All samples were collected by a self-made cylindrical plexiglass tube sampler, transferred into sterile sample tubes after being fully homogenized, stored in a 4°C–6°C incubator, and transported to the laboratory as soon as possible. The distribution of samples is presented in Figure 1 . The particle size of the sediments was determined using a laser particle size analyzer, and the sediment types were classified according to the modification of Folk’s classification of sediments: sand (greater than 10% of particles with a diameter less than 63 µm), muddy sand (10%–90% of particles with a diameter less than 63 µm), and mud (less than 10% of particles with a diameter less than 63 µm) (Folk et al., 1970). Strain M17^T was isolated from an intertidal mudflat (0–5 cm) collected from Qingdao, Shandong Provence (36°10′ N, 120°07′ E); strains M82^T and M415^T were isolated from two intertidal mudflats (0–5 cm) collected from Taizhou, Zhejiang Provence (28°27′ N, 121°37′ E and 29°4′ N, 121°37′ E, respectively).

Samples were serially diluted to 10⁻³ with sterile seawater using the standard dilution-plating method (Williams and Davies, 1965). Generally, a 100-μL aliquot of each dilution was spread on modified marine agar (per liter of distilled water: Bacto yeast extract 0.1 g, Bacto peptone 0.5 g, ferric citrate 0.1 g, NaCl 19.45 g, MgCl₂·6H₂O 12.6 g, MgSO₄ 3.24 g, CaCl₂ 1.8 g, KCl 0.55 g, NaHCO₃ 0.16 g, KBr 0.08 g, SrCl₂ 34.0 mg, NaSiO₃ 4.0 mg, NH₄NO₃ 1.6 mg, H₃BO₄ 22.0 mg, NaF 2.4 mg, Na₂HPO₄ 8.0 mg, agar 20 g) and incubated at 30°C for 3–7 days to a simulated oligotrophic environment. Afterward, the strains were isolated from different plates and purified by repeating streaking. All bacterial cultures were stored in Marine Broth 2216 (MB) medium containing 25% glycerol at −80°C. Six type strains, namely, Mangrovivirga cuniculi KCTC 72349^T, Pontibacter actiniarum KCTC 12367^T, Pontibacter litorisediminis KCTC 52252^T, Poritiphilus flavus MCCC 1K03853^T, Eudoraea chungangensis KCTC 42048^T, and Zeaxanthinibacter enoshimensis NBRC 101990^T, were purchased from the Korean Collection for Type Cultures (KCTC), the Marine Culture Collection of China (MCCC), and the NITE Biological Resource Center (NBRC), and used as reference strains in this study.

Strains M17^T, M82^T, and M415^T were cultured on Marine Agar (MA) medium for 3 days to observe their morphological characteristics, including colonial size, shape, edge, bulge, transparency, and color characteristics. Cell morphology, size, and special structure were examined using a transmission electron microscope (JEM-1230; JEOL). Gram staining reaction was performed according to the method described by Dong and Cai (Dong and Cai, 2001). Motility of the strains was assessed in semisolid MB medium containing 0.5% agar. The growth range and optimum temperature of the strains were determined in MB medium at 4°C, 10°C, 20°C, 25°C, 28°C, 30°C, 37°C, 40°C, 45°C, and 50°C, whereas their growth and optimal pH range were measured by adding appropriate buffer (40 mM) to MB medium (0.5 pH unit intervals), which include MES (pH 5.0–5.5), MOPS (pH 6.0–7.5), Tricine buffers (pH 8.0–8.5), and CAPSO (pH 9.0–10.0). By adding 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 6%, 8%, 10%, and 12% (w/v) NaCl to saltness-MB medium, the growth and optimal salinity range were determined. The optimal growth conditions and growth range were determined after 3 days and 1 month of culture, respectively. The growth condition of the strains was measured using a UV/visible spectrophotometer (Ultrospec 6300 Pro, Amersham Biosciences) at OD₆₀₀.

Sodium nitrate (20 mM) was used as a potential electron acceptor to assess the anaerobic growth of strains in the anaerobic system (AnaeroPack-MicroAero, 2.5 L, MGC, Japan) (Shi et al., 2017). Catalase activity was measured by dripping 3% (v/v) hydrogen peroxide solution to the colonies placed on sterile slides. Oxidase activity was determined by observing whether the cell color turned red within 1 min after dripping 1% p-amino dimethylaniline oxalate solution. Carotenoid was extracted by acetone/methanol (7:2, v/v) solution, and their absorption spectra were determined using a scanning UV/visible spectrophotometer (Bowman and Nichols, 2005). Strains were cultured in suitable medium containing sodium thiosulfate (5 g/L), and their hydrogen sulfide production capacity was determined using sterile filter strips soaked in the solution of lead acetate. Amylase, cellulase, and hydrolysis of Tweens 20, 40, 60, and 80 were carried out according to the previously described methods (Liu et al., 2019). Carbon source oxidation was tested using Biolog GEN III MicroPlates, and activity of other common bacterial enzymes was examined using the API ZYM kit. Other physiological characteristics of the strains were analyzed using the API 20NE kit. All of the BIOLOG and API tests were carried out according to the manufacturer’s instructions except for adjusting salinity to 2%.

Strains M17^T, M82^T, and M415^T and the six type strains were cultured on MB medium under optimal conditions, and the cells at the end of exponential growth stage were collected for chemotaxonomic analysis. Bacteria were collected and freeze-dried, then saponified, methylated, extracted, and washed to obtain the bacterial fatty acids (Sasser, 1990). Identification and quantification of the extracted cellular fatty acids of these strains were performed using a gas chromatograph (Agilent G6890N) and the Sherlock Microbial Identification System (MIDI database: Version 6.0). Polar lipids were extracted according to the procedure described by Minnikin et al. (1984), and composition analysis was performed on silica gel 60 F254 plates (10 × 10 cm, Merck) (Komagata and Suzuki, 1988). Isoprenoid quinones were extracted by a mixture of chloroform:methanol (2:1 v/v), and the further identification was performed by the HPLC-MS system.

A total of 352 strains were isolated from 34 sampling stations. The 16S rRNA gene was amplified by PCR using the universal primers 27F/1492R and sequenced by Guangdong Magigene Biotechnology Co., Ltd. (Guangzhou, China). The sequences were submitted to NCBI under the accession numbers OQ617539–OQ617890. The complete 16S rRNA gene sequences of strains M17^T, M82^T, and M415^T were extracted from their draft genomes. The similarities of all 16S rRNA gene sequences were identified by aligning these sequences against the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) and the EzBioCloud database (https://www.ezbiocloud.net/).

The 16S rRNA gene sequence alignment of strains M17^T, M82^T, and M415^T and their phylogenetically related taxa were performed by the ClustalW algorithm within MEGA11 v11.0.13 (Tamura et al., 2021), and the phylogenetic trees were reconstructed by the neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) algorithms within the MEGA11 software (Felsenstein, 1981; Saitou and Nei, 1987). The robustness of phylogenetic trees was assessed through bootstrap analysis based on 1,000 replications.

The genomes of strains M17^T, M82^T, and M415^T were extracted using a bacterial genomic DNA kit (Takara), and draft genomes were sequenced using the Illumina NovaSeq 6000 platform (PE150) in Guangdong Magigene Biotechnology Co., Ltd. (Guangzhou, China). The sequences were assembled using SPAdes v3.10.1 (Zou et al., 2020), and the completeness and contamination of the assembled draft genomes were accessed using CheckM v1.1.3 (Parks et al., 2015).

Phylogenomic analysis based on single-copy orthologous clusters (OCs) of strains M17^T, M82^T, and M415^T and the related type strains were performed as described (Xu et al., 2018). Briefly, the orthologous clusters were filtered based on the blastp+ program and 50% sequence identity using Proteinortho v5.16 (Lechner et al., 2011), and their formats were converted into OrthoMCL for subsequent analysis. The single-copy orthologous clusters were aligned through MAFFT v7.310 (Katoh and Standley, 2013), and the aligned sequences were concatenated after further refining by trimAL v1.4.1 (Capella-Gutierrez et al., 2009). The IQ-TREE v1.6.2 software was used to predict the best-fit models. The phylogenetic trees were constructed by maximum-likelihood algorithms based on the concatenated aligned single-copy orthologous clusters (Lam-Tung et al., 2015), and the best-fit models of strains M17^T, M82^T, and M415^T were LG+F+R6, LG+F+R5, and LG+F+R8, respectively. Finally, MEGA11 software was employed to visualize the phylogenetic trees.

Functional and metabolic pathway predictions were realized by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Evolutionary Genealogy of Genes with enhanced Non-supervised Orthologous Groups (EggNOG) (Kanehisa et al., 2017; Hernández-Plaza et al., 2023). The Average Nucleotide Identity (ANI) was calculated using the online ANI calculator based on the OrthoANIu algorithm, which is an improved iteration of the original OrthoANI algorithm (Yoon et al., 2017). Digital DNA–DNA hybridization (dDDH) and Two-way Average Amino Acid Identity (AAI) were calculated through Genome-to-Genome Distance Calculator 3.0 and the AAI calculator, respectively (Rodriguez-R and Konstantinidis, 2014; Meier-Kolthoff et al., 2022).

As for the potential in polysaccharide metabolism of strains M17^T, M82^T, and M415^T, carbohydrate-active enzymes (CAZymes) were predicted by the HMMER tools within dbCAN2 software based on Carbohydrate-Active enZYmes Database V11 (CAZy database), and SusC/D-like proteins (SusC, outer membrane TonB-dependent transporter; SusD, surface glycan-binding protein) and other auxiliary proteins and genes were predicted and annotated using PROKKA v1.12 and Rapid Annotation using Subsystem Technology (RAST) version 2.0 (Aziz et al., 2008; Seemann, 2014; Zhang et al., 2018; Drula et al., 2022). The PUL prediction relies on the identification in each genome of the PUL markers: the presence of adjacent genes encoding SusC/D-like proteins, and according to the position of CAZymes and SusC/D-like proteins in the genome, combined with the position of corresponding auxiliary proteins and genes (Terrapon et al., 2015). In addition, the substrates of these PULs were predicted by searching through the PUL database (PULDB) and BLAST the CAZymes through UniProt (Terrapon et al., 2018; Consortium, 2019).

The sampling stations are widely distributed in the bare tidal flats of China. The samples from 34 sampling stations were used for the isolation of strains ( Figure 1 ). A total of 352 bacterial strains were isolated. Compared with the 16S rRNA gene sequences with validly published species, the isolates were assigned to 180 species, belonging to 4 phyla, 7 classes, 22 orders, 37 families, and 94 genera; among them, 126 species and 52 genera had only one isolate ( Table 1 and Figure 2 ). In our study, a higher number of species were discovered among the culturable strains compared with mangrove sediments (116 pure culture strains distributed in 13 species, 1 sample; Sefrji et al., 2022), plant rhizosphere (59 pure culture strains distributed in 22 species, 4 samples; Brígido et al., 2019), and Tabernas Desert (236 strains distributed in 37 genera, 3 samples; Molina-Menor et al., 2021); our results showed that bare tidal flats had a greater diversity of culturable bacteria. Flavobacteriales and Bacillales represented two most abundant orders, accounting for approx. 50% of the total isolates ( Figure 3 ). A total of 17 strains were identified as Fictibacillus phosphorivorans, which was widely distributed in 12 sampling stations located in the different areas such as Jiangsu Province, Zhejiang Province, Guangxi Zhuang Autonomous Region, and Shanghai City. It became the most abundant and widely distributed culturable species in this study. Recent studies showed that Fictibacillus phosphorivorans was able to produce biosurfactant and displayed high nematicidal capability against root-knot nematodes (RKNs), which could infect almost all crops and lead to huge economic losses in agriculture around the world (Zheng et al., 2016; Pandey et al., 2021).

In addition, the effects of different depths and substrates on culturable bacteria were compared ( Table 1 ). Actinomycetota strains, a total of 13 species in 5 orders, were isolated only in 0–5-cm non-sandy sediment, some of which were reported to have great ecological functions and economic values; e.g., strains of Rhodococcus qingshengii and Brachybacterium paraconglomeratum have the ability to repair heavy metal pollution (Du et al., 2022; Harboul et al., 2022) and pesticide contamination (Chuang et al., 2021; Wang et al., 2021), strains of Arthrobacter pascens, known as indole-3-acetic acid (IAA)-producing bacteria, could regulate plant growth and development (Li et al., 2021), and strains of Cellulosimicrobium cellulans are able to produce ginsenoside Rg3, a known anticancer agent (Hu et al., 2019). Different from Actinomycetota species, Pseudomonadota species Psychrobacter nivimaris was the only species distributed in all three types of sediment ( Supplementary Figure 1 ), and strains in it may have the ecological function of repairing heavy metal pollution (Staloch et al., 2022).

In addition, a large number of novel species were discovered in bare tidal flats; a total of 47 strains showed less than 98.65% sequence similarities of the 16S rRNA gene with validly published species and may represent novel species (Kim et al., 2014). Most of them were assigned to Cytophagales and Flavobacteriales, both of which belong to the phylum Bacteroidota, with ratios of 46.2% and 30.2%, respectively. Three Bacteroidota strains (M17^T, M82^T, and M415^T) were chosen for further phylogenetic and functional characterization.

The morphological observations by transmission electron microscopy showed that the cells of strains M17^T, M82^T, and M415^T were slender and long (2.0–10.0 µm × 0.3–0.5 µm), ellipsoidal to ovoid (0.9–3.2 µm × 0.6–1.1 µm), and slender and long (1.8–8.0 µm × 0.3–0.5 µm), respectively ( Supplementary Figure 2 - Figure 4 ). For strain M17^T, after 3 days of cultivation, the colony was round, 1–3 mm in diameter, and orange in color; mobility was not observed in the semisolid MB medium; and anaerobic growth and carotenoid production were not detected. The colony of M82^T was round, 1–2 mm in diameter, and red in color after 3 days of incubation; mobility was not observed in the semisolid MB medium; and anaerobic growth and carotenoid production were both observed. The colony of M415^T was round, 0.5 mm in diameter, and orange in color; mobility was not observed; and anaerobic growth and carotenoid production were detected. All the three strains were positive in the oxidations of D-fucose, L-galactonic acid lactone, D-glucuronic acid, glucuronamide, and tetrazolium violet. Detailed differences between strains M17^T, M82^T, and M415^T and reference strains are summarized in Table 2 and Supplementary Table 1 .

The major respiratory quinone of strains M17^T and M82^T was MK-7, which is consistent with their reference strains Mangrovivirga cuniculi KCTC 72349^T, Pontibacter actiniarum KCTC 12367^T, and Pontibacter litorisediminis KCTC 52252^T. The major respiratory quinone of strain M415^T was MK-6, identical with that of Poritiphilus flavus MCCC 1K03853^T, Eudoraea chungangensis KCTC 42048^T, and Zeaxanthinibacter enoshimensis NBRC 101990^T.

The main polar lipids of strain M17^T were phosphatidylethanolamine (PE), aminoglycolipid (AGL), one unidentified phospholipid (PL), three unidentified aminolipids (ALs), three unidentified glycolipids (GLs), and six unidentified lipids (L1–6) ( Supplementary Figure 5 ). Compared with its reference strain M. cuniculi KCTC 72349^T, they both contained PE as main polar lipids, but strain M17^T comprised more polar lipids such as AGL, ALs, and GLs. The major polar lipids of strain M82^T were PE, two ALs, and nine unidentified lipids (L1–9), in which PE and plenty of unidentified lipids were also detected in other species of the genus Pontibacter (Nedashkovskaya et al., 2005; Zhang et al., 2008; Subhash et al., 2013; Park et al., 2016). The major polar lipids detected in M415^T were PE, phosphoglycolipid (PGL), aminophospholipid (APL), one GL, two ALs, and five unidentified lipids (L1–5).

Similar to M. cuniculi KCTC 72349^T, iso-C_15:0 and iso-C_17:0 3-OH were the main cellular fatty acids (>10%) in strain M17^T. Meanwhile, strain M82^T contained iso-C_15:0 and summed feature 4 (SF4) as the major cellular fatty acids (>10%), which was also found in its reference strains. However, some differences in the ratio of main fatty acids (such as iso-C_15:0) existed between the two novel isolates and their reference strains ( Table 3 ). Similar to its reference strains, strain M415^T contained iso-C_15:0 and iso-C_17:0 3-OH as the main cellular fatty acids, but the relatively higher ratio of iso-C_15:1 -G and the lower ratio of summed feature 3 (SF3) differed strain M415^T from its reference strains ( Table 3 ).

The 16S rRNA gene sequences between strain M17^T and M. cuniculi KCTC 72349^T shared the highest similarity of 99.28%, and less than 90.09% with other species ( Table 4 ). In both of the phylogenetic trees based on the 16S rRNA gene and single-copy orthologous clusters (concatenated protein sequences), strain M17^T formed a closest and robust cluster with M. cuniculi KCTC 72349^T ( Figure 4 and Supplementary Figure 6 ), indicating that strain M17^T was affiliated with the genus Mangrovivirga. Although sequence similarity of the 16S rRNA gene reached 99.28% between strains M17^T and M. cuniculi KCTC 72349^T, the dDDH, ANI, and AAI values between strain M17^T and M. cuniculi KCTC 72349^T were 57.9%, 84.0%, and 88.8%, respectively ( Table 4 ), all lower than the thresholds for species delimitation (Konstantinidis and Tiedje, 2005; Tindall et al., 2010; Kim et al., 2014), indicating that strain M17^T represents a new species within the genus Mangrovivirga. In addition, the cell morphology of the strain M. cuniculi KCTC 72349^T was short rod-shaped (1.0–1.2 µm × 0.3–0.5 µm; Sefrji et al., 2021), which was different from that of strain M17^T, showing a long rod shape (2.0–10.0 µm × 0.3–0.5 µm; Supplementary Figure 2 ). In addition, there were also a great number of differences between them in phenotypic and genomic properties ( Tables 2 - 5 ). Therefore, strain M17^T represents a novel species of the genus Mangrovivirga.

Strain M82^T shares the highest 16S rRNA gene sequence similarity of 97.85%, 97.43%, and 96.59%, respectively, with Pontibacter litorisediminis KCTC 52252^T, Pontibacter korlensis X14-1^T, and Pontibacter actiniarum KCTC 12367^T, lower than the threshold for species delimitation (Kim et al., 2014). Based on the phylogenetic analysis of 16S rRNA gene sequences and single-copy orthologous clusters, strain M82^T was closely clustered within Pontibacter strains ( Figure 5 and Supplementary Figure 7 ). The highest values of dDDH, ANI, and AAI between strain M82^T and the type strains within the genus Pontibacter were 35.6%, 81.1%, and 84.2%, respectively, which are lower than the thresholds of species delimitation ( Table 4 ).

Low 16S rRNA gene sequence similarities were found between strain M415^T and its type species of the genera Eudoraea (92.62%–93.68%), Zeaxanthinibacter (92.02%–92.91%), Muriicola (92.21%–92.83%), Robiginitalea (91.48%–92.74%), and Poritiphilus (92.44%). Phylogenetic analysis based on 16S rRNA gene sequences and single-copy orthologous clusters showed that strain M415^T was clearly separated from the related genera ( Figure 6 and Supplementary Figure 8 ), representing a novel species of a new genus within the family Flavobacteriaceae. The highest values of dDDH, ANI, and AAI between strain M415^T and its related species were 13.5%, 70.2%, and 69.4%, respectively ( Table 4 ).

The genomic features of strains M17^T, M82^T, and M415^T and their related strains were analyzed to further confirm the taxonomic status of these three novel strains ( Table 5 ). Strain M17^T showed a similar genome size and GC content with M. cuniculi KCTC 72349^T, but the latter has more protein-coding genes and obvious multicopy of the rRNA gene than strain M17^T. The genomic composition of strain M82^T was different from the related species, showing a higher GC content and a lower number of rRNA. Strain M415^T has the smallest genome size and the lowest protein-coding gene number, compared with its related species. According to the annotation result against the COG database, the most abundant category in strains M17^T and M82^T was cell wall/membrane/envelope biogenesis; however, in strain M415^T, it changed to amino acid transport and metabolism ( Supplementary Figure 9 ). The genome sequences of strains M17^T, M82^T, and M415^T were annotated against the KEGG database and 1,614 (39.43%), 1,775 (44.55%), and 1,368 (47.14%) genes were assigned to putative functions, respectively. These functions were mainly composed of carbohydrate metabolism, genetic information processing, signaling and cellular processing, and amino acid metabolism, and notably, strain M82^T contained much more function genes associated with energy metabolism and environmental information processing ( Supplementary Figure 10 ).

As indicated by KEGG pathway annotation, these three strains have many differences in metabolic pathways. As for sulfur metabolism, the assimilatory sulfate reduction (M00176) was devoid only in strain M415^T. In the lipid metabolism, phosphatidylcholine (PC) biosynthesis (M00091) was only found in strain M82^T; nevertheless, strain M82^T and strain M415^T both contained the threonine biosynthesis (M00018) which was not found in strain M17^T. In the metabolism of major nutrients, the complete β-oxidation pathway only existed in strain M17^T; the phosphate acetyltransferase-acetate kinase pathway was complete in strains M17^T and M415^T but incomplete in strain M82^T ( Figure 7 ). In addition, we also found that the glyoxylate cycle was presented in both strains M17^T and M82^T but not in strain M415^T ( Figure 7 ); the activation of the glyoxylate cycle could provide malic acid and NADH, which serve as precursors and energy sources for metabolic reactions to sustain cell survival under stress conditions (Schroeter et al., 2011; Yuan et al., 2019). Therefore, the existence of the glyoxylate cycle for resisting the stress condition in tidal flats might indicate the adaptation mechanism of strains M17^T and M82^T to such environment.

Considering the outstanding polysaccharide metabolic abilities of marine Bacteroidota (Krüger et al., 2019), especially Flavobacteriaceae strains (Kappelmann et al., 2019; Gavriilidou et al., 2020), and special geographical location of mudflats—the source of strains M17^T, M82^T, and M415^T, we further analyzed their CAZyme profiles and potential polysaccharide substrates. The result showed that strains M17^T, M82^T, and M415^T had 114, 155, and 103 CAZymes, respectively ( Figure 8A ), which were similar as the KEGG results, where 157, 175, and 153 genes for the carbohydrate metabolism pathway were detected, respectively. The detailed numbers of each CAZyme family presented in three strains are summarized in Supplementary Table 2 . GH and GT were the most abundant CAZyme classes, accounting for more than 70% of the total CAZymes; this finding was consistent with studies on carbohydrate-active enzymes in Flavobacteriaceae species by Gavriilidou et al., which also indicated that GH and GT were the most abundant CAZyme classes (Gavriilidou et al., 2020). Strain M82^T comprised the highest number of CAZymes assigned to different classes and SusC/D-like proteins, indicating that it may have great potential in polysaccharide metabolism. However, the CAZyme count per Mb genome in strain M415^T was similar with strain M82^T and much higher than that in strain M17^T, even though the total number of CAZymes of strain M415^T was smaller than strains M17^T and M82^T due to the smallest genome size of strain M415^T ( Figure 8B ). A great number of GT2 and GT4 were present in strain M415^T, and they were involved in critical glycan synthesis, such as cellulose, chitin, and mannan; in addition, a higher number of GH2, GH3, GH16, and GH30 revealed that strain M415^T can metabolize plant polysaccharides and oligosaccharides (Coutinho et al., 2003; Gómez-Silva et al., 2019).

Polysaccharide utilization loci (PULs) are specialized saccharolytic systems that exhibit functional homology to the paradigmatic starch utilization system; the number and type of PULs determine the potential of polysaccharide utilization (Xu et al., 2003; McKee et al., 2021). According to the annotation results of carbohydrate-active enzymes and the SusC/D-like protein complex, we manually sorted the PULs of three novel strains ( Figure 9 ); strains M17^T, M82^T, and M415^T have five, six, and two putative PULs, respectively. The PULs in strains M17^T and M82^T were specific for marine polysaccharide metabolism; for strain M17^T, PUL2 may be associated with the degradation of ulvan as the existence of GH2 (β-xylosidase), PUL3 may be associated with the degradation of laminarin as the existence of GH3 and GH16_3 (Tang et al., 2017; Chen et al., 2018), and PUL4 may be associated with the degradation of chitin as the existence of GH18 (Kappelmann et al., 2019). For strain M82^T, PUL1 may be associated with the degradation of fucoidan as the existence of GH29 (fucosidase) and PUL4 may be associated with the degradation of laminarin as the existence of GH16_3 (Tang et al., 2017; Chen et al., 2018). However, with the ability to utilize marine polysaccharides as strains M17^T and M82^T, strains M415^T can also utilize polysaccharides that are mainly present in land; for instance, it contained more glycogen and starch degrading CAZymes compared to another two strains (e.g., GH13, GH13_8; Berlemont and Martiny, 2015), and the PUL1 of strain M415^T may be related to the degradation of glycogen or starch.

Lentiprolixibacter (L. masc. adj. lentus, slow, delayed; L. masc. adj. prolixus, long, extended; N.L. masc. n. bacter, a rod; N.L. masc. n. Lentiprolixibacter, slowly growing long rod)

Cells are Gram-stain-negative, non-spore-forming, and non-motile aerobic rods. Catalase- and oxidase-positive. Predominant menaquinone is menaquinone 6 (MK-6). Major polar lipids are phosphatidylethanolamine, phosphoglycolipid, aminophospholipid, unidentified glycolipids, aminolipids, and lipids. The type species is Lentiprolixibacter aurantiacus.

Lentiprolixibacter aurantiacus (au.ran.ti.a’cus. N.L. masc. adj. aurantiacus, orange-colored)

Displays the following characteristics in addition to those given in the genus description. Cells are rod shaped and usually 0.3–0.5 µm wide and 1.8–8.0 µm long. Colonies (0.5 mm in diameter) are circular, convex, smooth, shiny, and orange pigmented after 3 days of incubation. Cells grow at 25°C–37°C (optimum, 25°C–30°C) in a medium of pH 6–8 (optimum, pH 6.5–7) and contain 1%–4% NaCl (optimum, 2%–3%). Catalase and oxidase are positive; nitrate reduction, carotenoid production, and H₂S production are negative. Starch is weakly hydrolyzed, but Tweens 20, 40, 60, and 80 are not. Dextrin, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turanose, β-methyl-D-glucoside, D-salicin, N-acetyl-D-glucosamine, α-D-glucose, fusidic acid, D-serine, D-glucose-6-PO₄, D-fructose-6-PO₄, pectin, glucuronamide, L-malic acid, bromo-succinic acid, nalidixic acid, and sodium butyrate are oxidized. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucosidase, and N-acetylglucosaminidase are positive and α-glucosidase is weakly positive. The major cellular fatty acids (>10%) are iso-C_15:0, iso-C_15:1 -G, and iso-C_17:0 3-OH. The polar lipids are phosphatidylethanolamine, phosphoglycolipid, aminophospholipid, one unidentified glycolipids, two unidentified aminolipids, and five unidentified lipids. The DNA G+C content of the type strain is 44.6%.

The type strain, M415^T (MCCC 1K08058^T = KCTC 92534^T), was isolated from an intertidal mudflat (0–5 cm) collected from Taizhou, Zhejiang Provence, PR China. The GenBank accession numbers for the 16S rRNA gene sequence and the draft genome sequence of strain M415^T are ON935779 and JAPFQP000000000, respectively.

Mangrovivirga halotolerans (ha.lo.to’le.rans. Gr. masc. n. hals, salt; L. pres. part. tolerans, tolerating, enduring; N.L. part. adj. halotolerans, salt-tolerating)

Cells are Gram-stain negative, non-motile, strictly aerobic rod-shaped, usually 0.3–0.5 µm wide, and 2–10 µm long, some more than 10 µm in length. Colonies (1–3 mm in diameter) are circular, convex, smooth, shiny, and orange pigmented after 3 days of incubation. Cells grow at 20°C–45°C (optimum, 20°C–28°C) in a medium of pH 6.5–9 (optimum, pH 6.5-7.5) and contain 0.5%–10% NaCl (optimum, 3.5%–6%). Catalase and oxidase activities are positive; nitrate reduction, carotenoid production, and H₂S production are negative. Starch is hydrolyzed, but Tweens 20, 40, 60, and 80 are not. Sucrose, stachyose, D-fucose, fusidic acid, L-alanine, glucuronamide, tetrazolium violet, α-keto-glutaric acid, L-malic acid, nalidixic acid, acetoacetic acid, propionic acid, acetic acid, formic acid, aztreonam, and sodium butyrate are oxidized. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucuronidase, β-glucosidase, and N-acetylglucosaminidase are positive. The predominant menaquinone is MK-7. The major cellular fatty acids (>10%) are iso-C_15:0 and iso-C_17:0 3-OH. The polar lipids are phosphatidylethanolamine, aminoglycolipid, one unidentified phospholipid, three unidentified aminolipids, three unidentified glycolipids, and six unidentified lipids. The DNA G+C content of the type strain is 35.9%.

The type strain, M17^T (MCCC 1K08105^T = KCTC 92592^T), was isolated from an intertidal mudflat (0–5 cm) collected from Qingdao, Shandong Provence, PR China. The GenBank accession numbers for the 16S rRNA gene sequence and the draft genome sequence of strain M17^T are ON935777 and JAPFQN000000000, respectively.

Pontibacter anaerobius (an.ae.ro’bi.us. Gr. pref. an-, not; Gr. masc. n. aêr, air; Gr. masc. n. bios, life; N.L. masc. adj. anaerobius, able to live in the absence of oxygen)

Cells are Gram-stain negative, non-motile, facultative aerobic rod-shaped, and usually 0.6–1.1 µm wide and 0.9–3.2 µm long. Colonies (1–2 mm in diameter) are circular, convex, smooth, shiny, and red pigmented after 3 days of incubation. Cells grow at 20°C–40°C (optimum, 28°C–37°C) in a medium of pH 6–9 (optimum, pH 6.5–7.5) and contain 0%–8% NaCl (optimum, 2%–3%). Carotenoid production and catalase and oxidase activity are positive. Nitrate reduction and H₂S production are negative. Starch and Tweens 20, 40, 60, and 80 are not hydrolyzed. Dextrin, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turanose, stachyose, D-raffinose, α-D-lactose, D-melibiose, β-methyl-D-glucoside, D-salicin, N-acetyl-D-glucosamine, α-D-glucose, D-mannose, D-galactose, D-fucose, L-fucose, fusidic acid, D-arabitol, myo-inositol, D-glucose-6-PO₄, D-fructose-6-PO₄, gelatin, glycyl-L-proline, L-aspartic acid, L-glutamic acid, L-serine, D-galacturonic acid, glucuronamide, acetoacetic acid, acetic acid, formic acid, and sodium butyrate are oxidized. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, α-galactosidase, α-glucosidase, β-glucosidase, and N-acetylglucosaminidase are positive; naphthol-AS-BI-phosphohydrolase and β-galactosidase are weakly positive. The predominant menaquinone is MK-7. The major cellular fatty acids (>10%) are iso-C_15:0, iso-C_17:0 3-OH, and summed feature 4 (ante-iso-C_17:1 B and/or iso-C_17:1 I). The polar lipids are phosphatidylethanolamine, two unidentified aminolipids, and nine unidentified lipids. The DNA G+C content of the type strain is 50.6%.

The type strain, M82^T (MCCC 1K08048^T = KCTC 92537^T), was isolated from an intertidal mudflat (0–5 cm) collected from Taizhou, Zhejiang Provence, PR China. The GenBank accession numbers for the 16S rRNA gene sequence and the draft genome sequence of strain M82^T are ON935778 and JAPFQO000000000, respectively.