Phenotypic characteristics
The most predominant cellular fatty acid (>10 % of the total fatty acids) of strain JC010T were summed feature 9 (iso-C17:1 ω9c) (24.9 %), C16:0 (22.2 %), iso-C15:0 (17.9 %), summed feature 3 (C16:1 ω6c/ C16:1 ω7c) (12.7 %), which was similar to all of the reference strains. A comparison of the cellular fatty acids of strain JC010T and the reference strains is shown in Table S3. The predominant isoprenoid quinone of strain JC010T was identified as menaquinone-7 (MK-7), which was the same as other strains in the genus Maridesulfovibrio. The polar lipids found in JC010T comprised one phosphatidylcholine (PC), one phosphatidylethanolamine (PE), one phosphatidylglycerol (PG), two phospholipids (PL) (Fig. S1).
The most predominant cellular fatty acid (>10 % of the total fatty acids) of strain JC022T were C16:0 (22.7 %), iso-C15:0 (16.0 %), summed feature 3 (C16:1 ω6c/ C16:1 ω7c) (13.1%), summed feature 9 (iso-C17:1 ω9c) (24.6 %), which was similar to all of the reference strains. A comparison of the cellular fatty acids of strain JC022T and the reference strains is shown in Table S3. The predominant isoprenoid quinone of strain JC022T was identified as menaquinone-7 (MK-7), which was the same as other strains in the genus Maridesulfovibrio. The polar lipids found in JC022T comprised one phosphatidylethanolamine (PE), two phosphatidylglycerols (PG), one phosphatidylcholine (PC), two phospholipids (PL) (Fig. S2).
Table 1. Differential characteristics between strain JC010T, JC022T and the members of their related species.
Characteristics
|
1
|
2
|
3 a
|
4 b
|
5 c
|
Morphology
|
Vibrios
|
Vibrios
|
Vibriosa
|
Curved
rodsb
|
Rodsc
|
Cell size (μm)
|
0.5-0.7×2.0-3.0
|
0.8-1.0×5.0-6.0
|
0.5-0.8×1.3-2.5
|
0.3×3.0
|
0.7-0.9×2.0-3.0
|
Temperature(°C)
(optimum Temperature)
|
4-37(28)
|
10-37 (28)
|
ND-45(30-36)
|
5–34.5(32.5-34.5)
|
15-45(40)
|
NaCl (%)
(optimum NaCl%)
|
1.0 -5.5
(4.0)
|
2.5-6.0
(3.0)
|
0.5-12
(2.0-4.0)
|
0-3.5
(1.2)
|
1.0-12
(3.0)
|
pH
(optimum pH)
|
4.0-9.0
(7.0)
|
4.0-8.0a
(7.0)
|
ND
(7.8)
|
5.5–7.5
(6.8-7.3)
|
6-8.5
(6.7)
|
Enzyme activity
|
|
|
|
|
|
alginate
|
+
|
-
|
-
|
-
|
-
|
Oxidase
|
-
|
-
|
-
|
-
|
+
|
casein
|
+
|
-
|
-
|
+
|
-
|
DNA G+Ccontent (mol %)
|
49.8
|
48.0
|
47.1
|
42.7
|
47.7
|
Electron acceptors
|
|
|
|
|
|
Sulfate
|
+
|
+
|
+
|
+
|
+
|
Sulfite
|
w
|
+
|
+
|
+
|
+
|
Thiosulfate
|
-
|
-
|
+
|
+
|
+
|
Nitrate
|
+
|
+
|
+
|
-
|
-
|
Fumarate
|
+
|
-
|
+
|
+
|
-
|
Fermentation
|
|
|
|
|
|
Lactate
|
w
|
+
|
+
|
+
|
+
|
Fumarate
|
+
|
-
|
+
|
+
|
-
|
Formate
|
+
|
w
|
+
|
-
|
ND
|
Malate
|
+
|
-
|
+
|
+
|
-
|
Pyruvate
|
-
|
-
|
-
|
+
|
-
|
Strains: 1. JC010T; 2. JC022T; 3. Maridesulfovibrio salexigens DSM 2638T; 4. Maridesulfovibrio zosterae DSM 11974T 5. Maridesulfovibrio salinus JCM 31065T. Data were obtained in this study unless indicated otherwise. +, positive; -, negative. W, weak reaction; ND, not detected; o, obligately anaerobic; f, facultatively anaerobic, WGS, whole-genome sequence survey. Data were taken from: a, Postgate J R et al. (1966); b, Nielsen JT et al. (1999); c, Zouhaier Ben Ali Gam et al. (2018).
Phylogenetic analysis based on 16S rRNA gene sequences
Strain JC010T showed the highest 16S rRNA gene sequence similarity to Maridesulfovibrio salexigens DSM 2638T (97.9 %), followed by Maridesulfovibrio zosterae lacT (96.8 %) and Maridesulfovibrio hydrothermalis AM13T (96.4 %). Phylogenetic analyses based on the NJ, MP, and ML algorithms showed that strain JC010T formed a distinct cluster within the genus Maridesulfovibrio and was different from all recognized species (Fig S4).
The 16S rRNA gene sequence similarity based on EzBioCloud data demonstrated that strain JC022T has the highest similarity to Maridesulfovibrio salexigens DSM 2638T (97.4 %), followed by Maridesulfovibrio lacT (96.9 %), and Maridesulfovibrio AM13T (96.7 %). Phylogenetic analyses based on the NJ, MP, and ML algorithms showed that strain JC022T formed a distinct cluster within the genus Maridesulfovibrio and was different from all recognized species (Fig S4). And there is a sequence similarity of 98.4 % between strain JC010T and JC022T based on the 16S rRNA gene sequence analysis.
Phylogenetic analysis based on whole-genome sequences
The ANI value of strain JC010T against its most closely related species Maridesulfovibrio salexigens DSM 2638T was 81.9 %, which was much lower than the cut-off value of 95–96 %. The digital DDH value between strain JC010T and Maridesulfovibrio salexigens DSM 2638T was 24.9 %, which was lower than the cut-off point of 70 % for the delineation of a novel species. The ANI value of strain JC022T against its related species Maridesulfovibrio salexigens DSM 2638T was 82.5 %, which was lower than the cut-off value of 95–96 %. The digital DDH value between strain JC022T and Maridesulfovibrio salexigens DSM 2638T was 25.6 %, which was much lower than the cut-off point of 70 % for the delineation of a novel species. Besides, phylogenetic tree based on the whole genome was constructed using UBCG pipeline (Seong-In Na et al 2018). The result shows that the strain JC010T and JC022T formed a distinct cluster within the genus Maridesulfovibrio and was different from all recognized species (Fig 2).
The genome of strain JC010T comprised 4 377 840 bp, with 3968 predicted genes, including 83 RNAs and 4051 genes. In the genome of JC010T, seventy-three tRNAs, four non-coding RNAs, and a total of three rRNA genes (one 23S rRNAs, one 16S rRNA, and three 5S rRNAs) were classified. The detailed draft genomic features of strain JC010T were summarized in Table S1. The genome of strain JC022T comprised 4 418 640 bp, with 3973 predicted genes, including 102 RNAs and 4075 genes. In the genome of JC022T, eighty tRNAs, four non-coding RNAs, and a total of three rRNA genes (six 23S rRNAs, three 16S rRNA, and nine 5S rRNAs) were classified. The detailed draft genomic features of strain JC022T are summarized in Table S2. In order to identify metabolic pathways related to the sulfate reduction system in strain JC010T and JC022T, the genome of strain JC010T and JC022T were annotated by the RAST server. Three genes, which coding sulfate-reduction-relating proteins (sulfate permease, sulfite reductase clustered protein, sulfur carrier protein) were found both in JC010T and JC022T under the analysis and classification.
Strain JC010T, JC022T, Maridesulfovibrio salexigens DSM 2638T (ACCN01000000) and Maridesulfovibrio zosterae DSM 11974T (AUDC00000000) were annotated based on clusters of orthologous groups (COG) database and the result are shown in Fig. S3. It was found that strain JC010T and JC022T were significantly in line with their reference strains. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, adenylylsulfate reductase subunit A (aprA), adenylylsulfate reductase subunit B (aprB), sulfite reductase (dsrA), and sulfite reductase (dsrB), which were identified as the key enzymes of dissimilatory sulfate reduction for SRB, were found in strain JC010T, JC022T and their reference strains. To confirm the respiration type of strain JC010T and JC022T, sodium bicarbonate, sodium sulphate, sodium thiosulfate, and sodium nitrate were added to the sterile fluid enrichment medium (anaerobic 2216E) respectively to detect their metabolic activity individually. The results showed that strain JC010T and JC022T could grow well under each condition, which was consistent with the result obtained from genome analysis.
Taxonomic conclusions
The major features of strain JC010T and strain JC022T were significantly in line with the strain in the genus Maridesulfovibrio, including colony morphology, the presence of catalase and oxidase activity, hydrolysis of gelatin, CM-cellulose, DNA, chitin, casein, Tweens 20, 40 and 80, the major respiratory quinone, the predominant cellular fatty acids (>10 %) and the DNA G+C content. However, there were some differences in the phenotypic characteristics between JC010T, strain JC022T, and their reference strains, including cell shape, temperature range for growth, hydrolysis, and utilization of some substrates, enzyme activities, electron acceptors and fermentative growth (Table 1). Overall consideration of phenotypic, phylogenetic, and genetic data, strain JC010T and strain JC022T were assigned as members of novel species in the genus Maridesulfovibrio, for which Maridesulfovibrio caeruleilacunae sp. nov and Maridesulfovibrio oucae sp. nov. are proposed respectively.
Description of Maridesulfovibrio caeruleilacunae sp. nov.
caeruleilacunae (cae.ru.le.i.la.cu’nae. L. masc. adj. caeruleus blue; L. fem. n. lacuna a hole; N.L. gen. fem. n. caeruleilacunae of a blue hole)
Cells are Gram-staining-negative, obligately anaerobic, motile, arcuation, and flagellated. Colonies on MA are round, beige, regular, and smooth. The cell size is approximately 2.0-3.0 μm in length and 0.5-0.7 μm in width after culture on MA for 72 h at 28 °C. The growth temperature is in the range of 4-37 °C (optimum 28 °C). The salinity range for growth is 1.0-5.5 % (w/v) NaCl (optimum 4.0 %), and the pH range is 4.0-9.0 (optimum 7.0). Oxidase and catalase are negative. Casein, alginate, and cellulose can be hydrolyzed, but DNA, gelatin, starch, chitin, and Tweens 20, 40, 80, cannot be hydrolysed. In the API 20A strip, acid is produced from glucose, sucrose, maltose, gelatin, glycerin, cellobiose, mannose, sorbitol, rhamnose, trehalose. In BIOLOG AN plate, it can use the N-acetyl-D-galactosamine, N-acetyl-β-D mannose, adonitol, D-arabitol, D-cellobiose, dextrin, dulcitol, D-fructose, L-fucose, D-galactose, gentiobiose, D-gluconic acid, D-glucosaminic acid, α-D-glucose, α-D-glucose-1-phosphate, D-glucose 6-phosphate, glycerin, DL-α-glycerophosphoric acid, m-inositol, maltose, maltotriose, D-maltotriose, D-mannose, D- melibiose, 3-methyl-D-glucose, α-methyl-D-galactoside, β-methyl-D-galactoside, β-methyl-D-glucoside, D-raffinose, L-rhamnose, sucrose, D-trehalose, turanose, acetic acid, formic acid, β-hydroxybutyric acid, DL-lactic acid, L-lactic acid, D-methyl lactate, D-malic acid, L-malic acid, propanoic acid, propanoic acid, methyl pyruvate, D-glucaric acid, succinic acid, alaninamide, L-alanine, L-alanyl-L-glutamine, L-alanyl-L-histidine, glycyl-L-proline, L-serine, L-threonine, L-valine, L-aspartic acid. Other substrates cannot be used. The major respiratory quinone is MK-7. The dominant fatty acids (>10 %) are C16:0, iso-C15:0, Summed features 3 (C16:1 ω6c/ C16:1 ω7c) and Summed features 9 (iso-C17:1 ω9c). The major polar lipids are one phosphatidylcholine (PC), one phosphatidylethanolamine (PE), one phosphatidylglycerol (PG), and two phospholipids (PL1-2).
The species belongs to the genus Maridesulfovibrio of the family Desulfovibrionaceae. The type strain, JC010T (= JCM 39061T = MCCC 1K03847T), was isolated from Yongle Blue Hole of the South China Sea at a water depth of 160m. The DNA G+C content of the type strain is 49.8 mol %. The Whole-Genome Shotgun project of strain JC010T have been deposited at DDBJ/ENA/GenBank under the accession VOIQ00000000. The version described in this paper is version VOIQ01000000. The GenBank accession number for the 16S rRNA gene sequence of strain JC010T is MK578583.
Description of Maridesulfovibrio oucae sp. nov.
oucae (ou'cae. N.L. gen. n. oucae of OUC, acronym of Ocean University of China)
Cells are Gram-staining-negative, facultatively anaerobic, non-motile, short rod-shaped, and non-flagellated. Colonies on MA are round, beige, regular, and smooth. The cell size is approximately 1.5-3.0 μm in length and 0.3-0.5 μm in width after culture on MA for 72 h at 16 °C. The growth temperature is in the range of 4-37 °C (optimum 16 °C). The salinity range for growth is 1.0-6.0 % (w/v) NaCl (optimum 3.0 %), and the pH range is 6.0-9.0 (optimum 7.0). Oxidase and catalase are negative. Gelatinase and starch can be hydrolyzed, but DNA, cellulose, Tweens 20, 40, 80, casein, and alginate cannot be hydrolysed. In the API 20A strip, acid is produced from urea, glucose, sucrose, maltose, melezitose, mannose, sorbitol, rhamnose. In BIOLOG AN plate, it can use the N-acetyl-D-galactosamine, adonitol, D-arabitol, D-cellobiose, dextrin, D-fructose, L-fucose, D-galactose, D-Galacturonic acid, gentiobiose, D-gluconic acid, D-glucosaminoic acid, α-D-glucose, α-D-glucose-1-phosphate, DL-α-glycerophosphoric acid, m-inositol, α-D- lactose, maltose, maltotriose, D-maltotriose, D-mannose, D- melibiose, 3-methyl-D-glucose, α-methyl-D-galactoside, β-methyl-D-glucoside, D-raffinose, L-rhamnose, D- sorbitol, sucrose, D-trehalose, turanose, acetic acid, formic acid, β-hydroxybutyric acid, DL-lactic acid, L-lactic acid, D-methyl lactate, D-malic acid, L-malic acid, propanoic acid, methyl pyruvate, glycine - L-aspartic acid, glyceryl-L-glutamine, glycyl-L-proline, L-serine, L-threonine, L-valine, L-aspartic acid, 2'-deoxyadenosine, inosine, thymidine, uridine. The major respiratory quinone is MK-7. The dominant fatty acids (>10 %) are C16:0, iso-C15:0, Summed features 3 (C16:1 ω6c/ C16:1 ω7c) and Summed features 9 (iso-C17:1 ω9c). The major polar lipids are one phosphatidylcholine (PC), one phosphatidylethanolamine (PE), one phosphatidylglycerol (PG), two phospholipids (PL1-2), and three unidentified lipids (L1-3).
The species belongs to the genus Maridesulfovibrio of the family Desulfovibrionaceae. The type strain, JC022T (= JCM 39062T = MCCC 1K03848T), was isolated from Yongle Blue Hole of the South China Sea at a water depth of 190m. The DNA G+C content of the type strain is 48.0 mol %. The Whole-Genome Shotgun project of strain JC022T have been deposited at DDBJ/ENA/GenBank under the accession VOPZ00000000. The version described in this paper is version VOPZ01000000. The GenBank accession number for the 16S rRNA gene sequence of strain JC022T is MK578584.