A marine sediment sample was collected off the coast of Xiaoshi Island, Weihai, PR China (122°00′58′′ E, 37°31′36′′ N) and subjected to an enrichment culture technique as described by Mu (Mu et al., 2018). The sample was collected at a depth of approximately 10 cm and was kept cold and in the dark during transportation to the laboratory. Incubation was performed at 25°C for 8 months using a 350 ml sealed glass bottle. The bottle was shaken twice a day and kept sealed during the incubation. The sediment sample was serially diluted to 10⁻³ in sterilized seawater and 0.1 ml aliquots of each dilution were spread on to the surface of modified marine agar (MA) 2216. The modified MA medium used for the characterization of strains contained the following components (g L⁻¹, pH 7.0): 5.0 tryptone, 1.0 yeast extract, 1.5 pyruvic acid sodium, and 2 ml vitamin mixture. The vitamin mixture contained the following components (mg L⁻¹): 5.0 thiamine, 5.0 riboflavin, 5.0 nicotinic acid, 5.0 D-Ca-pantothenate, 10.0 pyridoxine-HCl, 2.0 biotin, 2.0 folic acid, 0.1 cobalamin, 5.0 lipoic acid and 5.0 p-aminobenzoic acid. After incubation for 7 days at 33°C, colonies of the two strains were selected from the plate and re-streaked to obtain pure cultures. These strains were then cultivated on modified MA 2216 at 33°C for further physiological, biochemical, and chemical analyses. Pure cultures of the strains were preserved at −80°C in a sterile 1% (w/v) saline solution supplemented with 15% (v/v) glycerol.

The morphological and physiological features of strains D04^T and AAT^T were tested with cells grown on the modified MA at optimum growth conditions for 5 days. The experiment was repeated three times. Cell morphology and size were examined by light microscopy (E600; Nikon), transmission electron microscopy (JEM-1200, JEOL) and scanning electron microscopy (model Nova NanoSEM450; FEI). Gram reactions were carried out as described previously (CLSI, 2021). Gliding motility was examined in modified marine broth 2216 (MB; BD) supplemented with 0.3% agar as described previously (Bernardet et al., 2002). The growth temperature range was tested on the modified MA at various temperatures (4, 20, 25, 28, 30, 33, 37, 40, 45, and 50°C). To test for the pH range suitable for growth, bacterial strains were grown in modified marine broth 2216 with different buffers at a concentration of 20 mM. MES buffer was used for pH 5.5 and 6.0, PIPES buffer for pH 6.5 and 7.0, HEPES buffer for pH 7.5 and 8.0, Tricine buffer for pH 8.5, and CAPSO buffer for pH 9.0 and 9.5. For investigating growth at different NaCl concentrations, a medium containing 1 g/L⁻¹ yeast extract, 5 g/L⁻¹ peptone, and 20 g/L⁻¹ agar prepared with artificial seawater [0.22% MgCl₂, 0.15% CaCl₂, 0.32% MgSO₄, 0.02% NaHCO₃, and 0.07% KCl, with 3% NaCl (w/v)] was used. The bacterial strains were grown in this medium at NaCl concentrations ranging from 0% to 9% in increments of 0.5%.

The susceptibility of the bacterial strains D04^T and AAT^T to various antibiotics was tested using the disc diffusion method, as previously described (Du et al., 2014). A cell suspension of 0.5 McFarland standard was swabbed over the modified marine agar to create a uniform lawn before the aseptic placement of antibiotic discs onto the surface. After incubation for 5 days, the plates were examined for a clear zone of growth inhibition around the antibiotic discs. A total of 20 different antibiotic discs (with varying concentrations measured in micrograms per disc) were used to test the susceptibility of the bacterial strains D04^T and AAT^T: lincomycin (2), streptomycin (10), erythromycin (15), clarithromycin (15), kanamycin (30), ampicillin (10), rifampin (5), tetracycline (30), penicillin (10), chloramphenicol (30), cefotaxime sodium (30), gentamycin (10), ofloxacin (5), norfloxacin (30), neomycin (30), carbenicillin (100), ceftriaxone (30) tobramycin (10), vancomycin (30), and polymyxin B (300). CLSI standards were strictly followed for cultivation and inhibition zone diameter reading (Gcrhardt et al., 1994).

Bacterial growth was monitored at 600 nm using a spectrophotometer. Catalase and oxidase activity were tested by pouring 3% H₂O₂ solution onto the cells and by using an oxidase test reagent (bioMérieux), respectively. Anaerobic growth was tested for 15 days at 30°C on the modified MA with or without 0.1% (w/v) NaNO₃ in an anaerobic bag (hopebio). Hydrolyses of agar, starch, alginate, casein, CM-cellulose, DNA, and lipase (Tweens 20, 40, 60, and 80) were examined according to the method of CLSI (2021). API 20E and API 50CH tests (bioMérieux) were carried out according to the manufacturer’s instructions (except for salinity, which was adjusted to 3%). Enzyme activities were examined using the API ZYM test (bioMérieux). Oxidization of different compounds was tested in Biolog GEN III microplates according to the manufacturer’s instructions. All the API and Biolog tests were performed with three biological replicates each time and with two reference strains.

The 16S rRNA gene sequences were amplified using PCR with the primer pair 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) (Jordan et al., 2007; Du et al., 2014). Purified PCR products were ligated into the pMD18-T vector (Takara) and cloned by following the manufacturer’s instructions. Sequencing was performed by BGI Co. Ltd (Qingdao, PR China). The nearly complete 16S rRNA gene sequences of strains D04^T and AAT^T were obtained and compared with those available from the EzBioCloud ² (Yoon et al., 2017a) database for further phylogenetic analysis. Phylogenetic trees were constructed using MEGA version 11 employing the neighbor-joining (Saitou and Nei, 1987), maximum-likelihood (Felsenstein, 1981), and maximum-parsimony methods (Kumar, 1996). Bootstrap analyses were performed based on 1,000 replicates to estimate the confidence of branches in the generated phylogenetic trees (Felsenstein, 1985).

Genomic DNA of strains D04^T and AAT^T was extracted and purified using a bacteria genomic DNA kit (Takara). The draft genome sequences of strains D04^T and AAT^T were sequenced by Majorbio (MajorBio Co., Shanghai, China) using an Illumina MiSeq (Illumina, USA). The final genome was assembled with SOPA de novo version 2.04 (Luo et al., 2012). The resulting genomes were annotated using RAST (rapid annotation using subsystem technology online server) ³ (Aziz et al., 2008). The molecular functions of genes and proteins are associated with ortholog groups and stored in the KEGG orthology (KO) database (Kanehisa et al., 2016). The genes involved in metabolic pathways and ortholog groups of proteins were analyzed using the ⁴ and dbCAN2 meta servers ⁵ (Zhang et al., 2018). The presence of gene clusters encoding secondary metabolites was predicted using antiSMASH 6.1.1 ⁶ (Blin et al., 2021). The DNA G+C content was calculated based on the whole genome sequence. The AAI calculator estimates the average amino acid identity using both best hits (one-way AAI) and reciprocal best hits (two-way AAI) between two genomic datasets of proteins ⁷ . Average nucleotide identity (ANI) values were calculated using the ChunLab’s online ANI Calculator ⁸ (Lee et al., 2016). The genome-to-genome distance calculator (GGDC 3.0) ⁹ (Meier-Kolthoff et al., 2013) was used to calculate the digital DNA-DNA hybridization (dDDH).

The reference strain Saccharicrinis fermentans DSM 9555^T was obtained from the German Collection of Microorganisms and Cell Cultures GmbH and Labilibacter aurantiacus HQYD1^T was obtained from our laboratory. Both strains were cultured on modified MA at 33°C and studied in parallel with strains D04^T and AAT^T for physiological and chemotaxonomic comparisons. Cellular fatty acids and polar lipids were identified from a freeze-dried sample (40 mg and 50 mg) of cells grown to the exponential growth phase under optimal culture conditions on the modified MA. Cellular fatty acid methyl esters (FAMEs) were obtained from cells by saponification, methylation, and extraction following the MIDI protocol (Sasser, 1990). Cellular FAMEs were separated using gas chromatography and were identified and quantified using the Sherlock Microbial Identification System (version 6.1, MIDI 6890 with database TSBA6). Polar lipids were separated via two-dimensional silica gel TLC. Total lipid material was detected using molybdatophosphoric acid and the functional groups were determined using spray reagents specific for each group according to Fang et al. (2017). All polar lipid images were further analyzed as described by Minnikin et al. (1984). For respiratory quinone analyses, respiratory quinones were extracted from 200 mg of freeze-dried cell material using the two-stage method described by Minnikin et al. (1984). A silica-gel TLC plate (Merck Kieselgel 60 F254) was used to analyze the quinone type and the content of each quinone type was subsequently analyzed by HPLC according to the process described previously (Kroppenstedt, 1982).

The newly discovered strains D04^T and AAT^T of bacteria belonging to the Marinilabiliaceae family were facultative anaerobes, Gram-negative, rod-shaped, oxidase-positive, and catalase-negative. Strain D04^T had a width of 0.3 μm and a length of 3.4–4.1 μm, while strain AAT^T had a width of 0.4–0.5 μm and a length of 0.6–1.4 μm ( Supplementary Figure 1 ). Strain D04^T also demonstrated gliding motility, while strain AAT^T did not. Both strains grew as yellow-colored, smooth, and circular colonies on modified MA medium at 33°C. They were oxidase-positive, catalase-negative, and reduced nitrate to nitrite. The growth temperature range of the bacterial strains studied was between 20°C and 40°C and they were unable to grow at temperatures of 4°C and 45°C. Strains D04^T and AAT^T showed optimal growth at a temperature of 33°C. The pH range for growth of strain D04^T was 6.5–7.5, with optimal growth occurring at pH 7.0. Strain AAT^T was able to grow at pH values ranging from 6.0–7.5, with optimal growth occurring at a pH of 6.5. In terms of salt tolerance, strain D04^T was able to grow at NaCl concentrations ranging from 0% to 8.0% w/v, with an optimum NaCl concentration of 2.0%. Strain AAT^T was able to grow at NaCl concentrations ranging from 0.5% to 8.0% w/v, with an optimum NaCl concentration of 3.0%. The addition of a vitamin mixture to the medium increased their growth. The strains were positive for the hydrolysis of starch but negative for casein, CM-cellulose, alginate, and Tweens 20, 40, 60, and 80. Strain AAT^T was positive for DNA, while strain D04^T was negative. Both strains were resistant to antibiotics such as tobramycin, vancomycin, tetracycline, norfloxacin, neomycin, gentamycin, ofloxacin, streptomycin, kanamycin, and polymyxinB. However, strain D04^T was susceptible to erythromycin, ampicillin, penicillin, chloramphenicol, rifampin, and lincomycin, while strain AAT^T was between resistant and sensitive to cefotaxime sodium and carbenicillin.

Strains D04^T and AAT^T share similar morphological and biochemical characteristics with the other two species in the Marinilabiliaceae family, including being rod-shaped, Gram-stain-negative, and facultatively aerobic. They also showed positive results for various enzyme activities such as alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase, and naphthol-AS-BI-phosphohydrolase, as well as for tryptophan deaminase, Voges–Proskauer reaction, and arabinose. However, there were some differential phenotypic characteristics between strains D04^T and AAT^T and the two reference strains, as shown in Table 1 .

The nearly complete 16S rRNA gene sequences of strains D04^T (1480 bp) and AAT^T (1525 bp) were obtained. Sequence comparisons showed that strains D04^T and AAT^T had the highest 16S rRNA gene sequence similarity with the type strain of Saccharicrinis fermentans DSM 9555^T (94.1% and 94.9%, respectively), followed by Saccharicrinis marinus Y11^T (93.3% and 94.5%, respectively). The 16S rRNA gene sequences of strains D04^T and AAT^T were similar to those of Labilibacter aurantiacus HQYD1^T type strains (93.3% and 93.4%, respectively). The similarity of the 16S rRNA gene sequence between the two strains was 96.6%. The neighbor-joining, maximum-likelihood, and maximum-parsimony phylogenetic trees formed an independent sub-cluster supported by very high bootstrap values ( Figure 1 ). In the phylogenetic tree, strains D04^T and AAT^T are closely related to the members of the genera Saccharicrinis and Labilibacter. By using 16S rRNA gene sequence similarity and phylogenetic trees, Saccharicrinis fermentans DSM 9555^T and Labilibacter aurantiacus HQYD1^T were selected as reference strains.

The major fatty acids (>10%) of D04^T and AAT^T were iso-C_15:0 and anteiso-C_15:0, which was similar with the profiles of the two reference strains, although there were differences in the proportions of some fatty acids. Strain AAT^T also possesses major fatty acids, including iso-C_15:0 3-OH and iso-C_17:0 3-OH. Fatty acids (>0.5%) were listed in detail in Table 2 . The polar lipid profile of strain D04^T consisted of phosphatidylethanolamine (PE), three glycolipid (GL1-3) and one unknown polar lipid. The polar lipid profile of strain AAT^T consisted of phosphatidylethanolamine (PE), one glycolipid (GL), one aminophosphoglycolipid and one phosphoglycolipid. The presence of phosphatidylethanolamine (PE) was conserved in all strains. Further detailed polar lipid images with different specific strains are given in Supplementary Figure 6 . For respiratory quinone analyses, the results indicated that the major respiratory quinone of strains D04^T and AAT^T were identified to be MK-7, which matched other members in the family Marinilabiliaceae.

Plebeiobacterium marinum (ma.ri’num. L. fem. marinum, from the sea).

Cells are facultatively anaerobic, Gram-stain-negative, and rod-shaped, roughly 0.3 μm wide and 3.4–4.1 μm long. Gliding motility was not observed. Cells were observed to be yellow colored, flat, smooth, and with circular colonies at 33°C on the modified MA grown after 5 days. Strain was oxidase positive, catalase negative, and nitrate reduced to nitrite. Growth occurs at 20–40°C (optimum 33°C), pH 6.5–7.5 (optimum pH 7.0), and with 0–8% NaCl (optimum 2% w/v). The strain was positive for the hydrolysis of starch but negative for casein, DNA, CM-cellulose, alginate, and Tweens 20, 40, 60, and 80. The major fatty acids are iso-C_15:0 and anteiso-C_15:0. The predominant respiratory quinone is MK-7. The polar lipid profile consisted of a phosphatidylethanolamine, three glycolipids (GL1-3), and one unknown polar lipid. Cells are positive for alkali phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, and β-glucosidase. Cells are also positive for the utilization of tryptophan deaminase, Voges–Proskauer reaction, and arabinose. Acid is produced from d-arabinose, d-ribose, d-xylose, d-galactose, d-glucose, arbulin, aesculin ferric citrate, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-saccharose, starch, glycogen, d-gentiobiose, d-turanose, d-lyxose, potassium 2-keto-gluconate, potassium 5-keto-gluconate, l-arabinose, d-adonitol, methyl-β-d-xylopyranoside, d-fructose, d-mannose, l-rhamnose, dulcitol, inositol, inulin, d-raffinose, xylitol, d-tagatose, l-fucose, d-arabitol, and l-arabitol. In carbon source oxidation tests, positive results are obtained for dextrin, d-maltose, d-trehalose, d-cellobiose, gentiobiose, sucrose, d-turanose, stachyose, d-raffinose, α-d-lactose, d-melibiose, d-salicin, N-acetyl-β-d-mannosamine, N-acetyl-d-galactosamine, α-d-glucose, d-mannose, d-fructose, d-galactose, 3-methyl glucose, l-fucose, myo-inositol, d-glucose-6-PO_4, d-fructose-6-PO_4, gelatin, l-alanine, l-histidine, l-pyroglutamic acid, pectin, d-glucuronic acid, glucuronamide, α-keto-glutaric acid, l-malic acid, α-keto-butyric acid, propionic acid, acetoacetic acid, and acetic acid. The genomic DNA G+C content of the type strain is 36.8%.

The type strain D04^T (MCCC 1H00493^T = KCTC 92026^T) was isolated from coastal sediment collected from Xiaoshi Island, Weihai, China. Strain D04^T was stored in our own laboratory repository, ¹⁰ with code SDUM258008. The GenBank accession number for the 16S rRNA gene and the draft genome sequence of D04^T are OP745477 and JAPDPI000000000, respectively.

Plebeiobacterium sediminum (se.di.minum. L. gen. pl. n. sediminum of sediments, pertaining to source of isolation).

Cells are facultatively anaerobic, Gram-stain-negative, and rod-shaped, roughly 0.4–0.5 μm wide and 0.6–1.4 μm long. Gliding motility was observed. Cells were observed to be yellow colored, flat, smooth, and with circular colonies at 33°C on the modified MA grown after 5 days. Strain was oxidase positive, catalase negative, and nitrate reduced to nitrite. Growth occurs at 20–40°C (optimum 33°C), pH 6.0–7.5 (optimum pH 6.5), and with 0.5–8% NaCl (optimum 3% w/v). The strain was positive for the hydrolysis of starch, DNA, but negative for casein, CM-cellulose, alginate, and Tweens 20, 40, 60, and 80. The major fatty acids are iso-C_15:0, anteiso-C_15:0, iso-C_15:0 3-OH, and iso-C_17:0 3-OH. The predominant respiratory quinone is MK-7. The polar lipid profile consisted of phosphatidylethanolamine, one glycolipid (GL), one aminophosphoglycolipid, and one phosphoglycolipid. Cells are positive for alkali phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, leucine arylamidase, cystine arylamidase, trypsin, N-acetyl-β-glucosaminidase, α-glucosidase, and β-glucosidase. Cells are also positive for the utilization of tryptophan deaminase, Voges–Proskauer reaction, and arabinose. Acid is produced from d-arabinose, d-ribose, d-xylose, d-galactose, d-glucose, arbulin, aesculin ferric citrate, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-saccharose, starch, glycogen, d-gentiobiose, d-turanose, d-lyxose, potassium 2-keto-gluconate, potassium 5-keto-gluconate, erythritol, l-arabinose, d-fructose, d-mannose, l-rhamnose, N-acetylglucosamine, amygdalin, inulin, d-raffinose, xylitol, d-fucose, l-fucose, and l-arabitol. In carbon source oxidation tests, positive results are obtained for 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, α-d-glucose, d-mannose, d-fructose, d-galactose, l-rhamnose, d-glucose-6-PO₄, glycyl-l-proline, pectin, d-galacturonic acid, l-galactonic acid lactone, d-glucuronic acid, glucuronamide, methyl pyruvate, α-keto-butyric acid, and acetic acid. The genomic DNA G+C content is 33.4%.

The type strain AAT^T (MCCC1H00485^T = KCTC92028^T) was isolated from coastal sediment collected from Xiaoshi Island, Weihai, China. Strain AAT^T was stored in our own laboratory repository,¹⁰ with code SDUM258009. The GenBank accession number for the 16S rRNA gene and the draft genome sequence of AAT^T are OP745478 and JAPDPJ000000000, respectively.