Morphological, physiological, and chemotaxonomic characteristics

Morphological analysis of strain NBU2967T revealed that the cells are Gram-negative, rod-shaped, non-flagellated, and measure approximately 0.6–0.8 × 1.2–1.6 µm (Fig. S1). After 72 h of incubation on MA medium at 37.0°C, colonies were approximately 1 mm in diameter, circular, raised, and exhibited an orange-yellow color. Growth was observed within a pH range of 5.5–8.5, with an optimum at pH 6.5 (Fig. S2A); a temperature range of 10.0–40.0°C, with an optimum at 37.0°C; and NaCl concentrations of 1.0–5.0% (w/v), with an optimum at 2.0%. A pronounced exponential phase was observed between 24 and 36 h of incubation under optimal growth conditions (Fig. S2B). No growth occurred under anaerobic conditions, even after two weeks of incubation on modified MA medium supplemented with various electron acceptors, indicating that strain NBU2967T is strictly aerobic.

Biochemical tests indicated that strain NBU2967T was positive for catalase and oxidase activities, the methyl red test, hydrolysis of Tweens 20, 40, 60, and 80. Negative results were obtained for H2S production, Voges-Proskauer test, hydrolysis of starch and casein. Enzymatic activity profiling using the API ZYM test kit revealed positive reactions for alkaline phosphatase, esterase (C4), lipase (C8 and C14), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase, α-mannosidase, and β-fucosidase. The API 20 NE test kit showed positive activity for aesculin hydrolysis and β-galactosidase, while the API 50 CH test kit demonstrated positive utilization of a wide range of carbohydrates and glycosides, including D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, β-methyl-D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose, α-methyl-D-mannopyranoside, α-methyl-D-glucopyranoside, N-acetyl-β-D-glucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose, raffinose, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, and 2-ketogluconate. Antibiotic susceptibility testing revealed sensitivity to a broad spectrum of antibiotics, including lincomycin, clindamycin, tetracycline, doxycycline, erythromycin, ofloxacin, vancomycin, chloramphenicol, cephalexin, cefoxitin, amoxicillin, rifampicin, cephalothin, carbenicillin, ciprofloxacin, and ampicillin.

The major fatty acids (≥ 10%) were identified as iso-C15:0, iso-C17:0 3-OH, and Summed Feature 1 (iso-C15:1 H/C13:0 3-OH). A detailed composition of all fatty acids present at levels greater than 1% is provided in Table S1. The sole respiratory quinone was menaquinone-6. The polar lipid profile included phosphatidylethanolamine (PE), aminophospholipid (APL), two unidentified lipids (Ls), two unidentified phospholipids (PLs), and three unidentified aminolipids (Als) (Fig. S3). Key characteristics distinguishing strain NBU2967T from closely related genera within the family Flavobacteriaceae are summarized in Table 1.

Table 1 Characteristics differentiating strain NBU2967T from its reference strains within the family Flavobacteriaceae. The reference strains represent the six closest related generaPhylogenetic analysis based on the 16S rRNA gene sequences

The complete 16S rRNA gene sequence of strain NBU2967T (1,490 nt; GenBank accession number: MZ025914) was successfully obtained through PCR amplification and sequencing. A comparative analysis of this sequence using the EzBioCloud and NCBI GenBank databases revealed that strain NBU2967T exhibited the highest sequence similarity to M. algicola PoM-212T (94.88%), followed by M. cobaltidurans B1T (94.82%), M. aurantiacus CDA4T (94.33%), M. halichondriae Hal144T (94.27%), M. flavus KCTC 42508T (94.20%), and M. arenosus CAU 1321T (94.20%). Similarity to all other strains was less than 94.20%.

Phylogenetic trees constructed using the neighbor-joining, maximum-likelihood, and maximum-parsimony methods consistently demonstrated that strain NBU2967T formed a distinct lineage within the family Flavobacteriaceae (Fig. 2, Figs. S4, S5). Importantly, the 16S rRNA gene sequence similarities between strain NBU2967T and its closest phylogenetic neighbors were below the established thresholds for bacterial genus (< 95.0%) delineation [74, 75]. These results suggest that strain NBU2967T potentially represents a novel genus within the family Flavobacteriaceae.

Fig. 2

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences. Bootstrap values are shown at nodes as percentages of 1000 replicates. Values under 70% are hidden. Bar, 0.01 changes per nucleotide position

A total of 61 bacterial strains were isolated. Based on 16S rRNA gene sequence comparisons with validly published species, these isolates were assigned to 50 species belonging to 6 phyla, 9 classes, 16 orders, 26 families, and 33 genera. To investigate the evolutionary relationships among the isolates, a neighbor-joining phylogenetic tree was constructed based on aligned 16S rRNA gene sequences from all 61 strains (Fig. S6). The neighbor-joining tree revealed that these strains were distributed across multiple phylogenetically distinct lineages, forming several well-supported clades. Despite being recovered from the same sampling site, the strains exhibited considerable phylogenetic diversity, indicating a taxonomically complex microbial community. This phylogenetic topology reflects a heterogeneous and evolutionarily diverse bacterial population in the coastal tidal flat habitat.

Genomic characteristics

The draft genome of strain NBU2967T has a total length of 3,819,109 bp and is composed of 18 contigs, with a genomic DNA G + C content of 42.5% (Fig. S7 A). The draft genome comprises 3,412 genes, including 3,364 protein-coding genes, 14 pseudogenes, and 44 RNA genes (3 rRNA genes, 37 tRNA genes, and 4 non-coding RNA genes). A comparison of the genomic features of strain NBU2967T with 16 reference strains within the family Flavobacteriaceae is provided in Table S2. Genome annotation using the RAST subsystem categorization revealed that the identified genes are distributed across 22 functional categories, as illustrated in Fig. S7B.

RAST predictions showed that the genome of strain NBU2967T is involved in several important metabolic and biological processes. The most prominent categories were protein families: genetic information processing (173 genes) and genetic information processing (155 genes), suggesting that the strain plays a significant role in genetic information transfer and regulation. Additionally, carbohydrate metabolism (156 genes) indicates the strain's strong ability to metabolize carbon, allowing it to efficiently utilize carbon sources. The most prominent categories were protein families involved in genetic information processing (173 genes) and cellular functions related to genetic information (155 genes), suggesting that the strain has a strong capacity for genetic regulation and information processing.

Overall genomic relatedness and phylogenomics

The ANI, AAI, and dDDH values were calculated to identify the genomic similarities of strain NBU2967T with reference strains within the family Flavobacteriaceae (Fig. 3). The ANI values between strain NBU2967T and reference strains within the family Flavobacteriaceae ranged from 70.5% to 71.6%, which is below the genus-level threshold of 73.98% (95% confidence interval: 73.34–74.62%) [76]. Similarly, the AAI values ranged from 72.4% to 77.5%, consistent with the genus differentiation range of 60–80% within the family Flavobacteriaceae [77, 78]. The dDDH values were calculated to be between 16.8% and 19.6%, significantly lower than the proposed 70% threshold for species delineation [79]. Phylogenomic analysis based on whole-genome sequences further supports the distinct evolutionary position of strain NBU2967T, as it forms a separate lineage within the family Flavobacteriaceae (Fig. S8).

Fig. 3

Genomic similarities between strain NBU2967T and reference strains within the family Flavobacteriaceae. (A), The ANI values between strain NBU2967T and reference strains within the family Flavobacteriaceae; (B), The AAI values between strain NBU2967T and reference strains within the family Flavobacteriaceae; (C), The dDDH values between strain NBU2967T and reference strains within the family Flavobacteriaceae

Collectively, these findings strongly indicate that strain NBU2967T represents a novel taxon with unique genomic and evolutionary characteristics, justifying its classification as a new genus within the family Flavobacteriaceae.

Evaluation of the metabolic and ecological potentialMetabolic pathway analysis

The KEGG analysis of strain NBU2967T and reference strains within the family Flavobacteriaceae revealed a high degree of metabolic adaptability and diversity across this taxonomic group (Fig. 4). These include pathways involved in carbohydrate metabolism and energy production. Complete metabolic pathways, such as glycolysis (M00001), the citric acid cycle (M00009), and the pentose phosphate pathway (M00004), demonstrate the ability of these strains to efficiently utilize carbon sources (Fig. 5). Genome analysis revealed that strain NBU2967T encodes a complete cbb₃-type cytochrome c oxidase complex, along with additional cytochrome c oxidase subunits and heme-copper oxidases. These components constitute a functional terminal oxidase system that supports oxygen respiration [80]. The presence of these genes is consistent with the organism’s obligate aerobic phenotype observed in culture. Strain NBU2967T also exhibited a unique metabolic pathway not possessed by other strains: Gamma-Aminobutyric Acid (GABA) biosynthesis, eukaryotes, putrescine =  > GABA(M00135). It has been reported that GABA is widely utilized by microorganisms as an important signaling molecule and osmoprotectant in response to various abiotic stresses (e.g., high salinity, drought, darkness, and cytoplasmic acidification) [81]. Moreover, GABA metabolism is closely associated with the regulation of the cellular carbon/nitrogen balance, energy homeostasis, and redox state modulation [81]. Strain NBU2967T possesses the ability to biosynthesize GABA, suggesting that it may have enhanced capabilities for environmental stress response and osmoregulation, which could contribute to its survival and colonization in diverse ecological environments.

Fig. 4

The metabolic module integrity of strain NBU2967T, along reference strains. The solid circles and hollow circles indicate that the metabolic pathways were complete and incomplete, respectively

Fig. 5

Integrated pathways of β-(1,3)-glucan and β-(1,4)-xylan degradation with central carbon metabolism and terminal electron transport in bacteria. NADH/FADH2, Reduced cofactors that donate electrons to the respiratory chain; MFS, major facilitator superfamily; TBDT, TonB-dependent transporter; PPP, pentose phosphate pathway

In order to further understand its metabolic mechanisms in the marine tidal flat environment, its genome was analyzed and found to encode a variety of key enzymes related to carbon and sulfur metabolism, suggesting that the strain plays an important ecological role in organic matter transformation and nutrient cycling (Fig. 6). The analysis results showed that the strain contained α-glucuronidase, an enzyme that plays a crucial role in the degradation of plant cell wall polysaccharides, particularly in the hydrolysis of glucuronic acid-containing xylans [82]. Meanwhile, endo-β-(1,3)-glucanase was identified, an enzyme that targets β-(1,3)-glucan, a component of plant cell walls and fungal cell membranes [83]. This enzyme is critical for breaking down complex polysaccharides such as laminarin, a major carbohydrate in marine algae, and mixed-linkage glucans commonly found in cereals [84]. The presence of both α-glucuronidase and endo-β-(1,3)-glucanase suggests that this strain is well-equipped to degrade a broad spectrum of polysaccharides, thereby enhancing its ability to utilize organic matter from diverse sources. The strain also possesses endo-1,4-β-xylanase, which is capable of hydrolyzing the β-1,4-glycosidic bond in xylan, further enhancing its ability to degrade plant-derived carbon sources [85]. The combined action of these enzymes endowed the microorganism with strong adaptability and metabolic potential in the carbon-rich environment of marine tidal flats.

Fig. 6

Protein Structural Analysis. (A), Structure alignment of the α-glucuronidase from strain NBU2967T with that from Cellvibrio japonicus (PDB ID: 1GQK); (B), Structure alignment of the endo-β-(1,3)-glucanase from strain NBU2967T with that from Bacteroides uniformis (PDB ID: 6PAL); (C), Structure alignment of the endo-1,4-β-xylanase from strain NBU2967T with that from Bacteroides thetaiotaomicron (PDB ID: 3QZ4); (D), Structure alignment of the sulfatase from strain NBU2967T with that from Vibrio vulnificus (PDB ID: 6 VPU); (E), Structure alignment of the sulfite oxidase from strain NBU2967T with that from Gallus gallus (PDB ID: 3HBG); (F), Structure alignment of the PAPS reductase from strain NBU2967T with that from Bacteroides fragilis CAG:558 (PDB ID: 6USS). RMSD and TM-Score values are presented

In addition to its carbon metabolizing capacity, the strain showed equally remarkable potential in sulfur metabolism. The genome analysis revealed the presence of sulfatase, an enzyme capable of hydrolyzing a wide range of organic sulfate compounds, including sulfated marine polysaccharides, which are common in marine ecosystems [86]. This hydrolysis process releases inorganic sulfate and provides precursors for subsequent sulfur assimilation or reduction processes, thereby contributing to sulfur cycling in marine environments [87]. In addition, sulfite oxidase was identified. Sulfite oxidase plays a critical role in sulfur metabolism by catalyzing the oxidation of sulfite to sulfate, an essential step in the microbial sulfur cycle [88]. The mechanism not only helps detoxify sulfite, which is toxic to cells, but also facilitates sulfur cycling in microbial communities. Phosphoadenosine phosphosulfate (PAPS) reductase was also detected, which plays a key role in reducing PAPS to sulfide, an essential step in the assimilation of sulfate into biologically active sulfur compounds like cysteine [89]. The combined action of these sulfur-metabolizing enzymes allows the strain to significantly contribute to the sulfur cycle, aiding in the transformation and recycling of sulfur within marine tidal flat ecosystems.

Structural alignment of putative proteins from strain NBU2967T with verified enzymes revealed high conformity: putative α-glucuronidase exhibited a root mean square deviation (RMSD) of 1.09 Å compared to a validated α-glucuronidase (PDB ID: 1GQK); putative endo-β-(1,3)-glucanase exhibited a RMSD of 1.12 Å compared to a validated endo-β-(1,3)-glucanase (PDB ID: 6PAL); putative endo-1,4-β-xylanase exhibited a RMSD of 1.21 Å compared to a validated endo-1,4-β-xylanase (PDB ID: 3QZ4); putative sulfatase exhibited a RMSD of 0.56 Å compared to a validated sulfatase (PDB ID: 6 VPU); putative sulfite oxidase exhibited a RMSD of 1.38 Å compared to a validated sulfite oxidase (PDB ID: 3HBG); and putative PAPS reductase exhibited a RMSD of 1.57 Å compared to a validated PAPS reductase (PDB ID: 6USS) (Fig. 6). Structural modeling confirmed functional conservation of these enzymes.

Analysis of CAZymes predicted by dbCAN3

Strain NBU2967T and reference strains within the family Flavobacteriaceae harbored extensive reservoirs of CAZymes. Total CAZymes predicted by dbCAN3 across the genomes of these strains ranged from 115 to 277 per strain (Fig. 7). The most abundant CAZyme families in strain NBU2967T were Glycoside Hydrolases (GHs) (60 species), followed by Glycosyltransferases (GTs) (46 species), Carbohydrate Esterases (CEs) (17 species), Carbohydrate Binding Modules (CBMs) (13 species), Auxiliary Activities (AAs) (8 species), and Polysaccharide Lyase (PL) (1 species), respectively.

Fig. 7

Taxonomic annotation and distribution of CAZymes in strain NBU2967T and reference strains. The upper chord plot illustrates the number of CAZymes annotated in strain NBU2967T and reference strains within the family Flavobacteriaceae compared to the records in the dbCAN-seq database. The lower bubble plot illustrates the distribution of CAZymes families in strain NBU2967.T and reference strains within the family Flavobacteriaceae, and types containing more than 8 CAZymes per strain have been labeled in the plot. Detailed information can be found in Table S3

GH enzymes catalyze the cleavage of glycosidic bonds in a wide range of substrates, from small glucosinolates to complex polysaccharides, and are classified based on their catalytic mechanisms [90]. Among the 82 GH enzymes identified in the genomes of strain NBU2967T and reference strains within the family Flavobacteriaceae, GH188 enzymes accounted for the highest proportion, ranging from 2.9% to 72.7% (Table S3). GH188 enzymes, a specialized class of glycoside hydrolases, exhibit unique structural and catalytic properties. They are capable of breaking down sulfoquinovosyl diacylglycerols (SQDG) [91], thereby releasing sulfur and carbon sources that can be utilized by bacteria. This activity facilitates the decomposition of organic carbon, supports microbial food webs, and promotes coupled sulfur and carbon metabolism, thereby contributing to carbon cycling in tidal flats environments.

GT enzymes play a fundamental role in life processes by catalyzing the transfer of saccharide moieties from sugar nucleotide donors to acceptor molecules [92]. They are essential for the biosynthesis of oligosaccharides and N- and O-linked glycoconjugates. Among the GT families identified in strain NBU2967T and reference strains within the family Flavobacteriaceae, GT4 and GT2 accounted for the highest proportions (Table S3). These two families are believed to represent the ancestral sources of GH families [93]. According to the dbCAN-seq database, GT2 and GT4 constitute 30% and 28%, respectively, of the total GT enzymes in marine environments, underscoring their widespread presence among marine organisms [94]. The combined GH and GT gene ratio to total protein-coding genes in strain NBU2967T and reference strains within the family Flavobacteriaceae ranged from 2.3% to 5.4%, significantly higher than the typical ratio of 1–3% observed in other bacterial taxa [95]. This elevated ratio reflects an enhanced capacity for polysaccharide metabolism, suggesting a specialized adaptation for efficient carbohydrate utilization in marine environments.

CE enzymes release acyl or alkyl groups attached by ester linkage to carbohydrates and facilitate the degradation of complex polysaccharides [96], such as pectin and alginate [97]. Among the CE families identified in the genomes of strain NBU2967T and reference strains within the family Flavobacteriaceae, CE1 and CE14 were the most abundant (Table S3). These two families represent some of the largest and most diverse groups of CE enzymes, predominantly of bacterial origin [98]. They are recognized for facilitating the degradation of xylan, a component of plant cell walls, and chitin, a constituent of crustacean shells, respectively [98, 99]. It is presumed that strain NBU2967T could utilize CE1 enzymes to facilitate the hydrolysis of recalcitrant polysaccharides.

CBM enzymes are frequently associated with catalytic CAZymes and enhance their activity by binding to various carbohydrates [100]. Among the CBM families identified, CBM9 was the most abundant in the genomes of strain NBU2967T and reference strains within the family Flavobacteriaceae (Table S3). CBM9 proteins play a crucial role in the degradation of xylan [101].

PL enzymes cleave uronic acid-containing polysaccharides through a β-elimination mechanism, in contrast to the hydrolysis mechanism used by most other CAZymes [102]. This distinctive catalytic strategy allows PL enzymes to degrade a broader range of polysaccharides. Among the PL families identified in the genomes of strain NBU2967T and reference strains within the family Flavobacteriaceae, PL1, PL6, and PL7 were the most abundant (Table S3). These enzymes are crucial in breaking down polysaccharides derived from phytoplankton, such as alginate and pectin [103, 104]. This suggests that strain NBU2967T may be particularly adapted to degrade algal polysaccharides. Previous studies have shown that Flavobacteriaceae strains are efficient in utilizing high-molecular-weight compounds released by phytoplankton, converting these compounds into CO2 or bacterial biomass, which is subsequently incorporated into the marine food web [105]. This bacterial-mediated process is vital for the turnover of phytoplankton-derived organic carbon, influencing carbon flow within marine ecosystems and contributing to the global carbon cycle.

AA3 enzymes were the most abundant AA family identified in the genomes of strain NBU2967T and reference strains within the family Flavobacteriaceae (Table S3). AA3, which consist of four subfamilies (AA3_1, AA3_2, AA3_3, AA3_4), assists in lignocellulose degradation with its reaction products [106].

In summary, the abundant CAZymes identified in strain NBU2967T and reference strains within the family Flavobacteriaceae suggests that they have a great potential for utilizing marine-derived polysaccharides.

Prediction of secondary metabolites

Analysis of the predicted secondary metabolites from strain NBU2967T and reference strains within the family Flavobacteriaceae revealed a remarkable diversity and varying degrees of ubiquity in their biosynthetic pathways. Metabolites such as terpenes and aryl polyenes were detected in almost all strains (Fig. S9). As raw materials, terpenoids are extensively used in the pharmaceutical industry due to their antitumor, anti-inflammatory, antibacterial, antiviral, and antimalarial properties [107]. In contrast, secondary metabolite pathways such as non-ribosomal peptide synthetases (NRPS) and type III polyketide synthases (T3PKS) were identified only in a subset of strains. T3PKS are versatile enzymes involved in the biosynthesis of polyketides, which are secondary metabolites with diverse biological activities, including antimicrobial, antifungal, and anticancer properties [108]. This metabolic diversity highlights the potential ecological roles of strain NBU2967T and reference strains within the family Flavobacteriaceae in marine ecosystems.

Pangenome analysis

For strain NBU2967T and all reference strains, the pangenome contains 29,364 genes, of which the core genome represents 863 genes (approximately 2.9%), whereas the shell and unique genes represent 7,596 (approximately 25.9%) and 20,905 genes (approximately 71.2%), respectively. The number of core genes, shared genes, and unique genes in each strain is shown in Table S4. The remarkably low number of core genes in strain NBU2967T and reference strains within the family Flavobacteriaceae suggests that these microorganisms have undergone extensive horizontal gene transfer and gene loss events, resulting in highly distinct genetic compositions among different strains. This pronounced genetic diversity is likely a key factor in their ecological adaptability. Additionally, the substantial diversity of unique genes likely enhances the competitive fitness of individual strains within specific ecological niches [109]. This indicates a high degree of genetic diversity and some level of conservation in the genomes of strain NBU2967T and reference strains within the family Flavobacteriaceae (Fig. 8A). As shown by the Tettelin bets ft. curve (Fig. 8B), the increase in pangenome size was indeterminate until the accession of the last genome, so the pangenome of strain NBU2967T and reference strains within the family Flavobacteriaceae may be classified as ‘open’ for expansion.

Fig. 8

Pangenome analysis of strain NBU2967T and reference strains. (A), Clustering of genomes based on the presence/absence patterns of 29,364 pangenomic genes; (B), Pan and core genome sizes. Blue is pan genome size and red is core genome size. The numbers of pan and core genes are plotted as a function of the number of genomes (g) added sequentially. y-axis shows the change in core genome and pan genome sizes as a function of random addition of genomes plotted on x-axis. The sizes of core and pan genomes were computed using OMCL algorithm. The best fit regression represents the Tettelin curve for both core and pan genomes. CL, completeness. CN, contigs. GN, gene number. GL, genome length

Environmental distribution of the newly described genus Meishania

The MAP database was used to determine the worldwide distribution of the newly described genus Meishania. A total of 91,531 samples from 8,164 projects were analyzed to identify the representative sequence. The results of the biogeographic distribution analysis indicated that genus Meishania is widespread across different habitats, including aquatic, soil, animal, and plant environments. Specifically, genus Meishania were found in 49,916 aquatic samples (54.4%), 8,009 animal samples (8.8%), 3,437 soil samples (3.8%), and 701 plant samples (0.8%). Among the known environments, the primary habitats for genus Meishania were identified as the marine environment (22.4%) (Fig. 9A). The mapping of database sequencing reads to the standard operational taxonomic unit (OTU) sequence also showed that the marine environment had a predominant proportion (24.0%) of reads from the genus Meishania (Fig. 9B).

Fig. 9

Biogeographic distribution analysis of the newly described genus Meishania. (A), Frequency of samples with representative OTU sequence, by habitat and sub-habitat; (B), Abundance of sequencing reads mapping to the representative OTU sequence, by habitat and sub-habitat

This distribution suggests that genus Meishania may play an important role in aquatic ecosystems, especially in the oceans, and may be associated with specific ecological adaptations and environmental conditions in the waters. Given the detection of sequences in plant- and animal-associated samples, there is also potential for symbiotic or opportunistic interactions, which merits further investigation.