Study On The Molecular Mechanism Of The Enhancement Of Cadmium Resistance In Deep-sea Bacteria Via Cysteine

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 273by 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 Cd Cl₂ 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 E77could 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 Ton B-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.