16S rRNA phylogeny
The cloned sequence of the 16S rRNA gene of SZY PN-1T has been deposited in NCBI GenBank under accession number MW135304. Comparison of the 16S rRNA gene sequences of SZY PN-1T (1409 bp) with those of other species showed that the closest sequence was that of S. sibiricus strain RB16-17 (97.1 % sequence similarity), followed by S. neustonicus JCM 30734T (96.6 % sequence similarity) and other type strains with less than 94.2 % sequence similarity within the family Sphingosinicellaceae, which is lower than the threshold (98.65 %) for bacterial species demarcation (Kim et al 2014). The maximum-likelihood tree based on 16S rRNA gene sequences demonstrated that strain SZY PN-1T formed a monophyletic clade that was part of a branch with the genus Sandaracinobacter comprised of S. sibiricus RB16-17 and S. neustonicus JCM 30734T (Fig. 1). A similar result was obtained when using the neighbor-joining and maximum-parsimony algorithms (Fig. S1-2, available in the online version of this article), which also revealed that strain SZY PN-1T representing a novel species within the genus Sandaracinobacter.
The Whole Genome Shotgun project of strain SZY PN-1T has been deposited at DDBJ/ENA/GenBank under the accession JADCUC000000000, with the genomic size of 3.53 Mbp and the DNA G+C content of 65.0 %. The protein-concatamer tree based on 29 marker genes (frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf) indicated that the novel strain SZY PN-1T clustered within the genus Sandaracinobacter, forming a clade with the strain S. neustonicus JCM 30734T (Fig. 2). The threshold limits (95.0–96.0 % ANI and 95 % AAI) for delineation of bacterial species were considered as recommended (Chun et al. 2018; Thompson et al. 2013). The estimated results confirmed that strain SZY PN-1T represented a novel genomic species within the genus Sandaracinobacter, with ANI values ≤ 85.0 % and AAI values ≤ 76.0 % with the genome of S. neustonicus JCM 30734T, which was the closest relative comparing by 16S rRNA and whole-genome sequence. The detailed characteristics of the genomes of the strain SZY PN-1T, and other type strains within the genus Sandaracinobacter are listed in Table S1.
Strain SZY PN-1T showed good growth on R2A and BCYEα agar; weak growth on Columbia blood agar, MH agar, TSA and LB agar; but not on Haemophilus chocolate 2 agar, chocolate agar with PolyViteX (PVX agar, Bio-caring, China), CHAB agar and MacConkey agar (Bio-caring, China). After incubation for 72 h at 30 ℃ on R2A agar media, the colonies were 1–2 mm in diameter, circular, convex, a little hard and yellow coloured. Strains were able to grow at temperatures ranging between 10 and 37 ℃ (optimum, 30 ℃), pH 6.0–8.0 (optimum, pH 7.0) and in the presence of up to 1.0 % (w/v) NaCl with optimum growth at non-additional NaCl on R2A. Cell of strain SZY PN-1T was observed to be Gram-negative, aerobic, non-spore-forming and non-motile. The strain was enhanced by the presence of 5% CO2. Transmission electron microscopy image showed that the strain was a rod, 0.71–0.97 µm long and 0.53–0.63 µm wide without flagella, as shown in Fig. 3. The absorption spectrum of pigments extracted from the cells showed two peaks at 452 and 478 nm (Fig S3), indicating the presence of carotenoids. No peaks were detected above 600 nm, which showed that the strain SZY PN-1T was absent in BChl α.
The strain was positive for catalase activity but negative for oxidase activity and hydrolysed starch, casein, aesculin, gelatin, Tween 40, Tween 60 but not Tweens 20 and 80. Activities of urease, Voges–Proskauer test, H2S production and nitrate reduction were negative. The detailed phenotypic and physiological characteristics of strain SZY PN-1T are summarized and compared with P. fuscus CGMCC 1.12714T and closely related Sandaracinobacter species in Table 1.
Summed feature 8 (C18:1ω7c and/or C18:1ω6c; 41.8 %), C17:1 ω6c (12.5 %) and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c; 10.9 %) were the predominant cellular fatty acids (>10.0 %) in SZY PN-1T, which are in agreement with the composition of fatty acids in S. neustonicus. Detailed fatty acid compositions are shown in Table 2. Polar lipids of strain SZY PN-1T contained phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and sphingoglycolipids (SGLs) as major polar lipids (Fig. S4). Diphosphatidylglycerol (DPG), unidentified glycolipids (GLs) and unidentified lipids (Ls) were also present as minor polar lipids. The respiratory quinones comprised ubiquinones Q-10 (92.2 %) and Q-11 (7.8 %), whereas ubiquinone Q-11 was absent in S. sibiricus RB16-17 and S. neustonicus JCM 30734T within the genus Sandaracinobacter. Moreover, strain SZY PN-1T lacked ubiquinone Q-9, which was found to present in the strain of S. sibiricus RB16-17.
Based on the low 16S rRNA gene sequence and other phenotypic characteristics, strain SZY PN-1T could be distinguished from the related species within the genus Sandaracinobacter. The physiological and chemotaxonomic characteristics, including cellular fatty acid and polar lipid profiles, also indicated that strain SZY PN-1T was similar to the species within the genus Sandaracinobacter. These results revealed that strain SZY PN-1T should be classified as representing a novel species of the genus Sandaracinobacter, for which we propose the name Sandaracinobacter hominis sp. nov.
Description of Sandaracinobacter hominis sp. nov.
Sandaracinobacter hominis (ho'mi.nis. L. n. homo, -inis, a human, man, person; L. gen. masc. n. hominis, of man, signifying the isolation of strains from human skin).
Cells are Gram-negative, aerobic and rod-shaped, being 0.71–0.97 µm long and 0.53–0.63 µm wide without flagella. Growth occurs from 10 to 37 ºC (optimal at 30 ºC). Cells absorb at 430-490 nm, because of the presence of carotenoids. In the API ZYM strip, it is positive for alkaline phosphatase, esterase (C4), leucine arylamidase, trypsin, acidic phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase, and weakly positive for esterase lipase (C8) and α-chymotrypsin. Negative results are obtained for lipase (C14), valine arylamidase, cystine arylamidase, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. In the API 20NE strip, positive reactions are observed for hydrolysis of aesculin, β-galactosidase, assimilation of glucose and maltose, but reactions are negative for acid production from glucose, arginine dihydrolase, Assimilation of arabinose, mannose, mannitol, N-acetyl-d-glucosamine, gluconate, caprate, adipate, malate, citrate and phenylacetate. In the API 50CHB/E strip, positive reactions for esculin ferric citrate, negative reactions for glycerol, erythritol, d-arabinose, l-arabinose, d-ribose, d-xylose, L-xylose, d-adonitol, methyl β-d-xylopyranoside, d-galactose, d-glucose, d-fructose, d-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl α-d-mannopyranoside, methyl α-d-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-sucrose, d-trehalose, inulin, d-melezitose, d-raffinose, starch, glycogen, xylitol, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate are observed. The major cellular fatty acids are Summed feature 8 (C18:1ω7c and/or C18:1ω6c), C17:1 ω6c and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The polar lipids are composed of phosphatidylethanolamine, phosphatidylglycerol, two sphingoglycolipids, Diphosphatidylglycerol, five unidentified glycolipids and seven unidentified lipids. The major respiratory quinone is ubiquinone-10, whereas ubiquinone-11 is present in smaller amounts. The DNA G+C content of the type strain is 65.0 %.
The type strain SZY PN-1T (KCTC 82150T = NBRC 114675T), was isolated from a skin sample of a Chinese. The GenBank accession number for 16S rRNA gene sequence of the strain SZY PN-1T is MW135304. The GenBank/EMBL/DDBJ/PIR accession number for the Whole Genome Shotgun projects of the strains SZY PN-1T is JADCUC000000000.
All experiments involving human subjects were carried out according to the institutional review board protocols approved by Medical Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine in China (BE2019-165). Informed consent was obtained from all subjects.