西伯利亚Gudzhirganskoe盐湖土壤放线菌多样性、新颖性及抗菌活性研究

Diversity,novelty,and antimicrobial activity of actinobacterial strains isolated from soils in Gudzhirganskoe Saline Lake in Siberia

目的探究西伯利亚Gudzhirganskoe盐湖土壤放线菌多样性、新颖性和活性菌株的次级代谢产物,为放线菌新物种和新抗生素的发现储备菌种资源。方法采用10种分离培养基,利用稀释涂布法进行放线菌的分离纯化;通过PCR扩增和测序,比对16S rRNA序列进行放线菌多样性和新颖性分析;采用纸片扩散法对潜在放线菌新物种发酵液的酯相萃取物、水相和菌体相进行抗菌活性筛选;采用反相柱层析、葡聚糖凝胶Sephadex LH-20和半制备HPLC方法分离纯化菌株Hoyosella sp.S15b3-3粗提物的活性成分,通过ESI-MS、NMR波谱数据,并结合文献确定化合物的结构;采用微量稀释法评价化合物1和2的抗菌活性。结果从15份样品中分离获得1343株放线菌,分属于10目23科42属,其中涅斯捷连科菌属、拟诺卡菌属和链霉菌属为优势菌属;15株放线菌与近缘菌株的16S rRNA基因的最高相似度≤98.65%;5株潜在新物种对至少1株检定菌表现出抗菌活性;从Hoyosella sp.S15b3-3中分离鉴定2个已知化合物1(3-吲哚甲醛)和2(2-羟基-1-(3-吲哚)乙酮),化合物1对2株鲍曼不动杆菌(2799和ATCC19606)有一定的抑制作用,MIC值分别为64和32μg/mL。结论西伯利亚Gudzhirganskoe盐湖土壤含有丰富的放线菌资源,且新颖性较高,具有从中发现放线菌新物种和新抗生素的潜力,值得深入研究。

Objective To explore the diversity,novelty and active secondary metabolites of culturable actinobacteria from soils of Gudzhirganskoe Saline Lake in Siberia,and lay the foundation of finding new actinobacterial taxa and antibiotics.Methods Actinobacteria were isolated by using serial dilution plating method with 10 different media.The diversity and novelty of actinobacteria were analyzed by 16S rRNA gene sequences.The potential new species were selected to evaluate their antimicrobial activities by paper-disk diffusion method.The secondary metabolites produced by Hoyosella sp.S15b3-3 were isolated and purified by reversed-phase silica column chromatography,sephadex LH-20 and semi-preparation HPLC.The chemical structures were elucidated by ESI-MS,NMR spectral analysis and compared with the literature.The antibacterial activity of compound 1 and 2 was evaluated by micro-dilution method.Results Totally,1,343 actinobacterial strains affiliated with 42 genera in 23 families of 10 orders were obtained from 15 soil samples.The predominant genera were Nesterenkonia,Nocardiopsis and Streptomyces.Fifteen strains showed relatively low 16S rRNA similarities(≤98.65%)with validly described species.Five strains showed inhibitory activity against at least one of the indicator strains.Two known compounds were isolated and identified as 1(1H-indole-3-carboxaldehyde)and 2(2-hydroxy-1-(1H-indol-3-yl)ethanone).Compound 1 exhibited inhibitory activities against Acinetobacter baumannii 2799 and A.baumannii ATCC19606 with MIC value of 64 and 32μg/mL,respectively.Conclusion The study demonstrates that the soil in Gudzhirganskoe saline lake is rich of cultivable actinobacteria,which deserved to be further explored for the discovery of new actinobacterial species and antibiotics.