Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
|Place of Conferral||中国科学院海洋研究所|
|Keyword||深海冷泉 未培养微生物 蓝光感受 细菌多糖 抗肿瘤|
我们聚焦于蓝光处理获得的海绵杆菌新种菌株CSC3.9，对其基本生理生化性质等分类特征、蓝光感受机制和胞外多糖抗肝癌细胞Huh7.5生长增殖的作用进行了系统研究。通过对菌株CSC3.9的多相分类学和进化地位的鉴定分析，将其归为海绵杆菌属的新菌种。菌株CSC3.9表现为革兰氏染色阴性，异养、兼性厌氧生长，透射电镜下的形态表现为杆状，有单鞭毛。其最适生长温度为37℃，能够利用乙酸盐，果糖，葡萄糖，丙酮酸和蔗糖等底物。菌株CSC3.9的主要脂肪酸是C17:1 ω8c，主要的醌类化合物为泛素酮8（Q-8），最终我们将该菌株命名为Spongiibacter nanhainus CSC3.9。
在对Spongiibacter nanhainus CSC3.9基因组分析时发现，菌株CSC3.9中存在一个完整的多糖合成基因簇。对CSC3.9菌株胞外多糖EPS3.9进行了提取和活性评价，发现胞外多糖EPS3.9可以抑制肝癌细胞Huh7.5的生长。对所产多糖的结构进行了初步鉴定，发现EPS3.9主要是以葡萄糖和甘露糖组成的多糖，分子量是17.14 kDa。随后，对EPS3.9抗肝癌细胞增殖的机制进行了研究，通过蛋白质组学、扫描电镜和超薄切片透射电镜的形态学观察和Western blot的检测初步确定，EPS3.9是通过以GSDME介导的细胞焦亡途径对肝癌细胞Huh7.5进行杀伤。其后用免疫缺陷型肝移植瘤小鼠进行了EPS3.9的体内实验，结果表明EPS3.9在体内也有效的抑制了肝癌细胞瘤重量和体积的增长。
综上所述，我们通过变换不同的分离策略获得了多个深海冷泉未培养微生物的纯培养代表菌株，对突破难培养微生物培养技术瓶颈做出贡献；非光合微生物新种Spongiibacter nanhainus CSC3.9的感光机制的阐明扩展了对深海微生物特殊代谢和环境适应方式的认知；菌株CSC3.9胞外多糖的抗肿瘤活性研究为深海微生物资源的发掘和利用提供了新的视角。
The deep sea is the largest independent ecosystem on Earth, with unique geological structures such as upwelling alkanes seeping from cold seeps. These unique environmental conditions have fostered rich microbial and genetic diversity. However, due to the difficulty of sampling and culture, the unculturable deep-sea microorganisms are as high as 99%. The vast majority of archaea and bacteria have not obtained pure culture strains. Innovative culture methods and technologies to break through the barriers of microbial culture and obtain pure cultures of key microorganisms are of great importance to reveal the special metabolic patterns, the ecological role of driving element cycling of deep-sea microorganisms and the deep exploration of microbial resources.
In this study, 11 potential novel bacteria strains were successfully obtained from sediments and water samples in deep-sea cold seep environments through light incubation, antibiotic screening, and anaerobic culture. Based on the similarity of 16S rRNA sequences, and the construction of the phylogenetic tree, including one strain of Chloroflexi, one strain of Tenericutes, which are potential new orders. Besides, one potential novel family strain of Planctomycetes and one potential new genus strain of Bacteroidetes, one potential new species strain belonging to Proteobacteria, and six potential new species strains of Firmicutes were isolated.
Subsequently, we focused on a new species strain CSC3.9 obtained by blue light incubation. The basic physiological and biochemical characteristics of strain CSC3.9, the blue light sensing mechanism, and the effect of exopolysaccharide against the growth and proliferation of hepatoma cell Huh7.5 were studied in detail. Strain CSC3.9 was identified as a new species of Spongiibacter by multiphase taxonomy and evolutionary analysis. Cells of strain CSC3.9 were facultative anaerobic, Gram-reaction-negative, rod-shaped with single polar flagellum. The optimum temperature of strain CSC3.9 was 37℃. Growth was stimulated by the supplement of acetate, fructose, D-Glucose, pyruvate or sucrose. Major fatty acids and the main quinone of strain CSC3.9 were C17:1 ω8c and ubiquinone 8 (Q-8). Strain CSC3.9 was named Spongiibacter nanhainus CSC3.9.
Consistently, the blue light sensing mechanism of strain CSC3.9 was further explored. Growth of strain CSC3.9 was promoted by the illumination of blue light. We next determined that BLUF (a kind of typical blue light photoreceptor) was the most essential factor directing light sensing of strain CSC3.9 through a combined proteomic and genetic method. We demonstrated that BLUF protein activated high expression of chemotaxis and flagellar motility genes in the same gene cluster under blue light stimulation, and further changed the motility behavior of bacterial cells. Through the prokaryotic expression of BLUF protein, it was found that BLUF protein could specifically bind the cofactor FAD in vitro and showed a unique absorption peak, and the shift of absorption peak occurred after light irradiation. It was confirmed that BLUF protein had blue light response function in vitro. Notably, homologs of BLUF widely existed across the marine microorganisms (containing Spongiibacter species) derived from different environments including cold seeps. This strongly indicates that the distribution of light utilization by the non-phototrophic bacteria living in the ocean is broad and has been substantially underestimated.
The genome analysis of Spongiibacter nanhainus CSC3.9 showed that there was a complete exopolysaccharide synthesis gene cluster in strain CSC3.9. The exopolysaccharide EPS3.9 of strain CSC3.9 was extracted and tested. It was found that EPS3.9 could inhibit the growth of hepatoma cells Huh7.5. The structure of the exopolysaccharide was identified and it was found that EPS3.9 was mainly composed of glucose and mannose. The relative molecular weight of EPS3.9 was 17.14 kDa. Subsequently, the mechanism of EPS3.9 against proliferation of hepatoma cell was studied. Through proteomics, morphological observation of scanning electron microscopy, ultrathin transmission electron microscopy, and Western blot analysis, it was preliminarily confirmed that EPS3.9 killed hepatoma cell Huh7.5 through the DSDME-mediated pyroptosis pathway. The results showed that EPS3.9 also effectively inhibited the growth of hepatoma cell volume in vivo.
In summary, we obtained a number of pure cultured representative strains from deep-sea cold seep by changing different separation strategies, which contributed to breaking the bottleneck of culture technology for difficult-to-culture microorganisms. Elucidating the photosensitive mechanism of a novel species of non-photosynthetic microorganisms, Spongiibacter nanhainus CSC3.9, has opened up the understanding of the special metabolic and adaptive modes of deep-sea microorganisms. The antitumor activity of exopolysaccharide from strain CSC3.9 opens a new horizon for deep exploration and utilization of deep-sea microbial resources.
|Subject Area||地球科学 ; 海洋科学 ; 海洋生物学|
|MOST Discipline Catalogue||理学 ; 理学::海洋科学|
|Funding Project||Strategic Priority Research Program of the Chinese Academy of Sciences[XDA22050301] ; Strategic Priority Research Program of the Chinese Academy of Sciences[XDA22050301]|
|Table of Contents|
|单业奇. 深海冷泉未培养微生物的培养及一株海绵杆菌感光机制和代谢产物研究[D]. 中国科学院海洋研究所. 中国科学院大学,2022.|
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