Phylogenetic analysis
The almost-complete 16S rRNA gene sequence of strain T12T (1514 bp) was determined and confirmed with the draft genome sequence. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain T12T belonged to the genus Jeotgalibacillus, within the family Planococcaceae, sharing highly similarity with J. salarius ASL-1T (97.6%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain T12T was in the same cluster as J. campisalis SF-57T, J. proteolyticus 22-7T, J. marinus strain 581T and J. soli P9T in the neighbour-joining tree (Fig. 1), which was also shown in the maximum-likelihood tree (Supplementary Fig. S1). The low 16S rRNA gene sequence similarities and phylogenetic location indicated that strain T12T should be located within the genus Jeotgalibacillus as a novel species.
Genome features
General features of strain T12T genome were summarized in Supplementary Table S1. The genome size of strain T12T was 3506682 bp, which contained 4066 predicted genes of 26 scaffolds and the depth coverage of sequencing was 330×. The N50 and N90 value was 933824 bp and 135228 bp. There were 5S rRNAs of 6, tRNAs of 67 and sRNAs of 2. The DNA G+C content of strain T12T was 43.7 mol%, which was within the range of DNA G+C contents for species of the genus Jeotgalibacillus (39–44 mol%) as previously reported.
According to Cluster of Orthologous Groups (COG), the genome contained several genes coding for amino acid transport and metabolism, transcription, carbohydrate transport and metabolism, signal transduction mechanisms, inorganic ion transport and metabolism and a number of unknown functions, which played a role in several aspects of cell live activities (Supplementary Fig. S2).
Strain T12T contained 2041 genes related to the KEGG metabolic pathway, including metabolism (68.3%), environmental information processing (9.9%), genetic information processing (9.0%), cellular processes (7.5%), human diseases (3.1%) and organismal systems (2.2%) (Supplementary Fig. S3). RAST identified 915 subsystems from the strain T12T, in which amino acids and derivatives (250) had the highest counts, followed by carbohydrates (194) and protein metabolism (179) (Supplementary Fig. S4).
Furthermore, strain T12T contained one or more polar or peripheral flagella and 62 genes related to cell motility were found in the genome as well. In agreement with phenotypic data, a flagellar assembly pathway (02040) was found, which annotated genes coding for flagellar related proteins (flagellar basal-body rod protein FlgG; flagellar secretion chaperone FliS; flagellin; chemotaxis protein MotA and MotB). Besides, in carotenoid synthesis, a complete pathway synthesized from phytoene to lycopene was discovered. Among them, genes encoding for carotenoid biosynthesis such as crtI was found in strain T12T, which may be responsible for the phenotypic colour of strain T12T (Kallscheuer et al. 2019).
The results of the antiSMASH analysis showed that strain T12T had three biosynthetic gene clusters (BGCs) in its genome including lasso peptide (paeninodin), terpene (carotenoid) and siderophore cluster. The terpene biosynthetic gene cluster, which may be related to the colour pigmentation associated with the bacterial colony (Paniagua-Michel et al. 2012), was detected in most species of the genus Jeotgalibacillus.
ANI values between strain T12T and the type strains of recognized Jeotgalibacillus species were less than 74.9% (Supplementary Table S2), significantly lower than the threshold value of 95% for species and genus (Richter and Rossello-Mora 2009). AAI values (55.0–74.6%) were lower than 75% with all the closely related species, well below the proposed cut-off values for genus delineation in the family Planococcaceae. The dDDH relatedness comparison with the draft genome for strain T12T showed low percentage (19.0–20.6%) similarities with all the related species and were much lower than the boundary (70.0%) for species identification. ANI, AAI and dDDH analysis results suggest that strain T12T is a new species of the genus Jeotgalibacillus.
Morphology and Phenotypic characterization
Cells were Gram-stain-positive, flagellated, rod-shaped (0.4–0.6 µm in width, 1.6–3.4 µm in length) (Supplementary Fig. S5). Colonies were circular to slightly irregular, flat to raised, smooth, orange coloured and 0.8–1.4 mm in diameter after incubation on MA for 2 days at 37°C. Strain T12T had one or more polar or peritrichous flagella, which was similar to the reference strains. The novel strain was found to grow at a temperature range of 15–40°C (optimum temperature 37°C), pH 5.5–9.0 (optimum pH of 8.5) and NaCl tolerance of 0.0–9.0% (optimum 2%, w/v). It could grow at 0% NaCl condition, which was similar to the related strains. Strain T12T differed from the closely related strain J. salarius ASL-1T with respect to numerous phenotypic characteristics, such as lipase (C14), alkaline phosphatase, trypsin and α-chymotrypsin activities. Strain T12T showed negative for H2S, starch, alginate, cellulose, oxidase and catalase. Besides, it was sensitive to penicillin, ampicillin cefazolin, amikacin, gentamicin, erythromycin, norfloxacin, ciprofloxacin, compound sulfamethoxazole and chloramphenicol. Other characteristics of strain T12T are presented in the species description and Table 1.
Table 1 Differential phenotypic characteristics of strain T12T and other closely related members of the genus Jeotgalibacillus.
Characteristic
|
1
|
2
|
3
|
4
|
5
|
6
|
Oxidase
|
-
|
+
|
-
|
-
|
+
|
+
|
Growth range (optimum)
|
|
|
|
|
|
|
NaCl (w/v, %)
|
0–9% (2%)
|
0–20% (3–12%)
|
0–15% (2–5%)
|
0–10% (2%)
|
0–7% (3–3.5%)
|
0–18% (2%)
|
Temperature (°C)
|
15–40 °C (37 °C)
|
10–45 °C (30–35 °C)
|
4–39 °C (30 °C)
|
10–40 °C (33 °C)
|
5–30 °C (12–23 °C)
|
4–40 °C (30 °C)
|
Hydrolysis of
|
|
|
|
|
|
|
Gelatin
|
+
|
+
|
-
|
+
|
+
|
-
|
Starch
|
-
|
-
|
+
|
-
|
-
|
-
|
Acids from
|
|
|
|
|
|
|
Mannitol
|
-
|
+
|
+
|
-
|
-
|
+
|
D-Mannose
|
-
|
-
|
-
|
-
|
+
|
-
|
Enzyme activity
|
|
|
|
|
|
|
Esterase (C4)
|
+
|
+
|
-
|
+
|
-
|
+
|
β-Galactosidase
|
+
|
+
|
+
|
+
|
-
|
-
|
α-Glucosidase
|
+
|
+
|
-
|
-
|
-
|
-
|
Predominant menaquinone
|
MK-7
|
MK-7, MK-8
|
MK-7
|
MK-7
|
MK-7
|
MK-7, MK-8
|
Major fatty acid
|
iso-C15:0, anteiso-C15:0, C16:1ω7c alcohol, iso-C14:0
|
iso-C15:0, anteiso-C15:0
|
anteiso-C15:0, C16:1ω7c alcohol, iso-C14:0
|
anteiso-C15:0, iso-C15:0
|
anteiso-C15:0, iso-C15:0
|
anteiso-C15:0, C16:1ω7c alcohol
|
DNA G+C content (%)
|
43.7
|
44
|
41.8
|
41.6
|
39.3
|
42.9
|
Strains: 1, strain T12T; 2, J. alimentarius JCM 10872T; 3, J. campisalis JCM 11810T; 4, J. proteolyticus 1H00228T; 5, J. marinus DSM 1297T; 6, J. salarius KCTC 13257T. +, Positive; -, negative; w, weak. All data from this study except DNA G+C contents of the related strains, which were from the original species description: 2 (Yoon et al. 2010, Yoon et al. 2001), 3 (Cunha et al. 2012, Yaakop et al. 2015, Yoon et al. 2010), 4 (Li et al. 2018), 5 (Yoon et al. 2010, Yoon et al. 2001), 6 (Yoon et al. 2010).
Chemotaxonomy
The major polar lipids of strain T12T were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylcholine (PC) (Supplementary Fig. S6); these were different from J. proteolyticus 22-7T (aminophospholipid, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol) (Li et al. 2018). The predominant cellular fatty acids of strain T12T were summed iso-C15:0 (28.4%), anteiso-C15:0 (19.6%), C16:1ω7c alcohol (13.0%) and iso-C14:0 (10.1%), which were similar to those of the related strains and the detailed information was shown in Table 2. The sole quinone of strain T12T was identified as MK-7, which was similar to that of the other members of the genus Jeotgalibacillus. These chemotaxonomic data further confirm that strain T12T belongs to the genus Jeotgalibacillus.
Table 2 Cellular fatty acid compositions of strain T12T and the closest relatives.
Fatty acid
|
1
|
2
|
3
|
4
|
5
|
6
|
Saturated fatty acids
|
C10:0
|
–
|
–
|
TR
|
1.99
|
–
|
–
|
C14:0
|
1.31
|
TR
|
1.65
|
1.05
|
1.19
|
1.54
|
C16:0
|
2.30
|
TR
|
2.94
|
1.68
|
2.44
|
5.37
|
C18:0
|
TR
|
TR
|
1.63
|
TR
|
1.37
|
3.67
|
Unsaturated
|
C16:1ω7c alcohol
|
13.00
|
5.01
|
18.32
|
4.98
|
9.10
|
14.22
|
C16:1ω11c
|
2.24
|
2.04
|
2.06
|
2.84
|
1.84
|
2.02
|
C18:1ω9c
|
TR
|
–
|
1.25
|
–
|
TR
|
2.51
|
Branched fatty acids
|
iso-C14:0
|
10.10
|
1.60
|
16.26
|
1.47
|
8.83
|
9.09
|
iso-C15:0
|
28.40
|
52.22
|
3.20
|
15.98
|
19.61
|
8.57
|
anteiso-C15:0
|
19.63
|
14.65
|
31.05
|
39.15
|
31.19
|
30.62
|
iso-C16:0
|
6.56
|
TR
|
7.23
|
2.04
|
6.08
|
4.69
|
iso-C17:0
|
1.63
|
2.17
|
TR
|
4.61
|
1.54
|
TR
|
anteiso-C17:0
|
3.92
|
2.08
|
4.13
|
8.44
|
4.88
|
5.72
|
iso-C17:1ω10c
|
1.06
|
8.10
|
TR
|
2.66
|
1.33
|
TR
|
Summed Feature3a
|
TR
|
TR
|
TR
|
1.04
|
TR
|
TR
|
Summed Feature4b
|
2.34
|
5.06
|
3.97
|
5.36
|
4.83
|
3.51
|
Summed Feature8c
|
TR
|
TR
|
TR
|
TR
|
TR
|
2.22
|
Strains: 1, strain T12T; 2, J. alimentarius JCM 10872T; 3, J. campisalis JCM 11810T; 4, J. proteolyticus 1H00228T; 5, J. marinus DSM 1297T; 6, J. salarius KCTC 13257T. All data were taken from this study. Major components are indicated with bold text. TR, Traces (< 1.0%); -, not detected. Fatty acids amounting to < 1.0% of the total fatty acids in both strains are not shown.
aSummed feature 3, C16:1ω7c and/or C16:1ω6c
bSummed feature 4, iso-C17:1I and/or anteiso-C17:1B
cSummed feature 8, C18:1ω7c and/or C18:1ω6c