印度洋深海沉积物可培养放线菌多样性及抗革兰阴性菌活性物质研究

Diversity of culturable actinomycetes from the Indian Ocean deep-sea sediment and the identification of the antibacterial metabolites against Gram-negative bacteria

目的从印度洋深海沉积物中分离可培养海洋放线菌,对获得的一株海洋微生物进行抗革兰阴性菌活性物质研究。方法设计11种分离培养基进行海洋放线菌分离,通过16S r RNA基因序列比对分析确定菌株分类;利用多种色谱方法,对菌株IMB16-059产生的活性物质进行分离纯化,通过波谱学和化学方法鉴定其结构。结果根据菌落表型共分离得68株微生物,经16S r RNA分析表明,其中34株属于放线菌门,覆盖5科5属;其余属于厚壁菌门和变形菌门。25株发酵产物显示出抗革兰阴性菌活性。从赤杆菌IMB16-059中分离鉴定了6个化合物,其结构分别确定为L,L-环(脯氨酸-丙氨酸)(1),L,L-环(脯氨酸-酪氨酸(2)),D,L-环(脯氨酸-酪氨酸)(3),邻氨基苯甲酸(4),胸腺嘧啶(5),1H-喹啉-4-酮(6)。化合物1和4显示出抗铜绿假单胞菌活性,在样品浓度为20μg/片时,抑菌圈直径分别为13和20mm。结论印度洋深海沉积物蕴含丰富的微生物类群,其发酵产物显示出较好的抗革兰阴性菌活性,可为新抗生素发现提供重要的菌种资源。

Objective To investigate the diversity of culturable actinomycetes from the deep-sea sediment collected from the Indian Ocean and identify the antibacterial metabolites from one of the isolated marine strains. Methods The marine actinomycete strains were isolated by using 11 different media. The 16S rRNA sequence analysis was used to characterize the diversity of culturable actinomycetes. The antibacterial metabolites were isolated and purified by using a combination of chromatographic techniques. Their structures were elucidated using spectroscopic analysesand chemical methods. Results A total of 68 colonies with phenotypical bacteria were obtained and classified by the 16S rRNA sequence analysis. Among the identified bacteria, 34 strains were classified into five genera of five families while other strains were homologous to the phyla Firmicutes and Proteobacteria. The culture extracts of 25 strains displayed antibacterial activities against Gram-negative bacteria. Six compounds wereidentified from the fermentation broth of Erythrobacter sp. IMB 16-059. Their structures were determined as cyclo（L- Pro-l-Ala）（1）, cyclo（L-Pro-L-Tyr）（2）, cyclo（D-Pro-L-Tyr）（3）, anthranilic acid（4）, thymine（5）, quinolin-4（1/-/）-one（6）. Compounds 1 and 4 showed antibacterial activity against Pseudomonas aeruginosa with inhibition zones of 1 and 20mm at 20μg/disc, respectively. Conclusion The results indicated that the deep marine sediments from the India Ocean possess diverse and abundant actinomycetesa and antibacterial activities, which could provide potential sources for the discovery of new antibiotics.