深渊来源Citricoccus sp.strain NyZ702的分离培养及其降解4-羟基苯甲酸的特性

Isolation of a hadal trench-derived Citricoccus sp. strain NyZ702 capable of 4-hydroxybenzoate degradation

摘要:

[背景] 深渊沉积物中存在丰富的微生物细胞和活跃的微生物碳周转，因此，分离培养微生物资源对于认识深渊中的物质循环、能量代谢具有重要意义。芳香化合物在环境中广泛存在，基于组学分析揭示了深渊中具有潜在的芳香化合物代谢菌株，然而深渊来源的芳香化合物降解微生物纯培养和相关的代谢机理研究仍然缺乏。[目的] 从马里亚纳海沟沉积物样本中分离培养具有降解芳香化合物能力的微生物，对其代谢途径、中间产物和降解酶活力进行初步鉴定。[方法] 以4-羟基苯甲酸为唯一碳源对马里亚纳海沟沉积物样本中的降解菌株进行分离培养，结合形态观察、16S rRNA基因扩增与序列分析对菌株进行鉴定，通过底物生长实验验证其降解能力，通过高效液相色谱和超高效液相色谱-飞行时间质谱联用仪初步鉴定全细胞生物转化中间产物，利用紫外分光光度计测定其粗酶液催化4-羟基苯甲酸的活力，进而推测菌株降解4-羟基苯甲酸的代谢途径。[结果] 从深渊沉积物中分离培养获得一株好氧细菌，16S rRNA基因序列分析显示该菌株隶属于柠檬球菌属（Citricoccus），命名为Citricoccus sp.strain NyZ702。该菌株在LB固体培养基上经30℃培养4 d后呈柠檬黄色、不透明、表面光滑、边缘整齐、凸出于培养基表面、直径约为1—2 mm的圆形菌落。扫描电镜表明菌体呈球形，直径为0.4—0.6 μm，无鞭毛结构。该菌株为耐盐菌，最适生长盐浓度范围为2%—8%（质量体积分数）。该菌株可利用4-羟基苯甲酸为唯一碳源进行生长，可转化4-羟基苯甲酸至中间产物原儿茶酸，推测该菌株通过原儿茶酸途径降解4-羟基苯甲酸。菌株NyZ702的粗酶液具有4-羟基苯甲酸单加氧酶活力，对4-羟基苯甲酸的催化反应需要还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）作为辅因子。[结论] 从深渊沉积物样本分离得到一株4-羟基苯甲酸降解菌Citricoccus sp.strain NyZ702，该菌株以原儿茶酸为中间代谢产物降解4-羟基苯甲酸，丰富了深渊来源的微生物菌种资源，为深渊中的芳香化合物降解研究提供了一定的理论基础。

Abstract:

[Background] Hadal trench sediments have been reported to harbor abundant microbial cells and have active microbial carbon turnover. Therefore, isolation of microbial resources from trench sediments is of great significance for understanding the biogeochemical cycle and energy metabolism in the hadal trench. It is well known that aromatic compounds are widely spread in the environments. Omics analysis has revealed possible presence of aromatics-degrading microbes in the trench environment. However, there were little reports on the pure culture of trench derived aromatics-degrading microbes nor the possible degradation mechanism.[Objective] To isolate and cultivate bacteria capable of aromatics degradation from the Mariana Trench sediments and characterize its degradation pathway.[Methods] 4-Hydroxybenzoate (4HBA) was used as the sole carbon source to isolate bacteria from the Mariana Trench sediment. Morphological observation and phylogenetic analysis were used to identify the isolate. Growth curves were conducted to verify its optimum growth conditions and degradation ability. The metabolic intermediates during whole cells transformation and the activity of crude cell extracts against the substrate were analyzed, based on which a putative catabolic pathway was reconstructed. [Results] An aerobic bacterium was isolated from the trench sediment and belonged to Citricoccus genus, according to the phylogenetic analysis of its 16S rRNA gene. Colonies of this bacterium, designated Citricoccus sp. strain NyZ702, on LB agar plate were lemon yellowish, opaque, swell, neat edge and round with a diameter of 1—2 mm after 4-day cultivation at 30℃. Cells observed with Scanning Electric Microscope were spherical with a diameter of 0.4—0.6 μm without flagella. The strain was halotolerant with an optimal salt concentration of 2%—8% (W/V). The strain was able to utilize 4HBA as the sole carbon source and protocatechuate was detected as its catabolic intermediate, indicating it degraded 4HBA through the protocatechuate pathway. The cell extracts of this strain showed monooxygenase activity against 4HBA using NADPH as the cofactor. [Conclusion] A 4HBA degrading bacterium, Citricoccus sp. strain NyZ702 was isolated from the trench sediment and its 4HBA degradation was via protocatechuate as the intermediate metabolite. This study would enrich the microbial resources derived from the hadal trench, providing a certain theoretical foundation for the future research of aromatics degradation in the hadal trenches.