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

Actinobacteria, along with their abundant secondary metabolites, have become important sources 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 stains that producing novel natural medicine has become more difficult, which has greatly influen t the efficiency of the development 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 located in east Huinan of jilin province(China) was generated by the increasing of underground water aft er the volcanic eruption 200 thousand 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 the isolation and identification of novel actinobacteria, the species composition and the 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. 16 S rRNA gene sequencing was performed after the selection and classification of representative strains by observing cultural and morphological characteristics. Attribution was confirmed by comparing the similarities of the isolates with the type strains via Ez Taxon server online analysis. The analysis 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%), 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 16 S rRNA gene sequence showed that strain NEAU-C3 belongs to the genus Streptomyces and was found to share highest 16 S r RNA gene sequence similarities with Streptomyces hygroscopicus subsp. ossamyceticus JCM 4965T(97.7 %) and Streptomyces torulosus JCM 4872T(97.7 %). The comparative analysis of the 16 S rRNA gene sequences and the estimation of phylogenetic relationships showed that strain NEAU-C3 forms 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 4965 T and S. torulosus JCM 4872 T were below the threshold value of 70% for assigning bacterial strains to the same genomic species. Combined with phenotypic differences and physiological and biochemical characteristics, strain NEAU-C3 T 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 16 S r RNA gene sequence showed that strain NEAU-JM1 belongs to the genus Catellatospora and was found to share highest 16 S 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 based on the neighbour-joining algorithm showed that strain NEAU-JM1 T forms a separate subclade with NEAU-CL2 T and was close with C. coxensis DSM 44901 T, C. aurea NEAU-SH16 T, C. citrea DSM 44097 T, C. methionotrophica JCM 7543 T and C. chokoriensis JCM 12950 T, a relationship 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. Combined w ith phenotypic differences and physiological and biochemical characteristics, strain NEAU-JM1 T 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 16 S r RNA gene sequence showed that strain NEAU-JM2 belongs to the genus Micromonospora and was found to share highest 16 S 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 based on 16 S rRNA gene sequences showed that strain NEAU-JM2 T clustered with M. maoerensis NEAU-MES19 T and M. matsumotoense JCM 9104 T in the neighbour-joining tree, which was also recovered with the maximum-likelihood algorithm. The phylogenetic analysis of the gyr B gene sequence showed that strain NEAU-JM2 was found to share highest 16 S r RNA 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 evidence that strain NEAU-JM2 T 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 T 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 detection of the biological antibacteria activity test. Compared with the bacteria inhibition results, 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.