Screening And Identification Of Actinobacteria From Longwan Volcanic Lake And Study Of The Active Metabolites

The abundant secondary metabolites of actinobacteria is an important source of microbial natural medicine and their development and utilization are becoming a research focus that draw scientists' attention.With the development of human society,the appeal for natural medicine with high-activity and new structure becomes much stronger.However,with the further study of actinobacteria in conventional ecosystem,the acquirement of strains that producing novel natural medicine has become more difficult,which has greatly influence on the efficiency of thedevelopment of novel drugs.Therefore,searching for new isolation sources and the isolation of actinobacteria from special habitats is the efficient path that improves the new type of natural medicine efficiency.Longwan volcanic lake locating in east Huinan of Jilin province(China)was generated by the increasing of underground water after the volcanic eruption 200,000 years ago.The formation of specific wetlands ecosystem bases on its unique volcanic landforms and provides new resources for the richness of actinobacteria and novel bioactive natural compounds.Therefore,the purpose of this research is to study species composition,isolate and identify of novel actinobacteria and active metabolites of antagonism strains by using Longwan volcanic lake soil sediments.The specific research results are as follows:(1)158 actinobacteria of volcanic lake were obtained after isolation and purification.16S rRNA gene sequencing was performed after the selection and classification of representative strains by observing cultural and morphological characteristics.Classification was confirmed by comparing the similarities of the isolates with the type strains via EzTaxon server online analysis.The results showed that actinobacteria of volcanic lake were classified into 8 genera of the actinomycete:Streptomyces(76%),Micromonospora(14%),Nonomuraea(4%),Actinoplanes(2%),Catellatospora(1%),Geodermatophilu(1%),Nocardiopsis(1%)and Rhodococcu(1%).Evaluation analysis was performed to the novel actinobacteria candidate and 3 novel actinobacteria(NEAU-C3,NEAU-JM1 and NEAU-JM2)were identified by using polyphasic approach.(2)Characterization of strain NEAU-C3 using polyphasic approach:The phylogenetic analysis of the 16S rRNA gene sequence showed that strain NEAU-C3 belonged to the genus Streptomyces and was found to share the highest 16S rRNA gene sequence similarities with Streptomyces hygroscopicus subsp.ossamyceticus JCM 4965T(97.7%)and Streptomyces torulosus JCM 4872T(97.7%).The comparative analysis of the 16S rRNA gene sequences and the estimation of phylogenetic relationships showed that strain NEAU-C3 formed a separate subclade in the neighbour-joining tree and also supported by the maximum-likelihood algorithm.The levels of DNA-DNA relatedness between strain NEAU-C3 and S.hygroscopicus subsp.ossamyceticus JCM 4965T and S.torulosus JCM 4872T were below the threshold value of 70%for assigning bacterial strains to the same genomic species.Combining with phenotypic differences and physiological and biochemical characteristics,strain NEAU-C3 represents a novel species of the genus Streptomyces,for which the name Streptomyces vulcanius sp.nov.is proposed.(3)Characterization of strain NEAU-JM1 using polyphasic approach:The phylogenetic analysis of the 16S rRNA gene sequence showed that strain NEAU-JM1 belonged to the genus Catellatospora and was found to share the highest 16S rRNA gene sequence similarities with Catellatospora parii NEAU-CL2T(99.5%),Catellatospora.methionotrophica JCM 7543T(99.4%),Catellatospora.coxensis DSM 44901T(99.4%),Catellatospora.citrea DSM 44097T(99.3%)and Catellatospora.chokoriensis JCM 12950T(99.2%).The phylogenetic tree basing on the neighbour-joining algorithm showed that strain NEAU-JM1 formed a separate subclade with C.parii NEAU-CL2T and was close with C.coxensis DSM 44901T,C.aurea NEAU-SH16T,C.citrea DSM 44097T,C.methionotrophica JCM 7543T and C.chokoriensis JCM 12950T,which was also supported by the maximum-likelihood algorithm.The values of DNA-DNA relatedness between strain NEAU-JM1 and its close relatives were all below the threshold value of 70%for assigning bacterial strains to the same genomic species.Combining with phenotypic differences and physiological and biochemical characteristics,strain NEAU-JM1 represents a novel species of the genus Catellatospora,for which the name Catellatospora vulcania sp.nov.is proposed.(4)Characterization of strain NEAU-JM2 using polyphasic approach:The phylogenetic analysis of the 16S rRNA gene sequence showed that strain NEAU-JM2 belonged to the genus Micromonospora and was found to share the highest 16S rRNA gene sequence similarities with Micromonospora maoerensis NEAU-MES19T(99.5%),Micromonospora zeae NEAU-gq9T(98.9%),Micromonospora saelicesensis Lupac 09T(98.8%)and M.matsumotoense JCM 9104T(98.8%).The phylogenetic tree basing on 16S rRNA gene sequences showed that strain NEAU-JM2T clustered with M.maoerensis NEAU-MES19T and M.matsumotoense JCM 9104T in the neighbour-joining tree,which was also recovered with the maximum-likelihood algorithm.The phylogenetic analysis of the gyrB gene sequence showed that strain NEAU-JM2 was found to share the highest 16S rRNA gene sequence similarities with M.maoerensis NEAU-MES19T(94.5%),M.zeae NEAU-gq9T(94.0%),M.saelicesensis Lupac 09T(94.8%)and M.matsumotoense JCM 9104T(94.5%).However,it is evidenced that strain NEAU-JM2 formed a separate subclade away from any of them in the neighbour-joining tree.The relationships were also supported by the maximum-likelihood algorithm.In conclusion,the genotypic and phenotypic data showed that strain NEAU-JM2 represents a novel species of the genus Micromonospora,for which the name Micromonospora vulcania sp.nov.is proposed.(5)Antibacterial activity results showed that strain LW1-3 has a wide spectrum activity against 10 pathogenic fungi by performing the biological antibacteria activity test.Combining with the result of antibacteria test,strain LW1-3 was used for subsequent isolation of novel compounds.After isolation and purification of the fermentation products,two compounds,bafilomycin A1 and bafilomycin K,were isolated and identified from strain LW1-3.