Phenotypic features
The observed strain Kera-3T cells were rod-shaped, 0.8–1.4 µm in length, and 0.4–0.7 µm width, without flagellum (Fig. 1. A, B). Strain Kera-3T was identified as Gram-stain-positive using a Gram-stain kit (bioMerieux) according to manufacturer instructions. The colonies of strain Kera-3T were small, circular, flat, and white after seven days of incubation on R2A medium. The strain grew on commercial R2A (MB cell), TSB (BD difco), Nutrient Broth (BD difco), Luria-Bertani (BD difco) and 1/2-diluted R2A medium but did not grow on Reinforced clostridial medium. The best growth media was 1/2-diluted R2A. The growth temperature of strain Kera-3T was 18–37°C, and the pH was 6.0–8.0. The optimal temperature and pH were 25°C and 7.0, respectively. Salinity tolerance of strain Kera-3T was 0–1.0%. The growth temperature and pH condition of strain Kere-3T were similar with other strains within the family Iamiaceae. However, the salinity tolerance for strains belonging to family Iamiaceae was variable.
Chemotaxonomic Characterisation
The strain was positive for catalase, esterase (C4), esterase lipase (C8), leucine arylamidase, and naphthol-AS-BI-phosphohydrolase. Results were negative for nitrate reduction, alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, α–galactosidase, β–galactosidase, β–glucuronidase, α–glucosidase, β–glucosidase, N–acetyl-β-glucosaminidase, starch hydrolase, skim hydrolase, DNase, cellulase, and Tween 80. The strain could utilize carbon sources, D-glucose, sucrose, mannose, xylose, fructose, lactose, and pyruvate for growth. There were no particularly common enzyme activities characteristics or differentiating characteristics. Detailed characteristics of strain Kera-3T and its related strains are described in Table 1.
Table 1
ANI and AAI values between genomes of strain Kera-3T and related strains in the order Acidimicrobiales 1. Kera-3T (This study); 2. Actinomarinicola tropica SCSIO 58843T (previous study); 3. Rhabdothermincola salaria EGI L10124T (previous study); 4. Rhabdothermincola sediminis SYSU G02662T (previous study); 5. Desertimonas flava SYSU D60003T (previous study); 6. Ilumatobacter fluminis DSM 18936T (previous study); 7. Ilumatobacter nonamiensis YM16-303T (previous study); 8. Ilumatobacter coccineus YM16-304T (previous study); 9. Acidimicrobium ferrooxidans DSM 10331T (previous study); 10. Ferrimicrobium acidiphilum T23T (previous study); 11. Ferrithrix thermotolerans DSM 19514T (previous study); 12. Acidiferrimicrobium australe USS-CCA1T (previous study); 13. Coriobacterium glomerans PW2T (previous study).
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
11
|
12
|
13
|
1
|
*
|
72.6
|
72.1
|
71.2
|
70.5
|
70.3
|
69.6
|
69.7
|
68.0
|
65.8
|
66.4
|
71.8
|
65.3
|
2
|
59.8
|
*
|
72.8
|
72.5
|
71.1
|
70.7
|
69.5
|
70.3
|
68.8
|
66.3
|
66.4
|
72.2
|
66.0
|
3
|
59.5
|
62.0
|
*
|
73.4
|
70.4
|
70.2
|
69.1
|
69.7
|
67.2
|
65.9
|
66.9
|
72.3
|
65.5
|
4
|
60.1
|
63.1
|
65.4
|
*
|
69.8
|
69.6
|
68.7
|
68.7
|
67.5
|
65.8
|
65.8
|
72.0
|
64.8
|
5
|
56.6
|
57.6
|
56.8
|
57.7
|
*
|
72.9
|
71.3
|
72.3
|
68.1
|
65.5
|
65.1
|
70.7
|
65.9
|
6
|
56.4
|
57.5
|
57.1
|
57.6
|
62.1
|
*
|
72.5
|
72.9
|
67.6
|
65.9
|
65.7
|
70.2
|
65.3
|
7
|
56.1
|
56.9
|
56.8
|
56.9
|
61.3
|
63.6
|
*
|
76.5
|
67.6
|
65.4
|
65.1
|
69.1
|
65.7
|
8
|
55.8
|
57.1
|
56.8
|
56.9
|
61.7
|
63.3
|
73.5
|
*
|
67.5
|
65.4
|
65.1
|
69.9
|
65.2
|
9
|
53.2
|
53.8
|
54.2
|
54.8
|
53.4
|
53.1
|
52.7
|
52.5
|
*
|
66.9
|
65.6
|
68.7
|
66.5
|
10
|
53.1
|
53.7
|
54.0
|
54.2
|
52.9
|
52.8
|
52.8
|
52.8
|
59.8
|
*
|
67.2
|
66.3
|
65.5
|
11
|
54.2
|
54.6
|
54.9
|
55.7
|
53.8
|
53.9
|
53.4
|
53.3
|
56.6
|
57.9
|
*
|
66.4
|
65.8
|
12
|
58.1
|
59.7
|
60.4
|
60.9
|
57.3
|
57.0
|
56.7
|
56.8
|
55.9
|
56.1
|
56.5
|
*
|
65.5
|
13
|
50.1
|
50.3
|
50.3
|
50.4
|
49.8
|
49.6
|
49.2
|
49.7
|
49.9
|
49.6
|
50.1
|
50.3
|
*
|
Note: ANI (%, upper right) and AAI (%, lower left) of strain Kera-3T related genera were calculated. |
The cell wall of strain Kera-3T consisted of meso-DAP, alanine, and glutamic acid (Fig. S1). The major menaquinone of strain Kera-3T was MK-9 (H8). The major phospholipids of strain Kera-3T were diphosphatidylglycerol phosphoglycolipid, aminophospholipid, and aminophosphoglycolipid (Fig. S2).
The polar lipids of family Iamiaceae was found to consist of diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside, while that of strain Kera-3T has diphosphatidylglycerol and three unidentified glycolipids (Fig. S2).
The major fatty acids of strain Kera-3T were identified as C16:0 (46.82%) and C18:1ω9c (12.62%) (Table S1). C16:0 was consistent with results for all related strains within family Iamiaceae as major fatty acid but C18:1ω9c discriminated strain Kera-3T from related genera (Table 2).
Table 2
Differential characteristics of strain Kera-3T and the type strains of phylogenetically related genera 1. Kera-3T (This study); 2. Rhabdotherminicola sediminis SYSU G02662T (previous study); 3. Actinomarinicola tropica SCSIO 58843T (previous study); 4. Aquihabitans daechungensis CH22-21T (previous study); 5. Iamia majanohamensis F12T (previous study); 6. Ferrimicrobium acidiphilum T23T (previous study); 7 Desertimonas flava SYSU D60003T (previous study); 8. Ilumatobacter fluminis YM22-133T (previous study); Legend: Negative (-); positive (+); Not determined (ND).
Characteristic
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
Cell morphology
|
Rod
|
Short-rod
|
Short-rod
|
Short-rod
|
Rod
|
Rod
|
Coco-bacilli
|
Rod
|
Cell length(µm)
|
0.8–1.4
|
ND
|
1.0–2.0
|
0.7-1.0
|
1.2–1.7
|
1.0–3.0
|
1.1
|
1.3–1.6
|
Cell width(µm)
|
0.4–0.7
|
ND
|
0.3–0.6
|
0.3–0.5
|
0.3–0.5
|
0.5
|
0.7
|
0.4–0.5
|
Growth Conditions
|
|
Temperature (°C)
|
18–37 (25)
|
45
|
10–45 (35)
|
10–37 (30)
|
10–45 (30)
|
35–43 (35)
|
15–37 (28)
|
26–31 (28)
|
NaCl tolerance (w/v)
|
0–1.0
|
ND
|
0–5.0
|
0–1.0
|
0-12.5
|
ND
|
0.5
|
0–5
|
pH range
|
6.0–8.0
|
7.0
|
6.0–9.0
|
6.0–8.0
|
6.0–9.0
|
1.4-2.0
|
6.0–7.0
|
7.0–11.0
|
Carbon utilization
|
|
Glucose
|
+
|
ND
|
ND
|
ND
|
ND
|
-
|
ND
|
+
|
Maltose
|
ND
|
ND
|
-
|
+
|
-
|
-
|
ND
|
ND
|
Ribose
|
+
|
ND
|
ND
|
+
|
-
|
-
|
ND
|
-
|
Enzyme activity
|
|
|
Nitrate reduction
|
-
|
ND
|
+
|
-
|
+
|
ND
|
+
|
ND
|
DNA G + C contents (%)
|
73.2
|
70.5
|
72.5
|
71.8
|
74.4
|
54.9
|
70.2
|
68.0
|
Gene count
|
4,359
|
3.225
|
3,702
|
ND
|
ND
|
2,837
|
5,840
|
4,330
|
Genome size (bp)
|
4,661,060
|
3,293,621
|
3,715,454
|
ND
|
ND
|
3,088,650
|
6,220,580
|
4,775,416
|
Protein coding gene
|
4,265
|
3.052
|
3,596
|
ND
|
ND
|
2,715
|
5,730
|
4,253
|
Chemotaxonomic characteristics
|
|
Major menaquinones
|
MK-9(H8)
|
MK-9(H8)
|
MK-9(H8)
|
MK-9 (H6)
|
MK-9(H6).
|
MK-8(H10)
|
MK-9(H6),
MK-9(H8),
MK-9(H4)
|
MK-9(H8)
|
Diaminopimelic acid (DAP)
|
meso-DAP
|
meso-DAP
|
DD-DAP
|
meso-DAP
|
meso-DAP
|
meso-DAP
|
LL-DAP
|
LL-DAP,
|
Polar lipids
|
DPG, APL APGL, PGL
|
DPG, PI, PIM
|
DPG, PI, PC, PIM
|
DPG, PI, PIM
|
ND
|
ND
|
DPG
|
ND
|
Major fatty acids
|
C16:0, C18:1w9c
|
C16:0, C17:0, C18:0,
iso-C16:0
|
C17:1 ω8c, C17:0
|
C16:1 ω5c, C16:0,
C17:1 ω8c,
C18:1 ω9c
|
C17:0,
C17:1 w8c
|
iso-C16:0,
iso-C14:0,
iso-C15:0,
ai-C15:0,
C17:1 ω6c
|
C16:0,
iso-C16:0
|
iso-C16:0,
iso-C17:1 ω9c,
iso-C17:0
|
Based on phenotypic and chemotaxonomic features (Tables 1 and 2), strain Kera-3T could be distinguished from its related strains within the family Iamiaceae. The optimal growth temperature of strain Kera-3T was lower than other strains within the family Iamiaceae. It also can be differentiated by the chemotaxonomic property, particularly the presence of major fatty acids composition (C16:0, C18:1w9c) and main polar lipids (DPG, APL APGL, PGL). Details are available in the online version of this paper (Fig. S1, Fig. S2 and Table S1).
16s Rrna Gene Sequence Similarities And Phylogenetic Analysis
The 16S rRNA gene similarity calculations of strain Kera-3T indicate that this strain is a member of the family Iamiaceae, within the order Acidimicrobiales, with Rhabdothermincola sediminis SYSU G02662T (91.19%), Actinomarinicola tropica SCSIO 58843T (90.45%), Aquihabitans daechungensis CH22-21T (90.23%) and Iamia majanohamensis F12T (89.28%) and being the closest relative (Fig. 2). The phylogenetic tree constructed with the neighbor-joining and maximum-likelihood methods based on the 16S rRNA gene displays the Acidimicrobiales order divided into four family groups: Acidimicrobiaceae, Iamiaceae, ‘Microtrichaceae’, and Ilumatobacteraceae. The position of strain Kera-3T is located in Iamiaceae and is closest to Rhabdotherminicola sediminis SYSU G02662T.
The distinct phylogenomic lineage of strain Kera-3T was also supported by lower ANI and average AAI values than those obtained between the genomes of the strains within Family Iamiaceae. ANI values of strain Kera-3T against the genomes of related members within Order Acidimicrobiales were below 72.6% and AAI values of strain Kera-3T against the genomes of related members within Order Acidimicrobiales were below 60.1%. Following precedent research have suggested using > 95% of 16S rRNA gene identity to demarcate genus boundaries and 73.98% of average nucleotide identity (ANI) values to demarcate genus boundaries (Barco et al. 2020). In addition, strain Kera-3T was found to have a much larger genome size as compared to that of the genomes of strains within family Iamiaceae (Table 2).
Genome Sequencing And Genomic Characterisation
About 30,761 reads were obtained with 6,769 bp of mean subread length and assembled genome coverage was 44.0x. The obtained genome size was 4,938,963 bp in one circular chromosome and it contained 4,359 genes, 4,265 protein coding genes and one 16S rRNA gene. The G + C content of the genomic DNA was 70.5 % based on the complete genome sequence. The 16S rRNA gene sequence (1,411 bp) of strain Kera-3T from one circular chromosome has 99.9% similarity to 16S rRNA gene sequence with the sanger sequencing method.
Strain Kera-3T showed a very differential metabolic profile that clearly separates it from other members of the family Iamiaceae. These differences are especially significant on the carbohydrate and aminoacid metabolisms (Fig. 3). Genomic potential to utilize the various carbohydrates, such as carbohydrate deacetylation, ranging from monosaccharide to oligosaccharide, such as cellulose correlated significantly with the proposed species designations (Mantel test; r = 0.25, P value < 0.005) (Fig. 4). Combined genome information with phenotypic characteristics, these results suggest that carbohydrate metabolism is a key functional trait that delineates the species.
To better determine the evolutionary and taxonomical position of the new isolate, a phylogenomic tree was built on the PATRIC web server (https://patricbrc.org/) (Davis et al. 2020) using the RAxML tool v8.2.11 (Stamatakis 2014). Phylogenetic Tree Building Service using the ‘Codon Tree’ method on 1000 single-copy genes allows to identify that a total of 47 single-copy homology genes exist across the 14-given genome of the comparison species. A concatenated amino acid sequence of 21,731 length was used in the reconstruction of phylogenomic tree.
Based on the PATRIC web server, the phylogenomic tree provided further evidence showing that isolated the strain Kera-3T are close to the genus Rhabdothermincola (Fig. 5)(Table S2). Isolate the strain Kera-3T formed a distinct branch clustered with Rhabdothermincola salaria EGI L10124T, Rhabdothermincola sediminis SYSU G02662T, and Actinomarinicola tropica SCSIO 58843T, which was consistent with the 16S rRNA gene phylogenetic tree. Results of the single gene and genome-to-genome comparison analyses also support the classification based on 16S rRNA sequences. Strain Kera-3 belongs to the order Acidimicrobiales, but it was clearly distinguished from the family Ilumatobacteraceae in which the clade including Desertimonas flava SYSU D60003T and the family Acidimicrobiaceae in which the clades including Aciditerrimonas ferrireducens IC-180T and Acidimicrobium ferrooxidans ICP U75647T.
Taxonomic Conclusion
Phylogenetic and phylogenomic analysed results indicated that the strain Kera-3T formed a different cluster member of the family Iamiaceae, within the order Acidimicrobiales. Based on the above polyphasic taxonomic analysis, strain Kera-3T was confirmed as a novel genus in the family Iamiaceae. Therefore, those positions suggest that the new isolate is related to the family Iamiaceae and that it represents a new genus.
Description of Dermatobacter gen. nov.
Dermatobacter (Der.ma.to.bac.ter. N.L. derma (gen. dermatos), relating to skin; N.L. masc. adj. suff. Bacter; a small rod; N.L. masc. n. Dermatobacter; a skin derived bacterium).
Cells are rod-shaped, Gram-stain-positive, catalase-positive, non-motile, non-spore-forming, aerobic, and chemoheterotrophic. The major quinone detected is MK-9 (H8). The major polar lipids are diphosphatidylglycerol and phosphoglycolipid, aminophospholipid, and aminophosphoglycolipid. The peptidoglycan is composed of meso-DAP, alanine, and glutamic acids and is of type A1γ. Major fatty acids are C16:0, C18:1 ω9c. Phylogenetically, the strain is related to the family Iamiaceae in the Acidimicrobiales order. The type species is Dermatobacter hominis. The type strain, Kera-3T (= KACC 22415T = LMG 32493T), was isolated from human epidermal keratinocytes in Seongmam-si, Korea. The genomic DNA G + C content of the type strain is 73.2%.
Description of Dermatobacter homonis sp. nov
Dermatobacter hominis (ho’mi.nis. L. gen. masc. n. hominis, of a human being, pertaining to the host where this species is found). Cells are 0.4–0.7 µm in width and 0.8–1.4 µm in length, without flagellum. After 10 days of cultivation on growth medium, the colonies appear small and white. Growth occurs on R2A (MB cell), TSB (BD difco), NA (BD difco), and LB (BD difco) mediums at 18–37°C, while the optimal growth is observed on 1/2 diluted R2A medium at 25°C. Growth occurs at pH 6.0–8.0 and with NaCl concentrations of up to 1.0%, while growth is optimal at pH 7.0 and in the absence of NaCl. The strain is positive for catalase, esterase (C4), esterase lipase (C8), leucine arylamidase, and naphthol-AS-BI-phosphohydrolase; results were negative for nitrate reduction, alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, α–galactosidase, β–galactosidase, β–glucuronidase, α–glucosidase, β–glucosidase, N–acetyl-β-glucosaminidase, starch hydrolase, skim hydrolase, DNase, cellulase, and Tween 80. The strain could utilize carbon sources, D-glucose, sucrose, mannose, xylose, fructose, lactose, and pyruvate for growth. The type strain, Kera-3T (= KACC 22415T = LMG 32493T), was isolated from human skin (keratinocytes) in Seong-nam City, South Korea. The DNA G + C content of the genomic DNA was 70.5 % based on the complete genome sequence. The genome sequence for strain Kera-3T was deposited in the NCBI GenBank under the accession number CP085840.1.
The genome sequence for strain Kera-3T was deposited in the NCBI GenBank under the accession number CP085840. The NCBI GenBank accession number for the 16S rRNA gene sequence of strain Kera-3T is OL413447.