Genomic features
The draft genome of strain ASW11-19T contained 18 contigs with a total genome size of 3 547 440 bp and the N50 value was 503 268 bp. The sequencing depth was 263×. The genomic DNA G + C content was 44.4%. Strain ASW11-19T contained 3150 protein-coding genes, 16 pseudo genes and 62 RNA genes (2 rRNA, 5 ncRNA and 55 tRNA). Based on the genome annotation, the predicted functional genes of strain ASW11-19T, mainly belong to amino acid and derivatives (264), protein metabolism (184), cofactors, vitamins, prosthetic groups and pigments (130) and carbohydrates (118). The different genomic features of strain ASW11-19T and closely related strains based on the RAST results were listed in Table S1. In addition, 73 carbohydrate-active enzymes (CAZymes) genes, which include 27 glycoside hydrolases (12 families), 24 glycosyltransferases (12 families), 10 carbohydrate esterases (5 families), 6 auxiliary activities (3 families), 5 carbohydrate-binding modules (3 families) and 1 polysaccharide lyases (1 family), were predicted in the genome of strain ASW11-19T (Table S2).
The results of substrate hydrolysis experiments showed that strain ASW11-19T had stronger starch degradation capacity than reference strains A. hispanican and A. profundi (Fig. S3). RAST analysis of genome data of strain ASW11-19T revealed that there were four genes involved in starch degradation, including α-amylase (EC 3.2.1.1), pullulanase (EC 3.2.1.41), neopullulanase (EC 3.2.1.135), and α-1,6-maltotetraose-hydrolase (EC 3.2.1.196). Interestingly, the α-amylase gene was absence in reference strains A. hispanican and A. profundi. The α-amylase may play the crucial role in the starch degradation of strain ASW11-19T, and it could be further explored and utilized as potential marine enzyme for starch degradation.
The phylogenomic analysis based on genomic sequences showed that strain ASW11-19T and Alteromonas halophila JSM 073008T formed a distinct cluster within the genus Alteromonas (Figs S4, S5 and S6). The dDDH values between strain ASW11-19T and closely related species ranged from 19.0–23.4%, and the corresponding ANI values ranged from 70.6–78.6% (Table S3). The dDDH and ANI values were clearly below the species-delineating thresholds (70% and 95%), respectively (Chun et al. 2018; Colston et al. 2014), which indicating that strain ASW11-19T represented a putative novel species of the genus Alteromonas.
Morphology, physiology, and biochemical analysis
Strain ASW11-19T were aerobic, Gram-stain-negative and motile with flagella. Cells were short rod-shaped and 0.5–0.8 µm in diameter and 1.0–2.2 µm in length (Fig. S7) Colonies were yellowish-white, circular, opaque, flat convex with a smooth surface after incubation for 3 days. Strain ASW11-19T showed stronger starch degradation capacity than reference strains (Fig. S3). Strain ASW11-19T was sensitive to norfloxacin, levofloxacin, erythromycin, polymyxin B, chloramphenicol, ampicillin, roxithromycin, streptomycin, sulfaisoxazole, sulfamethoxazole and vancomycin, but resistant to cephalexin, lincomycin, kanamycin, oxytetracycline and tetracycline. The detailed results of phenotypical and biochemical characteristics are provided in Table 1 and the species description.
Table 1
Differential phenotypic and biochemical characteristics of strain ASW11-19T and closely related species of the genus Alteromonas.
Characteristic
|
1
|
2
|
3
|
4
|
5
|
Cell (µm)
|
0.5–0.8 × 1.0–2.2
|
ND *
|
0.75 × 1.0–2.0†
|
0.8–1.4 × 0.8–2.5
|
0.9 × 1.8
|
Growth:
|
|
|
|
|
|
Temperature (optimum, °C)
|
4–40 (37)
|
4–40 (25)*
|
4–40 (32)†
|
10–42 (25)
|
4–37
|
pH (optimum)
|
7.0-9.5 (7.5)
|
5.5–9.5 (6.5–7.5)*
|
5.0–10.0 (7.0–8.0)†
|
5.5–10.0 (6.0–9.0)
|
6.0-8.5 (7.0–8.0)
|
NaCl (optimum, %, w/v)
|
1.0–16.0 (3.0–5.0)
|
3.0–13.0 (5.5-6.0)*
|
7.5–15.0 (7.5–10.0)†
|
2.0–12.0 (2.0–4.0)
|
2.0–15.0
|
Hydrolysis of
|
|
|
|
|
|
Milk
|
+
|
−
|
+
|
ND
|
ND
|
Enzyme activity (API ZYM)
|
|
|
|
|
|
Lipase (C14)
|
W
|
W
|
+
|
−
|
W
|
Acid phosphatase
α-Galactosidase
|
−
−
|
+
+
|
−
W
|
+
+
|
+
−
|
β-Galactosidase
|
−
|
W
|
W
|
+
|
+
|
α-Glucosidase
|
−
|
+
|
−
|
+
|
−
|
β-Glucosidase
|
−
|
+
|
−
|
+
|
−
|
Utilization of (API 20NE)
|
|
|
|
|
|
Arginine
|
+
|
+
|
+
|
−
|
−
|
Urea
|
+
|
+
|
+
|
−
|
−
|
Mannitol
|
−
|
+
|
−
|
+
|
−
|
Maltose
|
−
|
+
|
−
|
+
|
−
|
Gluconate
|
−
|
+
|
−
|
−
|
−
|
Strains: 1, Alteromonas salexigens ASW11-19T (this study); 2, Alteromonas profundi 345S023T (this study); 3, Alteromonas hispanica F32T (this study); 4, Alteromonas fortis 1T (Barbeyron et al. 2019); 5, Alteromonas genovensis LMG 24078T (Vandecandelaere et al. 2008). |
All strains were positive for the following: hydrolysis of Tweens 20, 40, 60, 80, starch, casein and gelatin; activities of catalase, oxidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase, cystine arylamidase, and α-chymotrypsin. All strains were negative for N-acetyl-β-glucosaminidase, L-arabinose tryptophan, nitrate reduction, and for acidification of glucose. +, positive; −, negative; W, weakly positive; ND, not determined.
*Data from Shen et al. (2020)
|
†Data from Martínez-Checa et al. (2005) |
Chemotaxonomic analysis
The major fatty acids (> 10%) identified in strain ASW11-19T were summed featured 3 (24.0%, C16:1ω7c and/or C16:1ω6c), summed featured 8 (14.9%, C18:1ω7c and/or C18:1ω6c) and C16:0 (12.4%). The fatty acid profiles of strain ASW11-19T and closely related strains were similar, with summed featured 3 and C16:0 as the major fatty acids. However, the fatty acid profile of strain ASW11-19T presented some characteristics that differed from closely related species. For example, the contents of C16:0 of strain ASW11-19T (12.4%) was lower than that of A. profundi 345S023T (18.9%), A. hispanica F-32T (21.9%), A. fortis 1T (15.2%), and A. genovensis (17.1%), respectively. The differences in the relative amounts of other fatty acids are listed in Table 2. The sole respiratory quinone discovered in strain ASW11-19T was ubiquinone 8, which is in accordance with the description of genus Alteromonas. The major polar lipids detected in strain ASW11-19T, were phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phospholipid (PL) (Fig. S8), in line with reference strains A. hispanican and A. profundi. Additionally, two unidentified lipids (L1 and L2) were only found in strain ASW11-19T.
Table 2
Cellular fatty acid compositions (%) of strain ASW11-19T and closely related species of the genus Alteromonas.
Fatty acid
|
1
|
2
|
3
|
4
|
5
|
Saturated
|
|
|
|
|
|
C12:0
|
TR
|
3.7
|
3.1
|
1.7
|
2.9
|
C14:0
|
2.2
|
2.7
|
4.4
|
1.8
|
5.0
|
C16:0
|
12.4
|
18.9
|
21.9
|
15.2
|
17.1
|
C17:0
|
3.2
|
4.3
|
2.2
|
3.3
|
1.8
|
C18:0
|
2.6
|
1.2
|
1.3
|
−
|
1.8
|
Unsaturated
|
|
|
|
|
|
C15:1ω8c
|
2.2
|
1.8
|
1.3
|
1.7
|
TR
|
C17:1ω8c
|
8.2
|
5.3
|
4.0
|
7.5
|
3.5
|
C18:1ω9c
|
1.4
|
−
|
1.5
|
−
|
−
|
C18:3ω6, 9, 12c
|
1.4
|
TR
|
TR
|
−
|
−
|
Branched
|
|
|
|
|
|
iso-C16:0
|
2.6
|
2.0
|
0.7
|
1.0
|
1.1
|
iso-C18:0
|
1.5
|
TR
|
TR
|
−
|
TR
|
C16:0 N alcohol
|
2.0
|
TR
|
TR
|
6.7
|
3.1
|
10-methyl C17:0
|
5.8
|
TR
|
TR
|
8.6
|
2.9
|
Hydroxy
|
|
|
|
|
|
C10:0 3-OH
|
TR
|
1.5
|
1.3
|
1.1
|
2.1
|
C11:0 3-OH
|
1.4
|
1.8
|
1.0
|
1.0
|
TR
|
C12:0 3-OH
|
1.0
|
1.2
|
1.4
|
−
|
−
|
C12:1 3-OH
|
TR
|
−
|
1.4
|
−
|
1.3
|
Summed features*
|
|
|
|
|
|
2
|
3.2
|
4.2
|
4.0
|
2.7
|
−
|
3
|
24.0
|
29.7
|
33.1
|
21.3
|
28.2
|
8
|
14.9
|
9.6
|
9.7
|
14.5
|
12.1
|
Strains: 1, Alteromonas salexigens ASW11-19T (this study); 2, Alteromonas profundi MCCC 1K04570T (this study); 3, Alteromonas hispanica LMG 22958T (this study); 4, Alteromonas fortis 1T (Barbeyron et al. 2019); 5, Alteromonas genovensis LMG 24078T (Shen et al. 2020). |
Major fatty acids (> 10.0%) are indicated in bold type. TR Traces (< 1.0%); −, Not detected. |
*Summed features are groups of two or more fatty acids that could not be separated using the MIDI system. Summed feature 2 contains C12:0 aldehyde and/or an unknown fatty acid of equivalent chain-length 10.928. Summed feature 3 contains C16:1ω7c/C16:1ω6c. Summed feature 8 contains C18:1ω7c/C 18:1ω6c. |
Based on the results of phylogenetic, genomic, phenotypic and chemotaxonomic characteristics, it is proposed to classify strain ASW11-19T as a novel species, Alteromonas salexigens sp. nov.
Description of Alteromonas salexigens sp. nov.
Alteromonas salexigens (sa.lex′i.gens. L. n. sal salis salt, sea water; L. v. exigo to demand; N.L. part. adj. salexigens sea water-demanding). Cells are short rod-shaped with a size of 0.5–0.8 µm in diameter and 1.0–2.2 µm in length. Gram-staining reaction is negative. Motile by gliding. Colonies on TYS agar are yellowish-white, circular, convex, smooth and 1.0–2.0 mm in diameter after incubation for 3 days. Growth occurs at between 4 and 40°C with an optimum at 37°C. The pH range for growth is 7.0–9.5 (optimum pH 7.5). Growth was seen between 1.0–16.0% (w/v) NaCl with an optimum of 3.0–5.0% NaCl. Cells are positive for urease, oxidase and catalase. Nitrate is not reduced. Positive for hydrolysis of aesculin, starch, gelatin, casein, milk, Tweens 20, 40, 60 and 80, and negative for hydrolysis of cellulose. In the API 20NE system, cells are positive for the assimilation of arginine and urea but negative reaction to tryptophan, glucose, arginine, gelatin, N-acetyl-glucosamine, mannitol, L-arabinose, mannose, maltose, gluconate, malate, citrate, capric acid, adipic acid, and phenyl acetic acid. In the API ZYM system, cells are positive for alkaline phosphatase, esterase(C4), esterase lipase(C8), lipase(C14), valine, leucine, and cystine arylamidases, trypsin, naphthol-AS-BI-phosphohydrolase, α-chymotrypsin, but negative for acid phosphatases, N-acetyl-β-glucosaminidase, α-glucosidase, β-glucosidase, β-glucuronidase, α-galactosidase, β-galactosidase, α-mannosidase, β-fucosidase. The major fatty acids are summed featured 3 (C16:1ω7c and/or C16:1ω6c), summed featured 8 (C18:1ω7c and/or C18:1ω6c) and C16:0. The sole respiratory quinone is Q-8. The major polar lipids are phosphatidylglycerol, phosphatidylethanolamine and phospholipid. The DNA G + C content of the type strain is 444 %.
The type strain, Alteromonas salexigens (= MCCC 1K07239T = KCTC 92247T), was isolated from seawater sample collected from the Yellow Sea, PR China.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene and draft genome sequence of the type strain ASW11-19T is OP548644 and JAOTJC000000000, respectively.