根癌农杆菌GW4中磷酸盐调控蛋白PhoB介导的锑氧化调控机制
发布时间：2019-02-15 18:32
【摘要】：锑(Antimony)是一种剧毒的类金属,锑中毒会导致人体心肌衰竭、肝坏死等疾病。自然界中有些微生物能够通过氧化毒性强的Sb(III)至毒性弱的Sb(V)来解毒。本课题组前期发现在锑氧化型细菌Agrobacterium tumefaciens GW4中Sb(III)的氧化是锑氧化酶AnoA催化的酶促和H2O2介导的非酶促锑氧化共同组成的,但对于AnoA表达的调控机制还尚未阐明并且目前微生物Sb(III)压力下的响应机制也尚未报道。本论文开展了菌株GW4在不加锑与加50μM Sb(III)条件下的比较蛋白质组研究,然后利用分子生物学技术研究锑氧化酶AnoA的调控机制,并分析了锑压力下的代谢路径。通过蛋白质组学和质谱测序鉴定得到21个加锑差异表达蛋白质均上调表达,根据KEGG功能分类,分别属于锑氧化及外排、碳代谢、脂类代谢、氨基酸代谢及膦酸酯代谢和磷酸盐转运等。其中,我们发现在加锑条件下磷酸盐转运蛋白Pst S2的上调表达,前期报道PstS蛋白受磷酸盐调控蛋白PhoB的调控。PhoB为全局性调控蛋白,我们推测磷酸盐调控系统可能与Sb(III)氧化存在相关性。基因组分析发现菌种GW4有两套磷酸盐调控系统。将位于砷基因岛上一套命名为1,该基因簇中的磷酸盐调控蛋白基因phoB1位于磷酸盐转运系统pstBACS1的下游,方向相反;将远离砷基因岛的另一套命名为2,该基因簇中的磷酸盐调控蛋白基因phoB2位于磷酸盐转运系统pstBACS2的下游,方向相同。本论文主要结果如下:(1)通过报告基因融合实验,我们检测到GW4中的磷酸盐调控蛋白基因phoB1受Sb(III)诱导效果不明显,但phoB2能够受Sb(III)显著诱导;(2)在加Sb(III)条件下检测菌株GW4、GW4-ΔphoB1、GW4-ΔphoB2、GW4-ΔphoB1/phoB2中锑氧化酶基因anoA的酶活,结果发现较野生型GW4中anoA的酶活相比,缺失phoB2基因anoA的酶活性受到明显的抑制,phoB1基因的缺失使anoA的酶活性略有下降,而双敲除株GW4-ΔphoB1/phoB2中anoA酶活几乎丧失;(3)对各个菌株进行生长和锑氧化实验结果表明,单敲除株在50μM Sb(III)浓度下生长、锑氧化均不受影响,GW4-ΔphoB1/phoB2双敲菌株在不影响生长的情况下锑氧化效率明显降低;(4)对各个菌株在加Sb(III)条件进行胞内H2O2含量测定,结果显示GW4-ΔphoB1中H2O2含量较GW4显著升高,GW4-ΔphoB2中H2O2含量极显著升高,互补株均回复表型。这些结果表明单敲除株氧化速率没有降低很可能是由于H2O2发挥了Sb(III)氧化作用;而双敲除株中H2O2含量虽比野生型高,但仍低于GW4-ΔphoB2,并不足以弥补anoA的作用,因此氧化速率减慢;(5)细菌单杂交和EMSA等方法证明PhoB1、PhoB2能够与anoA启动子区DNA在体内和体外相互作用,并确定了结合位点。以上结果表明,磷酸盐调控蛋白PhoB1与PhoB2均对锑氧化酶基因anoA有一定的调控作用,但是PhoB2的调控作用更强。该研究阐明了菌株GW4中锑氧化酶基因anoA的调控机制,并解析了异养型细菌对锑的压力响应机制,为利用微生物进行锑污染治理具有十分重要的理论价值。
[Abstract]:Antimony (Antimony) is a highly toxic metal. Antimony poisoning can lead to heart failure, liver necrosis and other diseases. Some microorganisms in nature can detoxify by oxidizing highly toxic Sb (III) to weak Sb (V). Our previous study found that the oxidation of Sb (III) in antimony-oxidized bacteria Agrobacterium tumefaciens GW4 was composed of antimony oxidase AnoA catalyzed by enzyme and H2O2 mediated non-enzymatic antimony oxidation. However, the regulation mechanism of AnoA expression has not been elucidated and the response mechanism of microbial Sb (III) pressure has not been reported. In this paper, the comparative proteome of strain GW4 was studied without antimony and 50 渭 M Sb (III), then the regulation mechanism of antimony oxidase AnoA was studied by molecular biology, and the metabolic pathway of antimony under antimony pressure was analyzed. 21 differentially expressed antimony proteins were identified by proteomics and mass spectrometry sequencing. According to the functional classification of KEGG, they belong to antimony oxidation and efflux, carbon metabolism and lipid metabolism, respectively. Amino acid metabolism, phosphonate metabolism and phosphate transport. Among them, we found that the expression of phosphate transporter Pst S2 was up-regulated under antimony. It was reported that PstS protein was regulated by phosphate regulatory protein PhoB. PhoB was a global regulatory protein. We speculate that phosphate regulation system may be related to Sb (III) oxidation. Genomic analysis showed that GW4 had two phosphate regulatory systems. The phosphate regulatory protein gene phoB1 located on the arsenic gene island is located downstream of the phosphate transport system pstBACS1 in the opposite direction. Another set of genes far away from the arsenic gene island was named 2.The phosphate regulatory protein gene phoB2 in this gene cluster is located downstream of the phosphate transport system pstBACS2 in the same direction. The main results of this thesis are as follows: (1) through the reporter gene fusion experiment, we detected that the phoB1 gene of phosphate regulatory protein in GW4 was not significantly induced by Sb (III), but phoB2 could be significantly induced by Sb (III); (2) the enzyme activity of antimony oxidase gene anoA in strain GW4,GW4- 螖 phoB1,GW4- 螖 phoB2,GW4- 螖 phoB1/phoB2 was detected under the condition of Sb (III). The results showed that the enzyme activity of anoA in wild type GW4 was higher than that in wild-type GW4. The enzyme activity of anoA with deletion of phoB2 gene was obviously inhibited, the enzyme activity of anoA decreased slightly by the deletion of phoB1 gene, but the activity of anoA in GW4- 螖 phoB1/phoB2 of double knockout strain was almost lost. (3) the growth of each strain and antimony oxidation test showed that the antimony oxidation of single knockout strain at 50 渭 M Sb (III) concentration was not affected, and the antimony oxidation efficiency of GW4- 螖 phoB1/phoB2 double knock strain was obviously decreased without affecting the growth of the strain. (4) the intracellular H2O2 content of each strain was determined under the condition of adding Sb (III). The results showed that the H2O2 content in GW4- 螖 phoB1 was significantly higher than that in GW4, the H2O2 content in GW4- 螖 phoB2 was significantly higher than that in GW4, and the complementary strains returned to phenotype. These results suggested that the oxidation rate of single knockout plant did not decrease due to the Sb (III) oxidation of H2O2. Although the content of H2O2 in the double knockout plant was higher than that in the wild type, it was still lower than GW4- 螖 phoB2, which was not enough to make up for the effect of anoA, so the oxidation rate slowed down. (5) single hybridization and EMSA showed that PhoB1,PhoB2 could interact with anoA promoter DNA in vivo and in vitro, and the binding sites were determined. These results suggest that both phosphate regulatory protein PhoB1 and PhoB2 have certain regulatory effects on antimony oxidase gene anoA, but PhoB2 has a stronger regulatory effect. This study clarified the regulation mechanism of antimony oxidase gene anoA in strain GW4, and analyzed the pressure response mechanism of heterotrophic bacteria to antimony, which is of great theoretical value for antimony pollution control by microbes.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172
[Abstract]:Antimony (Antimony) is a highly toxic metal. Antimony poisoning can lead to heart failure, liver necrosis and other diseases. Some microorganisms in nature can detoxify by oxidizing highly toxic Sb (III) to weak Sb (V). Our previous study found that the oxidation of Sb (III) in antimony-oxidized bacteria Agrobacterium tumefaciens GW4 was composed of antimony oxidase AnoA catalyzed by enzyme and H2O2 mediated non-enzymatic antimony oxidation. However, the regulation mechanism of AnoA expression has not been elucidated and the response mechanism of microbial Sb (III) pressure has not been reported. In this paper, the comparative proteome of strain GW4 was studied without antimony and 50 渭 M Sb (III), then the regulation mechanism of antimony oxidase AnoA was studied by molecular biology, and the metabolic pathway of antimony under antimony pressure was analyzed. 21 differentially expressed antimony proteins were identified by proteomics and mass spectrometry sequencing. According to the functional classification of KEGG, they belong to antimony oxidation and efflux, carbon metabolism and lipid metabolism, respectively. Amino acid metabolism, phosphonate metabolism and phosphate transport. Among them, we found that the expression of phosphate transporter Pst S2 was up-regulated under antimony. It was reported that PstS protein was regulated by phosphate regulatory protein PhoB. PhoB was a global regulatory protein. We speculate that phosphate regulation system may be related to Sb (III) oxidation. Genomic analysis showed that GW4 had two phosphate regulatory systems. The phosphate regulatory protein gene phoB1 located on the arsenic gene island is located downstream of the phosphate transport system pstBACS1 in the opposite direction. Another set of genes far away from the arsenic gene island was named 2.The phosphate regulatory protein gene phoB2 in this gene cluster is located downstream of the phosphate transport system pstBACS2 in the same direction. The main results of this thesis are as follows: (1) through the reporter gene fusion experiment, we detected that the phoB1 gene of phosphate regulatory protein in GW4 was not significantly induced by Sb (III), but phoB2 could be significantly induced by Sb (III); (2) the enzyme activity of antimony oxidase gene anoA in strain GW4,GW4- 螖 phoB1,GW4- 螖 phoB2,GW4- 螖 phoB1/phoB2 was detected under the condition of Sb (III). The results showed that the enzyme activity of anoA in wild type GW4 was higher than that in wild-type GW4. The enzyme activity of anoA with deletion of phoB2 gene was obviously inhibited, the enzyme activity of anoA decreased slightly by the deletion of phoB1 gene, but the activity of anoA in GW4- 螖 phoB1/phoB2 of double knockout strain was almost lost. (3) the growth of each strain and antimony oxidation test showed that the antimony oxidation of single knockout strain at 50 渭 M Sb (III) concentration was not affected, and the antimony oxidation efficiency of GW4- 螖 phoB1/phoB2 double knock strain was obviously decreased without affecting the growth of the strain. (4) the intracellular H2O2 content of each strain was determined under the condition of adding Sb (III). The results showed that the H2O2 content in GW4- 螖 phoB1 was significantly higher than that in GW4, the H2O2 content in GW4- 螖 phoB2 was significantly higher than that in GW4, and the complementary strains returned to phenotype. These results suggested that the oxidation rate of single knockout plant did not decrease due to the Sb (III) oxidation of H2O2. Although the content of H2O2 in the double knockout plant was higher than that in the wild type, it was still lower than GW4- 螖 phoB2, which was not enough to make up for the effect of anoA, so the oxidation rate slowed down. (5) single hybridization and EMSA showed that PhoB1,PhoB2 could interact with anoA promoter DNA in vivo and in vitro, and the binding sites were determined. These results suggest that both phosphate regulatory protein PhoB1 and PhoB2 have certain regulatory effects on antimony oxidase gene anoA, but PhoB2 has a stronger regulatory effect. This study clarified the regulation mechanism of antimony oxidase gene anoA in strain GW4, and analyzed the pressure response mechanism of heterotrophic bacteria to antimony, which is of great theoretical value for antimony pollution control by microbes.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172
【参考文献】
相关期刊论文 前4条
1 王革娇;王倩;陈芳;李t熜，
本文编号：2423621
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