Strain TRM63209T was observed to grow optimally on ISP 3 and ISP 2, and showed moderate growth on ISP 1, ISP 4, ISP 5, nutrient agar and Gause’s synthetic agar no. 1, with slow growth on ISP 6, ISP 1 and Czapek’s medium. Light yellow soluble pigment was produced in ISP5 and Greenish White soluble pigment was produced in ISP6, the colour of other the aerial mycelium is white, other no diffusible pigment was produced on the media test, the color of ISP2 substrate mycelium is light yellow(Table 1). Morphological characteristics of strain TRM63209T were observed using SEM (Fig. 1). The strain was observed to form an abundant white aerial mycelium, occasionally twisted, which differentiates into spiral spore chains. Each spore was observed to be oval-shaped with a smooth surface (Fig. 1). Strain TRM63209T was found to grow only at 5–55 ℃, pH 4.0–12.0 and 0–20% (w/v) NaCl, with optimal growth at 28 ℃, pH 8.0 and with 1% (w/v) NaCl. Other physiological characteristics of strain TRM63209T are listed in the species description and in Table 1.
The whole-cell sugars of strain TRM 63209T were rhamnose, ribose, xylose, galactose, glucose and mannose, and the principal phospholipids were found to be diphosphatidylglycerol(DPG), Phos-phatidylethanolamine(PE), phosphatidylcholine(PC), phosphatidylinositol mannoside(PIM), phosphatidylinositol(PI) and an unknown phospholipid(L)(supplementary Fig. S1, Fig. S3). The diagnostic cell wall amino acid was identified as LL-diaminopimelic acid (supplementary Fig. S2). The predominant menaquinone was found to be MK-9(H6), MK-9(H2), MK-9(H8), MK-10(H2) (Supplementary Fig. S4). The major cellular fatty acids (> 5%) were identified as iso-C16:0(26.5%), 16:0 (21.4%), anteiso-C15:0 (9.8%), anteiso-C17:0 (9.7%), iso-C15:0 (6.7%) and Sum In Feature 3 (5.7%). Fatty acids present in smaller amounts (> 1%) were iso-C17:0 (4.3%), iso-C16:1 H (1.9%), iso-C14:0 (1.9%), 18:1 w9c (1.6%), 15:0(1.4%), Sum In Feature 9 (1.4%), anteiso-C17:1 w9c (1.4%), 14:0 (1.2%) and Sum In Feature 5 (1.7%)(supplementary Table S1). It utilized D-mannitol, trehalose, sucrose, L-rhamnose, galactose, raffinose, maltose, cellobiose, sorbose as carbon sources but not chitosan, xylan, L-arabinose, starch, melezitose, glucose, xylose, fructose, Inositol or xylitol. Comparison of the physiological properties of strain TRM 63209 T with other species of Streptomyces is shown in Table 2.
Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain TRM 63209 T belongs to the genus Streptomyces, with high sequence similarity to Streptomyces bungoensis DSM 41781T (GenBank accession no. KQ948892; 98.20%), Streptomyces PA1-07T (GenBank accession no. LC125632; 98.14%), Streptomyces longwoodensis DSM41677T (GenBank accession no. KQ948572; 98.00%) and Streptomyces caeruleatus NRRL B-24802T (GenBank accession no. KQ948975; 98.00%). Strain TRM63209T was found to form an unique clade that was different from other closely related species (Fig. 2). The apparent close relationship with Streptomyces bungoensis DSM 41781T did not receive high bootstrap support and was not supported by the other two (Maximum Likelihood and Minimum-Evolution) tree building methods (supplementary Fig. S5, Fig. S6). According to the guiding principles of Rong and Huang (2012), in multi-locus sequence analysis (MLSA) it is considered that pairs with evolutionary distances greater than 0.007 belong to different species. A MLSA of five house-keeping genes (atpD, gyrB, recA, rpoB, and trpB) indicated that the MLSA distances between strain TRM63209T and similar species were greater than the 0.007 threshold (Fig. S7). Therefore, it was concluded that TRM63209T represents a distinct species belonging to the genus Streptomyces.
The G + C content in the draft genome sequence of strain TRM63209T was determined to be 70.2 mol %. The complete genome of strain TRM63209T has a size of 8.49 Mb, did not distribute among chromosomes and plasmids. In its genome, 7804 genes were annotated, of which 7732 are putative protein-coding genes. The number of hypothetical proteins is 2367, corresponding to 31% of the total number of putatively annotated proteins. 60 tRNAs and seven copies of the 16S rRNA gene were identified. The genomic characteristics of the compared strains are quite heterogeneous (Table S3). The ANI relatedness between strain TRM63209T and the phylogenetically related strain Streptomyces bungoensis DSM 41781T, Streptomyces phyllanthi PA1-07T, Streptomyces longwoodensis DSM 41677T and Streptomyces caeruleatus NRRL B-24802T was respectively determined to be 82.76 %, 82.54%, 82.65%, 84.02%. This value is significantly lower than the widely accepted threshold for describing prokaryote species (95–96%; Kim et al. 2014). The dDDH value between strain TRM63209T and the phylogenetically related strain Streptomyces bungoensis DSM 41781T, Streptomyces phyllanthi PA1-07T, Streptomyces longwoodensis DSM 41677T and Streptomyces caeruleatus NRRL B-24802 was respectively determined to be 26.30%, 25.10%, 26.20%, 29.50%. These significantly lower than the 70% threshold value for delineation of prokaryotic genomic species (Wayne et al. 1987). It is thus proposed that strain TRM63209T can be differentiated from closely related Streptomyces species and represents a novel species. The supernatant of strain TRM63209T inhibited biofilm formation by both C. albicans, with inhibition ratios over 40% (Table S2). The anti-SMASH biosynthetic gene cluster prediction tool was used to investigate the draft genome sequence of strain TRM63209T and found one type I, two type III polyketide biosynthetic gene clusters, five nonribosomal peptide synthetase biosynthetic gene clusters and one NRPS-like fragment. In addition, five terpene, three siderophore, three Class I lanthipeptide clusters like nisin, one Non-alpha poly-amino acids like e-Polylysin(NAPAA), one ectoine, one Arylpolyene, one Other unspecified ribosomally synthesised and post-translationally modified peptide product (RiPP), one Redox-cofactors such as PQQ (NC_021985:1458906–1494876), one Oligosaccharide, two siderophore, one melamin and one indole biosynthetic gene clusters were detected. Numbers of secondary metabolite-associated gene clusters in TRM63209T in comparison to other species in the family that is shown in Table S4. A product of one of these clusters may be involved in the antibiofilm activity observed. Through anti-SMASH analysis, the 7-prenylisatin antibiotic biosynthesis gene cluster can be found, which can effectively inhibit the growth of fungi and the similarity to 60%, whereby the strain TRM63209T inhibits the formation of BF may be associated with this gene cluster (Liang D). In summary, the sequencing of the genome of strain TRM63209T further clarified the evolutionary relationship between strains and will guide the screening for active secondary metabolites.
Description of Streptomyces blattellae
Streptomyces blattellae (blat.tel'lae. N.L. gen. n. blattellae of the cockroach genus Blattella).
Aerobic, Gram-stain positive actinomycete. Forms abundant aerial mycelium, occasionally twisted, which differentiates into spiral spore chains. Each spore is oval-shaped with a smooth surface. The strain grew well and developed more abundant aerial mycelia on ISP3 and ISP2 than on ISP 1, ISP 4, ISP 5, NA and Gause’s synthetic agar no. 1, but poor growth was observed on ISP7 and Czapek’s medium. Growth was observed at 5–55, with 0–20 % (w/v) NaCl and at pH 4.0–12.0 and was found to grow optimally at 28℃, pH 7 and in the presence of 1% (w/v) NaCl. The whole-cell sugars of strain TRM 63209T were rhamnose, ribose, xylose, mannose galactose and glucose, and the principal phospholipids were found to be diphosphatidylglycerol(DPG), Phos-phatidylethanolamine(PE), phosphatidylcholine(PC),phosphatidylinositol mannoside(PIM), phosphatidylinositol(PI) and an unknown phospholipid(L). The diagnostic cell wall amino acid was identified as LL-diaminopimelic acid. The predominant menaquinone was found to be MK-9(H6), MK-9(H2), MK-9(H8), MK-10(H2). The major cellular fatty acids were identified asiso-C16:0, 16:0, anteiso-C15:0, anteiso-C17:0, iso-C15:0, Sum In Feature 3 and Summed Feature 3. The DNA G + C content of the strain was determined to be 70.2 mol %.
The type strain, TRM63209T (CCTCC AA 2018093T = TRM63209T ), was isolated from the in vivo of an Blattella germanica in Tarim University, Alar City, Xinjiang Province, The GenBank/ EMBL/ DDBJ accession numbers for the genome and 16S rRNA gene sequence of strain TRM63209T are WJBG00000000 and MK795724, respectively.