Molecular phylogenetic analysis
The almost-complete 16S rRNA gene sequence of YIM B01967T was 1540 bp (GenBank accession number MW386301). Strain YIM B01967T showed the highest 16S rRNA gene sequence similarity with Viridibacillus arvi (99.05%) and Viridibacillus arenosi (98.92%). The NJ tree, MP tree, and ML tree for the 16S rRNA shared the same topology and were presented in Fig 1, Fig S1, and Fig S2, respectively.
The draft genome of strain YIM B01967T contained 112 contigs, with a total length of 4,553,251 bp and an N50 length of 171,059 bp (GenBank accession number JAEOAH000000000), and genome coverage of 14.0´. The DNA G+C content of strain YIM B01967T was determined from the genome to be 36.3 mol%. Strain YIM B01967T genome was annotated with 4,444 genes, included 4,200 protein-coding genes, 60 rRNA genes, 50 tRNA genes, 5 ncRNA genes, and 184 pseudogenes. In contrast, the draft genome of the reference strain Viridibacillus arvi DSM 16317T consists of 4,758,570 bp with an N50 contig length of 244,670 bp and a G+C content of 35.0 mol%. The ANI value between strain YIM B01967T and Viridibacillus arvi DSM 16317T was 61.03% based on the draft genome sequence, which was lower than the 95.0% cut-off for species demarcation (Richter et al. 2016). The DNA-DNA hybridization values between strain YIM B01967T and Viridibacillus arvi DSM 16317T was 32.1%, which was much lower than the threshold value (70%) recommended for distinguishing novel prokaryotic species (Chun et al. 2018). The phylogenomics tree of YIM B01967T with the closely related strains was presented in Fig 2.
Morphological, physiological, and biochemical analyses
The YIM B01967T strain was Gram-positive, sporulating and active rod-shaped. The endospores were approximately round and are located in the sporangia with enlarged or slightly enlarged ends. (Fig S1). Cells can grow at pH 6.0-9.0 (optimum, pH 7.5) and in the presence of 0-2% (w/v) NaCl (optimum in 2% NaCl), Other physiological characteristics are given in Table 1. Strain YIM B01967T was Catalase-positive and oxidase-negative, and this feature was also present in Viridibacillus arvi DSM 16317T and Viridibacillus arenosi DSM 16319T.
Table 1
Phenotypic and chemotaxonomic characteristics that differentiate strain YIM B01967T from closely related reference strains
Characteristic
|
1
|
2
|
3
|
Colony colour
|
Light yellow
|
White
|
White
|
Growth conditions
|
|
|
|
Temperature (°C)
|
15-37
|
5-40
|
5-37
|
Growth in 5 % NaCl
|
-
|
-
|
+
|
Anaerobic growth
|
w
|
w
|
-
|
API 20NE
|
|
|
|
Urease
|
+
|
+
|
-
|
β-Glucosidase
|
+
|
-
|
-
|
Malic acid
|
+
|
-
|
-
|
Nitrate reduced
|
+
|
-
|
+
|
Gelatin hydrolysis
|
+
|
-
|
+
|
Aesculin hydrolysis
|
+
|
-
|
-
|
Arginine double hydrolysis
|
+
|
w
|
N
|
ONPG
API ZYM
|
-
|
w
|
-
|
Casein hydrolysis
|
-
|
w
|
-
|
Esterase
|
+
|
-
|
N
|
Lipid esterase
|
+
|
-
|
N
|
Chymotrypsin
|
-
|
+
|
N
|
β-Galactosidase
|
-
|
+
|
N
|
Acid production from(50CHB)
|
|
|
|
N-acetylglucosamine
|
-
|
w
|
-
|
D-fructose
|
-
|
+
|
-
|
Myo-inositol
|
+
|
-
|
-
|
D-arabitol
|
+
|
-
|
-
|
DNA G + C content (mol%)
|
36.3
|
35.0
|
35.0
|
Polar lipids
|
DPG, PME, PG, PE, 2PL
|
DPG, PG, PE, APL, 2PL
|
DPG, PG, PE, APL, 2PL
|
Major fatty acids(>10%)
|
iso-C15:0, anteiso-C15:0 , C16:1 ω10c
|
iso-C15:0, anteiso-C15:0
|
iso-C15:0, anteiso-C15:0
|
Strains: (1) YIM B01967T; (2) Viridibacillus arvi DSM 16317T (The data of API 20NE, API ZYM, acid production from (50CHB) and major fatty acids are from this research, other data from Heyrman et al. 2005; Albert et al. 2007); (3) Viridibacillus arenosi DSM 16319T (data from Heyrman et al. 2005; Albert et al. 2007).
+ Positive, - negative, w weak. All data were obtained from this study except where indicated.
|
Chemotaxonomic characterization
The cell wall amino acids of strain YIM B01967T contained aspartic acid, glutamic acid, alanine and lysine. As hese amino acids matches those of the reference strain Viridibacillus arvi DSM 16317T, we deduce that the peptidoglycan was the type A4a (Schleifer and Kandler 1972) with an L-Lys–D-Asp interpeptide bridge (Fig S4). Ribose, glucose, arabinose, galactose, and mannose were the major whole-cell sugars of strain. The quinone system consisted of the major compound MK-8 and moderate amounts of MK-7 in strain, which was identical to that found in members of the genus Viridibacillus (Albert et al. 2007). The cellular fatty acids profile consisted of iso-C15:0 (34.64%), anteiso-C15:0 (12.72%) and C16:1 ω10c (11.53%) as major fatty acids (>10%), and C16:1 ω7c alcohol(8.95%), anteiso-C17:0(6.15%), iso-C16:0(6.02%), Summed Feature 4(iso I-C17:1/anteiso B)(5.02%), iso-C14:0(3.68%), iso-C17:1 ω10c(3.06%), iso-C17:0(2.94%) and C16:0(2.21%) as minor fatty acids(>1%).(Table S1). The polar lipids of the strain YIM B01967T were diphosphatidylglycerol (DPG), phosphatidylmethylethanolamine (PME), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and two unidentified phospholipids (PL1, PL2). (Fig S5). Chemotaxonomic analyses including cell-wall peptidoglycan, whole-cell fatty acids, cell-wall sugars, and polar lipids exhibited the close similarity of strain YIM B01967T to type strains of the closely related species, which confirmed its affiliation to the genus Viridibacillus, with sufficient differences to warrant its proposal as representing a novel species of the genus Viridibacillus.
The detailed differentiating phenotypic and chemotaxonomic characteristics features between YIM B01967T and the reference strains were given in Table 1.
Consequently, based on the above findings, we characterized strain YIM B01967T as a novel species within the genus Viridibacillus, for which the name Viridibacillus soil sp.nov.is proposed.
Description of Viridibacillus soli sp.nov.
Viridibacillus soli (so′li. L. neut. gen. n. soli of soil, the source of the type strain)
Cells are straight, round-ended, Gram-positive, motile rods (0.6-0.8×2.5-3.0 mm), occurring singly and in pairs, the endospores are approximately round and are located in the sporangia with enlarged or slightly enlarged ends. Growth occurs at temperature range of 15-37 °C (optimum, 28 °C), pH 6.0-9.0 (optimum, pH 7.5) and in the presence of 0-2% (w/v) NaCl (optimum 2% NaCl). Catalase-positive and oxidase-negative. In API 20NE, positive for urease, β-glucosidase, and malic acid, nitrate is reduced, gelatin is hydrolyzed. In API ZYM, positive for alkaline phosphatase, esterase, lipid esterase, leucine aramidase, valine Aramidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. In the Biolog GEN III MicroPlate, positive for N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, D-galactose, 3-methyl-glucose, inosine, D-mannitol, D-arabitol, myoinositol, glycerol, D-glucose-6-PO4, D-fructose-6-PO4, gelatin, L-alanine, L-arginine, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, quinic acid, D-saccharic acid, D-lactic acid, methyl ester, L-lactic acid, α-keto-glutaric acid, L-malic acid, Tween 40, α-hydroxy-butyric acid, β-hydroxy-D, L-butyric acid, acetic acid, 1% sodium lactate, nalidixic acid, aztreonam. In the API 50CHB gallery, acid is not produced from any of the carbohydrate substrates. Cell wall peptidoglycan is presumably of the type A4a with an L-Lys–D-Asp interpeptide bridge. The cell-wall sugars are ribose, glucose, arabinose, galactose, and mannose. The quinone system consists of the major compound MK-8 and moderate amounts of MK-7. The fatty acids (>5% of total fatty acids) are iso-C15:0, anteiso-C15:0, C16:1 ω10c, C16:1 ω7c alcohol, anteiso-C17:0, iso-C16:0. The polar lipids profile include diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine phosphatidylethanolamine, and moderate to minor amounts of two unknown phospholipids (PL1, PL2). The DNA G+C content of the type strain is 36.3 mol% based on the draft genome sequence.
The type strain, YIM B01967T (= KCTC 43249 T = CGMCC 1.18436 T), was isolated from a soil sample collected from a forest in Ailaoshan National Nature Reserve, Yuxi City, Xinpin county, Yunnan province, China.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence and the draft genome sequence are MW386301 and JAEOAH000000000 respectively.