the National Center for Biotechnology Information (NCBI). GenBank/EMBL/DDBJ accession numbers of 16S rRNA gene sequences of the strains DS1-an-13321T and DS1-an-2312T are MZ851973 and MZ851974, respectively, and the genome sequences are CP081303 and CP082230, respectively. References Arnosti, C., et al. 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Supplementary Files Halocynthiibactersupplementary20240624.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 19 Aug, 2024 Reviews received at journal 18 Aug, 2024 Reviews received at journal 17 Aug, 2024 Reviewers agreed at journal 07 Aug, 2024 Reviewers agreed at journal 07 Aug, 2024 Reviewers invited by journal 02 Aug, 2024 Editor assigned by journal 02 Aug, 2024 Editor invited by journal 02 Jul, 2024 Submission checks completed at journal 01 Jul, 2024 First submitted to journal 24 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4632626","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":329447914,"identity":"26f5cdf1-64ed-4c80-99fb-571bda952e1e","order_by":0,"name":"Tra T.H. Nguyen","email":"","orcid":"","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Tra","middleName":"T.H.","lastName":"Nguyen","suffix":""},{"id":329447915,"identity":"457afa52-6024-4699-bc4a-cd66d361edeb","order_by":1,"name":"Tien Q. Vuong","email":"","orcid":"","institution":"Hanoi University of Science, Vietnam National University","correspondingAuthor":false,"prefix":"","firstName":"Tien","middleName":"Q.","lastName":"Vuong","suffix":""},{"id":329447916,"identity":"d45fcca9-e591-436f-b3f0-42a42fcaf42d","order_by":2,"name":"Ho Le Han","email":"","orcid":"","institution":"The University of Danang, University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ho","middleName":"Le","lastName":"Han","suffix":""},{"id":329447917,"identity":"29f8ad86-6465-4860-8d83-639f8ada29ed","order_by":3,"name":"Song-Gun Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYFACNoYDDBUJDGwSPGCuAZFazpCqhYGxLYGBgWgt8v3LEg/dnJeWzyfde4C5ooLB2LyBgBaDG88OHM7dlmPZJnMugfHMGQYzmQOEtEgcbwBqqTBgk8gxYGxsY7CRIOiwGSAtc2Ba/hGhheF8G9BhDTlQLQ0MZgS1GNxgSziccyzNgA3ol4MNxySMCTus/5jx55yaZAP52b0HHzbU2BjOIOgwiQQE+wCQS1ADAwP/ASIUjYJRMApGwcgGAAdHPb/VAjmmAAAAAElFTkSuQmCC","orcid":"","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Song-Gun","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-06-25 00:41:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4632626/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4632626/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-74787-6","type":"published","date":"2024-10-17T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60935182,"identity":"74f5b44b-ce58-4e35-806c-bb9c6279d2c2","added_by":"auto","created_at":"2024-07-23 18:35:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":135471,"visible":true,"origin":"","legend":"\u003cp\u003eOrigin, colony, and cell morphology of two novel isolates in the family \u003cem\u003eProlixibacteraceae. \u003c/em\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Sea squirt collected at a depth of 18 m under seawater at Sodol Port, East Sea; (\u003cstrong\u003eb\u003c/strong\u003e) position at which the isolated slice was cut; (\u003cstrong\u003ec\u003c/strong\u003e) and (\u003cstrong\u003ee\u003c/strong\u003e): colony morphology and SEM image of DS1-an-13321\u003csup\u003eT\u003c/sup\u003e cells; (\u003cstrong\u003ed\u003c/strong\u003e) and (\u003cstrong\u003ef\u003c/strong\u003e): colony morphology and SEM image of DS1-an-2312\u003csup\u003eT\u003c/sup\u003e cells. Scale bar: 1 cm (\u003cstrong\u003ea\u003c/strong\u003e); 1 mm (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e); 1 μm (\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure1Cellmorphology.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4632626/v1/8e414e9dfc99264aa0e5f3a4.jpg"},{"id":60935183,"identity":"f5b355c8-c2f2-4fea-ba8d-413f158df987","added_by":"auto","created_at":"2024-07-23 18:35:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":172345,"visible":true,"origin":"","legend":"\u003cp\u003e16S rRNA-based maximum-likelihood phylogenetic tree constructed wiht MEGA7 software (version 7.0.26) indicating the positions of two novel strains, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, with their closest representatives belonging to the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e and the closest family \u003cem\u003eMarinilabiliaceae\u003c/em\u003e. The strain \u003cem\u003eAgarivorans albus\u003c/em\u003e NBRC 102603\u003csup\u003eT\u003c/sup\u003e (GenBank accession number AB076561) was used as the outgroup. GenBank accession numbers are presented in parentheses. The 16S rRNA gene sequences were aligned by ClustalW, and the resulting file was trimmed in BioEdit software (version 7.2.5). Bootstrap values \u0026gt; 50% based on 1000 replicates are presented at nodes. The closed circles indicate the consensus of the nodes recovered by using three algorithms, ML, NJ, and MP, respectively. The open circles indicate the consensus of recovered nodes found from two out of three algorithms. Bar, 0.05 substitutions per nucleotide position.\u003c/p\u003e","description":"","filename":"Figure216Sphylogenetictree.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4632626/v1/9e295c6bef85465dec13b951.jpg"},{"id":60935184,"identity":"49b227bf-715a-46db-931e-6c81951d5906","added_by":"auto","created_at":"2024-07-23 18:35:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177065,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood phylogenetic tree showing the relationships among DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and their closely related species based on 92 core genes identified via the UBCG pipeline. GenBank accession numbers of the whole genome sequences are given in parentheses. \u003cem\u003eAgarivorans albus \u003c/em\u003eJCM 21469\u003csup\u003eT\u003c/sup\u003e (GCA_019670105.1) was used as the outgroup. Bootstrap values based on 1000 replicates are indicated at the branch nodes. Bar, 0.1 substitutions per site.\u003c/p\u003e","description":"","filename":"Figure3phylogenomictree.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4632626/v1/3c11ce301250a69f58bdfc83.jpg"},{"id":60935189,"identity":"6b0f5a54-adb8-4e22-a66a-6e15c8368f74","added_by":"auto","created_at":"2024-07-23 18:35:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80181,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on laminarin (\u003cstrong\u003ea\u003c/strong\u003e) and xylan (\u003cstrong\u003eb\u003c/strong\u003e) under anaerobic conditions.\u003c/p\u003e","description":"","filename":"Figure4Growthcurves.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4632626/v1/47a1f729d26d8d906fa01abf.jpg"},{"id":60935345,"identity":"b9435afa-a00e-41d0-8041-b4559d03af8f","added_by":"auto","created_at":"2024-07-23 18:43:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26143,"visible":true,"origin":"","legend":"\u003cp\u003eTLC plate analysis of hydrolysis products released from the reaction of cell-associated laminarin-degrading enzymes DS1-an-13321\u003csup\u003eT\u003c/sup\u003e on laminarin oligosaccharides and laminarin for 24 h at 30 ºC. Laminarin oligosaccharides: G, glucose; L2, laminaribiose; L3, laminaritriose; L4, laminaritetraose; L5, laminaripentaose; L6, laminarihexaose; Ln, laminarin. The solvent system: chloroform-acetic acid-water (6:7:1, v/v/v). e: enzyme reaction product. c: control reaction product. M: mixture of laminarin oligosaccharides.\u003c/p\u003e","description":"","filename":"Figure5HydrolysisproductlaminarinDS1an13321.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4632626/v1/5ae79ba0f70292016fd99737.jpg"},{"id":60935185,"identity":"dba108ed-ad55-40b8-a7ce-f4a98740e237","added_by":"auto","created_at":"2024-07-23 18:35:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13268,"visible":true,"origin":"","legend":"\u003cp\u003eTLC plate analysis of hydrolysis products released from the reaction of xylan-degrading enzyme DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on xylan after 24 h at 30 ºC. The solvent system: chloroform-acetic acid-water (6:7:1, v/v/v). e.Xn: crude enzyme reacting with xylan; ce: control of crude enzyme reacting without the presence of substrate. M: mixture of xylan oligosaccharides, including: X1, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose; X6, xylohexaose.\u003c/p\u003e","description":"","filename":"Figure6.HydrolyticproductxylanDS1an2312.pptx.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4632626/v1/47975831dc00b54ab6ccb391.jpg"},{"id":60935187,"identity":"f90f79f1-609c-4086-805d-bdb916e5e653","added_by":"auto","created_at":"2024-07-23 18:35:43","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":68851,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted schematic model of the xylan degradation and xylose utilization pathway in DS1-an-2312\u003csup\u003eT \u003c/sup\u003eunder anaerobic conditions. The enzymes in the pathway are endo-1,4-β-xylanase (GH10, GH5+CBM6+CBM6), xylose transporter XylE, xylose isomerase XylA, xylulose kinase (XK), phosphoketolase (PK).\u003c/p\u003e","description":"","filename":"Figure7Schematicmodelxylandegradation.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4632626/v1/bfb42ae5fb97415673b9e381.jpg"},{"id":67149161,"identity":"0c2db096-17cf-4775-9f7d-d9ccfcb1efa4","added_by":"auto","created_at":"2024-10-21 16:12:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2181657,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4632626/v1/b9d72142-a325-4b39-b2fb-ef8fc17be787.pdf"},{"id":60935346,"identity":"760307fb-b9af-468f-af98-7d2f3408a847","added_by":"auto","created_at":"2024-07-23 18:43:43","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":579453,"visible":true,"origin":"","legend":"","description":"","filename":"Halocynthiibactersupplementary20240624.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4632626/v1/03958c0c45bb526d6664d903.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Halocynthiibacter laminarini gen. nov., sp. nov. and Halocynthiibacter xylanolyticus sp. nov., marine anaerobic laminarin and xylan degraders in the phylum Bacteroidota","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolysaccharides constitute a significant proportion of the carbon inventory in marine organic matter and form a major structural component of both macro- and micro-algae [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Roughly half of this organic matter is released to the marine environment as dissolved and particulate organic matter (DOM, and POM) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This is further degraded by the activities of marine heterotrophic bacteria, which contribute to carbon cycling in marine ecosystems by producing smaller molecular weight compounds and minerals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among marine heterotrophic bacteria, \u003cem\u003eBacteroidota\u003c/em\u003e is one of the most abundant groups, after \u003cem\u003eCyanobacteria\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These bacteria engage in different strategies to contribute to regulate the carbon cycle. Indeed, \u003cem\u003eCyanobacteria\u003c/em\u003e are known as a primary producer because of their photoautotrophic ability in marine ecosystems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], whereas abundant members of marine \u003cem\u003eAlphaproteobacteria\u003c/em\u003e are known to preferentially utilize simple sugars and are found in the water column [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In contrast, \u003cem\u003eBacteroidota\u003c/em\u003e are known as polysaccharide degraders [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany studies have revealed the ability of \u003cem\u003eBacteroidota\u003c/em\u003e to degrade marine polysaccharides from dissolved or particulate organic matters, and the cell wall component of macro- and micro-algal cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This highlights the crucial role of this process in sequestering carbon within the marine environment, thereby significantly contributing to the carbon cycle in marine ecosystems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. To access this rich source of polysaccharides, \u003cem\u003eBacteroidota\u003c/em\u003e have evolved a unique apparatus for their polysaccharide degradation strategy [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This apparatus was first reported in \u003cem\u003eBacteroides thetaiotaomicron\u003c/em\u003e in the human gut microbiota [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It consists of SusD, an outer membrane-bound protein responsible for capturing polysaccharides [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and outer membrane-bound carbohydrate-active enzymes (CAZymes) that partly degrade target polysaccharides into oligosaccharides [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Subsequently, these oligosaccharides are imported into the cell periplasm via a membrane-bound SusC transporter [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Other CAZymes then further degrade these oligosaccharides to monosaccharides, which are transported into the cytoplasm through dedicated transporters [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This entire process is regulated by a regulator that senses the products of polysaccharide degradation. These SusC, SusD, CAZymes, transporters, and regulators are encoded by genes that are clustered together in a region of the genome and are known as polysaccharide utilization loci (PUL) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacteroidota\u003c/em\u003e was first described as a novel phylum by Krieg \u003cem\u003eet al.\u003c/em\u003e in 2010, in the second edition of Bergey\u0026rsquo;s Manual of Systematic Bacteriology [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The name \u003cem\u003eBacteroidota\u003c/em\u003e was revised (from \u003cem\u003eBacteroidetes\u003c/em\u003e) by Oren and Garrity in 2021 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. At the time of writing, the phylum \u003cem\u003eBacteroidota\u003c/em\u003e consists of six classes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://lpsn.dsmz.de/phylum/bacteroidota\u003c/span\u003e\u003cspan address=\"https://lpsn.dsmz.de/phylum/bacteroidota\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e): \u003cem\u003eBacteroidia\u003c/em\u003e Krieg 2012, \u003cem\u003eCytophagia\u003c/em\u003e Nakagawa 2012, \u003cem\u003eFlavobacteriia\u003c/em\u003e Bernardet 2012, \u003cem\u003eSphingobacteriia\u003c/em\u003e Kampfer 2012, \u003cem\u003eChitinophagia\u003c/em\u003e Munoz \u003cem\u003eet al.\u003c/em\u003e 2017, and \u003cem\u003eSaprospiria\u003c/em\u003e Hahnke \u003cem\u003eet al.\u003c/em\u003e 2018. Among these, three classes, \u003cem\u003eBacteroidia, Cytophagia\u003c/em\u003e, and \u003cem\u003eFlavobacteriia\u003c/em\u003e, are the largest, and a huge number of studies on the polysaccharide degradation capability of the member bacteria have been reported. To date, members of the class \u003cem\u003eFlavobacteriia\u003c/em\u003e are found to be abundant in the macroalgal phycosphere microbiome and in microalgal blooms and are well documented for macro- and microalgal polysaccharide degradation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Several studies have meanwhile demonstrated the ability of members of the class \u003cem\u003eCytophagia\u003c/em\u003e to degrade polysaccharides [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and predicted potential polysaccharide degradation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], although there are fewer available literature sources of marine \u003cem\u003eCytophagia\u003c/em\u003e bacteria degrading polysaccharides [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. On the other hand, members of the class \u003cem\u003eBacteroidia\u003c/em\u003e are mostly anaerobes and have been well studied for polysaccharide degradation in the mammalian gut microbiota [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Several studies on human gut \u003cem\u003eBacteroidia\u003c/em\u003e bacteria degrading marine polysaccharide under anaerobic conditions also have been reported [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, few studies on marine \u003cem\u003eBacteroidia\u003c/em\u003e that anaerobically degrade polysaccharides can be found in the literature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eXylan is the most abundant form of hemicellulose found in nature and is produced in marine and terrestrial environments [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Xylan from terrestrial plants contains a β-1,4-linkage D-xylopyranosyl backbone and units of acetate, ɑ-L-arabinofuranose, 4-O-methyl-glucuronic acid, or ferulic acid [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] whereas xylan from marine sources is homoxylan, which is found only in algae [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The backbone of marine xylan mainly consists of a 1,3-glycoside bond and a mixture of 1,3 and 1,4 glycoside bonds [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The molecular structure of xylan protects the cell walls of plants and algae from abiotic or biotic stresses, and it is very difficult to degrade xylan biologically. Biodegradation of xylan results in the production of xylose and xylooligosaccharides, which have potential roles in biotechnological applications such as biofuel, biomedicine, and food supplements [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Initially, xylan is degraded randomly by endoxylanases GH5, GH10, GH11, or GH30 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] to release unbranched xylooligosaccharides, which are then degraded into xylose by β-xylosidase GH39, or GH43 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Several studies have focused on the aerobic degradation of xylan by marine bacteria [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, to our knowledge, research on the anaerobic degradation of xylan by marine \u003cem\u003eBacteroidia\u003c/em\u003e bacteria has been limited.\u003c/p\u003e \u003cp\u003eLaminarin serves as the principal energy reserve glycan identified in brown algae [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and some microalgae [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], with an estimated annual production of approximately 12\u0026ndash;18 gigatons globally [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It is constructed by combining β-(1\u0026rarr;3)-linked glucose-based linear chains and a lower ratio of β-(1\u0026rarr;6)-linked side chains from glucose moieties [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Laminarin degradation results in the production of glucose and laminarin oligosaccharides, which can potentially be used in biotechnological applications in the biofuel, cosmetics, biomedical, and food industries [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To biologically degrade laminarin, endo-acting β-1,3-glucanases (glycoside hydrolase family 17, GH17) specifically break down the β-(1\u0026rarr;3)-linkage in the main chain, while exo-acting β-1,6-glucosidases (GH30) specifically hydrolyze the β-(1\u0026rarr;6)-linkage at the side chain of laminarin. Additionally, exo-acting β-1,3-glucosidases (GH3) specifically hydrolyze the β-(1\u0026rarr;3)-linkage of oligosaccharides into glucoses [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The ability of laminarin degradation is widely distributed in diverse marine heterotrophic bacteria. However, there has been limited research on the anaerobic degradation of laminarin by marine \u003cem\u003eBacteroidia\u003c/em\u003e bacteria.\u003c/p\u003e \u003cp\u003eThe family \u003cem\u003eProlixibacteraceae\u003c/em\u003e of the order \u003cem\u003eBacteroidales\u003c/em\u003e, class \u003cem\u003eBacteroidia\u003c/em\u003e, phylum \u003cem\u003eBacteroidota\u003c/em\u003e was first proposed by Huang \u003cem\u003eet al.\u003c/em\u003e 2014. At the time of writing, as described on the List of Prokaryotic Names with Standing in Nomenclature (LPSN, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bacterio.net/\u003c/span\u003e\u003cspan address=\"https://www.bacterio.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the family accommodates 12 valid genera, including \u003cem\u003eAquipluma\u003c/em\u003e Watanabe \u003cem\u003eet al.\u003c/em\u003e 2020, \u003cem\u003eDraconibacterium\u003c/em\u003e Du \u003cem\u003eet al\u003c/em\u003e 2014, \u003cem\u003eGaoshiqia\u003c/em\u003e Yu \u003cem\u003eet al.\u003c/em\u003e 2023, \u003cem\u003eMangrovibacterium\u003c/em\u003e Huang \u003cem\u003eet al.\u003c/em\u003e 2014, \u003cem\u003eMaribellus\u003c/em\u003e Zhou \u003cem\u003eet al.\u003c/em\u003e 2019, \u003cem\u003eMariniphaga\u003c/em\u003e Iino \u003cem\u003eet al.\u003c/em\u003e 2014, \u003cem\u003eMeniscus\u003c/em\u003e Irgens 1977, \u003cem\u003eProlixibacter\u003c/em\u003e Holmes \u003cem\u003eet al.\u003c/em\u003e 2007, \u003cem\u003ePuteibacter\u003c/em\u003e Sun \u003cem\u003eet al.\u003c/em\u003e 2020, \u003cem\u003eRoseimarinus\u003c/em\u003e Wu \u003cem\u003eet al.\u003c/em\u003e 2015, \u003cem\u003eSunxiuqinia\u003c/em\u003e Qu \u003cem\u003eet al.\u003c/em\u003e 2011, and \u003cem\u003eTangfeifania\u003c/em\u003e Liu \u003cem\u003eet al.\u003c/em\u003e 2014. They have been isolated from various habitats, particularly hypolimnion water [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], river sediment [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], mangrove sediment [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], marine sediment [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], seawater [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and crude oil [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Most of the members of the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e are Gram-staining negative, rod-shaped, non-spore-producing bacteria. They have no motility and their oxygen requirement ranges from aerobic to facultative anaerobic conditions. The predominant quinone component is menaquinone 7 (MK-7) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Several isolation methods have been applied to isolate members of the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e, including enrichment [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], a dilution technique [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and nitrogen-free medium cultivation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, we used another strategy to isolate novel bacteria by using a low nutrient isolation medium, which was prepared from 60% strength seawater (at the collection site) with 1.5% agar (BD) and a piece of filter paper placed on the surface of the agar as the sample carrier [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We isolated two novel bacteria from a sea squirt under anaerobic conditions.\u003c/p\u003e \u003cp\u003eIn this study, we identified and characterized two novel anaerobic isolates, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e (strictly anaerobic) and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e (facultative anaerobic), through genetic, morphological, biochemical, and chemotaxonomic analyses. These isolates were proposed as novel genus with two novel species within the family \u003cem\u003eProlixibacteraceae.\u003c/em\u003e Genome mining revealed that these isolates contain hundreds of CAZyme genes spanning 65 CAZyme families. Their glycoside hydrolases were the most abundant among CAZymes, and exhibited up to double frequency of GHs per genome compared to the average value in marine \u003cem\u003eBacteroidota.\u003c/em\u003e Additionally, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e was found to utilize laminarin as a sole carbon source under anaerobic conditions, while DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could utilize both laminarin and xylan. The final hydrolytic product of laminarin degradation was glucose, whereas xylan degradation primarily yielded xylotriose and xylotetraose. This study is the first to combine genome mining with \u003cem\u003ein vitro\u003c/em\u003e experiments to analyze xylan and laminarin degradation in anaerobic marine \u003cem\u003eBacteroidia\u003c/em\u003e, and the results enhance our understanding of the polysaccharide degradation strategies of anaerobic marine \u003cem\u003eBacteroidia\u003c/em\u003e and their role in marine carbon cycling.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEcology, isolation, and cultivation of isolates\u003c/h2\u003e \u003cp\u003eThe source of isolation was a sea squirt collected by a fisherman at a depth of 18 m beneath the surface of seawater in the East Sea, Republic of Korea (38\u0026ordm;38\u0026rsquo;48.5\u0026rdquo; N, 129\u0026ordm;44\u0026rsquo;29,2\u0026rdquo; E). The sample was then stored in an ice-pack container and delivered to the laboratory on the same day. For isolation, a low nutrient solid medium prepared by sixty percent strength seawater (collected from the sampling site) solidified with 1.5% agar (BD) was used. After autoclaving the medium, 50 mg/L cycloheximide (Aldrich Sigma) was added to the agar medium [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. All the steps for bacterial isolation were performed in an anaerobic chamber (Coy Lab Products, USA; N\u003csub\u003e2\u003c/sub\u003e: H\u003csub\u003e2\u003c/sub\u003e: CO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;94.5%:1.5%:5%). The plates were stored in an anaerobic chamber overnight to remove oxygen. A slice taken from the mouth of the squirt was placed on the top of a piece of filter paper (Whatman No.2) on the isolation plates. The plates were placed in an anaerobic jar with a bag of BD GasPak EZ anaerobe container system (BD) and incubated at 23 ℃. After 4\u0026ndash;5 days of incubation, the colony surrounding the filter paper was transferred and streaked onto fresh marine agar plates (BD marine agar 2216) under anaerobic conditions. After several transfers, single colonies of the two strains were obtained. The pure cultures of the two strains were preserved in 20% glycerol at -80 ℃, and lyophilized in ampoules stored at 4 ℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis based on the 16S rRNA gene sequence\u003c/h2\u003e \u003cp\u003eFor a phylogenetic analysis, the 16S rRNA gene sequences of the two isolates were determined. The 16S rRNA sequences of the two strains were sequenced using four universal primers: 27F (5\u0026rsquo;-AGAGTTTGATCCTGGCTCAG-3\u0026rsquo;), 518F (5\u0026rsquo;-CCAGCAGCCGCGGTAATAC-3\u0026rsquo;), 805R (5\u0026rsquo;-GACTACCAGGGTATCTAATC-3\u0026rsquo;), and 1492R (5\u0026rsquo;-TACGGYTACCTTGTTACGACTT-3\u0026rsquo;) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], via Sanger sequencing. The complete sequences were manually assembled using Vector NTI software (Invitrogen). Next, pairwise sequence alignment was performed on EzBioCloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/\u003c/span\u003e\u003cspan address=\"https://www.ezbiocloud.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The related sequences downloaded from EzBioCloud were aligned using ClustalW in BioEdit software (version 7.2.5). The trimmed sequences were then used to make phylogenetic trees in MEGA11 using three algorithms: neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The estimated matrix on MEGA11 based on the alignment fasta file showed that the optimal model for the MP tree was the general time reversible (GTR) model, and the rates and patterns were Gamma distributed with Invariant sites (G\u0026thinsp;+\u0026thinsp;I). Moreover, the Kimura two-parameter model was used for the NJ algorithm, and tree-bisection-reconnection (TBR) was used for the ML algorithm. The bootstrap resampling method with 1000 replicates was used to evaluate the phylogenetic tree. The sequence of strain \u003cem\u003eAgarivorans albus\u003c/em\u003e (accession number AB681878) was obtained from NCBI as the outgroup, and the sequence of \u003cem\u003eMarinilabilia salmonicolor\u003c/em\u003e NCIMB 2216\u003csup\u003eT\u003c/sup\u003e (accession number D12672) was retrieved from NCBI as the type strain of the closest family, \u003cem\u003eMarinilabiliaceae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003ePairwise alignment of 16S rRNA sequences revealed that the strains DS1-an-2312\u003csup\u003eT\u003c/sup\u003e and DS1-an-13321\u003csup\u003eT\u003c/sup\u003e had the highest similarity to \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e, with similarity values of 91.57% and 91.48%, respectively. Therefore, the isolated strains were considered to belong to a novel genus in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e or to a novel family in the order \u003cem\u003eBacteroidales.\u003c/em\u003e To delineate the two strains in terms of phylogeny, the type species of each genus in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e and the type strains of the second closest family, \u003cem\u003eMarinilabiliaceae\u003c/em\u003e, were included in a phylogenetic tree. The resulting phylogenetic tree, reconstructed based on three algorithms, revealed that the two novel isolates belonged to a cluster within the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e and formed a separate cluster from other genera in this family (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003cb\u003e)\u003c/b\u003e. Based on the phylogenetic position of the two isolates on the phylogenetic tree and their top hit similarity, three strains, \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e KCTC 25263\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eProlixibacter bellariivorans\u003c/em\u003e KCTC 25261\u003csup\u003eT\u003c/sup\u003e, and \u003cem\u003eSunxiuqinia elliptica\u003c/em\u003e KCTC 32215\u003csup\u003eT\u003c/sup\u003e, were selected as the reference strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological characterization\u003c/h2\u003e \u003cp\u003eThe morphology of colonies of the two novel isolates was observed on MA plates after three days of cultivation under anaerobic conditions. Gram staining was performed according to the standard protocol, and the prepared slices were observed under a light microscope (Nikon Eclipse 80i). Scanning electron microscopy (SEM, JEOL JSM 7600F) was used to observe the cell morphology [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To determine their temperature range, the two strains were cultured in marine broth in test tubes purged with nitrogen gas and then incubated over a temperature range from 10\u0026ndash;30 ℃ for three days and one week at 4 ℃ and 35 ℃. To determine the pH range, marine broth in test tubes purged with nitrogen gas was used to determine the growth of the two novel isolates using the following buffer systems at 50 mM: 5.5-6.0 (MES), 6.5-7 (PIPES), 7.5-8.0 (HEPES), 8.5 (Bis-Tris), and 9.0\u0026ndash;10.0 (CHES) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In a NaCl tolerance experiment, Zobell broth [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] in artificial seawater [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] was supplemented with NaCl concentrations of 0, 0.5, and 1\u0026ndash;10% (with intervals of 1.0%, w/v). To determine the oxygen requirements, the two isolates were cultivated on MA plates in anaerobic jars under anaerobic conditions provided by a bag of BD GasPak EZ anaerobe container system (BD) and under microaerophilic conditions provided by a bag of BD GasPak EZ CO\u003csub\u003e2\u003c/sub\u003e container system. The plates were inoculated for three days at 28 ℃.\u003c/p\u003e \u003cp\u003eThe biochemical characteristics of the two novel isolates and their reference strains were determined on marine agar as the basal media at 28 ℃ for three days under anaerobic conditions unless otherwise specified. The hydrolysis of starch was tested by supplying 0.2% starch to MA plates and detecting a clear zone after staining with iodine solution. The hydrolysis of cellulose was assessed on carboxymethyl cellulose agar plates [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] using artificial seawater [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] with 2% NaCl instead of distilled water, and a clear zone was detected after embedding in Congo Red and washing with 1% (w/v) NaCl solution. The hydrolysis of Tween 20 and Tween 80 was performed by adding 0.1% (v/v) Tweens 20 and 80 to MA plates, respectively. Catalase activity was determined by dropping a 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution onto the surface of cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Oxidase activity was tested by reacting cells with an oxidase reagent (bioMerieux). All five strains were cultured on DNase agar (Difco) using artificial seawater with 2% NaCl instead of distilled water to test DNase activity. Chitin (1%, w/v) was added to MA plates, and chitin hydrolysis activity was detected by the appearance of a clear zone [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Gelatinase activity was evaluated on nutrient gelatin (Remel Gelatin medium) in which distilled water was replaced with artificial seawater [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] supplemented with 2% (w/v) NaCl for one week at 25 ℃, and a positive result was recognized by liquidation of the medium [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo determine anaerobic metabolism, the growth of the two novel isolates on a variety of carbon sources was assessed. An inoculum of the strains was prepared in 5 mL of marine broth (MB, Difco) supplemented with 20 mM HEPES (Sigma) and adjusted to pH 7.0 using 2N NaOH. The strains were cultivated on basal media (BS) supplemented with carbon substrates (0.2%, w/v). The composition of the BS was as follows: 23 g NaCl, 1.3 g KCl, 1 g MgCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO, 0.1 g CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO, 0.5 g NH\u003csub\u003e4\u003c/sub\u003eCl, 0.2 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 3.82 g Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 0.08 g FeCl\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO, 1 mL trace elements (KCTC Media No. 918, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kctc.kribb.re.kr/access/search/viewMedia?sn=918\u003c/span\u003e\u003cspan address=\"https://kctc.kribb.re.kr/access/search/viewMedia?sn=918\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), 1 mL vitamin solution [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], 0.1 g yeast extract, 2.5 g NaHCO\u003csub\u003e3\u003c/sub\u003e, 4.76 g HEPES, and 1 L deionized H\u003csub\u003e2\u003c/sub\u003eO, adjusted to pH 7.0 using 2N NaOH. The following carbon substrates were tested: fructose, galactose, glucose, xylose, maltose, lactose, sucrose, and starch. BS without substrates was used as the control. For fermentation conditions, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was omitted from the composition of the basal medium. For sulfate reduction conditions, additional carbon sources (10 mM), which served as an electron donor, were evaluated, including acetate, benzoate, formate, fumarate, hexanoate, octanoate, lactate, propionate, and pyruvate [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and sulfate (27 mM) was tested as an electron acceptor. For an additional anaerobic respiration test, nitrate (10 mM), nitrite (10 mM), sulfite (10 mM), and thiosulfate (10 mM) were added to BS to replace sulfate (27 mM) as electron acceptors.\u003c/p\u003e \u003cp\u003eThe growth of the strains was monitored under a microscope (Nikon Eclipse 80i) and compared with that of the control. The concentration of sulfate in the culture broth was measured [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The utilization of mono-, di-, and polysaccharides was quantified by measuring the production of reducing sugars during growth using a DNS assay [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Fermentation products were determined after five days of cultivation using high-performance liquid chromatography (HPLC), as described previously [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The chromatograph of the 5-day culture was compared with that of the 0-hour culture. Different and high peaks were identified by aligning the retention times with those of candidate standard compounds. All experiments were performed in duplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eChemotaxonomic characterization\u003c/h2\u003e \u003cp\u003eThe fatty acid profiles of strains DS1-an-2312\u003csup\u003eT\u003c/sup\u003e and DS1-an-13321\u003csup\u003eT\u003c/sup\u003e, as well as their reference strains, were determined after they were grown on MA plates for three days. Cells were harvested for a fatty acid analysis following the procedure outlined in the MIDI protocol (version 6.2). Subsequently, the extracted fatty acid methyl esters were injected into a gas chromatograph system, and their fatty acid components were identified based on the RTSBA 6.0 database.\u003c/p\u003e \u003cp\u003eTo determine the quinone type, freeze-dried DS1-an-2312\u003csup\u003eT\u003c/sup\u003e and DS1-an-13321\u003csup\u003eT\u003c/sup\u003e cells were mixed with chloroform-methanol (2:1, v/v) and shaken overnight. The extract was collected through paper filtration, concentrated via evaporation, and recovered by adding acetone. The acetone suspension was then applied to a thin-layer chromatography (TLC) plate (Kieselgel 60F\u003csub\u003e254\u003c/sub\u003e, 20 \u0026times; 20 cm, Merck) and separated using a combination of petroleum ether-diethyl ether (9:1, v/v). The quinone band on the TLC plate was visualized with UV light, and the samples were harvested and recovered in 100% acetone. The quinone extracts were further analyzed via reversed-phase chromatography using a mobile phase of methanol-isopropanol (7:5, v/v) and a wavelength of 270 nm to detect the quinone components [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe polar lipids of strains DS1-an-2312\u003csup\u003eT\u003c/sup\u003e and DS1-an-13321\u003csup\u003eT\u003c/sup\u003e were extracted from their freeze-dried cells following the detailed methods of Komagata and Suzuki [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The extracted lipids were then applied onto a silica gel TLC plate and developed on the plate in two dimensions. The first dimension involved a combination of chloroform-methanol-water (65:25:4, v/v/v), and the second a combination of chloroform-methanol-acetic acid-water (80:15:12:4, v/v/v/v) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. To identify the polar lipid profiles of the two novel isolates, TLC plates were sprayed with individually appropriate reagents: molybdatophosphoric acid for total lipids, ninhydrin for lipids with amino groups, molybdenum blue for lipids with phosphate groups, and ɑ-naphthol in a sulfuric acid solution for lipids with sugar groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGenome analysis\u003c/h2\u003e \u003cp\u003eFor genomic analysis, genomic DNA was extracted based on the method of Vengadesh \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] with several modifications. Briefly, the isolated strains cultured anaerobically for two days in marine broth were harvested. The cell mass was mixed with proteinase K (20 mg/mL, 20 mM Tris-HCl, pH 8.0) in cetyltrimethylammonium bromide (CTAB) solution. The mixtures were incubated at 37 ℃ for one hour. Next, one volume of phenol-chloroform-isoamyl alcohol (PCI, 25:24:1, v/v/v) was added to the mixture, followed by centrifugation at 11 000 \u0026times; g. The aqueous phase was collected and transferred to a new tube, and the nucleic acids were precipitated by adding a 0.6 volume of isopropyl alcohol and a one-tenth volume of 3 M sodium acetate. The tubes were inverted to ensure a consistent mixture and then incubated at 4 ℃. After 1\u0026ndash;2 hours of incubation, the tubes were centrifuged at 16 000 \u0026times; g for 20 minutes at 4 ℃. Next, the suspension was removed and replaced twice with 100 \u0026micro;L of 70% ethanol (cooled at -20 ℃), twice. The pellet was dried at room temperature and re-dissolved in DNase-free water. RNA was then removed from the solution by treatment with RNase A at 40 ℃ for one hour. The DNA component subsequently was separated by one volume of PCI and precipitated with alcohol as aforementioned. The DNA was re-dissolved in DNase-free water and used for genome sequencing.\u003c/p\u003e \u003cp\u003eThe whole-genome sequences of both novel strains were obtained by a combination of two sequencing methods, short-read Illumina sequencing (Macrogen, Inc., Seoul, Republic of Korea) and Nanopore sequencing. For Illumina sequencing, the short-length DNA of each strain was used to construct a library based on the protocol of the TruSeq DNA PCR-Free Sample Preparation Guide, part #15036187 Rev. D. For nanopore sequencing, high-molecular-weight DNA was used to prepare a library for nanopore sequencing according to the SQK-LSK109 protocol (version GDE_9063_v109_revN_14Aug2019). The genomes of both strains were \u003cem\u003ede novo\u003c/em\u003e assembleed by Canu (version 2) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] based on a combination of raw data from Nanopore and Illumina sequencing. Medaka \u003cb\u003e(\u003c/b\u003eversion 1.3.2, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/nanoporetech/medaka\u003c/span\u003e\u003cspan address=\"https://github.com/nanoporetech/medaka\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used as a polishing tool for assembly by counting the occurrences of each nucleotide at each position on the assembled sequence to predict the true base at that position. The quality of the assembled genome and annotation completeness were assessed by BUSCO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://busco.ezlab.org/\u003c/span\u003e\u003cspan address=\"https://busco.ezlab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. CheckM (version 1.1.3) was used to estimate the contamination and completeness of the genome [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The genome was annotated on Prokka (version 1.12) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The average nucleotide identity (ANI) tool on EzBioCloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/tools/ani\u003c/span\u003e\u003cspan address=\"https://www.ezbiocloud.net/tools/ani\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and the genome-to-genome distance calculator (version 2.1) on DSMZ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ggdc.dsmz.de/ggdc.php#\u003c/span\u003e\u003cspan address=\"https://ggdc.dsmz.de/ggdc.php#\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] were used for digital DNA-DNA hybridization to distinguish the novel isolates from their closest valid taxa. The whole-genome sequences of the reference strains, including \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e (GCA_005217565), \u003cem\u003eProlixibacter bellariivorans\u003c/em\u003e JCM 13498\u003csup\u003eT\u003c/sup\u003e (GCF_000621705), \u003cem\u003eSunxiuqinia elliptica\u003c/em\u003e CGMCC 1.9156\u003csup\u003eT\u003c/sup\u003e (GCF_900113005), \u003cem\u003eMaribellus luteus\u003c/em\u003e XSD2\u003csup\u003eT\u003c/sup\u003e (GCA_003576475), \u003cem\u003eDracinibacterium orientale\u003c/em\u003e FH5\u003csup\u003eT\u003c/sup\u003e (GCA_000626635), \u003cem\u003eAquipluma introreducens\u003c/em\u003e MeG22\u003csup\u003eT\u003c/sup\u003e (AP018694), \u003cem\u003eMangrovibacterium diazotrophicum\u003c/em\u003e DSM 27148\u003csup\u003eT\u003c/sup\u003e (GCF_003610535), \u003cem\u003eMariniphaga anaerophila\u003c/em\u003e DSM 26910\u003csup\u003eT\u003c/sup\u003e (GCF_900129025), \u003cem\u003eTangfeifania diversioriginum\u003c/em\u003e DSM 27063\u003csup\u003eT\u003c/sup\u003e (GCF_900141875), and \u003cem\u003eGaoshiqinia sediminis\u003c/em\u003e A06\u003csup\u003eT\u003c/sup\u003e (GCF_025907915.1), were retrieved from NCBI. The average amino acid identity (AAI) was calculated using the AAI calculator from the Kostas laboratory (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://enve-omics.ce.gatech.edu/aai/\u003c/span\u003e\u003cspan address=\"http://enve-omics.ce.gatech.edu/aai/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Pairwise comparisons for the percentage of conserved proteins (POCP) were calculated based on the method provided by Qin \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The amino acid FASTA sequences of the reference strains were retrieved from NCBI. Functional genes within each genome were also annotated using KEGG and deciphered to pathways using KEGG Decoder [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] and KEGG-Expander (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/bjtully/BioData/tree/masterEGGDecoder\u003c/span\u003e\u003cspan address=\"https://github.com/bjtully/BioData/tree/masterEGGDecoder\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Other databases were used for annotation including the Clusters of Orthologous Genes (COGs) [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and Gene Ontology (GO) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] databases. Carbohydrate-active enzymes were identified through the CAZy database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cazy.org/\u003c/span\u003e\u003cspan address=\"http://www.cazy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] and the dbCAN server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bcb.unl.edu/dbCAN2/blast.php\u003c/span\u003e\u003cspan address=\"https://bcb.unl.edu/dbCAN2/blast.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Biosynthesis gene clusters (BGCs) and metabolic gene clusters (MGCs) were predicted by antiSMASH 7.1.0 [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] and gutSMASH [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], respectively. Prophages were predicted in the genomes using PHASTER [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. CRISPR-Cas in the genomes was predicted using CRISPRCasFinder [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The subcellular location of proteins in prokaryotes was predicted via PSORTb (version 3.0.3) [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The predicted model of xylan degradation and xylose utilization was drawn by using Affinity Designer (version 1.10.6.1665).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePolysaccharide-degrading ability\u003c/h2\u003e \u003cp\u003eTo test polysaccharide utilization, two novel strains were anaerobically grown on BS media supplemented with individual polysaccharides (0.1\u0026ndash;0.2%, w/v), including alginate, cellulose, chitin, κ-, λ-, and ι-carrageenan, fucoidan, laminarin, starch, and xylan. Ten microliters of each culture was observed under a light microscope (Nikon Eclipse 80i) every two days to monitor growth. By comparing the cell numbers each day and with those of the control (no carbon source), the growth of both strains was recorded. The production of reducing sugars in the supernatant was detected by reacting the cell-free supernatant with 3,5-dinitrosalicylic acid (DNS) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eGrowth of DS1-an-13321\u003c/b\u003e\u003csup\u003e\u003cb\u003eT\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eand DS1-an-2312\u003c/b\u003e\u003csup\u003e\u003cb\u003eT\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eon laminarin and xylan\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe ability of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e to grow on laminarin and the ability of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e to grow on xylan were further studied. During the growth of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on laminarin and xylan, black particles and clumped cells were produced in the culture broth, and the density of the cells in culture could not be measured by a spectrophotometer. The growth of DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on the corresponding substrates was therefore monitored under a light microscope (Nikon Eclipse 80i). We counted the cells in culture every 6 h under a microscope to track the growth of both strains on laminarin and xylan to make growth curves. Briefly, every 6 h, 100 \u0026micro;L of broth culture was harvested and diluted tenfold. Subsequently, 10 \u0026micro;L aliquots of diluted solution were dropped onto glass slides, stained with safranine (BD), and cells were counted under 400\u0026times; magnification by a microscope (Nikon Eclipse 80i). Five frames were randomly selected for statistical analysis. Subsequently, the cells in each frame were counted, and the average cell number and standard deviation were calculated considering the dilution factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLocation of laminarin and xylan-degrading enzymes\u003c/h2\u003e \u003cp\u003eFor laminarin-degrading enzymes, cells were harvested by centrifugation from the 4-day cultures of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on laminarin. The cell pellet was washed and resuspended in phosphate buffer solution (PBS, pH 7.2). Subsequently, the cell suspension was incubated with laminarin (final concentration of 0.1%, w/v) for 6 h at 30 \u0026ordm;C. The supernatant was stirred with 50% (w/v) (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (final concentration) to precipitate the extracellular protein. Overnight dialysis was applied to remove the salt. The resulting solution was incubated with laminarin (at a final concentration of 0.1%, w/v) for 6 h at 30 \u0026ordm;C. The enzyme reaction was assessed by detection of reducing sugars by a DNS assay [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The change of reducing sugars from the beginning to the end of the enzyme reaction was determined. The procedure used to determine the location of the active xylan-degrading enzymes was the same as that used for laminarin-degrading enzymes. However, in the enzyme reaction, the final concentration of xylan was 0.2% (w/v).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDegradation of laminarin and xylan by whole cell enzymes\u003c/h2\u003e \u003cp\u003eFour-day old strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e cells grown on laminarin (0.1%, w/v) were harvested. The cells were harvested by centrifugation, washed three times, and resuspended in PBS. The cell suspension was then used as a crude enzyme of cell-associated laminarin-degrading enzymes to test for enzyme activity. In the case of DS1-an-2312\u003csup\u003eT\u003c/sup\u003e grown on xylan (0.2%, w/v), 4-day old DS1-an-2312\u003csup\u003eT\u003c/sup\u003e cells grown on xylan were harvested by centrifugation. The cell pellet and remaining insoluble xylan were washed three times to remove remaining soluble sugars and resuspended in PBS. The cell suspensions were used as crude enzymes, and enzyme activity was directly tested. The DNS assay was applied to detect the production of reducing sugars during the hydrolysis of laminarin and xylan. The products of the hydrolysis reaction were further analyzed in the next step.\u003c/p\u003e \u003cp\u003eThe hydrolysis products of the crude enzymes were assessed by thin layer chromatography (TLC) according to the method of Lee \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. In brief, the crude enzymes from strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e were incubated with each substrate (1 mg/mL, final concentration) of glucose (G), laminaribiose (L2), laminaritriose (L3), laminaritetraose (L4), laminaripentaose (L5), laminarihexaose (L6), and laminarin (Ln) at 30 \u0026ordm;C for 24 h. The reaction of the crude enzymes without substrates and the reaction of each substrate without the crude enzymes were included as controls. The enzyme-reaction tubes were centrifuged at 8000 rpm for 2 min at room temperature to harvest the degradation products of the reaction. The supernatant was applied to a TLC plate without concentration. The TLC plate was then developed in a chloroform-acetic acid-water (6:7:1, v/v/v) solvent system [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. The TLC plates were visualized by spraying a mixture of ethanol-sulfuric acid (95:5, v/v), followed by drying in an oven at 150 \u0026ordm;C for 5 to 10 min. For hydrolysis products from xylan degradation of DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, the cell pellet was washed three times with PBS to remove the remaining soluble sugar from the culture. Subsequently, to assess the mode of action of the DS1-an-2312\u003csup\u003eT\u003c/sup\u003e xylan-degrading enzyme, the reducing sugars present at the initial crude enzyme solution and the reducing sugars that were produced during the enzyme reaction were compared. The limitation of this test is the remaining insoluble xylan in the cell pellet; thus, during enzyme incubation with the whole cell, this remaining xylan that had not yet been consumed was further degraded, and the resulting reducing sugars was released into the suspension. Thus, the presence of hydrolytic products during enzyme reaction of crude enzyme was detected via TLC in the same manner as in the analysis of the laminarin-hydrolytic products.\u003c/p\u003e \u003cp\u003eThe oligosaccharides of xylan and laminarin were purchased from Megazyme, and xylan and laminarin were purchased from Sigma.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIsolation, cultivation, and identification\u003c/h2\u003e \u003cp\u003eTwo strains were isolated from a sea squirt (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) collected at a depth of 18 m of the East Sea, Republic of Korea. A portion of the feeding area of the sea squirt (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) was cut and placed on the surface of filter paper on a low nutrient agar medium that was prepared by sixty percent seawater collected from the same sampling location. Pure cultures of the two novel strains were obtained by transferring colonies exhibiting gliding traits around the filter paper. The strains were transferred several times onto fresh marine agar (MA) plates until no contaminants were detected. The morphologies of the pure cultures of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e are presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cb\u003ed\u003c/b\u003e, respectively. Strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e formed nearly round colonies with a tortilla color in the center and a cream color at the periphery, while strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e exhibited irregular colonies with a round tortilla color in the center and broad swarming at the outer edge of the colonies. The cells of both strains were long rods with a length exceeding 20 \u0026micro;m during the log-phase of growth and a width ranging from 0.25\u0026ndash;0.5 \u0026micro;m (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f, and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Interestingly, both strains showed a transition from a long rod to a spherical shape at the end of the stationary phase (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e were maintained on MA plates at 23 \u0026ordm;C and 28 \u0026ordm;C, respectively. For long-term preservation, two novel isolates were preserved by using a liquid-dry method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative physiological characteristics of two novel strains, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, and their representative reference strains in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e. Strains: 1, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e; 2, DS1-an-2312\u003csup\u003eT\u003c/sup\u003e; 3, \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]; 4, \u003cem\u003eProlixibacter bellariivorans\u003c/em\u003e JCM 13498\u003csup\u003eT\u003c/sup\u003e [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]; 5, \u003cem\u003eSunxiuqinia elliptica\u003c/em\u003e CGMCC 1.9156\u003csup\u003eT\u003c/sup\u003e [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. * Data obtained from [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColony\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNearly round shape colony and bright brown color\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIrregular shape, bright brown color\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCreamy white*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSlightly pink*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell diameter (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLong-rod shape, long-shape: 0.25\u0026ndash;0.5 x\u0026thinsp;~\u0026thinsp;20, Spherical shape: 0.48\u0026ndash;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLong-rod shape long-shape: 0.25\u0026ndash;0.5 x\u0026thinsp;~\u0026thinsp;20, Spherical shape: 0.48\u0026ndash;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRod shape,\u003c/p\u003e \u003cp\u003e1-3x0.3-0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFilamentous-rod shape, 0.33 x 10.5\u0026ndash;12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRod shape, 0.4 x 0.8\u0026ndash;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature range (℃)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u0026ndash;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026ndash;32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u0026ndash;37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u0026ndash;42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u0026ndash;42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOptimum growth temperature (℃)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u0026ndash;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH range\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.0-8.5 (6.5\u0026ndash;8.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.0\u0026ndash;8.0 (7.0-7.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0\u0026ndash;9.0 (7.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.0\u0026ndash;9.0 (7.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.0\u0026ndash;9.0 (7.0\u0026ndash;8.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaCl (%) range(optimum)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u0026ndash;4 (2\u0026ndash;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026ndash;4 (2\u0026ndash;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u0026ndash;6 (3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5-8 (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5\u0026ndash;10 (3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxygen requirement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMicroaerophilic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicroaerophilic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFacultative anaerobic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFacultative anaerobes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStrictly aerobic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH2S production\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHydrolysis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGelatin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+ \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+ \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e- \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e- \u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+ \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e- \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+ \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e- \u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDNase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+ \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e- \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e- \u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUtilization\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFructose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGalactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXylose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource of isolation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSea squirt\u003c/p\u003e \u003cp\u003e(-18 m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSea squirt\u003c/p\u003e \u003cp\u003e(-18 m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSeawater at a depth of 90 m at Yongle blue Hole in the South China sea.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSurface of an electricity-harvesting electrode incubated in marine sediments.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSediment of a seashore pond for sea cucumber culture in Jimo, Qingdao, on the east coast of China.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe 16S rRNA gene sequences of the pure cultures were determined. The analysis of the sequences on the EzBioCloud server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/\u003c/span\u003e\u003cspan address=\"https://www.ezbiocloud.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) revealed that the highest 16S rRNA gene sequence similarities to \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e for strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e was 91.26% and 91.37%, respectively. Moreover, 16S rRNA similarities of the two novel isolates with type strains of all existing genera in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e, which is the parent taxon of \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e, were calculated (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). This analysis demonstrated that the 16S rRNA similarity between the two isolates and existing members of the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e fell within the range of 86.70 to 91.37%, while a similarity of 98.75% was observed between the two isolates. Based on these results, we hypothesized that the two novel isolates represent a novel genus and two novel species. Therefore, 16S rRNA-based phylogenetic tree, genome-based phylogenetic tree, genomic indices, and polyphasic taxonomic study were performed to evaluate this hypothesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e16S rRNA-based and genome-based phylogeny\u003c/h2\u003e \u003cp\u003eA phylogenetic tree based on 16S rRNA gene sequences by combining three algorithms, ML, NJ, and MP, showed the taxonomic position of the two novel isolates within the families \u003cem\u003eProlixibacteraceae\u003c/em\u003e and \u003cem\u003eMarinilabiliaceae\u003c/em\u003e in the phylum \u003cem\u003eBacteroidota\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e formed a single cluster with strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, but the two strains were separated from all other representatives in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e, even \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e (approximately 91% of 16S rRNA similarity).\u003c/p\u003e \u003cp\u003eBased on 92 core genes from their genomes, a genome-based phylogenetic tree was constructed under the UBCG pipeline using the ML algorithm to clarify the taxonomic position of the two isolates within the families \u003cem\u003eProlixibacteraceae\u003c/em\u003e and \u003cem\u003eMarinilabiliaceae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The two novel isolates clustered with their closest relative, \u003cem\u003ePuteibacter caerulelacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e, with a high bootstrap value of 92%. However, this cluster was separated from other representatives in the families \u003cem\u003eProlixibacteraceae\u003c/em\u003e and \u003cem\u003eMarinilabiliaceae.\u003c/em\u003e Considering the results from both the 16S-based and genome-based phylogenetic trees, the two novel strains could be classified as a novel genus within the family \u003cem\u003eProlixibacteraceae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the taxonomic proposal of the two novel isolates, the genomic indices of the two isolates against the type species in all genera of the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e, including the ANI, dDDH, AAI, and POCP values, were calculated (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The ANI and dDDH values between strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e were 73.99% and 20.9%, respectively. These values were under the cutoff values of the ANI (95%) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and dDDH (70%) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] for species demarcation. In addition, the ANI and dDDH values between the two isolates and other representatives in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e ranged from 66.17\u0026ndash;67.76% and 24.0-37.4%, respectively. All these values were much lower than the cutoff values of the ANI (95%) and dDDH (70%). Hence, the two novel isolates were deemed candidates for two novel species classified in the family \u003cem\u003eProlixibacteraceae.\u003c/em\u003e For a comparison of the amino acid sequences, the AAI and POCP values between the two novel isolates and representatives in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e ranged from 48.33\u0026ndash;52.35% and 29.01\u0026ndash;37.37%, respectively. Both of these ranges fell under the cutoff values for genus demarcation of AAI (60%) [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] and POCP (50%) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] but strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e shared 71.34% and 73.55% for the AAI and POCP values, respectively, with strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e. Therefore, the genomic indices provide strong evidence for the proposal of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e as candidates for two novel species in a novel genus in the family \u003cem\u003eProlixibacteraceae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANI values calculated using the EzBioCloud service, digital DNA-DNA hybridization values calculated with Genome-to-Genome Distance Calculator 3.0, AAI values calculated using the AAI calculator (Kostas lab), POCP values calculated among novel strains and their reference strains: 1, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e (CP081303); 2, DS1-an-2312\u003csup\u003eT\u003c/sup\u003e (CP082230); 3, \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e (SJCO00000000); 4, \u003cem\u003eProlixibacter bellariivorans\u003c/em\u003e JCM 13498\u003csup\u003eT\u003c/sup\u003e (JHXO00000000); 5, \u003cem\u003eSunxiuqinia elliptica\u003c/em\u003e CGMCC 1.9156\u003csup\u003eT\u003c/sup\u003e (FONW00000000); 6, \u003cem\u003eMaribellus luteus\u003c/em\u003e XSD2\u003csup\u003eT\u003c/sup\u003e (QWGR00000000); 7, \u003cem\u003eDraconibacterium orientale\u003c/em\u003e FH5\u003csup\u003eT\u003c/sup\u003e (CP007451); 8, \u003cem\u003eAquipluma nitroreducens\u003c/em\u003e MeG22\u003csup\u003eT\u003c/sup\u003e (AP018694); 9, \u003cem\u003eMangrovibacterium diazotrophicum\u003c/em\u003e DSM 27148\u003csup\u003eT\u003c/sup\u003e (RAPN00000000); 10, \u003cem\u003eMariniphaga anaerophila\u003c/em\u003e DSM 26910\u003csup\u003eT\u003c/sup\u003e (FQUM00000000); 11, \u003cem\u003eTangfeifania diversioriginum\u003c/em\u003e DSM 27063\u003csup\u003eT\u003c/sup\u003e (FQZE00000000); 12, \u003cem\u003eGaoshiqia sediminis\u003c/em\u003e A06\u003csup\u003eT\u003c/sup\u003e (JAPAAF000000000).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"13\" nameend=\"c13\" namest=\"c1\"\u003e \u003cp\u003eANI value (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e66.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e67.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e67.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e67.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e66.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e66.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e66.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e66.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e66.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e66.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e66.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e66.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e66.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e66.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e66.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"13\" nameend=\"c13\" namest=\"c1\"\u003e \u003cp\u003eDNA-DNA hybridization (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e26.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e30.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e29.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e31.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e24.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e26.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e28.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e28.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e31.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"13\" nameend=\"c13\" namest=\"c1\"\u003e \u003cp\u003eAAI values (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e48.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e48.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e48.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e49.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e49.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e48.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e49.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e48.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e48.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e48.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e49.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e48.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e48.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e49.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"13\" nameend=\"c13\" namest=\"c1\"\u003e \u003cp\u003ePOCP values (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e29.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e31.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e32.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e31.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e33.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e28.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e30.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e31.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e30.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e32.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological characterization\u003c/h2\u003e \u003cp\u003eThe two novel strains showed differences with other representatives in the family \u003cem\u003eProlixibacteraceae.\u003c/em\u003e Both strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could hydrolyze starch and DNA, while the closest strain \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] cannot hydrolyze starch or DNA, \u003cem\u003eProlixibacter bellariivorans\u003c/em\u003e JCM 13498\u003csup\u003eT\u003c/sup\u003e [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e] cannot hydrolyze DNA, and \u003cem\u003eSunxiuqinia elliptica\u003c/em\u003e CGMCC 1.9156\u003csup\u003eT\u003c/sup\u003e [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] cannot hydrolyze starch (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Only strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e is strictly anaerobic, while most others are facultative anaerobic, and only strain \u003cem\u003eSunxiuqinia elliptica\u003c/em\u003e CGMCC 1.9156\u003csup\u003eT\u003c/sup\u003e is strictly aerobic [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Interestingly, even though DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an02312\u003csup\u003eT\u003c/sup\u003e belong to one genus, only strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could utilize xylose (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The detailed characteristics of the two novel isolates and representatives in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChemotaxonomic characterization\u003c/h2\u003e \u003cp\u003eThe fatty acid profiles of the two novel isolates and their reference strains are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The two novel isolates were distinguished from each other and from their closest valid species by the different major fatty acid components and their proportions. The major fatty acid components (\u0026gt;\u0026thinsp;10% of the total fatty acids) of strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e were \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e (28.08%), \u003cem\u003eanteiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e (21.27%), and \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e 3OH (13.58%). In addition, the predominant fatty acid components (\u0026gt;\u0026thinsp;10% of the total fatty acids) of strain DS1-an2312\u003csup\u003eT\u003c/sup\u003e were \u003cem\u003eanteiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e (20.08%), \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e (16.51%), \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e17:0\u003c/sub\u003e 3OH (12.83%), and \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e 3OH (10.4%). The primary components (\u0026gt;\u0026thinsp;10% of the total fatty acids) of their three reference strains included \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e, \u003cem\u003eanteiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e, and \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e17:0\u003c/sub\u003e 3OH. The primary isoprenoid quinone of both novel strains was menaquinone-7 (MK-7), which is common to other species in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e. The polar lipids of strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e included phosphatidylethanolamine (PE), phosphatidylserine (PS), two amino-lipids (AL1-2), an aminophospholipid (APL), a phospholipid (PL), and two unidentified lipids (L1-2) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. a\u003c/b\u003e). The polar lipid components of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e consisted of phosphatidylethanolamine (PE), three aminophospholipids (APL1-3), and two unidentified lipids (L1-2) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. b\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the cellular fatty acid compositions (%) of two novel strains, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, and their closest relatives in the family \u003cem\u003eProlixibacteraceae.\u003c/em\u003e Strains: 1, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e; 2, DS1-an-2312\u003csup\u003eT\u003c/sup\u003e; 3, \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e; 4, \u003cem\u003eProlixibacter bellariivorans\u003c/em\u003e JCM 13498\u003csup\u003eT\u003c/sup\u003e; 5, \u003cem\u003eSunxiuqinia elliptica\u003c/em\u003e CGMCC 1.9156\u003csup\u003eT\u003c/sup\u003e. The percentages of the fatty acids are presented as numbers. -, not detected. Values\u0026thinsp;\u0026lt;\u0026thinsp;1% are not shown. Values\u0026thinsp;\u0026gt;\u0026thinsp;10% are in bold. Summed features 1: C\u003csub\u003e15:1\u003c/sub\u003e iso H/C\u003csub\u003e13:0\u003c/sub\u003e 3OH; Summed features 2: C\u003csub\u003e14:0\u003c/sub\u003e 3OH/C\u003csub\u003e16:1\u003c/sub\u003e iso I; Summed features 3: C\u003csub\u003e16:1\u003c/sub\u003e ω7c/C\u003csub\u003e16:1\u003c/sub\u003e ω6c; Summed features 4: C\u003csub\u003e17:1\u003c/sub\u003e iso I/anteiso B; Summed features 8: C\u003csub\u003e18:1\u003c/sub\u003e ω7c; Summed features 9: C\u003csub\u003e16:0\u003c/sub\u003e 10-methyl.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatty acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStraight-chain saturated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e14:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e16:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e17:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e18:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStraight-chain unsaturated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e15:1\u003c/sub\u003e ω6c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e16:1\u003c/sub\u003e ω5c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e17:1\u003c/sub\u003e ω6c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e18:1\u003c/sub\u003e ω9c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBranched\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e12:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eiso-C\u003c/b\u003e\u003csub\u003e\u003cb\u003e13:0\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e14:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e15:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e16:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e16:1\u003c/sub\u003e H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e16:0\u003c/sub\u003e H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e17:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanteiso-C\u003csub\u003e13:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanteiso-C\u003csub\u003e15:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanteiso-C\u003csub\u003e17:0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHydroxylated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e14:0\u003c/sub\u003e 2-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e15:0\u003c/sub\u003e 2-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003e15:0\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e3-OH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e16:0\u003c/sub\u003e 3-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e17:0\u003c/sub\u003e 2-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e17:0\u003c/sub\u003e 3-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003e18:0\u003c/sub\u003e 3-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e14:0\u003c/sub\u003e 3-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eiso-C\u003c/b\u003e\u003csub\u003e\u003cb\u003e15:0\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e3-OH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e16:0\u003c/sub\u003e 3-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eiso-C\u003csub\u003e17:0\u003c/sub\u003e 3-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSummed features\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAnaerobic metabolism\u003c/h2\u003e \u003cp\u003eThe type of anaerobic metabolism of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e was assessed. Under fermentation conditions, both strains were able to utilize galactose, glucose, lactose, maltose, sucrose, and starch. Strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e was also able to utilize xylose, whereas DS1-an-13321\u003csup\u003eT\u003c/sup\u003e was not able to utilize xylose. The presence of sulfate did not change the carbon utilization pattern of either strain, as also observed under fermentation conditions, and a decrease of sulfate was not observed (data not shown). Additionally, strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e could weakly grow on pyruvate, but grew actively on glucose. Strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could grow on lactate or pyruvate. The growth of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on glucose was also observed in the presence of nitrate and thiosulfate, but no reduction of nitrate or thiosulfate was observed. Interestingly, only strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could grow in the presence of sulfite. However, according to the API 20NE test, neither DS1-an-13321\u003csup\u003eT\u003c/sup\u003e nor DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could perform nitrate/nitrite reduction under anaerobic conditions. Moreover, in the presence of sulfite, the growth of DS1-an-13321\u003csup\u003eT\u003c/sup\u003e was suppressed, and in the presence of nitrite, the growth of both strains was suppressed. These results indicated that strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e have fermentation metabolism even in the presence of a high concentration of sulfate (27 mM). This high concentration of sulfate in the basal medium mimicked the sulfate concentration in natural sea water [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe fermentation products of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e from the above experiments were analyzed through HPLC [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. By comparing the retention times of standard SCFAs under the same analysis conditions, the major fermentation products of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on galactose, glucose, maltose, lactose, sucrose, and starch and those of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on xylose were acetic acid and propionic acid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePolysaccharide utilization\u003c/h2\u003e \u003cp\u003eFor polysaccharide utilization, strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e were tested for growth on alginate, cellulose, chitin, κ-, λ-, and ι-carrageenan, fucoidan, laminarin, starch, and xylan in basal media. Their growth was observed under a microscope. Strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e was found to utilize laminarin and starch as the sole carbon source, while strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could utilize xylan in addition to laminarin and starch (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, a DNS assay could not detect any reducing sugars from the culture broth. This observation might be related to the \u0026ldquo;selfish\u0026rdquo; lifestyle of polysaccharide degraders in the phylum \u003cem\u003eBacteroidota\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on organic substrates under anaerobic conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDS1-an-13321\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDS1-an-2312\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMonosaccharide\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFructose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGalactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXylose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDisaccharide\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaltose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePolysaccharide\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlginate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellulose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eκ-Carrageenan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eλ-Carrageenan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eι-Carrageenan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFucoidan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaminarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXylan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSeveral studies have reported that members of the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e can utilize polysaccharides. For instance, \u003cem\u003eMangrovibacterium lignilyticum\u003c/em\u003e is enriched in media containing lignin [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], \u003cem\u003eTangfeifania diversioriginum\u003c/em\u003e is capable of hydrolyzing starch, and \u003cem\u003eSunxiuqinia indica\u003c/em\u003e [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] and \u003cem\u003eGaoshiqia sediminis\u003c/em\u003e [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e] contain 176 genes and 297 genes related to CAZymes, respectively. However, there has not yet been a study based on a combination of \u003cem\u003ein silico\u003c/em\u003e analysis and \u003cem\u003ein vitro\u003c/em\u003e experiments to assess polysaccharide utilization among members of the family \u003cem\u003eProlixibacteraceae.\u003c/em\u003e Interestingly, both novel isolates could utilize laminarin, which was not previously reported for members of the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e, and strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could degrade xylan. Therefore, in this study, we further investigated laminarin and xylan utilization by the isolates through \u003cem\u003ein vitro\u003c/em\u003e examination and \u003cem\u003ein silico\u003c/em\u003e genomic mining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eGrowth of DS1-an-13321\u003c/b\u003e\u003csup\u003e\u003cb\u003eT\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eand DS1-an-2312\u003c/b\u003e\u003csup\u003e\u003cb\u003eT\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eon laminarin and xylan\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eBased on the cell counting method, with counts taken every six hours for growth on laminarin and every 24 hours for growth on xylan, the growth curves of DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on laminarin and xylan are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The growth of strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e on laminarin reached the late log phase on day 3, while the late log phase of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e occurred on day 4. The growth of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on xylan reached the late log phase on day 3. Based on these results, cultures at their late log phase were harvested for further enzyme activity studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLaminarin degradation was detected in enzyme reaction of cell-associated proteins of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e with laminarin, but it was not found in the supernatant of their broth cultures. This indicated that laminarin-degrading enzymes were cell-associated proteins. Likewise, the xylan-degrading enzymes of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e were also associated with cells.\u003c/p\u003e \u003cp\u003eTo trace the mode of action of the laminarin- and xylan-degrading enzymes of DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, cell-associated enzymes were harvested from each strain and the degradation products were determined by a TLC analysis. Based on the RF values of the enzyme reactant and standard compounds, the major hydrolytic product on the laminarin or laminarin oligosaccharides of both strains was identified as glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cb\u003eand Figs. S3a, b\u003c/b\u003e). These results indicated that the laminarin-degrading enzymes of both strains exhibited exo-hydrolytic activities. For the xylan-degrading enzyme, the hydrolytic products by the crude enzyme of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e on xylan were xylosetriose and xylotetraose (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating that the cell-associated xylan-degrading enzyme of DS1-an-2312\u003csup\u003eT\u003c/sup\u003e exhibited endo-hydrolytic enzyme activities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eGenome analysis\u003c/h2\u003e \u003cp\u003eThe whole genomes of DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, determined by a combination of Nanopore and Illumina platforms, were obtained with high completeness (BUSCO values: 94.3% and 93.5%, respectively). Both the DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e genomes comprised a single circular chromosome with sizes of 4,465,088 bp and 5,187,288 bp, respectively, and had G\u0026thinsp;+\u0026thinsp;C content 35.9% and 36.5%, respectively (\u003cb\u003eTable S2)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe whole-genome sequence of strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e contained 3,545 predicted genes including 3,341 coding genes and eight pseudogenes. Among these, there were 158 tRNAs, five noncoding RNAs, and 41 rRNA genes (15 5S rRNAs, 13 16S rRNAs, and 13 23S rRNAs) (\u003cb\u003eTable S2\u003c/b\u003e). On the other hand, genome analysis of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e revealed that the strain harbored 3,807 predicted genes, consisting of 3,634 coding genes and six pseudogenes. Among these, there were 128 tRNAs, five noncoding RNAs, and 40 rRNA genes (14 5S rRNAs, 13 16S rRNAs, and 13 23S rRNAs) (\u003cb\u003eTable S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAntiSMASH and gutSMASH revealed that the genome of strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e encodes one BGC belonging to the nonribosomal peptide synthetase family and nine MGCs, while the genome of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e encodes one BGC belonging to the linear azoline-containing peptides family and nine MGCs. In strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e, the three COGs with the greatest number of genes were related to cell wall/membrane/envelope biogenesis, the mobilome (prophases, transposons), translation, ribosomal structure, and biogenesis. Moreover, in strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, the three COGs with the greatest number of genes were related to cell wall/membrane/envelope biogenesis, inorganic ion transport and metabolism, and carbohydrate transport and metabolism (\u003cb\u003eFig. S4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGenome mining revealed that the genomes of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e contain a high number of genes encoding carbohydrate-active enzymes (CAZymes). The CAZy database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cazy.org/\u003c/span\u003e\u003cspan address=\"http://www.cazy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which contains information about carbohydrate-active enzymes, including glycoside hydrolase (GH, cleavage of glycosidic bonds), glycosyl transferase (GT, construction of glycosidic bonds), polysaccharide lyase (PL, nonhydrolytic hydrolysis of glycosidic bonds), carbohydrate esterase (CE, cleavage of carbohydrate esters), auxiliary activity (AA, a redox enzyme that works in conjunction with other CAZymes), and carbohydrate-binding modules (CBM, adhesion to carbohydrates), was used to assess the detailed composition of CAZymes in the genomes of the two isolates, and the results are presented in \u003cb\u003eTable S3\u003c/b\u003e. Strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e encoded a total of 155 CAZymes consisting of 84 GHs, 32 GTs, 13PLs, seven CEs and 19 CBMs, while strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e encoded a total of 249 CAZymes, an amount 1.6 times greater than that of DS1-an-13321\u003csup\u003eT\u003c/sup\u003e, consisting of 128 GHs, 37 GTs, 27 PLs, 35 CEs, and 22 CBMs. The number of GH genes per genome in strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e was 18.81 (GHs/Mb), while in strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e it was 24.46 (GHs/Mb); both of these values are significantly greater than the average value of 12 GHs/Mb in the genomes of other members of marine bacteria of the class \u003cem\u003eBacteroidia\u003c/em\u003e [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. The genomes of DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e contained 27 and 34 PULs, respectively, which are approximately one-third lower than the average number of PULs in human gut \u003cem\u003eBacteroides\u003c/em\u003e and similar to the number of PULs found in the genus \u003cem\u003ePrevotella\u003c/em\u003e (average 23 PULs/genome) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genomes of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e contained genes predicted to be involved in laminarin degradation. The genome of strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e harbored genes encoding for four GH3, one GH16, and one GH30, while strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e harbored genes encoding for seven GH3, two GH16, and one GH30 (\u003cb\u003eTable S4)\u003c/b\u003e, which has been reported to contribute to the degradation of laminarin in other marine bacteria [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Unlike other laminarin degraders of \u003cem\u003eGramella\u003c/em\u003e spp. [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e], \u003cem\u003eFormosa\u003c/em\u003e spp. [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e] (class \u003cem\u003eFlavobacteriia\u003c/em\u003e) or \u003cem\u003eBacteroides\u003c/em\u003e spp. [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e] (class \u003cem\u003eBacteroidia\u003c/em\u003e), neither strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e nor DS1-an-2312\u003csup\u003eT\u003c/sup\u003e harbored genes encoding GH3 and GH16, which in general collocate with each other and collocate with SusC/SusD (a signature for the PUL structure). Instead, GH3 and GH16 of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e were located separately, and each gene was collocated with SusC/SusD in the genomes. For strain DS1-an13321\u003csup\u003eT\u003c/sup\u003e, we found that PUL8 contained a tandem of SusD/TBDR, an unknown protein, and two copies of GH3. Additionally, these two GH3s were predicted to be located in the periplasmic space (PSORTb scores of 9.44 and 9.76). Furthermore, strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e harbored an unidentified PUL (21_un_PUL), and the gene cluster contained SusD (K4L44_09375), SusC (K4L44_09380), TonB-dependent receptor (TBDR) (K4L44_09385), two unknown proteins (K4L44_09390, K4L44_09405), superoxide dismutase, Ni (K4L44_09395), GH3 (K4L44_09400), IS4 family transposase (K4L44_09410), and GH30 (K4L44_09415) (\u003cb\u003eFig. S5a\u003c/b\u003e). Within this gene cluster, we detected GH3 (cleavage β-1,3-glucan) and GH30 (cleavage β-1,6-glucan), which PSORTb could predict at multiple locations on the cell. In contrast, strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e consisted of three PULs (PUL9, PUL29, and an unidentified PUL (12_un_PUL)), which contained GH3 without the presence of GH16 and GH30. We also found that the GH10 and GH5 genes in PUL9 and PUL29, respectively, were predicted to degrade the xylan main chain, indicating that these two PULs may play a role in xylan degradation rather than laminarin degradation. Additionally, in the 12_un_PUL (\u003cb\u003eFig. S5b\u003c/b\u003e), we detected several genes of SusC/SusD and GH3. The GH3 was predicted to locate in the periplasmic space with PSORTb score 9.44. To identify the active gene cluster responsible for laminarin degradation, further transcriptomic analysis is required.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, the novel strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e was identified as an anaerobic bacterium capable of utilizing xylan as a sole carbon source. Whole-genome analysis of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e revealed the presence of CAZymes involved in the effective degradation of the natural polymer xylan (\u003cb\u003eTable S4\u003c/b\u003e, and \u003cb\u003eS6\u003c/b\u003e). We found that strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e harbored genes encoding two GH5, one GH10, one GH30, and three GH141 enzymes (\u003cb\u003eTable S4)\u003c/b\u003e. Notably, using PULDB, all four potential xylan utilization loci were identified, PUL8, PUL9, PUL25, and PUL29 (\u003cb\u003eFig. S5c\u003c/b\u003e). A detailed analysis of these PUL indicated that PUL9 and PUL29 would have greater potential for xylan degradation in strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e (\u003cb\u003eTable S5\u003c/b\u003e). Specifically, PUL9 contained SusC and SusD, which are responsible for capturing polysaccharides and delivering oligosaccharides into the cytoplasm [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It also contained a GH10 enzyme, which exhibited the highest amino acid similarity of 26% (covering 80% of the sequence) to endo-1,4-β-xylanase (UniProt accession code G4MLU0) and 24.4% (covering 81% of the sequence) to a reported GH10 module glycoside hydrolase of \u003cem\u003eCaldicellulosiruptor danielii\u003c/em\u003e (PDB accession code 6D5C_A). Additionally, the PUL9 of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e contained a GH3 enzyme, which exhibited the highest similarity (43.5%, covering 83% of the sequence) to β-xylosidase of \u003cem\u003eFormosa agariphila\u003c/em\u003e (UniProt accession code T2KMH0). Similarly, PUL29 contained the SusC, SusD, and a multidomain protein consisting of one GH5 subfamily 46 domain and two CBM6 modules. The multidomain protein exhibited 32.8% similarity to endoglucanase C of \u003cem\u003eAcetivibrio thermocellus\u003c/em\u003e (UniProt accession code A3DJ77). Moreover, these two enzymes, GH5 and GH10, were not found in the genome of strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e, which cannot utilize xylose and xylan as a sole carbon source under anaerobic conditions (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, through genome analysis, physiological characterization, and TLC analysis, it was inferred that strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e exhibited strong xylan degradation capabilities (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cb\u003eFigs. S5\u003c/b\u003e and \u003cb\u003e6\u003c/b\u003e). In the xylan degradation process, endo-1,4-β-xylanase and β-xylosidase enzymes degrade xylan to xylooligosaccharides (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The genome of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e encoded both endo-1,4-β-xylanase (GH10) and β-xylosidase (GH3) enzymes (\u003cb\u003eTable S5\u003c/b\u003e). Additionally, arabinofuranosidase GH30, a multisubstrate-specific family enzyme, acts as an endo-1,4-β-xylanase and degrades xylooligosaccharides. Subsequently, xylooligosaccharides are transported into the cell membrane. Bacterial strains typically use active transport mechanisms, with some routes utilizing high and low affinity transporters. Only the genome of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, not DS1-an-13321\u003csup\u003eT\u003c/sup\u003e, encoded the xylose transporter (XylE), which is a low-affinity transporter associated with xylooligosaccharide transportation via a proton motive force [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e], and xylose isomerase (XylA), which facilitates the reversible conversion of D-xylose into D-xylulose [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe whole-genome sequence of DS1-an-2312\u003csup\u003eT\u003c/sup\u003e was analyzed and the xylose metabolic pathway of the strains was modelled (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). It was hypothesized that the metabolic pathway of the novel species involves xylose isomerase, as indicated by the presence of genes such as xylose isomerase (K5X82_00205) in its genome. In the isomerase pathway, the xylose transporter XylE (K5X82_00210) is responsible for the uptake of xylooligosaccharides. Xylooligosaccharides are degraded into D-xylose at the periplasm under the function of GH3 (K5X82_03105) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This model hypothesis was supported by a TLC experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e), where no detectable D-xylose was detected in the enzyme-reaction supernatant after removing the whole-cell-associated proteins of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e. The xylose isomerase xylA (K5X82_00205) enzyme converts D-xylose to D-xylulose, which is phosphorylated to D-xylulose-5-phosphate by the xylulokinase enzyme [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. The phosphoketolase enzyme further degrades D-xylulose-5-phosphate (a 5-carbon compound) into acetyl phosphate (a 2-carbon compound) and glyceraldehyde-3-phosphate (a 3-carbon compound). Some anaerobic bacteria, such as \u003cem\u003eClostridium\u003c/em\u003e sp, and lactic acid bacteria, can cleave xylulose-5-P by phosphoketolase into these compounds [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan additionalcitationids=\"CR91 CR92\" citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaken together, the results of this study not only support two novel strains that represent a novel genus with two novel species in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e, class \u003cem\u003eBacteroidia\u003c/em\u003e, phylum \u003cem\u003eBacteroidota\u003c/em\u003e but also expand our understanding of the strategies employed by marine \u003cem\u003eBacteroidia\u003c/em\u003e bacteria to access and degrade polysaccharides anaerobically. By mimicking natural nutrient conditions for isolation, pure cultures of the type strains of the two novel species were obtained. Both strains were capable of fermenting glucose, galactose, maltose, lactose, sucrose, and starch, with only DS1-an-2312\u003csup\u003eT\u003c/sup\u003e exhibiting the ability to utilize xylose. The major fermentation products of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e were acetic acid and propionic acid. Genome mining revealed that both novel species contained rich sources of CAZymes. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrated that both novel species could degrade laminarin and starch, with only DS1-an-2312\u003csup\u003eT\u003c/sup\u003e capable of utilizing xylan under anaerobic conditions. Both strains possessed cell-associated laminarin-degrading enzymes, exhibiting exo-hydrolytic enzyme activity and producing glucose as the major final product. In addition, strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e possessed a cell-associated xylan-degrading enzyme with endo-hydrolytic enzyme activity, producing xylotriose and xylotetraose as the major final products. These results highlight the potential biotechnological applications of the two novel species and their strategies for adaptation under anoxic conditions in marine ecosystems through fermentation and polysaccharide degradation. For further study of the molecular mechanism of laminarin and xylan degradation in both novel species, future work will involve transcriptomic and proteomic analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription of\u003c/b\u003e \u003cb\u003eHalocynthiibacter\u003c/b\u003e \u003cb\u003egen. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eHalocynthiibacter\u003c/em\u003e gen. nov. (Ha.lo.cyn.thi.i.bac'ter. N.L. fem. n. \u003cem\u003eHalocynthia\u003c/em\u003e, an animal genus, N.L. masc. n. \u003cem\u003ebacter\u003c/em\u003e, a rod; N.L. masc. n. \u003cem\u003eHalocynthiibacter\u003c/em\u003e, a rod from \u003cem\u003eHalocynthia\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eCells are Gram-stain-negative, anaerobic, rod-shaped, and oxidase- and catalase-negative. Prominent fatty acid components are \u003cem\u003eiso-\u003c/em\u003eC\u003csub\u003e15:0\u003c/sub\u003e, \u003cem\u003eanteiso-\u003c/em\u003eC\u003csub\u003e15:0\u003c/sub\u003e, \u003cem\u003eiso-\u003c/em\u003eC\u003csub\u003e15:0\u003c/sub\u003e 3-OH, and \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e17:0\u003c/sub\u003e 3-OH. The major respiratory quinone type is menaquinone-7 (MK-7). Glucose, galactose, maltose, lactose, sucrose, and starch are fermented to produce a mixture of acid as major products. The genus \u003cem\u003eHalocynthiibacter\u003c/em\u003e belongs to the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e, phylum \u003cem\u003eBacteroidota.\u003c/em\u003e The type species is \u003cem\u003eHalocynthiibacter xylanolyticus.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription of\u003c/b\u003e \u003cb\u003eHalocynthiibacter laminarini\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eHalocynthiibacter laminarini\u003c/em\u003e sp. nov. (la.mi.na.ri'ni. N.L. gen. n. \u003cem\u003elaminarini\u003c/em\u003e, of laminarin, referring to its ability to hydrolyze laminarin).\u003c/p\u003e \u003cp\u003eCells are Gram-strain-negative, mesophilic, neutrophilic, strictly anaerobic, long rod-shaped at the log phase, and spherical at the end of the stationary phase of growth. They are oxidase- and catalase-negative. Round and bright brown colonies appeared on the surface of MB agar plates. Growth occurs at 15\u0026ndash;30 ℃ (optimum, 20\u0026ndash;30 ℃), at pH 6.0-8.5 (optimum, 6.5\u0026ndash;8.5), and with 2\u0026ndash;4% NaCl (optimum, 2\u0026ndash;3%). H\u003csub\u003e2\u003c/sub\u003eS is produced. Positive for hydrolysis of gelatin, DNA, laminarin, and starch. Galactose, glucose, maltose, lactose, sucrose, and starch are fermented to produce acetic acid and propionic acid as the major products. The major fatty acid components are \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e, \u003cem\u003eanteiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e, \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e 3-OH, and \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e17:0\u003c/sub\u003e 3-OH. Menaquinone 7 (MK-7) is the major quinone. The polar lipid profile comprises phosphatidylethanolamine (PE), two unidentified amino-lipids (AL1-2), one unidentified aminophospholipid (APL), one unidentified phospholipid (PL), one identified lipid (L), and one phosphatidylserine (PS).\u003c/p\u003e \u003cp\u003eThe type strain DS1-an-13321\u003csup\u003eT\u003c/sup\u003e (=\u0026thinsp;KCTC 25031\u003csup\u003eT\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;DSM 115329\u003csup\u003eT\u003c/sup\u003e) was isolated from a sea squirt at a depth of 18 m under the surface of seawater. The genome contains one circular chromosome that is 4.47 Mb long. The G\u0026thinsp;+\u0026thinsp;C content is 35.9%, as calculated from whole-genome sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescription of\u003c/b\u003e \u003cb\u003eHalocynthiibacter xylanolyticus\u003c/b\u003e \u003cb\u003esp. nov.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eHalocynthiibacter xylanolyticus\u003c/em\u003e sp. nov. (xy.la.ni.ly'ti.cus. N.L. neut. n. \u003cem\u003exylanum\u003c/em\u003e, xylan; Gr. masc. adj. \u003cem\u003elytikos\u003c/em\u003e, dissolving; N.L. masc. adj. \u003cem\u003exylanilyticus\u003c/em\u003e, xylan-dissolving).\u003c/p\u003e \u003cp\u003eCells are gram-stain-negative, mesophilic, neutrophilic, long rod-shaped at the log phase and spherical at the end of the stationary phase of growth. They are oxidase- and catalase- negative. Irregularly shaped and bright brown colonies appeared on the surface of MB agar plates. Growth occurs at 10\u0026ndash;32 ℃ (optimum, 20\u0026ndash;30 ℃), at pH 6.0\u0026ndash;8.0 (optimum, 7.0-7.5), and with 1\u0026ndash;4% NaCl (optimum, 2\u0026ndash;3%). H\u003csub\u003e2\u003c/sub\u003eS is produced. Positive for hydrolysis of gelatin, DNA, laminarin, starch, and xylan. Galactose, glucose, xylose, maltose, lactose, sucrose, and starch are fermented to produce acetic acid and propionic acid as the major products. The major fatty acid components are \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e, \u003cem\u003eanteiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e, \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e15:0\u003c/sub\u003e 3-OH, and \u003cem\u003eiso\u003c/em\u003e-C\u003csub\u003e17:0\u003c/sub\u003e 3-OH. Menaquinone 7 (MK-7) is the major quinone. The polar lipid profile comprises phosphatidylethanolamine (PE) and three unidentified aminophospholipids (APL1-3).\u003c/p\u003e \u003cp\u003eThe type strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e (=\u0026thinsp;KCTC 25032\u003csup\u003eT\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;DSM 115328\u003csup\u003eT\u003c/sup\u003e) was isolated from a sea squirt at a depth 18 m under the surface of sea water. The genome contains one circular chromosome that is 5.19 Mb long. The G\u0026thinsp;+\u0026thinsp;C content is 36.52%, as calculated from whole-genome sequencing.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT.T.H.N. performed experiments of isolation, identification, and characterization of the bacterial strains including anaerobic metabolism, polysaccharide degradation, and genome analysis. T.T.H.N also wrote the manuscript. T.Q.V. analyzed genomes, constructed a UBGC genome tree, determined POCP indices, and drew the xylan degradation pathway. H.L.H carried out the genome analysis for xylose utilization and proposed the schematic model of xylose utilization, and wrote the paragraphs regarding xylose utilization. S-G.K. supervised the experiments and finalized the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors thank Prof. Dr. Bernhard Schink from the University of Konstanz (Germany) and Prof. Dr. Aharon Oren from Edmond J. Safra Campus, The Hebrew University of Jerusalem (Israel), for their help with the nomenclature of the new genus and two novel species names. The authors also thank Mrs. Mi-Kyung Eom and Dr. Li Zhun at KCTC for maintaining the two novel isolates and for providing the SEM images, respectively. This research was supported by The Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5232423) and a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2021M3H9A1030164).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe DNA sequences generated from the study are available at the National Center for Biotechnology Information (NCBI). GenBank/EMBL/DDBJ accession numbers of 16S rRNA gene sequences of the strains DS1-an-13321T and DS1-an-2312T are MZ851973 and MZ851974, respectively, and the genome sequences are CP081303 and CP082230, respectively.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArnosti, C., \u003cem\u003eet al.\u003c/em\u003e The biogeochemistry of marine polysaccharides: Sources, inventories, and bacterial drivers of the carbohydrate cycle. Ann. Rev. Mar. Sci. 13, 81\u0026ndash;108 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrause-Jensen, D. and Duarte, C. M. Substantial role of macroalgae in marine carbon sequestration. Nat. Geosci. 9, 737\u0026ndash;742 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB\u0026auml;umgen, M., \u003cem\u003eet al.\u003c/em\u003e A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan. J. Biol. 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Biotechnol. 60, 160\u0026ndash;167 (2002).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Polyphasic taxonomy, KEGG, CAZyme, polysaccharide degradation, Prolixibacteraceae, PUL","lastPublishedDoi":"10.21203/rs.3.rs-4632626/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4632626/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe bacterial group of the phylum \u003cem\u003eBacteroidota\u003c/em\u003e greatly contributes to the global carbon cycle in marine ecosystems through its specialized ability to degrade marine polysaccharides. In this study, it is proposed that two novel facultative anaerobic strains, DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e, which were isolated from a sea squirt, represent a novel genus, \u003cem\u003eHalocynthiibacter\u003c/em\u003e, with two novel species in the family \u003cem\u003eProlixibacteraceae.\u003c/em\u003e The 16S rRNA sequence similarities of these two strains were 91.26% and 91.37%, respectively, against \u003cem\u003ePuteibacter caeruleilacunae\u003c/em\u003e JC036\u003csup\u003eT\u003c/sup\u003e, which is the closest recognized neighbor. The complete genomes of strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e each consisted of a single circular chromosome with a size of 4.47 and 5.19 Mb, respectively. The average amino acid identity and the percentage of conserved proteins against the type species of the genera in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e ranged from 48.33\u0026ndash;52.35% and 28.34\u0026ndash;37.37%, respectively, which are lower than the threshold for genus demarcation. Strains DS1-an-13321\u003csup\u003eT\u003c/sup\u003e and DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could grow on galactose, glucose, maltose, lactose, sucrose, laminarin, and starch, and only DS1-an-2312\u003csup\u003eT\u003c/sup\u003e could grow on xylose and xylan under fermentation conditions. These strains produced acetic acid and propionic acid as the major fermentation products. Genome mining of the genomes of the two strains revealed 27 and 34 polysaccharide utilization loci, which included 155 and 249 carbohydrate-active enzymes (CAZymes), covering 57 and 65 CAZymes families, respectively. The laminarin-degrading enzymes in both strains were cell-associated, and showed exo-hydrolytic activity releasing glucose as a major product. The xylan-degrading enzymes of strain DS1-an-2312\u003csup\u003eT\u003c/sup\u003e was also cell-associated, and had endo-hydrolytic activities, releasing xylotriose and xylotetraose as major products. The evidence from phenotypic, biochemical, chemotaxonomic, and genomic characteristics supported the proposal of a novel genus with two novel species in the family \u003cem\u003eProlixibacteraceae\u003c/em\u003e, for which the names \u003cem\u003eHalocynthiibacter laminarini\u003c/em\u003e gen. nov., sp. nov. and \u003cem\u003eHalocynthiibacter xylanolyticus\u003c/em\u003e sp. nov. are proposed. The type strain of \u003cem\u003eHalocynthiibacter laminarini\u003c/em\u003e is DS1-an-13321\u003csup\u003eT\u003c/sup\u003e (=\u0026thinsp;KCTC 25031\u003csup\u003eT\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;DSM 115329\u003csup\u003eT\u003c/sup\u003e) and the type strain of \u003cem\u003eHalocynthiibacter xylanolyticus\u003c/em\u003e is DS1-an-2312\u003csup\u003eT\u003c/sup\u003e (=\u0026thinsp;KCTC 25032\u003csup\u003eT\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;DSM 115328\u003csup\u003eT\u003c/sup\u003e).\u003c/p\u003e","manuscriptTitle":"Halocynthiibacter laminarini gen. nov., sp. nov. and Halocynthiibacter xylanolyticus sp. nov., marine anaerobic laminarin and xylan degraders in the phylum Bacteroidota","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-23 18:35:38","doi":"10.21203/rs.3.rs-4632626/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-19T05:31:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-18T15:50:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-17T13:51:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283679485141009292287135904949309065678","date":"2024-08-08T03:13:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"911828219595306761702911307961496374","date":"2024-08-07T15:43:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-03T03:28:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-03T02:24:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-02T12:59:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-01T04:22:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-25T00:40:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"efe2c526-4131-4cf4-8d26-549ab08e7785","owner":[],"postedDate":"July 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34877811,"name":"Biological sciences/Biotechnology"},{"id":34877812,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2024-10-21T16:06:48+00:00","versionOfRecord":{"articleIdentity":"rs-4632626","link":"https://doi.org/10.1038/s41598-024-74787-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-10-17 15:57:16","publishedOnDateReadable":"October 17th, 2024"},"versionCreatedAt":"2024-07-23 18:35:38","video":"","vorDoi":"10.1038/s41598-024-74787-6","vorDoiUrl":"https://doi.org/10.1038/s41598-024-74787-6","workflowStages":[]},"version":"v1","identity":"rs-4632626","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4632626","identity":"rs-4632626","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}