河北九莲城淖尔可培养放线菌多样性及抗菌活性筛选

【目的】勘探干涸的九莲城淖尔土壤放线菌多样性并进行活性筛选,以期发现药用微生物资源,为新抗生素的发现奠定基础。【方法】采用15种分离培养基,以稀释涂布法分离放线菌;根据分离菌株的16S rRNA基因序列同源性分析放线菌多样性;发酵液经乙酸乙酯萃取,菌丝体经丙酮浸提,获得提取浓缩物样品;样品通过纸片扩散法进行抗菌活性初筛;抗菌阳性菌株采用PCR技术进行Ⅰ型聚酮合酶(PKS I)KS域、Ⅱ型聚酮合酶(PKS II)KS域和非核糖体多肽合成酶(NRPS)A结构域抗生素生物合成基因的检测。【结果】从11份盐湖土壤样品中分离纯化到251株放线菌,其分布于放线菌纲的10个目15个科31个属,其中优势菌属为链霉菌属和拟诺卡氏菌属;251株放线菌中包括57株耐(嗜)盐放线菌,其优势菌属为拟诺卡氏菌属(22株)和涅斯捷连科氏菌属(15株)。基于16S r RNA基因序列的系统发育分析显示,菌株J11Y309为糖霉菌科潜在新属,菌株J12GA03为分枝杆菌科潜在新种。96株放线菌活性检测结果显示,56株至少对1株检定菌具有抗菌活性,阳性率为58.3%;56株有活性的放线菌中,47株至少含有1种抗生素生物合成基因,其中17株同时具有3种抗生素生物合成基因。【结论】干涸的九莲城淖尔土壤中含有较为丰富的药用放线菌资源,具有从中发现放线菌新物种和新抗生素的潜力。

Biodiversity and antimicrobial activity of culturable actinobacteria isolated from Jiuliancheng Nur in Hebei Province

[Objective] To study the diversity and antimicrobial activity of culturable actinobacteria from dried Jiuliancheng Nur in Hebei Province, China. [Methods] Actinobacteria were isolated by using dilution plating technique with 15 media. Isolates were analyzed by comparison of 16S rRNA gene sequences. Crude extracts from fermented broth were obtained using ethyl acetate and from mycelia by acetone. Disk diffusion was used in antibacterial and antifungal assay and positive strains were further screened for PKS I, PKS II and NRPS antibiotics biosynthetic genes by PCR. [Results] A total of 251 actinobacterial strains, isolated from 11 soil samples, belonged to 31 genera affiliated with 15 families in 10 orders; and Streptomyces and Nocardiopsis are dominant genera. Out of these isolates, 57 were halotolerant or halophilic isolates, including 22 Nocardiopsis isolates and 15 Nesterenkonia isolates. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain J11Y309 is a prospective novel genus affiliated with the family Glycomycetaceae and J12GA03 is a prospective novel member affiliated with the family Mycobacteriaceae. Out of 96 fermented strains, 56 exhibited positive activity in antibacterial or antifungal assay, and the total positive rate reached 58.3%. Out of 56 positive strains, 47 carried at least 1 antibiotic biosynthetic gene and 17 strains carried all the 3 genes. [Conclusion] Jiuliancheng Nur shows a rich biodiversity of culturable medicinal actinobacteria, and it is a valuable source of novel antibiotics.