Isolation Of Microorganisms From The Gut Of Macrotermes Barneyi And Identification Of Antimicrobial Substances From Gut Related Streptomyces GM5

Macrotemes barneyi is a kind of fungus-growing termite that widely lives in southern China.It can specially cultivate Termitomyces as cultivar.In natural environment,termites and their comb are threatened by many kinds of pathogenic fungi,such as Xylaria.Symbiotic microorganisms in fungus-growing termites may produce a variety of active metabolites to help termites resist fungal infection.In order to explore the antibacterial mechanism of intestinal microorganisms of fungus-growing termites and isolate antibacterial active strains,we used M.barneyi as research materials,detected the activity of intestinal strains.A strong antibacterial activity strain GM5 was obtained,its genome and biosynthetic gene clusters of compounds was sequenced and predicted.Active compounds produced of GM5 strain were separated and purified.Firstly,the guts of M.barneyi were used as the sample to isolate the strains,and the antibacterial activity of the strain was detected.35 strains were tested to have antibacterial activity against Xylaria or E.coli.Among of them,19 strains belonged to Streptomyces,and 3 potential new strains were obtained.Streptomyces sp.HF5 was taken as a putative novel strain based on the 16S rRNA sequence.Polyphasic taxonomy was employed to further identify the potential new species.This study not only provided a better understanding of the active role of intestinal microorganisms from termites,but also provided new resources for exploring new antimicrobial active strains.Then,due to the strain GM5 with strong activity against both Xylaria and E.coli.We observe the morphology and identify molecular biological of Streptomyces GM5.The supernatant of GM5 performed high antibacterial activity against many bacterias and fungus.Explore the culture time of strain GM5,compound was extracted by liquid fermentation,resin adsorption,Sephadex gel chromatography,TLC chromatography,HPLC and semi HPLC preparation.LC-MS and NMR were used to analyze the structure of the compound.Finally,we analyzed the genome of GM5 by antiSMASH 6.0 software.The strain contained 33 clusters encoding the synthesis of secondary metabolites,including PKS,Nrps,Terpene,Ectoine,Butyrolactone and other 14 types.Combined with the LC-MS results,the compound was presumed to be actinomycin D.In summary,33 strains of the gut of M.barneyi with antibacterial activity were isolated and identified.A new strain belongs to Streptomyces(Streptomyces sp.HF5)was identified.Meanwhile,the genomic of GM5 strain,which produces the highest antibacterial activity against many bacterias and fungus,gene cluster for biosynthesis of secondary metabolite was predicted.Finally,the putative active compound was purified and identified as speculated as structural analog of actinomycin D.In summary,the results showed that gut of termites would be a promising treasure-house for screening microorganisms with antibacterial activity.This study deepened our understanding of the antibacterial activity microorganisms in the gut of termites,showed the role of intestinal microorganisms and their metabolites in the symbiotic relationship between termites and fungi in the nursery.This study laid a foundation for further study on the ecological significance and molecular mechanism of termites intestinal microorganisms,and provided strain resources for exploring new antibacterial compounds.