中国科学院海洋研究所机构知识库

重金属污染是全球性的环境问题，在海洋环境中尤为严峻。我国工业的迅速发展导致重金属污染比较严重，而在各类重金属中汞的毒性比较大，并且难以降解，汞污染已经严重影响到了人类健康，如何健康合理地治理汞污染已经成为人类社会的热点。

汞介导的毒性是微生物生存的最大障碍之一，尽管抗汞微生物在汞污染环境的生物修复中担当着重要的角色,但细菌对汞胁迫的遗传和生理适应性机制仍然不清楚。在本研究中，我们发现一株海洋细菌Pseudomonas stutzeri 273能耐受50 µmol/L的Hg²⁺，而且在该浓度下对Hg²⁺的脱除率高达94%，所以该菌株有潜力发展成为有效脱除Hg²⁺的工程菌。

通过对P.stutzeri 273的全基因组分析，发现该菌含有一个汞抗性基因簇，该基因簇包括三个编码跨膜转运Hg²⁺蛋白的基因（merT、merE和merF），一个编码细胞膜上Hg²⁺结合蛋白的基因(merP)，一个编码汞还原酶的基因（merA），两个编码转录调节蛋白的基因（merR和merD）。进而我们通过基因同源重组等分子遗传学方法研究了P. stutzeri 273的汞抗性分子机制：逐个敲除了P. stutzeri 273中的7个汞抗性基因（merE、merD、merA、merF、merP、merT和merR）。通过观察野生型菌株和突变菌株在不同Hg²⁺浓度（0、20和50 μmol/L）的固体LB平板上的生长状况，我们发现野生型菌株P. stutzeri 273可以在50 µmol/L Hg²⁺的平板上正常生长，而ΔmerA和ΔmerT这两个突变体无法在Hg²⁺浓度为20 µmol/L的培养基中生长；同时ΔmerP和ΔmerD突变株在含Hg²⁺的培养基中生长受到了显著的抑制，而其它突变体则没有太大变化。由此我们推断merT、merP、merA和merD是该细菌抗汞作用中的关键基因。

此外，汞胁迫还能够抑制P. stutzeri 273的鞭毛发育、运动、趋化性和生物膜的形成。通过生理实验分析我们发现：随着Hg²⁺浓度的升高，P. stutzeri 273的鞭毛在逐渐变短，运动能力明显下降，生物膜形成受到抑制；同时在Hg²⁺存在下，它趋向无Hg²⁺环境的方向生长。推测是因为细菌感受到周围环境中存在重金属汞的威胁，然后通过调节鞭毛的形成来降低游动能力，或者调节鞭毛来改变细菌的运动方向，从而降低环境中重金属汞对它的危害。另外，细菌的游动能力跟生物膜的形成也有一定关系，游动能力强的细菌更容易形成生物膜，正是因为细菌周围环境中存在重金属汞的威胁，P. stutzeri 273不再向外游动或者游动缓慢，因此导致生物膜的形成受到影响。通过转录组进一步分析，发现在汞的胁迫下，P. stutzeri 273的鞭毛发育、运动性、趋化性等相关基因的表达都受到显著下调，这与生理学实验结果是一致的。

通过遗传学和生理实验分析，我们发现缺失了merF基因的菌株，不再长出鞭毛，在固体LB平板上也丧失了游动能力，生物膜形成也受到了显著抑制。通过转录组和蛋白组数据分析发现，merF基因的缺失显著改变了P.stutzeri 273中鞭毛基因和蛋白的表达量。因此，我们推断Hg²⁺转运蛋白MerF可以决定鞭毛的发育、运动和生物膜的形成，并且在P.stutzeri 273适应汞胁迫过程中起着重要作用。此外，在一些人类病原菌中也存在着MerF同源蛋白，鉴于MerF在鞭毛和生物膜形成中的重要作用，可以将其作为靶标筛选抑制相应病原菌的药物。

总而言之，我们的结果对研究海洋微生物对汞胁迫适应机制提供了一个独特视角，也为发展相应重金属脱除生物制品提供了理论依据和候选微生物。

Global heavy metal pollution is becoming more and more serious, especially in the marine environment. With the rapid development of industry in China, heavy metal pollution is also serious, among which mercury is more toxic and difficult to degrade. Mercury pollution has seriously affected the health of human beings, and how to control mercury pollution healthily and reasonably has become a hot spot in human society.

Mercury-mediated toxicity remains one of the greatest barriers against microbial survival, even though bacterial resistance to mercury compounds can occur. However, the genetic and physiological adaptations of bacteria to mercury stress still remain unclear. Here, we show that the marine bacterium P. stutzeri 273 is resistant to 50 µmol/L Hg²⁺ and removes up to 94% Hg²⁺ from culture. Therefore, it is presumed that P. stutzeri 273 may be an engineering bacterium that can effectively degrade the toxicity of heavy metal mercury.

Through analyzing the whole genome of P. stutzeri 273, we found that the bacterium contained a whole set of mercury-resistant genes. It was consisted with merT, merE and merF (encoding the Hg²⁺-transporter), merP (encoding the binding protein of Hg²⁺), merA (encoding the mercuric reductase), merD and merR (encoding the regulatory protein of mer operon). A series of genetic methods were used to study the mercury resistance analysis mechanism of P. stutzeri 273, such as homologous recombination of genes and complementary experiments. We successfully knocked out the seven mercury-resistant genes of P. stutzeri 273 and obtained the corresponding mutants (ΔmerE, ΔmerD, ΔmerA, ΔmerF, ΔmerP, ΔmerT and ΔmerR). We observed the growth status of wild type and mutant strains on solid LB medium in the absence or presence of 20 or 50 μmol/L of HgCl_2. As expected, the wild type showed normal growth in the concentration of 50 μmol/L Hg²⁺. However, the mutants with deletion of merA or merT exhibited the greatest sensitivity to the low concentration of Hg²⁺, such that the cells could not even grow in the agar plate containing 20 μmol/L Hg²⁺. The deletion of merD affected bacterial growth to a certain extent in the presence of 20 μmol/L Hg²⁺, but cellular growth was completely inhibited by 50 μmol/L Hg²⁺. The deletion of merP only affected bacterial growth under the 50 μmol/L Hg²⁺ condition but not the low level Hg²⁺ condition.Thus,we show that merT, merP, merA and merD are essential for bacterial mercuric resistance when challenged with Hg²⁺.

Further, mercury stress inhibited flagellar development, motility, chemotaxis and biofilm formation of P. stutzeri 273. Through physiological experiments, we found that with the increase of mercury concentration, the flagella of P. stutzeri 273 gradually became shorter and its mobility decreased greatly. At the same time, bacterial biofilm formation is related to its swimming ability, as bacteria with strong swimming ability are more likely to form biofilms. Because the threat of heavy metal mercury in the surrounding environment, P. stutzeri 273 will no longer move or move slowly, which affects the formation of biofilms. From the analysis of transcriptome, it indicates that P. stutzeri 273 physiologically adapted to the mercury stress by down-regulating motility and positive chemotaxis, which is consistent with the results of physiological experiments.

Notably, we discovered the mutant ΔmerF did not grow flagella, nor did it move upstream of the solid LB plate, and the biofilm formation was also greatly affected.   Through the analysis of transcriptional and proteomic data, it was found that the   merF gene significantly changed the expression of flagellum gene and protein in p.stutzeri 273. Our results strongly indicate that MerF determines flagellar development, motility and biofilm formation in P. stutzeri 273 and plays an integral role in P. stutzeri 273 to develop physiological responses to mercury stress. Notably, MerF homologues are also prevalent in different human pathogens. It can be used as a target to screen drugs that inhibit the corresponding pathogens, given the role of MerF in flagella and biofilm formation.

 In summary, our results provide a unique perspective for studying the adaptation mechanism of marine microorganisms to mercury stress, and provide theoretical basis and candidate microorganisms for the development of biological products for heavy metal removal.