【作者】 李晶晶；

【导师】 焦念志； 荀鲁盈； 夏永振；

【作者基本信息】 山东大学 ， 生物学， 2019， 硕士

【摘要】 H2S及硫化物在自然界中广泛存在,是生物地球化学循环中重要的组成部分。H2S是一种有毒气体,能够通过抑制呼吸链而影响中枢神经系统和呼吸系统的活动,从而产生毒性作用,然而随着相关研究的深入,H2S也被认为是继CO和NO之后的第三类气体信号分子,既可以对细胞的生理起到重要的调控作用,也能作为一种还原性的物质直接参与细胞清除自由基的过程,抵抗氧化压力。异养细菌可以在好氧环境中氧化有机质产生H2S,在异养菌中发现的SQR-PDO途径是已知的最重要的H2S氧化途径之一,其能将硫化物经多硫化物氧化成亚硫酸盐和硫代硫酸盐。硫酸盐是硫化合物中氧化程度最高的盐类,也是植物和多数微生物生成半胱氨酸的主要硫源。高浓度的含硫有机工业废水,也会严重影响国家生态安全及环境健康。因此,研究硫及其化合物代谢在生物地球化学循环中有很重要的作用。BigR、CstR和SqrR是分别来自三种不同菌的负调控因子,负调控下游H2S氧化相关基因的转录,进一步的研究发现罗尔斯通氏菌中存在一个新型的σ54因子依赖的调控因子FisR,对sqr和pdo基因起到正调控作用。我们通过生物信息学分析发现在地衣芽孢杆菌(Bacillus licheneformis)MW3中,sqr和pdo基因的上游存在编码双组分调控系统的nreBC基因和yrkD调节基因,但其调控sqr和pdo的机制是未知的,并且调控因子YrkD和双组分系统NreBC如何协同发挥调控作用以及感应的信号分子是不清楚的,因此研究此H2S氧化途径的调控功能十分必要。本文中我们首先构建了nre 和nreC敲除株,发现其在诱导后丧失了氧化H2S的能力,这表明双组分系统NreBC对H2S氧化途径是正调节的激活型转录因子。然而同时敲除yrkD和nreBC基因之后,sqr基因不能正常转录,但在只敲除yrkD基因时sqr基因在不受诱导的条件下能够正常转录,结合qPCR分析结果表明YrkD能抑制双组份调控系统nreBC的表达,进而影响sqr基因的转录。随后,提取了野生株的RNA并进行共转录分析,结果发现yrkD-yrkE-yrkF-yrkH-yrkI-yrkJ-ydfQ-nreB-nreC-sqr-pdo存在一个共转录文本。同时通过构建sqr-pdo反转株,将sqr和pdo整体反转过来,发现在硫化物诱导的情况下sqr基因仍然可以表达,这说明sqr-pdo前面也有独立的启动子。进一步通过5’ RACE分析转录起始位点,结果证实了yrk 和sqr前面有转录起始位点。进而构建了绿色荧光蛋白报告系统,在野生菌中研究发现多硫化物能诱导菌株中绿色荧光蛋白的产生,而在yrkD基因敲除株中,发现多硫化物并不能诱导绿色荧光蛋白产生,而NaHS可以诱导绿色荧光蛋白的表达,结果说明了YrkD和NreB的感应信号不同,YrkD感应多硫化物,而NreB的感应信号是HS-，这是一种新型的感应信号,对于H2S氧化有重要的作用。本文在B.licheniformis中发现了一条新型H2S氧化途径,其中包含已知的SQR-PDO途径,研究揭示了 SQR-PDO途径受到一种新型的YrkD和NreBC双调控因子的级联调控;同时,NreBC作为一个双组份系统也是首次被发现参与了H2S氧化途径的调控;与已知的多硫化物感应信号不同,本文中发现能感应HS-信号的调控因子。本文深入揭示了这条新型H2S氧化途径的调控机理,这将丰富我们对异养细菌硫氧化多样性的认知,为开展H2S的生物处理及其应用提供一定的依据,具有一定的生态意义。更多还原

【Abstract】 Hydrogen sulfide and its derivatives,an important part of biogeochemical cycle.widely exist in nature.Hydrogen sulfide is a kind of toxic gas,which can affect the activity of central nervous system and respiratory system by inhibiting respiratory chain.With the deepening of relevant studies,H2S is also considered as the third gas signal molecules after CO and NO,which can not only play an important regulatory role on cell physiology,but also resist oxidative pressure by directly participated in the process of cell scavenging free radicals as a reducing substance.Heterotrophic bacteria can produce H2S by oxidized organic matter in aerobic environment.The SQR-PDO pathway is important for the H2S oxidation of heterotrophic bacteria.which can oxidize sulfide into sulfite and thiosulfate through polysulfide.Sulfates are the salts with the highest degree of oxidation among sulfur compounds,they are also the major sulfur source for cysteine synthesis in plants and most microorganisms Industrial waste water with high concentration of sulfur also seriously affect the national ecological and environmental.Therefore,the research about sulfur oxidation play an important role in biogeochemical cycles.BigR.CstR and SqrR are reported as negative regulators from three different bacteria respectively,which negatively regulate the transcription of H2S oxidation-related genes.In addition.FisR,a new type of σ54-dependent regulator in Cupriavidus pinatubonensis JMP134.positively regulates the expression of sqr and pdo.Bioinformatics analysis showed that a gene cluster was located in upstream of sqr and pdo genes in Bacillus licheniformis MW3,which contained the two-component system NreBC and a series of sulfur transport-related genes.However,the mechanism of the regulatory factor yrkD and the two-component system NreBC regulating sqr and pdo is not clearIn this paper.the ability to oxidize H2S after induction was lost after deleted nreB and nreC genes,indicating that NreBC is an activated transcription factor.However,SQR could transcribe without induction when only yrkD was knocked out.SQR could not normally transcribe after the knockout of yrkD and nreBC strains.The comparison showed that YrkD could inhibit the expression of nreBC.After the establishment of the green fluorescent protein reporting system in MW3,it was found that the generation of green fluorescent protein could be induced by polysulfide,while the generation of green fluorescent protein could be induced by hydrogen sulfide instead of polysulfide on the basis of the knockout of yrkD strain.The results showed that the induction signals of YrkD and NreB are different.The induction of YrkD is sulfane sulfide,while the induction signal of NreB is HS-,which is a new type of induction signal and plays an important role in the sulfide metabolism.The RNA of the wild-type strain was extracted and analyzed for co-transcription.it was found that the co-transcription text existed in yrkD-yrkE-yrkF-yrkk-yrkI-yrkJ-ydfq-nreB-nreC-sqr-pdo.The sqr-pdo reversed strain was constructed,it was found that the SQR could still be expressed after the induction of sulfide when the sqr and pdo were reversed as a whole,which indicated that there were independent promoters in front of sqr-pdo.Transcription initiation sites were identified by the 5’ RACE analysis,the results confirmed that transcription initiation sites were located in front of yrkD and sqr.In B.licheniformis,a novel H2S oxidation pathway was found in this paper.This total pathway includes the known SQR-PDO pathway,which was cascade regulated by YrkD and NreBC.Meanwhile.NreBC,as a two-component system,has been found to be involved in the regulation of H2S oxidation pathway for the first time.In addition,known regulatory signals are polysulfide,and regulatory factors that can sense HS-signal has been found for the first time.The regulation mechanism of sulfide oxidation pathway revealed in this paper would enrich our cognition of the diversity of sulfur oxidation in heterotrophic bacteria,provide certain basis for the application of biological processing of H2S,and have certain ecological significance.更多还原