| Objectives The exploration of the diversity of actinomycetes and further selection of representative strains for antibacterial activity studies were performed through Isolating and identifying of culture-able actinomycetes from nodules and the rhizosphere soil of Hippophae rhamnoides in Nyingchi and Shannan regions as well as a plant and the rhizosphere soil from Motuo region of Tibet.Complete genome sequencing,gene cluster prediction of secondary metabolite synthesis and metabolite analysis were conducted for some strains,which could be potential bacterial sources for separating the natural products from microorganisms.Methods Actinomycetes were isolated from 5 root nodules and the rhizosphere soil of Hippophae rhamnoides,1 sample of Balanophora sp.and its rhizosphere soil by diluting and coating method.The naked eye discernible colony was selected,transferred and purify to ISP2 plate;the 16 S r RNA sequence of the strain was obtained by PCR amplification,and the phylogenetic tree of adjacency Neighbor-Joining method(N-J)was constructed to analyze the diversity of actinomycetes from Tibet.After 16 S r RNA sequence comparison,the strains were frozen and stored in the refrigerator at-80? in glycerol tube.Representative actinomycetes were fermented with 12 kinds of fermentation medium in96-well plate,and 7 kinds of detected bacteria were selected to evaluate the antibacterial activity of the strains,and the representative strains were selected to collect mass spectrometry data by Ultra high performance liquid chromatography-mass spectrometry(UPLC-MS)to analyze their metabolites.The genome of some strains were sequenced,and the secondary metabolites were predicted by anti SMASHV5.0 software.Furthermore,the(SSN)network of sequence similarity was analyzed by BIG-SCAPE algorithm to explore the biosynthesis potential of secondary metabolites of actinomycetes from Tibet.Results 1 A total of 385 strains of actinomycetes were isolated from root nodules and rhizosphere soil of Hippophae rhamnoides in Nyingzhi and Shannan,Tibet.112 strains of actinomycetes were obtained by 16 S r RNA sequence,which were distributed in 7 orders,12 families and 23 genera,of which Streptomyces accounted for 41%,Micromonospora accounted for 9.8%,and Microbacterium accounted for 8.9%.The Amycolatopsis and Nocardia each account for 5.3%.These five genera of actinomycetes are the dominant actinomycetes that can be cultured in this area.2 The results of antibacterial activity of 46 representative strains showed that 17 strains of actinomycetes had antibacterial activity against at least one kind of tested bacteria,and the positive inhibition rate was 36.9%.16 strains of actinomycetes had strong antibacterial activity against methicillin-resistant Staphylococcus aureus(MRSA),and 13 strains of actinomycetes had higher inhibition rate of MRSA than vancomycin.In addition,7 strains of actinomycetes had antibacterial activity against carbapenem-resistant Acinetobacter baumannii(CRAB).Among them,Streptomyces sp.XZ19-363 had significant bacteriostatic effect on CRAB,and the bacteriostatic rate was 91.3%.3 The metabolites of two strains of Streptomyces sp.XZ19-122 and Streptomyces sp.XZ19-015 were analyzed by UPLC-MS,and it was found that these two strains of Streptomyces could produce ion chelating agent Nonactin compounds with antibacterial activity,and it was speculated that their antibacterial activity might be produced by these compounds.4 The secondary metabolite synthesis gene clusters of three strains Nocardia sp.XZ19-231,Nocardia sp.XZ19-369 and Nocardia sp.XZ19-385 were predicted.A large number of unknown orphan gene clusters were found,which predicted the potential to produce novel active substances.5 Actinomycetes isolated from a plant of Balanophora sp.and its rhizosphere soil in Motuo area,37 strains of different actinomycetes were obtained by 16 S r RNA sequence comparison,which were distributed in 3 orders,8 families and 16 genera.The dominant strain was Streptomyces,According to Kim,the critical value of 16 S r RNA was lower than 98.65% as the standard for potential new species.There were 5 potential new bacteria in the samples in Motuo area.It needs to be confirmed by a number of follow-up classification studies.Conclusions 1 Actinomycetes isolated from the root nodules and the rhizosphere soil of Hippophae rhamnoides in Nyingzhi and Shannan area of Tibet,Balanophora sp.and the rhizosphere soil in Motuo area are rich and diverse,there is a high proportion of potential new actinomycetes from Balanophora sp.plant and its rhizosphere soil in Motuo area of Tibet.2 The actinomycetes isolated from the root nodules and rhizosphere soil of Hippophae rhamnoides in Nyingchi and Shannan area have good antibacterial activity and can provide high quality bacteria source for screening new bioactive substances.3 Two strains of Streptomyces with broad-spectrum antibacterial activity were found to contain rich metabolites by UPLC-MC analysis,which further proved that they had medicinal microbial value.4 The three strains of Nocardia isolated from the rhizosphere soil of Hippophae rhamnoides in Nyingchi and Shannan region contain a large number of unknown orphan gene clusters,which has the potential to synthesize new compounds.It has important research and application value.Figure12;Table 5;Reference 122... |
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