深海冷泉生态系统是地球上独特且尚未被充分探索的极端环境之一，该生态系统中的微生物在海洋元素循环中发挥着重要作用。冷泉中富含甲烷、硫酸盐等物质，微生物通过化能自养或异养的方式驱动着物质循环，然而其中的许多机制尚待揭示。在海水中，微生物能够将硫酸盐转化为有机硫，再将其矿化为硫酸盐，这一过程在地球复杂的生物地球化学循环体系中占据着关键地位，对地球的硫循环而言至关重要。微生物在硫元素于不同环境介质间的迁移、转化与分配过程中发挥着关键作用，维持着硫循环的动态平衡。这不仅对地球生态系统的稳定、气候调节具有重要影响，也在生物演化过程中起到了深远而持久的作用。因此，深入研究微生物的硫代谢机制，有助于更全面理解深海生态系统的功能，以及微生物在硫循环中的核心贡献。近年来，深海微生物的光响应机制成为了研究的热点。由于深海光照条件独特，微生物已经进化出特有的光感受蛋白，能够感知微弱的光信号并将其转化为生化信号，从而优化能量获取途径并适应极端环境。然而，深海微生物的代谢网络、光响应机制及其调控通路仍需进一步深入研究。此前的研究中，我们从南海冷泉中分离出了一株硫氧化细菌Erythrobacter flavus 21-3，实验结果表明，在实验室及深海原位条件下，该菌株通过一条新的硫代硫酸盐氧化途径生成单质硫（ZVS）。偶然的实验发现，蓝光显著增强了E. flavus 21-3产生ZVS的能力。然而，在深海环境中，波长较长的地质光可能更为常见。

为此，本研究深入研究了波长较长的红外光对E. flavus 21-3菌株产生单质硫的影响。实验结果显示，红外光能够显著促进E. flavus 21-3产生单质硫。这一过程涉及到细菌光敏色素BPHP-15570及其下游的双鸟苷酸环化酶DGC-0450这两个关键的蛋白。在红外光的刺激下，BPHP-15570发生自磷酸化，进而激活DGC-0450，使得胞内c-di-GMP的浓度升高。随后，c-di-GMP与其受体蛋白mPilZ-1753结合，激活硫代硫酸盐脱氢酶（TsdA）和硫氧化酶（SoxB）的活性，最终驱动ZVS的生成。

当环境中硫代硫酸盐浓度过高时，E. flavus 21-3会过量产生单质硫，从而对细胞自身造成毒性。已有研究证实，第二信使c-di-GMP在此单质硫的形成通路中起关键的正向调控作用。我们注意到，多聚磷酸激酶2（polyphosphate kinase 2，PPK2）与c-di-GMP合成酶需要相同的底物GTP。基于此，我们提出一个科学假设：PPK2可能通过竞争消耗细胞内的GTP，间接降低c-di-GMP的合成水平，从而对单质硫的生成产生负调控。因此，本研究旨在揭示PPK2的这一潜在负调控功能。研究结果发现，PPK2通过竞争胞内的GTP抑制c-di-GMP的合成，进而对单质硫的产量和生物被膜形成产生负调控作用。当敲除ppk2基因后，在红外光照射下，菌株的c-di-GMP浓度显著升高，单质硫的产量也相应增加，且生物被膜形成更为明显。进一步的蛋白质组学分析表明，ppk2缺失导致多种蛋白的表达上调，包括含有GGDEF结构域、具有潜在双鸟苷酸环化酶功能的蛋白，以及与鞭毛组装和趋化性相关的蛋白。这些结果表明，ppk2的缺失导致了E. flavus 21-3胞内c-di-GMP浓度的升高。为了进一步验证PPK2的功能，本研究在Escherichia coli BL21(DE3)和Pseudomonas aeruginosa PAO1中过表达了此蛋白。结果显示，PPK2的过表达显著降低了两种细菌胞内的c-di-GMP和GTP的浓度，并有效抑制了E. coli BL21(DE3)和P. aeruginosa PAO1生物被膜的形成。这一发现揭示了微生物通过底物竞争实现代谢稳态的新机制，为深入解析深海细菌如何适应环境、采取何种生存策略提供了新的见解。

综上所述，本研究运用多种生物学技术（组学、遗传学和分子生物学等），研究了深海冷泉细菌E. flavus 21-3在硫元素循环中的红外光响应机制、代谢稳态调控机制。揭示了深海细菌利用红外光驱动硫代谢的分子机制，并提出PPK2通过GTP竞争调控c-di-GMP的代谢模型。这些成果不仅深化了对深海微生物生态功能的认知，还为生物地球化学模型的完善提供了实验依据。

The deep-sea cold seep ecosystem is one of the Earth’s unique and still poorly explored extreme environments, where microorganisms play a crucial role in oceanic element cycles. Cold seeps are rich in substances such as methane and sulfate, and microorganisms drive material cycling through chemolithoautotrophic or heterotrophic processes. However, many of these mechanisms remain to be elucidated. In seawater, microorganisms can convert sulfate into organic sulfur and subsequently mineralize it back into sulfate. This process is central to the Earth’s complex biogeochemical cycles and is vital for the global sulfur cycle. Microorganisms play a key role in the migration, transformation, and distribution of sulfur across different environmental media, maintaining the dynamic balance of the sulfur cycle. This not only has significant implications for the stability of Earth’s ecosystems and climate regulation but has also had profound and lasting effects on biological evolution. Therefore, in-depth studies of microbial sulfur metabolism are essential for a more comprehensive understanding of the functions of deep-sea ecosystems and the pivotal contributions of microorganisms to the sulfur cycle. Additionally, in recent years, the photoreception mechanisms of deep-sea microorganisms have become a research hotspot. Due to the unique light conditions in the deep sea, microorganisms have evolved specialized photoreceptor proteins capable of sensing faint light signals and converting them into biochemical signals, thereby optimizing energy acquisition pathways to adapt to the deep-sea environment. However, the metabolic networks, photoreception mechanisms, and regulatory pathways of deep-sea microorganisms are still to be explored in greater detail. In prior research endeavors, scientists successfully isolated a sulfur-oxidizing bacterium named Erythrobacter flavus 21-3 from a cold-seep in the South China Sea. Both under laboratory-controlled conditions and in the native in situ deep-sea environment, this particular bacterial strain is capable of oxidizing thiosulfate via a newly identified sulfur oxidation pathway, resulting in the production of S₈ in the form of zero-valent sulfur (ZVS). An unexpected experiment revealed that blue light significantly enhanced the ability of E. flavus 21-3 to produce ZVS, although longer wavelengths of geological light are more commonly found in the deep sea.

Therefore, the present study investigated the impact of infrared light on ZVS production by E. flavus 21-3. The results showed that infrared light significantly enhances ZVS generation in E. flavus 21-3 through a regulatory pathway involving the bacteriophytochrome BPHP-15570 and its downstream diguanylate cyclase DGC-0450. Upon infrared stimulation, BPHP-15570 undergoes autophosphorylation, activating DGC-0450 to elevate intracellular cyclic di-GMP (c-di-GMP) levels. c-di-GMP then binds to its receptor protein mPilZ-1753, which in turn activates thiosulfate dehydrogenase (TsdA) and sulfur oxidase (SoxB), ultimately driving ZVS production.

When the concentration of thiosulfate in the environment is excessively high, E. flavus 21-3 produces an excess of ZVS, which has toxic effects on the cells. It has been reported that c-di-GMP plays a key role in the ZVS formation pathway. Additionally, polyphosphate kinase 2 (PPK2) is known to share the same substrate (GTP) as c-di-GMP and performs a similar function. Therefore, this study aims to explore the negative regulatory role of PPK2 in ZVS formation. The study found that PPK2 inhibits the synthesis of c-di-GMP by competing for intracellular GTP, thereby negatively regulating the production of ZVS and biofilm formation. After knocking out the ppk2 gene, the c-di-GMP levels in the strain significantly increased under infrared light exposure, while the production of ZVS also correspondingly increased, and biofilm formation became more pronounced. Further proteomic analysis revealed that the deletion of ppk2 led to the upregulation of various proteins, including those containing the GGDEF domain with potential diguanylate cyclase activity, as well as proteins associated with flagellar assembly and chemotaxis. These results indicated that the deletion of ppk2 leads to elevated intracellular c-di-GMP levels in E. flavus 21-3. To further validate the function of PPK2, this study overexpressed this protein in Escherichia coli BL21(DE3) and Pseudomonas aeruginosa PAO1. The results demonstrated that PPK2 overexpression significantly reduced intracellular c-di-GMP and GTP levels in both bacterial strains and effectively inhibited biofilm formation in E. coli BL21(DE3) and P. aeruginosa PAO1. These findings uncover a novel mechanism by which microorganisms achieve metabolic homeostasis through substrate competition, providing theoretical insights into the environmental adaptation strategies of deep-sea bacteria.

In summary, this study combines multidisciplinary approaches to explore infrared-driven sulfur cycling, metabolic regulation, and genetic tools in the deep-sea bacterium E. flavus 21-3. We reveal the molecular mechanism of infrared-driven sulfur metabolism in deep-sea bacteria and propose a PPK2-GTP competition model for c-di-GMP regulation. These findings deepen understanding of deep-sea microbial ecology and provide insights for biogeochemical modeling.

第1章 绪 论... 1

1.1 深海微生物参与的元素循环... 1

1.2 深海冷泉生态系统及其微生物组成... 1

1.3 海洋微生物介导的硫循环... 3

1.3.1 异化硫酸盐还原... 3

1.3.2 硫化物及单质硫氧化... 4

1.3.3 硫代硫酸盐氧化... 5

1.4 光营养硫细菌的硫代谢... 6

1.4.1 紫色硫细菌硫化合物的转化... 6

1.4.2 绿色硫细菌硫化合物的转化... 7

1.4.3 紫色非硫细菌硫化合物的转化... 8

1.5 深海光源与光感微生物的响应机制... 8

1.5.1 深海光源... 8

1.5.2 深海微生物的感光机制... 13

1.5.3 蓝光促进深海硫氧化细菌Erythrobacter flavus 21-3 形成单质硫的机制研究... 16

1.6 双组分调控系统在细菌代谢中的作用... 17

1.7 环二鸟苷酸（c-di-GMP）在微生物中的调控作用... 19

1.7.1 第二信使c-di-GMP的发现与基本特性... 19

1.7.2 c-di-GMP的合成与降解机制... 20

1.7.3 c-di-GMP效应子的多样性及其介导的生理表型调控网络... 22

1.8 研究内容及意义... 23

第2章 红外光促进E. flavus 21-3形成单质硫的通路... 24

2.1 前言... 24

2.2 实验材料与方法... 24

2.2.1 实验试剂及主要培养基... 24

2.2.2 菌株培养... 27

2.2.3 单质硫含量测定... 28

2.2.4 蛋白结构预测与注释... 28

2.2.5 血红素加氧酶（HO）和细菌光敏色素（BPHP-15570）的克隆、表达和纯化... 29

2.2.6 细菌光敏色素（BPHP-15570）蛋白的点突变... 30

2.2.7 蛋白组测定与分析... 31

2.2.8 E. flavus 21-3基因敲除株的构建... 31

2.2.9 胞内c-di-GMP浓度的测定... 33

2.2.10 细菌双杂交实验... 34

2.2.11 相对基因表达量测试... 34

2.3 实验结果... 36

2.3.1 在红外光和黑暗条件下E. flavus 21-3的单质硫产量存在差异... 36

2.3.2 细菌光敏色素蛋白（BPHP-15570）和血红素加氧酶（HO）的表达纯化... 40

2.3.3 双组分系统BPHP-RS15570/DGC-0450响应红外光释放c-di-GMP. 41

2.4 讨论... 47

2.5 本章小结... 50

第3章 多聚磷酸激酶2（PPK2）负调控E. flavus 21-3单质硫形成... 51

3.1 前言... 51

3.2 实验材料与方法... 51

3.2.1 实验试剂及主要培养基... 51

3.2.2 菌株培养... 51

3.2.3 E. flavus 21-3基因敲除株的构建... 52

3.2.4 胞内GTP浓度的测定... 52

3.2.5 胞内c-di-GMP浓度的测定... 52

3.2.6 单质硫浓度的测定... 52

3.2.7 生物被膜的测定... 52

3.2.8 蛋白组测定与分析... 53

3.2.9 P. aeruginosa PAO1感受态的制备... 53

3.2.10 多聚磷酸激酶2（PPK2）在P. aeruginosa PAO1以及E.coli BL21(DE3)中的过表达... 53

3.3 实验结果... 53

3.3.1 多聚磷酸激酶2（PPK2）通过影响c-di-GMP的生物合成负调控E. flavus 21-3生物被膜的形成和ZVS的产生... 53

3.3.2 在P. aeruginosa PAO1以及E.coli BL21(DE3)中验证多聚磷酸激酶2（PPK2）的负调控作用... 58

3.4 讨论... 60

3.5 本章小结... 63

第4章 总结与展望... 64

参考文献... 67

附录 本研究使用的引物序列... 82

致谢... 86

作者简历及攻读学位期间发表的学术论文与其它相关学术成果    88