Polyphasic Taxonomy And Secondary Metabolites Analysis Of Three Streptomyces Strains From Saline Lakes

Actinomycetes is an important source for the discovery of bioactive natural products.Streptomyces is the largest genus of the phylum of Actinomycetota.Most of the natural products from actinomycetes were reported to be produced by the genus of Streptomyces.In recent years,many new species of Streptomyces have been discovered,which have yielded novel metabolic products.Therefore,the isolation and identification of new Streptomyces species not only enriches the species resources of Streptomyces,but also holds great significance for the discovery of new active secondary metabolites.In this study,a polyphasic taxonomic approach was employed to test and analyze three novel Streptomyces strains（XMNu-215,XMNu-224,and XMNu-361）isolated from Xilin Gol Plateau saline lakes,Inner Mongolia,involved in molecular phylogenetic analysis,morphological and cultural characteristics,physiological and biochemical properties,and cellular chemotaxonomy,which aims to determine their taxonomic status.The whole genome sequences of strain XMNu-215,XMNu-224,and XMNu-361 were also obtained,and the sequences were used in analyzing their biosynthetic gene clusters（BGCs）of secondary metabolites by anti SMASH,which aims to predict their ability of synthesizing secondary metabolites.Strain XMNu-215 and XMNu-361 showed the highest 16S r RNA gene similarity of 97.73%and 97.86%to the closely related type strain S.lopnurensis TRM 49590^T,respectively.Strain XMNu-224 shared the highest16S r RNA gene similarity of 97.65%to S.sodiiphilus YIM 80305^T.Strain XMNu-215,XMNu-224 and XMNu-361 shared the 16S r RNA gene similarity levels about 98%between each other.The phylogenetic trees constructed based on 16S r RNA gene sequences showed that strain XMNu-215,XMNu-224 and XMNu-361 clustered with S.lopnurensis TRM 49590^Tand S.sodiiphilus YIM 80305^T forming a stable subclade.Multilocus sequence analysis（MLSA）based on concatenated sequences of the atp D-gyr B-rec A-rpo B-trp B genes and phylogenetic analysis based on the whole genome further confirmed their close relationship.In addition,the digital DNA–DNA hybridization（d DDH）values between the genomes of strain XMNu-215,XMNu-224,XMNu-361 and the reference type strains S.lopnurensis TRM 49590^T were 33.70%,34.20%and 32.50%,respectively.The d DDH values between the genomes of strain XMNu-215,XMNu-224,XMNu-361 and S.sodiiphilus YIM 80305^T were 24.50%,24.60%and23.10%,respectively.The average nucleotide identity-by blast（ANIb）values between the genomes of strain XMNu-215,XMNu-224,XMNu-361and S.lopnurensis TRM 49590^T,S.sodiiphilus YIM 80305^T ranged from 79%to 87%,79%to 88%and 78%to 87%,respectively.The d DDH values between the genomes of strain XMNu-215,XMNu-224 and XMNu-361ranged from 34%to 36%;the ANI values ranged from 87%to 88%.The d DDH values and the ANI values were below the thresholds of 70%and 95%for prokaryotic conspecific assignation.Therefore,based on the comprehensive analysis of 16S r RNA genes,housekeeping genes,and genomic characterization,it was concluded that strain XMNu-215,XMNu-224 and XMNu-361 should be three different novel species of the genus Streptomyces.Strain XMNu-215,XMNu-224 and XMNu-361 were all Gram-staining-positive,and could grow at 15℃～37℃and with 0～3%（w/v）Na Cl.All the three strains had abundant aerial mycelium,and the growth states of strain XMNu-215,XMNu-224 and XMNu-361 were observed to be similar,with long filamentous mycelium,microhelical ends of mycelium,and slightly expanded ends of mycelium under scanning electron microscopy.The unique structure of strain XMNu-361 was the formation of expansion at the branches of the mycelium.Strain XMNu-215,XMNu-224 and XMNu-361 were positive for catalase production and nitrate reduction tests.The diagnostic diamino acid of strain XMNu-215,XMNu-224 and XMNu-361was LL-diaminopimelic acid.The whole-cell sugars of strain XMNu-215were mannose,glucose and ribose.The whole-cell sugars of strain XMNu-224 were mannose,glucose,ribose and galactose.The whole-cell sugars of strain XMNu-361 were glucose and ribose.The major fatty acids of strain XMNu-215 were anteiso-C_15:0,iso-C_16:0,C_16:0 and anteiso-C_17:0.The major fatty acids of strain XMNu-224 were anteiso-C_15:0,iso-C_16:0 and C_16:0.The major fatty acids of strain XMNu-361 were anteiso-C_15:0,iso-C_16:0 and iso-C_15:0.The major polar lipids were diphosphatidylglycerol,phosphatidylethanolamine,phosphatidylinositol and phosphatidylinositol mannoside.The predominant menaquinones were MK-9（H₆）and MK-9（H₈）.The three strains showed the typical cellular chemotaxonomic characteristics similar to the genus Streptomyces,but they could be distinguished from each other and differentiated from other closely related type strains by some biochemical reactions and cellular chemotaxonomic components.Thus,strain XMNu-215,XMNu-224 and XMNu-361 are considered to represent three novel species of the genus Streptomyces.A total of 22 BGCs were identified by anti SMASH in the genomes of strain XMNu-215,and the main clusters were PKS,NRPS and terpene,etc.In the genome sequence of strain XMNu-215,four putative BGCs（ectoine,geosmin,citrulassin D and pristinol）shows 100%similarity to the existing gene clusters,eight putative BGCs showed moderate similarity（30%～70%of genes showed similarity）,nine putative BGCs showed low similarity（<30%of genes showed similar）and one putative BGCs were not conserved relative to any known clusters.A total of 23 BGCs were identified by anti SMASH in the genomes of strain XMNu-224,and the main clusters were PKS,terpene,NRPS and ectoine,etc.In the genome sequence of strain XMNu-224,four putative BGCs showed high similarity（>70%of genes showed similarity,including three BGCs showed 100%similarity）,seven putative BGCs showed low similarity（<30%of genes showed similar）and twelve putative BGCs were not conserved relative to any known clusters.A total of 50 BGCs were identified by anti SMASH in the genomes of strain XMNu-361,and the main clusters were PKS,lanthipeptide and NRPS,etc.In the genome sequence of strain XMNu-361,three putative BGCs（livipeptin,naringenin and ectoine）shows 100%similarity to the existing gene clusters,seventeen putative BGCs showed low similarity（<30%of genes showed similar）and thirty putative BGCs were not conserved relative to any known clusters.The existence of these high and moderate similarity BGCs indicated that the strain XMNu-215,XMNu-224 and XMNu-361 has the ability to produce these metabolites.The existence of these cryptic BGCs implied that strain XMNu-215,XMNu-224 and XMNu-361 could be a potential source for secondary metabolites with novel structure.