Genome Analyses
The draft genome sequence of strain 2E275T consisted of 4,565,251 bp in length, producing 156 contigs which length was between 301 bp and 299,961 bp, and the N50 value was 79,060. The sequencing depth of coverage was 150×. 3901 genes, with 3788 protein-coding genes, 3 rRNAs and 64 tRNAs, were identified. The DNA G + C content was 38.2%, which was different from that of C. agarivorans YM01T (44.8 %) and C. sediminis D2T (40.4 mol%). One 16S rRNA gene sequence (1525 bp) was extracted from the genome of strain 2E275T, showing 100% similarity with that from traditional Sanger sequencing. The ANI value between genomes of strain 2E275T and C. agarivorans YM01T, C. sediminis D2T were 73.5% and 72.2% respectively, and the dDDH value were 22.9% and 21.2% respectively (Table S1). Both of them were lower than the species differentiation thresholds of 95% and 70%, respectively (Thompson et al. 2013; Meier-Kolthoff et al. 2013). Furthermore, we calculated the ANI values between Catenovulum species and Algibacillus agarilyticus which are in the same node and found that the values were both less than 75. It was also useful for distinguishing strain Algibacillus agarilyticus from the genus Catenovulum.
Based on secondary metabolite analysis predicted by antiSMASH, strain 2E275T has 5 putative biosynthetic gene clusters (BGCs), including ectoine cluster, NRPS-like cluster, RiPP-like biosynthetic cluster, one arylpolyene region and one hserlactone region, which suggested strain 2E275T might synthesize these active compounds. The similarity between ectoine and the known cluster was 83%. We also found that strain 2E275T shares four secondary metabolites with its most similar strain C. agarivorans YM01T. Furthermore, 2,187 entries (56.3%) were annotated and assigned to functional categories by KEGG. The genes narB, narD, narG, narH and narL have been predicted, and the annotation results showed that strain 2E275T had dissimilatory nitrate reduction metabolism pathway. During the dissimilatory nitrate reduction, nitrate was reduced to nitrite under the function of genes narG, narH and narL, and then to ammonia under the action of genes narB and narD. In addition, strain 2E275T contained the complete pathway of assimilatory sulfate reduction. During this reaction, sulfate is reduced to H2S.
For the polysaccharides utilization, the genome of strain 2E275T has been analyzed by the CAZy database. Strain 2E275T contains 117 carbohydrate-active enzymes, glycoside hydrolases (GHs) are the largest number of enzymes. In the GH family, there are one α-agarase (EC 3.2.1.158) and four β- agarases (EC 3.2.1.81). α-agarase belongs to GH96 family and β- agarases belong to GH50 family. According to report, they can hydrolyze the α-1,3 glycosidic bond and β-1,4 glycosidic bond between two agarbiose respectively (Michel et al. 2006). This discovery could explain why strain 2E275T can degrade agar significantly (Fig. 2).
Physiology
Cells of strain 2E275T were Gram-stain-negative, strictly aerobic, rod-shaped and motile by means of a single polar flagellum. Strain 2E275T was approximately 0.4-1.0 µm width and 1.0–3.0 µm length (Fig. S2). Strain 2E275T could grow at 4–42°C (optimum, 37°C), at pH 5.5–10.5 (optimum, pH 6.5-7.0) and with 0–12% (w/v) NaCl (optimum, 3%). Strain 2E275T hydrolyze Tweens (20, 60 and 80), starch, agar, alginate, and cellulose, but not DNA and Tween 40. Strain 2E275T was positive for catalase and oxidase. Nitrate is reduced to ammonia. Strains 2E275T was resistant to cefotaxime sodium (30 µg), vancomycin (30 µg), gentamycin (10 µg), ampicillin (10 µg), chloramphenicol (30 µg), ceftriaxone (30 µg), ofloxacin (5 µg), norfloxacin (10 µg), tobramycin (10 µg), erythromycin (15 µg), rifampin (5 µg), penicillin (10 µg), clarithromycin (15 µg), kanamycin (30 µg), but susceptible to streptomycin (10 µg), neomycin (30 µg), tetracycline (30 µg), lincomycin (2 µg). Strains 2E275T could be distinguished from the closely related strains by a few physiological and biochemical characteristics, including the results of API and Biolog tests (Table 1). The complete morphological, physiological and biochemical characteristics of strains 2E275T were provided in the species description and Table 1.
Chemotaxonomy
Fatty acid analyses revealed that the major fatty acids of strain 2E275T were summed feature 3 (42.1%, C16:1ω7c and/or C16:1ω6c), C16:0 (26.1%) and summed feature 8 (18.4%, C18:1ω7c). Although C. agarivorans YM01T and C. sediminis D2T share the same major cellular fatty acids summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 8 (C18:1ω7c) and C16:0 with strain 2E275T, the proportion of the major cellular fatty acids differentiate strain 2E275T from the others, as shown in Table 2. Ubiquinone-8 was detected as the predominant respiratory quinone of strain 2E275T, which is consistent with other Catenovulum strains. The polar lipids of strains 2E275T were phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one phosphoaminolipid (PN), two unidentified phospholipids (PL) and three unknown lipids (Fig. S3).
Description of Catenovulum algicola sp. nov.
Catenovulum algicola (al.gi’co.la. L. fem. n. alga, seaweed, alga; L. masc./fem. n. suff. -cola, inhabitant, dweller; N.L. masc./fem. n. algicola, alga-dweller).
Cells are Gram-stain-negative, motile by means of a single polar flagellum, strictly aerobic. Cells are straight to slightly curved rods, 0.4–1.0 µm in width and 1.0–3.0 µm in length. Colonies on MA are pale white colored, smooth, circular and 1.0–1.5 mm in diameter after 2 days incubation at 37°C. Growth occurs at 4–42°C, pH 5.5–10.5 and in the presence of 0–12% (w/v) NaCl. Optimal growth is observed at 37°C, pH 6.5-7.0 and in the presence of 3% (w/v) NaCl. Nitrate is reduced to nitrite. The genomic DNA G + C content of the type strain is 38.2%. Agar, starch, alginate, Tweens (20, 60 and 80), cellulose are hydrolysed, but DNA and Tween 40 are not. Cells are positive for catalase, oxidase activity, but negative for utilization of citrate, indole and H2S production. Voges–Proskauer reaction are positive, glucose can be fermented. Alkaline phosphatases, esterase (C4), esterase lipase (C8), leucine arylamidases, acid phosphatases, α-glucosidase, α-galactosidase and naphthol-AS-BI-phosphohydrolase are present, but lipase (C14), valine arylamidases, cystine aminopeptidase, trypsin, chymotrypsin, lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, urease, gelatinase, tryptophan deaminase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and β-fucosidase are absent. Acid can be produced from l-arabinose, d-xylose, methyl β-d-Xylopyranoside, d-xylose, d-glucose, d-fructose, d-mannose, l-Rhamnose, inositol, amygdalin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-sucrose, inulin, d-raffinose, d-turanose, d-melezitose, salicin, glycogen, starch, d-gentiobiose, d-lyxose and potassium 5-ketogluconate. According to Biolog GEN III MicroPlates assays, strain 2E275T can utilize the following substrates as carbon and energy sources: Dextrin, d-Maltose, 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, 3-Methyl Glucose, d-Fucose, l-Fucose, l-Rhamnose, d-Fructose-6-PO4, l-Histidine, Pectin, d-Galacturonic, l-Galactonic, d-Gluconic Acid, d-Glucuronic Acid, Glucuronamide, α-Keto-Glutaric Acid, Acetoacetic Acid. The predominant isoprenoid quinone is ubiquinone-8. The polar lipids are PE and PG as major components, two unidentified phospholipids (PL), aminophospholipid (PN) and three unidentified lipids (L) as moderate to minor components. The major cellular fatty acids are summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 8 (C18:1ω7c) and C16:0.
The type strain, 2E275T (= MCCC 1H00528T = KCTC 92643T), was isolated from kelp seedlings collected from a kelp nursery pond of Weihai, China.