Identification And Antibacterial Activity Of Three Novel Species From The Genus Streptomyces And New Insights Into General Description Of Streptomyces Species

Actinomycetes,as the main antibiotic-producing bacteria,have play a tremendous role in in agriculture,industry and medical field.In recent decades,endophytic actinomycetes,especially members of the genus Streptomyces,have attracted much attention because of their ability to produce natural metabolites with similar properties to the host,but little is known about endophytic actinomycetes from Xanthium sibiricum.Therefore,it is of great practical significance to isolate and identify endophytic actinomycetes from X.sibiricum.In this study,three novel Streptomyces strains were identified using a polyphasic taxonomic approach currently accepted by microbial taxonomists.At the same time,according to the prediction of metabolite synthesis gene cluster and plate confrontation culture,the strain with strong antibacterial ability was selected for LC-MS detection.In addition,the definition of Streptomyces species was revised based on comparative genomic analysis of 80 pairs of Streptomyces species(including heterotypic synonyms)in order to more quickly and accurately determine the taxonomic status of a Streptomyces strain.The main results are as follows.(1)Strains CRXT-Y-14~T,CRXT-G-22~T and CRPJ-33~T were identified as three novel Streptomyces species based on phenotypic,chemotaxonomic and molecular genetic characteristics.Strain CRXT-Y-14~Twas isolated from leaves of X.sibiricum collected in Shaoshan,Hunan Province,for which the name Streptomyces xanthii was proposed;Strains CRXT-G-22~T was isolated from seeds of X.sibiricum collected in Shaoshan,Hunan Province,for which the name Streptomyces roseirectus was proposed;Strain CRPJ-33~T was isolated from leaves of X.sibiricum collected in Pingjiang,Hunan Province,for which the name Streptomyces genisteinicus was proposed.(2)The biosynthetic gene clusters of strains CRXT-Y-14~T,CRXT-G-22~T and CRPJ-33~Twere predicted using anti SMASH.Results indicated that all three strains had abundant gene clusters and showed great potential for exploitation.In addition,the results from antagonism experiment showed that strain CRXT-Y-14~Thad a slight antagonistic effect on Mucor sp.Strain CRPJ-33~Thad a slight antagonistic effect on Staphylococcus aureus in ISP3 and the size of the inhibition ring was about 0.3 cm.Strain CRXT-G-22~T had antagonistic effect on Nigella,Aspergillus niger,Penicillium sp and Mucor sp.The antagonistic effect was most obvious to Nigella.Based on the preliminary identification of metabolites,it was found that strain CRXT-G-22~T contains abundant active substances,especially among the identified known compounds,the relative contents of genistein and indole-3-carbinol were higher,up to 33.8%and 18.4%,respectively.Clinically,these two substances are mainly derived from plants.The former could prevent and inhibit the formation of tumor by promoting apoptosis and antiangiogenesis.It also has the effects of antibacterial and lipid-lowering.The latter has a variety of function such as prevention and resistance to cancer,antiviral,anti-inflammatory,antioxidant activity.Compared with extraction from plants,the production of genistein and indole-3-carbinol by microbial fermentation is not restricted by external conditions such as sunlight and temperature.It also has short production cycle and simple operation.So,the discovery of strain CRXT-G-22~T has important practical significance.(3)Based on correlation among MLSA,d DDH and ANI values of a total of 80 pairs of Streptomyces species(including heterotypic synonyms),we found,on the one hand,a 70%d DDH value did not corresponded to a 95-96%ANIm value but approximately to a 96.7%ANIm value in the genus Streptomyces.On the other hand,we proposed that when the MLSA distance value between a novel Streptomyces strain and a reference strain was less than 0.008,the novel strain could be considered as a heterotypic synonym of the reference strain;when the MLSA distance value was greater than or equal to 0.014,the novel strain could be regarded as a new Streptomyces species;when the MLSA distance value was between 0.008 and 0.014(not included),ANIm or d DDH value between a new strain and a reference strain must be calculated in order to determine the taxonomic status of a novel strain.Although 70%d DDH or 96.7%ANIm value could act as the threshold value in delineating Streptomyces species,if d DDH or ANIm value was less than but close to 70%or 96.7%cut-off point,the taxonomic status of a novel strain could be only determined by a combination of phenotypic characteristics,chemotaxonomic characteristics and genome phylogenetic analysis.