Sreening And Identification Of Endosymbiotic Actinobacteria From Four Kinds Of Insects From Agricultural Habitat And Study Of Their Antibacterial Activity

Natural products, an important source of bioactive substances, are ubiquitious in the natural ecosystem and play a pivotal role in antibiotic drug discovery with most antibacterial drugs being derived from a natural product or natural product lead. With emergence of multi-drug resistant bacteria, the efficiency of the discovery of new drugs need to improve continuously.However, the new natural products from the traditional separation source have gradually cut down. Researchers have more interest in the new separation source for discovering the natrual products. The insects, as the most abundant animals in the world, exist in the various habitats on the earth. The unique metabolites of insect’s symbiotic microorganisms have aroused great interest. During these researches, the researchers have paid more attention on the metabolites from the symbiont actinobacteria of the insects. Hence, we choose the beetles of the order Coleoptera as the specimens to investigate the diversity of symbionts actinobacteria. After that, the isolates seem as the new speices are identified in a polyphasic approach and a bioactivity-guided approach is then employed to isolate and determine the chemical identity of bioactive constituents with antimicrobial activity from these symbionts actinobacteria. The results are as follows:(1) A total of 41 symbionts actinobacteria were successfully isolated from several insects’ specimens. According to the phenotypic characteristics, cultural characteristics, phylogenetic analysis based on 16 S r RNA, these strains were classified into four genera, and the dominant genus was Streptomyces(27), while Micromonospora(11), Streptosporangium(2) and Nonomuraea(1) were also recovered.(2) Strain NEAU-CX1 T was characterized using a polyphasic approach. 16 S r RNA gene sequence similarity studies showed that the strain NEAU-CX1 T belonged to the genus Micromonospora, being most closely related to Micromonospora coxensis JCM 13248T(99.3 %),Micromonospora purpureochromogenes DSM 43821T(99.1 %) and Micromonospora siamensis TT2-4 JCM 12769T(99.1 %), similarities to other type strains of the genus Micromonospora were lower than 99 %. The neighbour-joining phylogenetic tree based on 16 S r RNA gene and gyr B sequences showed that the isolate formed a distinct phyletic line with M. purpureochromogenes DSM 43821 T. This branching pattern was also supported by the tree constructed with the maximum-likelihood method. A comparative study between strain NEAU-CX1 T and the closest related Micromonospora type strains revealed that it differed from them in morphological, physiological and biochemical characteristics and phylogenetic analysis. Therefore, it is proposed that strain NEAU-CX1 T represents a novel Micromonospora species, for which the name Micromonospora palomenae sp. nov. is proposed. The type strain of Micromonospora palomenae is NEAU-CX1T(=CGMCC 4.7175T=JCM 30056T).(3) Strain NEAU-JC6 T was characterized using a polyphasic approach. 16 S r RNA gene sequence similarity studies showed that the strain NEAU-JC6 T belonged to the genus Micromonospora, being most closely related to Micromonospora haikouensis DSM 45626T(99.3 %), Micromonospora carbonacea JCM 3139T(99.1 %) and Micromonospora krabiensis JCM 12869T(99.1 %), similarities to other type strains of the genus Micromonospora were lower than 99 %. The neighbour-joining phylogenetic tree based on 16 S r RNA gene and gyr B sequences showed that the isolate formed a distinct phyletic line with M. haikouensis DSM 45626 T. This branching pattern was also supported by the tree constructed with the maximum-likelihood method. A comparative study between strain NEAU-JC6 T and the closest related Micromonospora type strains revealed that it differed from them in morphological, physiological and biochemical characteristics and phylogenetic analysis. Therefore, it is proposed that strain NEAU-JC6 T represents a novel Micromonospora species, for which the name Micromonospora harpali sp. nov. is proposed. The type strain of Micromonospora harpali is NEAU-JC6T(=CGMCC 4.7173T=JCM 30055T)(4) Strain NEAU-FHSN1 T was characterized using a polyphasic approach. 16 S r RNA gene sequence similarity studies showed that the strain NEAU-FHSN1 T belonged to the genus Streptosporangium, being most closely related to Streptosporangtum shengliensis NEAU-GH7T(98.7 %), similarities to other type strains of the genus Streptosporangium were lower than 98.7%. The neighbour-joining phylogenetic tree based on 16 S r RNA gene sequences showed that the isolate formed a distinct phyletic line with S. roseum JCM 3005T(98.6%),S. vulgare DSM 43802T(98.6%),S. canum HBUM 170018T(98.5%) and S. album DSM 43023T(98.4%), far distant with S. shengliensis NEAU-GH7 T.This branching pattern was also supported by the tree constructed with the maximum-likelihood method. A comparative study between strain NEAU-FHSN1 T and the closest related Streptosporangium type strains revealed that it differed from them in morphological, physiological and biochemical characteristics and phylogenetic analysis. Therefore, it is proposed that strain NEAU-FHSN1 T represents a novel Streptosporangium species, for which the name Streptosporangium lutulentum sp. nov. is proposed. The type strain of Streptosporangium lutulentum is NEAU-FHSN1T(=CGMCC 4.7141T= DSM 46740T)(5) The bioactivity of isolated symbiont actinobacteria was performed with using an in vitro screening technique, two strains with good biological activity were found from these symbionts actinobacteria. The fermentation products of strain C7(NEAU-TLV1) showed good anti-bacteria activity and the fermentation products of strain C24(NEAU-T4) revealed well antifungal activity. After separation and purification of the fermentation products, two known compounds, staurosporine and bafilomycins B1, were isolated and identified from C7 and C24, respectively.