根癌農(nóng)桿菌GW4中磷酸鹽調(diào)控蛋白PhoB介導(dǎo)的銻氧化調(diào)控機(jī)制
發(fā)布時(shí)間：2019-02-15 18:32
【摘要】：銻(Antimony)是一種劇毒的類金屬,銻中毒會(huì)導(dǎo)致人體心肌衰竭、肝壞死等疾病。自然界中有些微生物能夠通過(guò)氧化毒性強(qiáng)的Sb(III)至毒性弱的Sb(V)來(lái)解毒。本課題組前期發(fā)現(xiàn)在銻氧化型細(xì)菌Agrobacterium tumefaciens GW4中Sb(III)的氧化是銻氧化酶AnoA催化的酶促和H2O2介導(dǎo)的非酶促銻氧化共同組成的,但對(duì)于AnoA表達(dá)的調(diào)控機(jī)制還尚未闡明并且目前微生物Sb(III)壓力下的響應(yīng)機(jī)制也尚未報(bào)道。本論文開(kāi)展了菌株GW4在不加銻與加50μM Sb(III)條件下的比較蛋白質(zhì)組研究,然后利用分子生物學(xué)技術(shù)研究銻氧化酶AnoA的調(diào)控機(jī)制,并分析了銻壓力下的代謝路徑。通過(guò)蛋白質(zhì)組學(xué)和質(zhì)譜測(cè)序鑒定得到21個(gè)加銻差異表達(dá)蛋白質(zhì)均上調(diào)表達(dá),根據(jù)KEGG功能分類,分別屬于銻氧化及外排、碳代謝、脂類代謝、氨基酸代謝及膦酸酯代謝和磷酸鹽轉(zhuǎn)運(yùn)等。其中,我們發(fā)現(xiàn)在加銻條件下磷酸鹽轉(zhuǎn)運(yùn)蛋白Pst S2的上調(diào)表達(dá),前期報(bào)道PstS蛋白受磷酸鹽調(diào)控蛋白PhoB的調(diào)控。PhoB為全局性調(diào)控蛋白,我們推測(cè)磷酸鹽調(diào)控系統(tǒng)可能與Sb(III)氧化存在相關(guān)性。基因組分析發(fā)現(xiàn)菌種GW4有兩套磷酸鹽調(diào)控系統(tǒng)。將位于砷基因島上一套命名為1,該基因簇中的磷酸鹽調(diào)控蛋白基因phoB1位于磷酸鹽轉(zhuǎn)運(yùn)系統(tǒng)pstBACS1的下游,方向相反;將遠(yuǎn)離砷基因島的另一套命名為2,該基因簇中的磷酸鹽調(diào)控蛋白基因phoB2位于磷酸鹽轉(zhuǎn)運(yùn)系統(tǒng)pstBACS2的下游,方向相同。本論文主要結(jié)果如下:(1)通過(guò)報(bào)告基因融合實(shí)驗(yàn),我們檢測(cè)到GW4中的磷酸鹽調(diào)控蛋白基因phoB1受Sb(III)誘導(dǎo)效果不明顯,但phoB2能夠受Sb(III)顯著誘導(dǎo);(2)在加Sb(III)條件下檢測(cè)菌株GW4、GW4-ΔphoB1、GW4-ΔphoB2、GW4-ΔphoB1/phoB2中銻氧化酶基因anoA的酶活,結(jié)果發(fā)現(xiàn)較野生型GW4中anoA的酶活相比,缺失phoB2基因anoA的酶活性受到明顯的抑制,phoB1基因的缺失使anoA的酶活性略有下降,而雙敲除株GW4-ΔphoB1/phoB2中anoA酶活幾乎喪失;(3)對(duì)各個(gè)菌株進(jìn)行生長(zhǎng)和銻氧化實(shí)驗(yàn)結(jié)果表明,單敲除株在50μM Sb(III)濃度下生長(zhǎng)、銻氧化均不受影響,GW4-ΔphoB1/phoB2雙敲菌株在不影響生長(zhǎng)的情況下銻氧化效率明顯降低;(4)對(duì)各個(gè)菌株在加Sb(III)條件進(jìn)行胞內(nèi)H2O2含量測(cè)定,結(jié)果顯示GW4-ΔphoB1中H2O2含量較GW4顯著升高,GW4-ΔphoB2中H2O2含量極顯著升高,互補(bǔ)株均回復(fù)表型。這些結(jié)果表明單敲除株氧化速率沒(méi)有降低很可能是由于H2O2發(fā)揮了Sb(III)氧化作用;而雙敲除株中H2O2含量雖比野生型高,但仍低于GW4-ΔphoB2,并不足以彌補(bǔ)anoA的作用,因此氧化速率減慢;(5)細(xì)菌單雜交和EMSA等方法證明PhoB1、PhoB2能夠與anoA啟動(dòng)子區(qū)DNA在體內(nèi)和體外相互作用,并確定了結(jié)合位點(diǎn)。以上結(jié)果表明,磷酸鹽調(diào)控蛋白PhoB1與PhoB2均對(duì)銻氧化酶基因anoA有一定的調(diào)控作用,但是PhoB2的調(diào)控作用更強(qiáng)。該研究闡明了菌株GW4中銻氧化酶基因anoA的調(diào)控機(jī)制,并解析了異養(yǎng)型細(xì)菌對(duì)銻的壓力響應(yīng)機(jī)制,為利用微生物進(jìn)行銻污染治理具有十分重要的理論價(jià)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：X172
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 王革嬌;王倩;陳芳;李t熜，
本文編號(hào)：2423621
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