Strains SKP4-8T and SKP8-2T were Gram-stain-positive, strictly aerobic and spore-forming rods. Endospores are produced at terminal position in swollen sporangia (Supplementary Fig. S1). Colonies were smooth, circular, low convex and cream in the color (1–2 mm in dimeter) after grew on modified JCM medium no. 377 agar at 37°C for 2 days. Motile by peritrichous flagella. No growth in anaerobic condition. Strain SKP4-8T grew in 1–16 % (w/v) NaCl (optimum 5–10 %), at 25–45°C (optimum 37°C) and at pH 6.0–9.0 (optimum 7.0). Strain SKP8-2T grew in 0–20 % (w/v) NaCl (optimum 3–10 %), at 25–45°C (optimum 37°C) and at pH 6.0–9.0 (optimum 8.0). The results of phenotypic characteristics are given in the species description and listed in Table 1. The strains SKP4-8T and SKP8-2T were sensitive to chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), novobiocin (5 µg), penicillin G (20 U), rifampicin (5 µg) and tetracycline (30 µg) but were resistant to ampicillin (10 µg) and streptomycin (10 µg).
Table 1
Differential characteristics of strains SKP4-8T, SKP8-2 and Allobacillus halotolerans LMG 24826T. All data are from this study. +, positive reaction; -, negative reaction.
Characteristics
|
SKP4-8T
|
SKP8-2T
|
LMG 24826T
|
Temperature range for growth (optimum) (°C)
|
25–45 (37)
|
25–45 (37)
|
20–45 (37)
|
NaCl range for growth (optimum) % (w/v)
|
1–16 (5–10)
|
0–20 (3–10)
|
1–20 (5–10)
|
Nitrate reduction
|
+
|
+
|
-
|
Hydrolysis of Tween 80
|
+
|
-
|
-
|
API ZYM tests
|
|
|
|
C14 Lipase
|
+
|
-
|
-
|
Trypsin
|
+
|
-
|
-
|
Acid phosphatase
|
+
|
+
|
-
|
Acid production from:
|
|
|
|
D-Fructose
|
-
|
+
|
-
|
D-Galactose
|
|
+
|
|
D-Mannose
|
-
|
+
|
-
|
D-Ribose
|
-
|
-
|
+
|
Strain SKP4-8T and SKP8-2T contained meso-diaminopimelic acid in the cell wall peptidoglycan and the major menaquinone was MK-7. The major fatty acids found in strain SKP4-8T were iso-C15:0 (54.4 %), antesio-C15:0 (11.9 %), and iso-C17:0 (11.6 %). In addition, the other fatty acids, C16:0 (0.8 %), iso-C14:0 (1.8 %), iso-C16:0 (3.2 %), anteiso-C17:0 (4.5 %), C16:1ω11c (0.6 %) C16:1ω7c alcohol (5.7 %) and summed feature 4 (1.7 %) were also detected and are different from strain SKP8-2T (Table 2). The major fatty acids found in strain SKP8-2T contained antesio-C15:0 (36.3%), iso-C15:0 (19.2 %), iso-C16:0 (16.5 %) and antesio-C17:0 (16.3 %). In addition, the other fatty acids, C16:0 (0.7 %), iso-C14:0 (4.4 %), iso-C17:0 (3.2 %), C16:1ω7c alcohol (1.4 %) and summed feature 4 (0.6 %) were also detected (Table 2). The polar lipids of strain SKP4-8T were phosphotidylglycerol (PG), diphosphatidylglycerol (DPG), three unknown phospholipids (PL1, PL2 and PL3) and unknown glycolipid (GL) (Supplementary Fig. S2). Strain SKP8-2T showed major polar lipid similar to strain SKP4-8T which contained of PG, DPG, unknown glycolipid (GL), but showed two unknown phospholipids (PL1 and PL2) (Supplementary Fig. S3).
Table 2
Cellular fatty acids composition (%) of strains SKP4-8T, SKP8-2T and A. halotolerans LMG 24826T. Data are determined in this study.
Fatty acids
|
SKP4-8T
|
SKP8-2T
|
LMG 24826T
|
Saturated straight-chain
|
|
|
|
C16:0
|
0.8
|
0.7
|
0.7
|
Saturated branched-chain
|
|
|
|
iso-C14:0
|
1.8
|
4.4
|
5.7
|
iso-C15:0
|
54.4
|
19.2
|
5.2
|
anteiso-C15:0
|
11.9
|
36.3
|
52.4
|
iso-C16:0
|
3.2
|
16.5
|
21.0
|
iso-C17:0
|
11.6
|
3.2
|
0.4
|
anteiso-C17:0
|
4.5
|
16.3
|
12.1
|
Unsaturated straight-chain
|
|
|
|
C16:1ω11c
|
0.6
|
-
|
-
|
C16:1ω7c alcohol
|
5.7
|
1.4
|
0.8
|
Summed Feature 4
|
1.7
|
0.6
|
0.1
|
Fatty acids comprising less than 0.5 % are omitted. -, not detected. |
Summed feature 4 contained iso-C17:1 I and/or anteiso-C17:1 B. |
The draft genome sequence of strain SKP4-8T was 2.59 Mb in size with an average in silico DNA G + C content of 38.8 mol%. The draft genome sequence of strain SKP8-2T was 2.54 Mb in size with an average in silico DNA G + C content of 39.0 mol%. The draft genomic features of the strains SKP4-8T and SKP8-2T are described in Table 3. The ANIb and ANIm values of the draft genomes between strain SKP4-8T and its closest related species, A. halotolerans LMG 24826T (86.6 and 88.1 %, respectively) are shown in Table 4. The ANIb and ANIm values of the draft genomes between strain SKP8-2T and its closest related species, A. halotolerans LMG 24826T (87.1 and 88.3 %, respectively) are shown in Table 4. Strain SKP4-8T exhibited 93.2 and 93.8 % of ANIb and ANIm with SKP8-2T. Both ANI values are clearly lower than the 95–96 % cut-off for species delineation (Meier-Kolthoff et al. 2013). The digital DNA-DNA hybridization (dDDH) value between the genomes of strain SKP4-8T and A. halotolerans LMG 24826T was 32.5 % (C.I. model 30.1–35.1 %). The digital DNA-DNA hybridization (dDDH) values between the genomes of the strain SKP8-2T and A. halotolerans LMG 24826T were 33.0 % (C.I. model 30.6–35.5 %) which are significantly lower than the threshold value of 70 % commonly used to delineate separated species status (Richter and Rosselló-Móra 2009) (Table 4), which are significantly lower than the threshold value of 70 % commonly used to delineate separated species status (Wayne et al. 1987). It is concluded from the above results that strains SKP4-8T and SKP8-2T should be recognized as a new species within the genus Allobacillus.
Table 3
Genomic statistics of strains SKP4-8T (VMHE00000000), SKP8-2T (JAGSIE000000000) and A. halotolerans LMG 24826T (JAHLZF000000000)
Attribute
|
SKP4-8T
|
SKP8-2T
|
LMG 24826T
|
Genome size (bp)
|
2,595,246
|
2,543,747
|
2,726,708
|
G + C content (mol%)
|
38.8
|
39.0
|
39.5
|
Number of contigs
|
102
|
80
|
96
|
Total genes
|
2,666
|
2,622
|
2,820
|
Number of coding sequences
|
2,603
|
2,563
|
2,759
|
RNA genes
|
63
|
59
|
61
|
Table 4
ANIb and ANIm values (%) and the digital (in silico) DNA-DNA hybridization (dDDH) values between the draft genomes of strains SKP4-8T, SKP8-2T and its closely related type strains.
Query genome
|
Reference genome
|
ANIb
|
ANIm
|
% dDDH (Formula 2*)
|
Model C.I.
|
Distance
|
Prob. Digital DNA-DNA hybridization > 70%
|
G+C difference
|
1
|
2
|
93.2
|
93.8
|
53.2
|
[50.5 - 55.9%]
|
0.0
|
28.9
|
0.1
|
1
|
3
|
86.6
|
88.1
|
32.5
|
[30.1 - 35.1%]
|
0.1
|
0.2
|
0.7
|
2
|
3
|
87.1
|
88.3
|
33.0
|
[30.6 - 35.5%]
|
0.1
|
0.3
|
0.5
|
Draft genomes: 1, Strain SKP4-8T; 2, SKP8-2T; 3, A. halotolerans LMG 24826T. All data were determined in this study. |
*Recommended formula (identities/HSP length), which is liberated of genome length and is thus prosperous against the use of incomplete draft genome |
On the basis of 16S rRNA gene sequence analysis, strains SKP4-8T (1471 nt) and SKP8-2T (1441 nt) were closely related to A. halotolerans LMG 24826T with 98.8 and 99.3 %, respectively (Fig. 1, Figs.S4 and S5). Strains SKP4-8T exhibited 99.5 % 16S rRNA gene sequence similarity with SKP8-2T. In addition, strains SKP4-8T and SKP8-2T were differentiated from A. halotolerans LMG 24628T by growth in NaCl, nitrate reduction, aesculin hydrolysis, and acid production from D-fructose, D-galactose, D-glucose and D-mannose (Table 1), the amount of fatty acids of iso-C15:0 and anteiso-C15:0 (Table 2) and DNA-DNA relatedness. Strain SKP4-8T and SKP8-2T should represent the novel species of the genus Allobacillus for which the name Allobacillus salarius sp. nov. and Allobacillus saliphilus sp. nov. respectively, are proposed.
Description of Allobacillus salarius sp. nov.
Allobacillus salarius (sa.la’ri.us. L. fem. adj. salarius of, or belonging to salt).
Cells are strictly aerobic Gram-stain-positive rods (0.25–0.30 x 0.8–3.5 µm). Motile by peritrichous flagella. Spores with oval shape are located at terminal position. Colonies are circular, convex, entire and cream in color (1–2 mm in diameter). Growth occurs at 25-45°C, pH 6.0–9.0 and 1–16 % (w/v) NaCl (optimally at 37°C, at pH 7.0 and in 5–10 % NaCl). Positive for oxidase, catalase, urease and hydrolysis of aesculin, Tween 20, Tween 40, Tween 60, and Tween 80. Negative for nitrate reduction and hydrolysis of arginine, gelatin, skim milk, starch and acid production from L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, D-melibiose, D-raffinose, L-rhamnose, D-ribose, D-salicin, D-trehalose and D-xylose. In the API ZYM system positive reaction for alkaline phosphatase, C4 esterase, C8 esterase, C14 lipase, leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, α-chymotrypsin, acid phosphatase,naphthol-AS-BI-phosphohydrolase, while α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acertyl-β-glucosaminidase, α-mannosidase and α-fucosidase. The polar lipid profile contains phosphotidylglycerol, diphosphatidylglycerol, three unknown phospholipids and one glycolipids. Contains meso-diaminopimelic acid in the peptidoglycan. The major cellular fatty acids are iso-C15:0, antesio-C15:0, and iso-C17:0 and the major isoprenoid quinone is MK-7.
The type strain is SKP4-8T (= KCTC 33905T = LMG 30016T = TISTR 2499T), isolated from shrimp paste (Ka-pi) collected from a market in Samut Sakhon province, Thailand. The in silico DNA G + C content of the type strain is 38.8 mol%. The DDBJ accession numbers for the 16S rRNA gene sequence of strains SKP4-8T is LC215448. The GenBank accession numbers of the draft genome of strain SKP4-8T is VMHE00000000.
Description of Allobacillus saliphilus sp. nov.
Allobacillus saliphilus (sa.li.philus.L. n. sal, salis salt; Gr. adj. philos loving; N.L. masc. adj. saliphilus salt-loving)
Cells are strictly aerobic Gram-stain-positive, rods (0.2–0.3 x 0.6–4.9 µm). Motiles by peritrichous flagella. Colonies are convex, circular, entire and cream in color (1–2 mm in diameter). Spores with oval shape are located at terminal position. Growth occurs at 25-45°C (optimum 37°C), pH 6.0–9.0 (optimum 8) and 0–20 % (w/v) NaCl (optimum 3–10 % NaCl). Positive for oxidase, catalase, nitrate reduction and hydrolysis of aesculin, Tween 20, Tween 40, Tween 60 and urea, but are negative for hydrolysis of arginine, gelatin, skim milk, starch, and Tween 80. Produces acid from D-fructose, D-galactose, D-glucose, D-mannose and sucrose but no acid from L-arabinose, D-cellobiose, lactose, maltose, D-mannitol, D-melibiose, D-raffinose, L-rhamnose, D-ribose, D-salicin, sorbitol, D-trehalose and D-xylose. In the API ZYM system, acid phosphatase, N-acetyl-β-glucosaminidase, alkaline phosphatase, cystine arylamidase, α-galactosidase and α-glucosidase (w) are positive while α-chymotrypsin, esterase lipase (C 4), esterase lipase (C 8), α-fucosidase, β-galactosidase, β-glucosidase, β-glucuronidase, leucine arylamidase, lipase (C14), α-mannosidase, naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase are negative. The polar lipid profile contains phosphotidylglycerol, diphosphatidylglycerol, two unknown phospholipids and an unknown glycolipid. Meso-diaminopimelic acid is presented in the cell wall peptidoglycan. The major cellular fatty acids are antesio-C15:0, iso-C15:0, iso-C16:0 and antesio-C17:0. The major isoprenoid quinone is MK-7.
The type strain is SKP8-2T (= KCTC 33906T = LMG 29682T = TISTR 2558T), isolated from shrimp paste (Ka-pi) collected from a market in Samut Sakhon province, Thailand. The in silico DNA G + C content of the type strain is 39.0 mol%. The DDBJ accession numbers for the 16S rRNA gene sequence of strains SKP8-2T is LC215449. The GenBank accession numbers of the draft genome of strain SKP8-2T is JAGSIE000000000.