Streptomyces blattellae, a novel actinomycete isolated from the in vivo of a Blattella germanica

During a screening for novel and useful actinobacteria in desert animal, a new actinomycete was isolated and designated strain TRM63209T. The strain was isolated from in vivo of a Blattella germanica in Tarim University in Alar City, Xinjiang, north-west China. The strain was found to exhibit an inhibitory effect on biofilm formation by Candida albicans ATCC 18,804. The strain was observed to form abundant aerial mycelium, occasionally twisted and which differentiated into spiral spore chains. Spores of TRM63209T were observed to be oval-shaped, with a smooth surface. Strain TRM63209T was found to grow optimally at 28 °C, pH 8 and in the presence of 1% (w/v) NaCl. The whole-cell sugars of strain TRM63209T were rhamnose ribose, xylose, mannose, galactose and glucose, and the principal polarlipids were found to be diphosphatidylglycerol, phos-phatidylethanolamine, phosphatidylcholine, phosphatidylinositol mannoside, phosphatidylinositol 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) (14.64%), MK-9(H2) (19.65%), MK-9(H8) (22.34%), MK-10(H2) (25.37%). The major cellular fatty acids were identified as iso-C16:0, 16:0, anteiso-C15:0, anteiso-C17:0, iso-C15:0 and Sum in Feature 3. Analysis of the 16S rRNA sequence showed that strain TRM63209T exhibits high sequence similarity to Streptomyces bungoensis strain DSM 41781T 98.20%. A multi-locus sequence analysis of five house-keeping genes (atpD, gyrB, rpoB, recA and trpB) and phylogenomic analysis also illustrated that strain TRM63209T should be assigned to the genus Streptomyces. The DNA G + C content of the strain was determined to be 70.2 mol%. Average nucleotide identity (ANI) between strain TRM63209T and S. bungoensis DSM 41781T, Streptomyces phyllanthi PA1-07T, Streptomyces longwoodensis DSM 41677T and Streptomyces caeruleatus NRRL B-24802T were 82.76%, 82.54%, 82.65%, 84.02%, respectively. Digtal DNA-DNA (dDDH) hybridization were 26.30%, 25.10%, 26.20%, 29.50%, respectively. Therefore, it is concluded that strain TRM63209T represents a novel species of the genus Streptomyces, for which the name Streptomyces blattelae is proposed. The type strain is TRM63209T (CCTCC AA 2018093T = LMG 31,403 = TRM63209T).


Introduction
The genus Streptomyces was initially described in 1943 (Waksman and Henrici 1943) and more than 850 species with valid names of Streptomyces have been published (https://www.bacterio.net/streptomyces. html) (Zhang et al. 2017). Streptomyces are aerobic Gram-positive bacteria with well-developed mycelia, which can produce a large number of conidia for reproduction. The members of the genus are with high DNA G ? C content, and species are widely distributed in aquatic and terrestrial ecosystems. Many Streptomyces undergo morphological differentiation and also have a mycelial phase. Streptomyces are highly versatile and produce an abundant array of bioactive secondary metabolites (Genilloud 2017) that have been used as antibiotic, anti-carcinogenic, antihelminthic and antifungal compounds. Consequently, they are very important for biotechnology, medicine and agriculture (Barka et al. 2015).
Strain TRM63209 T isolated from in vivo of an Blattella germanica in Tarim University in Alar City, Xinjiang, north-west China(40°55 0 N,81°29 0 E) This strain inhibits the formation biofilm of Candida albicans ATCC 18,804. On the basis of phylogenetic, phenotypic and genetic data, TRM63209 T considered to a novel species of the genus Streptomyces, for which the name Streptomyces blattelae is proposed.

Isolation of Streptomyces strain and culture conditions
As part of a program to unravel the diversity of symbiotic actinomycetes in insect-microbe and to discover novel actinomycetes and novel natural products, strain TRM63209 T was isolated from the in vivo of a Blattella germanica, the Tarim University, Alar, Xinjiang Province, north-west China. Blattella germanica is washed with sterile distilled water to remove surface impurities. The surface was sterilized in 70% ethanol for 60 s and then washed three times in sterile distilled water. Grind it to a powder and suspend in sterile distilled water incubated on a rotary shaker at 180 rpm 37°C for 30 min (Liu et al. 2017), ultrasound 3 min, and the suspension was appropriately diluted before being spread onto Czapek's agar (Wiese et al. 2008) supplemented with nystatin (100 mg/ml) and nalidixic acid (50 mg/ml) (Arocha-Garza et al. 2017). After 21 days of incubation at 28°C, the isolate was transferred and purified on International Streptomyces Project (ISP) 4 medium (Shirling and Gottlieb 1966) and the spore and mycelia maintained as glycerol suspensions (20%, v/v) at -80°C.

Chemotaxonomy
Isomers of diaminopimelic acid were analysed following the method of Hasegawa et al. (1983). The whole cell sugar composition was analysed following the method of Staneck and Roberts (1974). Polar lipids in cells of strain TRM63209 T were extracted and examined by two-dimensional TLC and identified following the methods of Minnikin et al. (1984). Menaquinones were extracted using the method of Collins (1985) and subjected to HPLC analysis (Groth et al. 1997). The cellular fatty acid composition was determined using the Microbial Identification System (MIDI Sherlock version 6.0) (Sasser 1990).

Phylogenetic analyses
Genomic DNA extraction and PCR amplification of the 16S rRNA gene from strain TRM63209 T were performed following Chun and Goodfellow (1995). The purified PCR product was cloned into the vector pMD19-T (Takara) and sent to Sangon for gene sequencing. Multiple alignments with sequences from closely related Streptomyces species and calculations of sequence similarity were performed using the EzTaxon-e server (Kim et al. 2012). Phylogenetic analyses were performed using MEGA version 7.0 (Kumar et al. 2016) selecting the neighbour-joining (Saitou and Nei 1987), Maximum-Evolution (Rzhetsky and Nei 1993) and maximum-likelihood (Felsenstein 1981) algorithms. Topologies of the resultant trees were evaluated using the Felsenstein's (1985) resampling method with 1000 replications. AtpD, gyrB, rpoB, recA and trpB genes were obtained using primers and amplification conditions as previously described (Guo et al. 2008;Hatano et al. 2003). Phylogenetic relationships were reconstructed using the Neighbour-Joining algorithm as described above. Phylogenomic analysis was performed online by Type (strain) Genome Server (TYGS) (Meier-Kolthoff et al. 2019).
The whole genome of TRM63209 T was sequenced by Oxford Nanopore technologies. The DNA G ? C content of strain TRM63209 T was obtained by whole genome sequencing. The Average nucleotide identity (ANI) was determined as described by Lee et al. (2015). DNA-DNA relatedness values were determined online according to the method of Meier- Kolthoff et al. (2013). DNA-DNA hybridization (dDDH) values were calculated at the Genome-to Genome Distance Calculator (GGDC) website using formula 2, as originally described by Auch et al. (2010) and updated by Meier-Kolthoff et al. (2013). Anti-SMASH was used to predict the biosynthetic gene clusters of strain TRM63209 T (Blin et al. 2013).
Antifungal and antibacterial activity C. albicans ATCC 18,804 was obtained from China Center for Type Culture Collection, and was cultured with Sabouraud dextrose agar/broth (SDA/SDB). Unless specified otherwise, ISP 3 was used to culture TRM63209 T strain.
A 4% (v/v) inoculum of well-growing strain TRM63209 T was used to culture strain TRM63209 T in ISP 3 liquid culture medium (20 g oatmeal and 1 ml trace salt per L distilled water) and incubated at 28°C with shaking at 180 rpm for 10 days. Cells were removed by centrifugation to leave the supernatant, which was kept at 4°C for further screening of biofilm inhibition. The effect of the strain TRM63209 T growth supernatants on static biofilm formation measured according to Balasubramanian et al. (2017). Briefly, test organism cells were diluted 1:100 with fresh SDB to bring the test cell suspension to a concentration of 1 9 10 8 cells per mL. 100 lL aliquots of cells were added to the wells of a 96 well plate and 100 lL of supernatants was added, then the plates inoculated with C. albicans were incubated at 37°C for 72 h. Wells without the supernatant (100 lL SDB) was used as blank control. After crystal violet staining, the absorbance was measured at 490 nm by an enzymelinked immunosorbent assay reader (Bio-Rad). Relative activity of biofilm formation was indicated as Relative Biofilm Formation % (RBF %) calculated the following formula: RBF % = Treated OD 490 /Untreated OD 490 9 100%.

Results and discussion
Strain TRM63209 T 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 ISP 5 and greenish White soluble pigment was produced in ISP 6, the colour of other the aerial mycelium is white, other no diffusible pigment was produced on the media test, the color of ISP 2 substrate mycelium is light yellow ( Table 1). The growth and cultural characteristics of strain TRM63209 T related type strains are listed in the species description and in Table S1.
Morphological characteristics of strain TRM63209 T 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 TRM63209 T was found to grow only at 5-55°C, pH 4.0-12.0 and 0-20% (w/v) NaCl, with optimal growth at 28°C, pH 8.0 and with 1% (w/v) NaCl. Other physiological characteristics of strain TRM63209 T are listed in the species description and in Table 1.
Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain TRM63209 T belongs to the genus Streptomyces, with high sequence similarity to Streptomyces bungoensis DSM 41781 T (GenBank accession no. KQ948892; 98.20%), Streptomyces phyllanthi PA1-07 T (GenBank accession no. LC125632; 98.14%), Streptomyces longwoodensis DSM41677 T (GenBank accession no. KQ948572;  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 TRM63209 T and similar species were greater than the 0.007 threshold (Fig. S7). Result of phylogenomic analysis also supported that strain TRM63209 T belonged to genus Streptomyces (supplementary Fig. S8). The DNA G ? C content in the draft genome sequence of strain TRM63209 T was determined to be 70.2 mol %. The complete genome of strain TRM63209 T 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. S4). The ANI relatedness between strain TRM63209 T and the phylogenetically related strain Streptomyces caeruleatus NRRL B-24802 T , Streptomyces bungoensis DSM 41781 T , Streptomyces longwoodensis DSM 41677 T and Streptomyces phyllanthi PA1-07 T were respectively determined to be 84.02%, 82.76%, 82.65%,82.54%. 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 TRM63209 T and the phylogenetically related strain Streptomyces bungoensis DSM 41781 T , Streptomyces phyllanthi PA1-07 T , Streptomyces longwoodensis DSM 41677 T and Streptomyces caeruleatus NRRL B-24802 was respectively determined to be 26.30%, 25.10%, 26.20%, 29.50%. significantly lower than the 70% threshold value for delineation of prokaryotic genomic species (Wayne et al. 1987). It is thus proposed that strain TRM63209 T can be differentiated from closely related Streptomyces species and represents a novel species. The supernatant of strain TRM63209 T inhibited biofilm formation by both C. albicans, with inhibition ratios over 40% (Table S3). The anti-SMASH biosynthetic gene cluster prediction tool was used to investigate the draft genome sequence of strain TRM63209 T 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 nonalpha poly-amino acids like e-Polysin (NAPAA), one ectoine, one arylpolyene, one other unspecified ribosomally synthesised and post-translationally modified peptide product (RiPP), one redox-cofactors such as  Table 2 Differential characteristics between strain TRM63209 T and phylogenetically related species of the genus Streptomyces   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 TRM63209 T inhibits the formation of BF may be associated with this gene cluster (Liang D). In summary, the sequencing of the genome of strain TRM63209 T further clarified the evolutionary relationship between strains and will guide the screening for active secondary metabolites.
The type strain, TRM63209 T (CCTCC AA 2018093 T = LMG 31,403), was isolated from the in vivo of a 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 TRM63209 T are WJBG00000000 and MK795724, respectively.