Morphological and physio-chemical characteristics
Strain HUAS 5T produced grey aerial mycelium that differentiated into spiral spore chains with smooth-surfaced round or oval spores on Gause's synthetic No. 1 medium (No. 1) at 28°C after incubation 14d, which indicated that it had typical morphological traits of the genus Streptomyces (Fig. 1). Strain HUAS 5T grew well on No. 1, Reasoner' 2A (R2A), ISP 2–7 media without diffusible pigment. The strain grew at 15–40°C (optimum, 28°C), pH 6.0–9.0 (optimum, pH 7) and in presence of 0–5.0% (w/v) NaCl.
Strain HUAS 5T was positive for nitrate reduction and hydrolysis of Tweens (20, 40 and 80), but negative for hydrolysis of starch and Tween 60. Strain HUAS 5T could utilize d-fructose, d-glucose, d-ribose, d-xylose, malt sugar, lactose, l-rhamnose, raffinose, and saccharose as sole carbon sources, but could not utilize l-arabinose, d-cellobiose, d-galactose, d-mannose and myo-inositol. Aspartate, histidine, l-alanine, l-glycine, l-hydroxyproline, l-ornithine, l-phenylalanine, l-proline, l-serine, tyrosine and valine, were utilized as sole nitrogen sources, but not for arginine, l-cysteine and l-glutamic acid.
Chemotaxonomic characteristics
The diagnostic amino acid of stain HUAS 5T contained ll-diaminopimelic acid (ll-DAP); Glucose, ribose and mannose were presented in whole-cell hydrolysates. The primary cellular fatty acids (> 5.0%) of strain HUAS 5T were iso-C16:0 (30.3%), iso-C14:0 (14.4%), anteiso-C15:0 (11.7%), iso-C15:0 (9.9%), C16:0 (6.8%), iso H-C16:1 (5.5%), Sum in Feature 3 (C16:1 ω7C/C16:1 ω6C) (5.1%). Other fatty acids present in smaller amounts (> 1.0%) were anteiso-C17:0 (2.1%), C14:0 (1.5%), Sum in Feature 5 (C18:2 ω6,9c/C18:0 ante) (1.5%), Sum in Feature 9 (10-methyl C16:0) (1.5%), anteiso-C17:1 ω9c (1.2%). The phospholipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphotidylinositol (PI), phosphatidylinositol mannosides (PIM), phosphatidyl methyl ethanolamine (PME), phospholipids (PLS), phospholipids choline (PC), phospholipids of unknown structure containing glucosamine (NPG) and unidentified phospholipids (L1, L2, L3 and L4) (Fig. S1). All these results confirmed that stain HUAS 5T should belong to the genus Streptomyces of (Williams et al., 1989).
Genomic characteristics and phylogenetic analysis
The result of Rapid Annotation by using Subsystems Technology indicated that, strain HUAS 5T had 305 subsystems, consist of 23 categories, and the subsystem coverage was 18%. The identified subsystem features were ‘amino acids and derivatives’(380 CDSs), ‘carbohydrates’(331 CDSs), ‘protein metabolism’ (214 CDSs), ‘cofactors, vitamins, prosthetic groups, pigments’ (176 CDSs), ‘fatty acids, lipids, and isoprenoids’ (170 CDSs), ‘respiration’ (118 CDSs), ‘nucleosides and nucleotides’ (95 CDSs), ‘DNA metabolism’ (80 CDSs), ‘stress response’ (65 CDSs), ‘metabolism of aromatic compounds’ (52 CDSs), ‘virulence, disease and defense’ (50 CDSs), ‘RNA metabolism’ (50 CDSs), ‘cell wall and capsule’ (42 CDSs), ‘membrane transport’ (33 CDSs), ‘nitrogen metabolism’ (33 CDSs), ‘miscellaneous’ (30 CDSs), ‘phosphorus metabolism’ (27 CDSs), ‘regulation and cell signaling’ (26 CDSs), ‘iron acquisition and metabolism’ (22 CDSs), ‘secondary metabolism’ (18 CDSs), ‘dormancy and sporulation’ (9 CDSs), ‘potassium metabolism’ (6 CDSs) and ‘sulfur metabolism’ (6 CDSs). The 21 biosynthetic gene clusters were terpene, thiopeptide, LAP [linear azol(in)e-containing peptides], RiPP-like [post-translationally modified peptide product (RiPP) cluster], T1PKS (type 1 polyketide synthase), PKS-like (other types of PKS), NRPS-like (NRPS-like fragment), lanthipeptide-class-I, NI-siderophore, ladderane, arylpolyene, NRPS (non-ribosomal peptide synthetase), lassopeptide, crocagin, butyrolactone, redox-cofactor, ectoine, T2PKS (Type II PKS), betalactone, NAPAA (non-alpha poly-amino acids like e-polylysin) and RiPP-like [post-translationally modified peptide product (RiPP) cluster]. Meanwhile, vanW gene in vanI cluster, HelR and Streptomyces venezuelae rox were discoveried by CRAD analysis, which might lead to resistance to glycopeptide antibiotic rifamycin antibiotic. Finally, 15 CRISPR repeats and 20 genetic islands with a size range from 6,001 to 39,486 bp were found in the genome of strain HUAS 5T.
The blast analysis of the 16S rRNA gene sequence (1529 bp) of strain HUAS 5T indicated that it belonged to the genus Streptomyces and exhibited highest sequence similarity to Streptomyces hirsutus NRRL B-2713T (97.3%). A ML Phylogenetic analysis of 16S rRNA gene sequence indicated that strain HUAS 5T formed an independent lineage, which suggested that it belonged to a potential novel species (Fig. 2). This relationship was recovered in the NJ and MP trees (Figs. S2 and S3). Stackebrandt and Ebers (2006) have pointed out that when the 16S rRNA gene similarity between a new isolate and its relatives was in the threshold range of 98.7–99%, DNA–DNA relatedness studies should be mandatory between them. However, in this work, the highest 16S rRNA gene similarity between strain HUAS 5T and other type strains with validly published names was 97.3%, which far less than 98.7%, therefore there's no need to carry out average nucleotide identity (ANI) analysis and the digital DNA-DNA hybridization (dDDH) or DDH values for evaluating their DNA–DNA relatedness in delineating new species.
Based on all of these data above, strain HUAS 5T is characteristically a Streptomyces and represents a novel species of the genus Streptomyces, for which the name Streptomyces chenzhouensis sp. nov. is proposed.
DESCRIPTION OF STREPTOMYCES CHENZHOUENSIS SP. NOV.
Streptomyces chenzhouensis (chen.zhou.en'sis.N.L. fem. adj. chenzhouensis, pertaining to chenzhou, a city of Hunan Province in China).
The strain produces grey aerial mycelium that differentiated into spiral spore chains with smooth-surfaced round or oval spores on Gause's synthetic No. 1 medium. Grows well on No. 1, Reasoner' 2A, ISP 2–7 without diffusible pigment. Growth occurs at 15–40°C (optimum, 28°C), pH 6.0–9.0 (optimum, pH 7) and in presence of 0–5.0% (w/v) NaCl. Positive for nitrate reducation and hydrolysis of Tweens (20, 40 and 80), but negative for hydrolysis of starch and Tween 60. Utilizes d-fructose, d-glucose, d-ribose, d-xylose, malt sugar, lactose, l-rhamnose, raffinose, saccharose, as sole carbon sources, but not l-arabinose, d-cellobiose, d-galactose, d-mannose and myo-inositol. Utilizes aspartate, histidine, l-alanine, l-glycine, l-hydroxyproline, l-ornithine, l-phenylalanine, l-proline, l-serine, tyrosine and valine, as sole nitrogen sources, but not arginine, l-cysteine and l-glutamic acid. The diagnostic amino acid is ll-diaminopimelic acid (ll-DAP), glucose, ribose and mannose are present in whole-cell hydrolysates. The menaquinones are MK-9(H4), MK-9(H6) and MK-9(H8). The phospholipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphotidylinositol (PI), phosphatidylinositol mannosides (PIM), phosphatidyl methyl ethanolamine (PME), phospholipids (PLS), phospholipids choline (PC), phospholipids of unknown structure containing glucosamine (NPG) and unidentified phospholipids (L1, L2, L3 and L4). The DNA G + C content of the type strain is 71.3%.
The type strain, HUAS 5T (= MCCC 1K08552T = JCM 36055T), was isolated from the root tissue of Cathaya argyrophylla collected in Chenzhou city of Hunan Province, PR China. The NCBI accession numbers for the full-length 16S rRNA gene sequence and genome sequence of strain HUAS 5T are OR237570 and CP121682, respectively.