| Sulfide has inextricably link to life on earth, and the interaction of sulfide with acidic iron-containing water promotes the appearance of colloidal iron-sulfur protein membranes, which are thought to greatly enhance the chance of the first organic synthesis event. It can be found from the discovery of ancient fossils, there are many chemolithoautotroph organisms used the sulfur-containing compounds as the sole energy source. Until now, sulfide continues affecting the growth of many organisms, together with the fact that sulfur is the 6th abundant element in microbial biomass, indicating that sulfur metabolism is essential for the biogeochemical cycle.Hydrogen sulfide is mainly produced due to sulfate reduction, volcanoes and so on in the environment, and it is also naturally produced in the cell body, mainly resulting from the metabolism of sulfur-containing amino acids. The producing H2S can also be released into the air. Many gases, for example H2S and SO2, are produced in the ocean also released into the air. Excess hydrogen sulfide pollutes the air, and SO2 causes acid rain as well as other environmental problems. Finally, H2S is oxidized to sulfate, which then used by plants to complete the critical step of the sulfur cycle, and the oxidation of H2S is primarily dependent on the action of microorganisms, especially some sulfur oxidizing bacteria and archaea.The toxicity of hydrogen sulfide is closely related to its concentration, which can produce neurotoxicity at high concentrations and have important physiological functions at low concentrations. As in mammals, high concentrations of H2S can lead to physiological poisoning till death, but a low concentration of H2S can regulate a variety of physiological functions for medical purpose. Based on these physiological functions, H2S has been considered to be the third gasotransmitters following NO and CO.Maintaining the concentration of hydrogen sulfide is crucial for organisms, where excess hydrogen sulfide is removed primarily by the combined action of enzymes. A set of important enzyme systems was found from the detailed research of sulfide oxidation in mammalian mitochondria, including sulfide:quinone oxidoreductase (SQR), persulfide dioxygenase (PDO) and sulfurtransferase (ST). Although the metabolic pathway of H2S oxidized to thiosulfate has been reported, there are some unclear questions. (1) What is the specific form of sulfane sulfur which is produced via SQR oxidizing H2S? The SQR in eukaryotic mitochondria requires a sulfane sulfur receptor; is this common to all organisms? If so, what is the receptor? Glutathione, cysteine and sulfite are likely as the receptor, the real physiological receptor is still not clear. (2) Whether the SQR/PDO/ST system exists in heterotrophic bacteria? If so, do they have similar functions? (3) Is there a metabolic pathway of sulfide oxidation different from that of eukaryotes in heterotrophic bacteria? What is the physiological function? (4) At present, most of the studies in microbes are concentrated in autotrophic bacteria. However, due to the wide distribution and sheer aboundance as well as the rapid growth rates of heterotrophic bacteria, it is possible for heterotrophic bacteria to speed the rate of H2S metabolism, which may has a greater ecological significanceIn order to solve the above-mentioned problems, this thesis focuses on the oxidation of H2S in heterotrophic bacteria. To study the enzymology of SQR/PDO/ST system, the function, the pathway, and the products of H2S oxidation using recombinant E.coli strains. Also, the metabolism of H2S was studied in Cupriavidus pinatubonensis JMP134. The following questions are studied in detail:(1) The distribution and diversity of persulfide dioxygenases (PDOs) in the bacteria were searched by the BLAST program in the sequenced bacterial genome using the previously identified mammalian PDOs as the query sequences. The results showed the homologous proteins of the PDO are widely distributed in the bacteria, and mainly in the proteobacteria and cyanobacteria, accounting for about 20% of the total sequenced bacterial genomes. According to multiple sequence alignment, the newly discovered and reported proteins werew used to construct the phylogenetic tree of PDOs. The tree make the PDOs divided into three subgroups:PDO1, PDO2 and Blh, and these three subgroups of PDOs are phylogenetically related to GloB1 and GloB2, which are glyoxalases ?. The analysis of the structures of MxPDO1 and PpPDO2 shows the difference between PDO1 and PDO2 in substrate binding sites, which supports the classification of PDOs in this thesis. Fourteen proteins were choose from PDO1, PDO2, Blh, GloB1 and GloB2 subgroups, and to test their activity with GSSH as the substrate. The results showed that there were 8 enzymes had persulfide dioxygenase activity, which belonged to PDO1, PDO2 and Blh subgroup, but those enzymes in subgroup GloB 1 and GloB2 had no activity, in agreement with the results predicted by our phylogenetic analysis. Among them, MxPDO1a had the highest catalytic efficiency. ICP-MS result showed that ferrous ion could be used as a cofactor in PDOs since every PDO containing equal concentration of ferrous ion, several PDOs can use manganese (Mn2+) instead of ferrous ion, as confirmed by metal dialysis and pre-incubated experiments. The feature of PDOs using metal as the cofactor is in accordance with the characteristic of the metallo-?-lactamase family. Further, according to the sequence similarity, we used BLASTP program to search for the possible sulfur oxygenases in gram-positive bacteria. Three possible genes were ligated with Cpsqr gene, and cloned into plasmid pBBR1MCS2, then transformed into E. coli BL21 (DE3), and the activity of the strains were analyzed. The results showed that the heterologous expression strains could rapidly metabolize H2S, indicating that the persulfide dioxygenase-like proteins of these gram-positive bacteria had the ability to oxidize the sulfane sulfur produced by CpSQR but could not determine whether they used the GSSH as substrate. At last, the knockout strain of PDO in the Pseudomonas aeruginosa PAO1, APaPDO2, resulted in the accumulation of more H2S compared with wild strain, which indicated that PDOs could played an important physiological role in the bacterial H2S oxidation.(2) Two genes coding for CpSQR and CpPDO2 of Cupriavidus pinatubonensis JMP134 were cloned into E. coli BL21 (DE3), and the heterologous expression strains were used to study their metabolic pathway of H2S. Because CpSQR contains two domains, HcaD and DUF442, of which the HcaD domain was a sulfide:quinone oxidoreductase according to early reports and the DUF442 domain was a putative sulfur transferase. It was found that the product of CpSQR oxidized H2S mainly were disulfide and trisulfide confirmed by HPLC and MS, which is different from the product of SQRs from eukaryotic mitochondria; In addition, we demonstrated that CpDUF442 domain was a sulfur transferase, which played a catalytic role in the three reaction involved the H2S oxidation. One of the new functions of CpDUF442 was that it enhanced the reaction of polysulfides and GSH, which implied CpDUF442 can promote the rate of sulfane sulfur transfer. Moreover, CpDUF442 also catalyzed the reaction of GSSH and sulfite, which is consistent with that reported in eukaryotes. We also found that polysulfides reacts with GSH or sulfites spontaneously, and their spontaneous reaction rates were determined by spectroscopic methods as 8.4±0.7 ?M min-1 and 6.3±0.5?M min-1, respectively. And when CpDUF442 was added, the reaction rate of the polysulfides and GSH increased to 13.3±1.2 ?M min-1, while reaction rate of polysulfides and sulfite had no significant change. Using biochemical experiments and analysis data in vivo, it was found that CpDUF442 could not oxidized the thiosulfate, which was inconsistent with the conclusion that sulfur transferase could convert thiosulfate and GSH to GSSH and sulfite in yeast and human. We also study the metabolites of H2S and polysulfides oxidation and presented a flux balance analysis model. The end-products of CpSQR and CpPDO2 were a mixture of sulfite and thiosulfate with little sulfane sulfur accumulation. All the results show that the function of CpDUF442 in catalyzing GSSH and sulfite in this SQR/PDO/ST system is not significant. On the contrary, the function of CpDUF442 may be primarily to promote the distribution of sulfane sulfur, such as the reaction of polysulfides and GSH to produce GSSH. Finally, the H2S oxidation pathway in recombinant bacteria was proposed according to the flux balance analysis.(3) Finally, we systematically analyzed the genes related to the metabolism of hydrogen sulfide and other sulfur compounds using the sequenced genome of Cupriavidus pinatubonensis JMP134 at NCBI website. C. pinatubonensis JMP134 had multiple systems related to the sulfur oxidation, namely the SQR/PDO/ST system, SOX system, SO system, and FCSD system. The key genes of these systems were knocked out, and the knock-out strains were used to analyze the products of the oxidation, of sulfide, sulfite, or thiosulfate. The results showed the SQR/PDO/ST system was responsible for the oxidation of H2S. The SOX system could complement the SQR function after inactivation of SQR, but the efficiency was not high. The detectable end-product of the SQR/PDO/ST system for H2S oxidation may only be thiosulfate, which is different from the products (SO32- and S2O32- mixture) of sulfide oxidation by the recombinant E. coli with SQR/PDO/ST, possibly due to different PDO activity. The main exogenous sulfite oxidation was by the SO system, the product was sulfate, and the consumed sulfite and generated sulfate were close to 1:1; furthermore, the SOX system and SoxCD cannot play the role of oxidizing exogenous sulfite. The SOX system was responsible for both endogenous and exogenous thiosulfate oxidation. The oxidation product of thiosulfate was sulfate, and each thiosulfate produced about two sulfates. SoxF (FCSD) may participate in the SOX system as an important part to oxidize thiosulfate, but SoxF alone did not function on the oxidation of H2S. Sulfite and thiosulfate oxidation pathways are consistent with the law of mass balance, so that cells will not accumulate too much these sulfur compounds by transforming to sulfate and released back to the environment. Based on the results of genome analysis and whole-cell analysis, we also proposed a metabolic model of sulfur compounds in the heterotrophic bacteria C. pinatubonensis JMP134, which start with the assimilation sulfate reduction, and including H2S production in the cell, H2S is mainly oxidized by the SQR/PDO/ST system, the production of thiosulfate, and the thiosulfate and sulfite oxidation by the SOX system and SO system, respectively. Further, the model includes the free transformation between thiosulfate and sulfite dependent on the sulfane sulfur pool and sulfurtransferase. This work provides the foundation for using heterotrophic bacteria with SQR and PDO for sulfide bioremediation. |
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