Morphological and phenotypical characteristics
Cells of strain XSD401T were facultative anaerobic, Gram-stain-negative, no flagella, coccus to rod shaped, 0.35–0.5 µm in diameter and 1.2–3 µm in length (Fig. S1). The motility was not detected under the microscope by hanging drop methods. The gliding motility was detected under the microscope and was also confirmed using semi-solid agar. The shape of the cells and the color of the colonies are the same as most Psychroserpensstrains. Growth occurs at 20–37 °C (optimal 33 °C), pH 5.5–9.5 (optimal pH 6.5–7.5) and 0.5–5% (w/v) NaCl (optimal NaCl 3.0%). Strain XSD401T was positive for catalase and hydrolysis of Tween 20, 40 and 80, casein, sodium alginate, but negative for the hydrolysis of oxidase, agar, starch, CM-cellulose, DNase and Tween 60. Strain XSD401T is susceptible to ampicillin (10), rifampin, but resistant to tobramycin (10), tetracycline (30), norfloxacin (30), neomycin (30), gentamycin (10), ofloxacin (30), kanamycin (30). All of them contain esterase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase activity. Comprehensive morphological, biochemical and physiological traits of strain XSD401T are summarized in the species description. The test results distinguishing strain XSD401T from the related strains are shown in Table 1.
Phylogenetic analysis of 16S rRNA sequences
The 16S rRNA gene of strain XSD401T was amplified by PCR using universal primers (27F and 1492R).16S rRNA (1488 bp) was basically complete and stored in the NCBI GenBank database. The 16S rRNA accession number of strain XSD401T was MN330029. The results showed that strain XSD401T had the highest similarity (96.88%) to the 16S rRNA gene of P. damuponensis KCTC 23539T.
Only one 16S rRNA gene sequence (1524 bp) was extracted from the draft genome, shared 99.9 % similarity with the nearly complete (1488 bp) 16S rRNA gene sequence obtained by conventional Sanger sequencing. The complete sequence (1524 bp) matched with a counterpart of PCR amplification (1483 bp).
The results of phylogenetic analysis also indicated that strain XSD401T was affiliated to the genus Psychroserpens, but formed a unique clade separated from other species, indicating that this strain represents a novel species (Fig. 1). The results showed that the strain XSD401T was located in Psychroserpens, and was associated with P. damuponensis KCTC 23539T and P. burtonensis DSM 12212T. The phylogenetic tree was constructed based on maximum likelihood and maximum parsimony. The phylogenetic data indicated that the strain XSD401T was a new species of Psychroserpens.
Genomic analysis
Draft genome sequencing of strain XSD401T yielded a genome of 3.7 Mb in length after assembly, producing 60 contigs, and the N50 value was 706,489 bp, with the largest being 1,282,496 bp and the smallest 298 bp. The genomic DNA G+C content of strain XSD401T is 33.9 mol%. The ANI values between strain XSD401T and P. damuponensis KCTC 23539T, P. burtonensis DSM 12212T were 76.91%, 76.88%, respectively. The dDDH values between strain XSD401T and P. damuponensis KCTC 23539T,P. burtonensis DSM 12212T, were 20.40% and 20.30%, respectively.
Further, we focused on the genes of XSD401T that were predicted by KEGG pathway annotation (https://www.kegg.jp). Among the 3512 protein-coding genes, strain XSD401T pathway modules showed that thirty-nine of them were complete, including carbohydrate metabolism, energy metabolism, lipid metabolism, nucleotide metabolism, amino acid metabolism, glycan metabolism, metabolism of cofactors and vitamins, biosynthesis of terpenoids and polyketides. To be specific, the genome of strain XSD401T contains a complete set of genes of the citrate cycle (TCA) pathway that transform acetyl-CoA to oxaloacetate. Known as pilE, pilB, that encode protein pilB and protein pilE in the genome for the synthesis of type IV Pilus. Several sugar transporters were tagged in the genome, providing additional support for the sugar-dependent growth of strain XSD401T. The Embden-Meyerhof pathway and the Semi-phosphorylative Entner-Doudoroff pathway are incomplete because they lack the necessary genes. The formaldehyde assimilation, dissimilatory nitrate reduction and assimilatory sulfate reduction are incomplete due to the lack of the necessary genes.
After annotating with KEGG, the results showed the strain XSD401T has 39 complete metabolic pathways, P. burtonensis DSM 12212T has 44, and P. damuponensis KCTC 23539T has 41 (Fig. 2). In these three strains, 37 of the metabolic pathways are shared, strain XSD401T and P. damuponensis KCTC 23539T each have 2 distinct metabolic pathways, and P. burtonensis DSM 12212T has 5 distinct metabolic pathways. For example, it is found that strain XSD401T lacks the assimilatory sulfate reduction pathway, and P. burtonensis DSM 12212T lacks a block, but the assimilatory sulfate reduction pathwayof P. damuponensis KCTC 23539T is complete. Strain XSD401T and P. burtonensis DSM 12212T have no glycogen biosynthesis pathway, but P. damuponensis KCTC 23539T has a complete glycogen biosynthesis pathway. Strain XSD401T lacks the polyamine biosynthesis pathway, while P. burtonensis DSM 12212T and P. damuponensis KCTC 23539T all have a complete polyamine biosynthesis pathway.
Chemotaxonomic characteristics
The respiratory quinone of strain XSD401T was Menaquinone-6 (MK-6), which is typical of the genus Psychroserpens. The major polar lipids of strain XSD401T consist of phosphatidylethanolamine (PE) (Fig. S2), which also was typical of the genus Psychroserpens. Minor amounts of three unidentified aminolipids (AL1, AL2, AL3) and one unidentified lipid (L) were also detected. Strain XSD401T differed from P. damuponensis KCTC 23539T by the absence of AL4 and L2-L3. Strain XSD401T differed from P. burtonensis DSM 12212T by the absence of L4.
The predominant cellular fatty acids found in strain XSD401T were iso-C15:0 (26.73%), iso-C15:1 G (18.4%), iso-C17:0 3-OH (14.51%), iso-C15:0 3-OH (11.35%), C16:0 (6.99%), along with the lesser amounts of summed feature 3 (C16:1 ω7cand/or C16:1 ω6c) (4.38%), C18:1 ω9c (3.19%), C16:0 3-OH (2.09%), C16:1 ω9c (1.3%), anteiso-C15:0 (1.26%), C18:0 (1.25%), iso-C16:0 3-OH (1.19%).
Taxonomic conclusion
The respiratory quinone of strain XSD401T was Menaquinone-6 (MK-6), which is typical of the genus Psychroserpens. The major polar lipids of strain XSD401T consist of phosphatidylethanolamine (PE), which also was typical of the genus Psychroserpens. Strain XSD401T contained iso-C15:0 (26.73%) as predominant fatty acids (≥10.0 %) in common with closely related strains. The fatty acids composition of strain XSD401T were somewhat different from P. damuponensis KCTC 23539T and P. burtonensis DSM 12212T.The fatty acid differences could be observed between strain XSD401T and its closely strains (Table 2). These findings suggest that strain XSD401T is most closely related to P. damuponensis KCTC 23539T, rather than P. burtonensis DSM 12212T.
Based on the phenotypic, chemotaxonomic and phylogenetic results, strain XSD401T represents a novel species within the genus Psychroserpens, for which the name Psychroserpens luteus sp. nov. is proposed.
Description of Psychroserpens luteus sp. nov.
Psychroserpens luteus(lu’te.us L. masc. adj. luteus, orange-coloured).
Cells are facultative anaerobic, Gram-stain-negative, non-motile, no flagella, but gliding motility is present, coccus to rod shaped, 0.35–0.5 µm in diameter and 1.2–3 µm in length. After 2 days of incubation on MA at 33 °C, the colonies were circular, smooth, yellow, with diameters of 1.0–2.0 mm. Growth occurs at 20–37 °C (optimal 33 °C), pH 5.5–9.5 (optimal pH 6.5–7.5) and 0.5–5% (w/v) NaCl (optimally 3.0%). Positive for catalase and nitrate reduction, negative for oxidase activities. Hydrolysis of Tween 20, 40 and 80, casein, sodium alginate, but not starch, agar, CM-cellulose, DNase and Tween 60. The reaction of tryptophan deaminase(TDA), Voges-Proskauer reaction(VP), gelatinase (GEL) are positive, while the other reactions are negative. Activities of alkaline phosphatase, esterase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase were positive, and the others were negative. Acids are produced from d-ribose, esculin ferric citrate, d-tagatose, and potassium 5-ketogluconate. Positive for the oxidation of dextrin, d-maltose, d-trehalose, d-cellobiose, gentiobiose, sucrose, d-turanose, stachyose, d-raffinose, d-lactose, d-melibiose, β-methyl-d-glucoside, d-salicin, α-d-glucose, d-mannose, d-fructose, d-galactose, 3-methyl glucose, d-sorbitol, d-mannitol, d-gluconic acid, and l-lactic acid can all be assimilated by carbon sources. Sensitive to ampicillin, rifampin, but resistant to tobramycin, tetracycline, norfloxacin, neomycin, gentamycin, ofloxacin, kanamycin. The main fatty acids are iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH, iso-C15:0 3-OH, C16:0. Phosphatidylethanolamine (PE), three unidentified aminolipids (AL1, AL2, AL3) and one unidentified lipid (L) were the major polar lipids. The major respiratory quinone is MK-6.
The type strain, XSD401T (=MCCC 1H00396T =KCTC 72684T =JCM 33931T), was isolated from red algae in the Xiaoshi Island, Weihai, China. The DNA G+C content of the type strain is 33.91 mol%.