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半胱氨酸增强深海细菌镉抗性的分子机制研究
马宁
Subtype博士
Thesis Advisor孙超岷
2020-11-18
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士学位
Keyword深海微生物,镉抗性,L-半胱氨酸,CdS纳米颗粒,生物矿化
Abstract

重金属镉在深海环境中广泛存在,深海微生物进化出了成熟且多样的镉耐受机制以维持在高镉环境中的生长和繁殖。与此同时,环境中与镉共存的化合物如半胱氨酸可显著影响深海微生物的镉抗性。本研究分别从冲绳海槽热液沉积物、东海深海沉积物和马里亚纳海沟深海沉积物中筛选出了三株具有镉耐受能力的深海细菌Idiomarina sp. OT37-5bPseudomonas stutzeri 273 Pseudoalteromonas sp. MT33b,并对半胱氨酸提高其镉抗性的分子机制进行了研究。

Idiomarina sp. OT37-5b镉耐受机制的研究中发现L-半胱氨酸(Cys)通过促进硫化镉(CdS)纳米颗粒的生成显著提高了Idiomarina sp. OT37-5b的镉抗性和脱除率。甲硫氨酸-γ-裂解酶通过催化Cys脱硫生成硫化氢(H2S),在CdS纳米颗粒生物矿化中发挥重要作用。蛋白质组学结果显示Cys有效阻止了镉胁迫条件下镉离子进入Idiomarina sp. OT37-5b进入细胞内部,并且促进了胞内ROS的清除以及氮元素的还原和能量的产生。同时,CdS纳米颗粒显著促进了Idiomarina sp. OT37-5b在光照下的生长速度和胞内ATP的合成,表明该细菌可通过CdS纳米颗粒进行光能的利用。蛋白质组分析表明在光能利用过程中发挥关键作用的细胞组分有电子传递体、细胞色素复合物以及F-ATP酶。以上结果表明,热液生态系统深海细菌已经形成了巧妙的对抗重金属胁迫的方法。本研究为深海环境中可能存在的微生物光能利用现象提供了有力的证据,并为研究深海生态系统中非光合细菌的光能利用以及光能转化为化学能的生物过程提供了良好模型。

P. stutzeri 273镉耐受机制的研究中发现Cys同样通过促进CdS纳米颗粒的生成显著提高了P. stutzeri 273的镉抗性和脱除率。通过蛋白质组学和基因敲除等方法首次发现P. stutzeri 273中的苏氨酸脱水酶(threonine dehydrase, TD)具有半胱氨酸脱硫酶活性,该酶通过催化Cys脱硫生成H2S来参与P. stutzeri 273的镉耐受过程。进而,我们通过TD的异源表达和分离纯化进一步确定了重组苏氨酸脱水酶(rTD)在胞外仍具有半胱氨酸脱硫酶活性,并分析了其催化反应的过程。rTD催化的Cys脱硫过程分为两步:首先,CysH2O反应生成L-丝氨酸(L-serine)和H2S;进而,L-丝氨酸进行脱氨作用生成丙酮酸(pyruvate)和NH3。同时,建立了CdS纳米颗粒生物合成单酶体系,该体系以rTD作为催化酶、以Cys作为硫供体、以CdCl2作为镉供体,可高效的催化CdS纳米颗粒的合成。其中rTD除了作为催化酶控制反应进程之外,还具有包被物的作用,能够控制CdS纳米颗粒的形成速度和颗粒粒径。随后,我们对rTD的晶体结构进行了解析。rTD是非对称二聚物,第77位精氨酸(R77)在TD发挥半胱氨酸脱硫酶作用中发挥重要作用。NH4+可通过改变R77的构象抑制rTD的活性,由于NH4+rTD催化Cys脱硫的产物之一,该现象可能是一种产物的反馈抑制现象。将R77突变为谷氨酸(E)得到的突变体R77E活性较野生型明显降低,表明带负电荷侧链的E77可以通过TD表面的负电势间隙阻挡小分子进入活性位点。因此,R77可能作为rTD中小分子交换的开关,吸收带负电荷的小分子进入rTD的活性位点,如5’-磷酸吡哆醛(PLP)和Cys。本部分研究表明深海沉积物生态系统中的微生物通过代谢Cys和介导CdS生物矿化参与了所在生境的硫循环和含硫金属矿物的形成。同时,本研究首次发现了苏氨酸脱水酶的半胱氨酸脱硫酶活性,并为生物合成CdS纳米颗粒提供了新方法和高效的生物酶资源。

Pseudoalteromonas sp. MT33b镉耐受机制的研究中发现Cys同样可通过促进CdS纳米颗粒的生成显著提高Pseudoalteromonas sp. MT33的镉抗性和脱除率。同时,我们通过扫描电子显微镜和透射电子显微镜观察到除CdS沉淀的生物合成外,生物膜的形成同样是Pseudoalteromonas sp. MT33耐受镉胁迫的重要方式之一。在含镉条件下添加Cys进一步促进了Pseudoalteromonas sp. MT33b生物膜的形成,表明Cys同时通过促进Pseudoalteromonas sp. MT33b生物膜的形成提高了该细菌的镉抗性。转录组分析结果显示,Cys有效阻止了镉胁迫条件下镉离子进入Pseudoalteromonas sp. MT33b细胞内部,并且促进了胞内能量的产生。同时,鞭毛组装、群体感应、细菌趋化、双组分系统、TonB依赖型受体等相关基因在Pseudoalteromonas sp. MT33b生物膜的形成过程中发挥重要作用。本部分研究结果表明,Cys脱硫生成H2SCys提高Pseudoalteromonas sp. MT33b镉抗性的主要方式,并且生物膜的形成是Pseudoalteromonas sp. MT33b耐受环境胁迫的重要途径。

本文通过研究发现来自不同生境的三株深海细菌均将CdS纳米颗粒的形成作为耐受重金属镉的重要方式,说明将有毒的镉离子转变为毒性较低的CdS沉淀是深海环境中细菌耐受镉胁迫的普遍方式,也表明深海微生物广泛参与了不同生境中的含硫重金属矿物的生物矿化。Cys脱硫在此三种深海细菌中均为CdS形成的重要供硫途径,说明有机硫代谢在不同生境深海细菌的生长代谢中均占据重要地位,并且反映了深海微生物是热液、深渊等不同深海生境有机硫循环的重要参与者。

Other Abstract

The heavy metal cadmium widely exists in the deep-sea environment, and deep-sea microbes have evolved sophisticated and diverse cadmium tolerance mechanisms to maintain the growth and reproduction in the high cadmium environment. At the same time, compounds such as cysteine that coexist with cadmium in the environment can significantly influence the cadmium resistance of deep-sea microorganisms. Three cadmium tolerant deep-sea bacteria including Idiomarina sp. OT37-5b, Pseudomonas stutzeri 273, and Pseudoalteromonas sp. MT33b were isolated from the hydrothermal sediments of Okinawa Trough, the deep-sea sediments of the East China Sea, and the sediments of the Mariana Trenth, respectively, and their cadmium tolerance mechanisms were studied.

Both Cd-resistance and removal efficiency of Idiomarina sp. OT37-5b were significantly promoted by the supplement of L-cysteine (Cys) and meanwhile large amount of cadmium sulfide (CdS) nanoparticles were observed. Production of hydrogen sulfide (H2S) from Cys catalyzed by methionine gamma-lyase was further demonstrated to contribute to the formation of CdS nanoparticles. Proteomic results showed Cys effectively prevented Cd2+ from entering into the cells of Idiomarina sp. OT37-5b under cadmium stress, and improved the activities of ROS scavenging enzymes, and thereby boosted the nitrogen reduction and energy production of Idiomarina sp. OT37-5b. Notably, the existence of CdS nanoparticles obviously promoted the growth and intracellular ATP synthesis of Idiomarina sp. OT37-5b when exposed to light, indicating this bacterium might grab light energy through CdS nanoparticles. Proteomic analysis revealed the expression levels of essential components for light utilization including electron transport, cytochrome complex and F-type ATPase were significantly up-regulated, which strongly suggested the formation of CdS nanopaticles promoted light utilization and energy production. These results suggest that deep-sea bacteria in hydrothermal ecosystem have developed ingenious ways to combat heavy metal stress. Our results provide a strong evidence for the existence of microbial light energy utilization in the deep-sea environment, and provide a good model to investigate the uncovered mechanisms of self-photosensitization of nonphotosynthetic bacteria for light-to-chemical production in the deep biosphere.

Both Cd-resistance and removal efficiency of P. stutzeri 273 were also significantly promoted by the supplement of Cys and meanwhile large amount of CdS nanoparticles were observed. Threonine dehydrase (TD) in P. Stutzeri 273 was first found to have cysteine desulfhydrase activity by proteomic and gene knockout methods. The enzyme participated in the cadmium tolerance process of P. Stutzeri 273 by catalyzing Cys desulfurization to generate H2S. Furthermore, the cysteine desulfhydrase activity of TD was further determined through purified protein, and the substrates of catalytic reaction were also analyzed. The TD-catalyzed Cys desulfurization process was divided into two steps: first, the reaction of Cys and H2O generated L-Serine and H2S; L-serine then performed deamination to produce pyruvate and NH3. At the same time, a single enzyme system for CdS nanoparticle biosynthesis was established, in which rTD was used as the catalytic enzyme, Cys as the sulfur donor and CdCl2 as the cadmium donor. The single enzyme system could effectively catalyze the synthesis of CdS nanoparticles. rTD not only acted as the catalytic enzyme to control the reaction process, but also acted as a capping agent to control the formation speed and particle size of CdS nanoparticles. Then, we analyzed the crystal structure of rTD. rTD was a dimer, and arginine at position 77 (R77) played an important role in the catalysis of Cys desulfurization by rTD. NH4+ could inhibit TD activity by changing the structure of R77. Since NH4+ was one of the products of TD-catalyzed Cys desulfurization, this phenomenon might be a feedback inhibition of the product. The mutant R77E obtained by mutating R77 to glutamate (E) showed significantly lower activity than the wild type, indicating that the negatively charged side chain of E77 could block small molecules from entering the active site through the negative potential gap on the rTD surface. Therefore, R77 might act as a switch for rTD small molecule exchange, absorbing negatively charged small molecules into the active sites of rTD, such as pyridoxal 5’-phosphate (PLP) and Cys. This study showed that microorganisms in deep-sea sediments were involved in the sulfur cycle and the formation of metal sulfide mineral of the habitat. At the same time, this study first discovered the cysteine desulfhydrase activity of threonine dehydratase, and provided a new way and efficient biological resources for the CdS nanoparticles biosynthesis.

Adding Cys also significantly increased the cadmium resistance and the removal rate of Pseudoalteromonas sp. MT33b by promoting the generation of CdS nanoparticles. Meanwhile, scanning electron microscopy and transmission electron microscopy observation showed that, in addition to CdS precipitation biosynthesis, biofilm formation was also one of the important ways for Pseudoalteromonas sp. MT33b to withheld cadmium stress. The addition of Cys under cadmium-containing conditions further promoted the formation of Pseudoalteromonas sp. MT33b biofilm to enhance the cadmium resistance of the bacterium. Transcriptomic analysis showed that Cys effectively prevented Cd2+ from entering into the cells of Pseudoalteromonas sp. MT33b under cadmium stress, and promoted energy production. Meanwhile, related genes such as flagella assembly, quorum sensing, bacterial chemotaxis, two-component system, and TonB-dependent receptor played important roles in Pseudoalteromonas sp. MT33b biofilm formation. This study indicated that the formation of H2S from Cys was the main way of Cys increasing cadmium resistance of Pseudoalteromonas sp. MT33b, and biofilm formation was an important pathway for Pseudoalteromonas sp. MT33b to cope with environmental stresses.

Taken togerher, this study found that the formation of CdS nanoparticles was one important way of cadmium tolerance in all the three deep-sea bacteria from different habitats, indicating that transforming poisonous cadmium ion into less toxic CdS precipitation was a widespread way in deep-sea bacteria to tolerant cadmium stress, and the deep-sea microbes extensively involved in metal sulfide mineral biomineralization in different habitats. Cys desulfurization was an important sulfur supply pathway of CdS formation in these three deep-sea bacteria. The results suggested that organic sulfur metabolism played an important role in the metabolism of deep-sea bacteria, and revealed that the deep-sea microorganisms were important components involved in organic sulfur cycle in deep-sea habits.

Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/168717
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
马宁. 半胱氨酸增强深海细菌镉抗性的分子机制研究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.
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