Genomic and Phenotypic Characterization of Streptomyces sirii sp. nov., Amicetin-Producing Actinobacteria Isolated from Bamboo Rhizospheric Soil

Abstract

In our large-scale search for antimicrobial-producing bacteria, we isolated an actinomycete strain from rhizospheric soil of Bambusa vulgaris. The strain designated BP-8 showed noticeable antibacterial activity. BP-8 was subjected to a whole-genome analysis via a polyphasic taxonomy approach, and its antibacterial metabolite was identified by HRLS-MS. The results of the physiological and morphological analyses indicated that BP-8 is an aerobic, neutrophilic, mesophilic organism that is tolerant to 8% NaCl and can use a wide range of carbohydrates. It forms curly sporophores with a warty surface. The results of the phylogenetic and average nucleotide identity analyses and in silico DNA-DNA hybridization calculation indicated that BP-8 represents the type strain of a novel Streptomyces species. A comparative in silico analysis of the genome sequences of BP-8 and its closest related strains revealed the presence of genes encoding chemotaxonomic markers characteristic of Streptomyces. The antibacterial compound was identified as amicetin. Genomic mining also revealed more than 10 biosynthetic gene clusters that have not been described previously and may lead to the discovery of new valuable compounds. On the basis of these results, strain BP-8^T (=VKM Ac-3066^T = CCTCC AA 2024094^T) is proposed as the type strain of the novel species Streptomyces sirii sp. nov.

Keywords: BGCs; Streptomyces; amicetin; polyphasic taxonomy; rhizosphere soil.

Figure 5

Analysis of bioactive metabolites from the strain BP-8^T: (a) a comparison of amicetin BGC (BGC0000953) with region 7 in the BP-8^T complete genome sequence, generated using clinker tool [46]. Homologous genes are highlighted with colors, and labels indicate identity of the genes. (b) MS2 fragmentation spectrum of [M+H]⁺ ion (m/z 619.3078) of the isolated compound; structure of amicetin and characteristic [45] fragmentation patterns.

Figure 1

Phylogenetic trees of Streptomyces sp. BP-8^T and type strains of the related species: (a) tree inferred with FastME 2.1.6.1 [5] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 91.5%. The tree is rooted at the midpoint. (b) Part of phylogenomic tree for Streptomyces genera constructed with de novo workflow of GTDB-tk.

Figure 2

Cultural and morphological properties of BP-8^T on ISP3 media after 10 days at 28 °C: (a) color of aerial mycelium, (b) color of substrate mycelium, (c) spiral chains of spores (×1000 magnification).

Figure 3

Scanning electron micrographs of strain BP-8^T on ISP3 agar for 12 days at 28 °C: (a) spiral chains of spores; (b) warty surface of non-mature spore chain among smooth aerial hyphae.

Figure 4

Induction of a two-color reporter system sensitive to inhibitors of ribosome progression and DNA replication. Erythromycin (Ery), levofloxacin (Lev), and the compounds obtained during the broth culture purification via solid-phase extraction were placed on an agar plate with E. coli ΔtolC cells transformed with a pDualrep2 plasmid. The fluorescence of the E. coli lawn was scanned at 553/574 nm for RFP fluorescence and 588/633 nm for Katushka2S. The induction of Katushka2S expression is triggered by translation inhibitors, and RFP is regulated by the SOS response to DNA damage. BC—broth culture of BP-8^T; 0, 10, 20, 25, 30, 35, 40, and 50%—elution with acetonitrile solutions in water of indicated volume concentrations.