Genome analysis
The whole genomes of DS1-an-13321T and DS1-an-2312T, 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-13321T and DS1-an-2312T 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-13321T 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-2312T 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-13321T encodes one BGC belonging to the nonribosomal peptide synthetase family and nine MGCs, while the genome of strain DS1-an-2312T encodes one BGC belonging to the linear azoline-containing peptides family and nine MGCs. In strain DS1-an-13321T, 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-2312T, 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-13321T and DS1-an-2312T 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-13321T encoded a total of 155 CAZymes consisting of 84 GHs, 32 GTs, 13PLs, seven CEs and 19 CBMs, while strain DS1-an-2312T encoded a total of 249 CAZymes, an amount 1.6 times greater than that of DS1-an-13321T, consisting of 128 GHs, 37 GTs, 27 PLs, 35 CEs, and 22 CBMs. The number of GH genes per genome in strain DS1-an-13321T was 18.81 (GHs/Mb), while in strain DS1-an-2312T 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-13321T and DS1-an-2312T 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-13321T and DS1-an-2312T contained genes predicted to be involved in laminarin degradation. The genome of strain DS1-an-13321T harbored genes encoding for four GH3, one GH16, and one GH30, while strain DS1-an-2312T 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-13321T nor DS1-an-2312T 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-13321T and DS1-an-2312T were located separately, and each gene was collocated with SusC/SusD in the genomes. For strain DS1-an13321T, 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-13321T 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-2312T 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-2312T was identified as an anaerobic bacterium capable of utilizing xylan as a sole carbon source. Whole-genome analysis of strain DS1-an-2312T 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-2312T 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-2312T (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-2312T 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-13321T, 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-2312T 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-2312T 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-2312T, not DS1-an-13321T, 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-2312T 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-2312T. 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-2312T exhibiting the ability to utilize xylose. The major fermentation products of strains DS1-an-13321T and DS1-an-2312T 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-2312T 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-2312T 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-C15:0, anteiso-C15:0, iso-C15:0 3-OH, and iso-C17:0 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%). H2S 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-C15:0, anteiso-C15:0, iso-C15:0 3-OH, and iso-C17:0 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-13321T (= KCTC 25031T = DSM 115329T) 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%). H2S 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-C15:0, anteiso-C15:0, iso-C15:0 3-OH, and iso-C17:0 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-2312T (= KCTC 25032T = DSM 115328T) 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.