菲降解菌分离鉴定、降解基因克隆与表达及菲跨膜作用研究
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摘要
本文通过选择性富集培养，从上海炼油厂附近污染土壤中分离到两株菲降解细菌，及一株可加速菲降解菌的菲降解速率的细菌，并对所分离菌株进行了生化鉴定以及系统发育研究，同时进行了降解特性、代谢途径、降解基因克隆及表达方面的研究，对菲的膜毒性机制进行了初步的探讨性研究。以期为建立有效的菲污染预警指标体系和PAHs污染的生物修复提供有益的参考。
本研究所获主要结论如下：
1、从石油污染土壤中筛选分离到两株菲降解菌株，分别命名为ZP1和ZP2菌株，同时分离到一株可加速菌株ZPl对菲降解速率的菌株，命名为ZP5菌株。通过革兰氏染色、细菌形态学观察、生理生化测试、抗生素抗性实验、碳源利用实验、全细胞脂肪酸分析、G+C mol％含量分析以及16S rDNA序列同源性分析，证明ZP1、ZP2和．ZP5菌株这三株菲降解细菌应分别属于鞘氨醇单胞菌属、假单胞菌属及tistrella属。其中菌株ZP1被鉴定为少动鞘氨醇单胞菌种(Sphingomonaspaucimobilis)的又一菌株，ZP2被鉴定为施氏假单胞菌种(Pseudomonas stutzeri)，这是首次报道施氏假单胞菌种菌株具有菲降解活性。
2、ZP1和ZP2两株菲降解菌都有很好的温度、pH及菲含量适应性。少量蛋白胨、酵母膏及葡萄糖的加入可不同程度的加速两株菌对菲的降解；氯化铵、硝酸铵和硫酸铵为氮源时对这两株菌的菲降解速率几乎没有影响；菲浓度范围从250 ppm到1000 ppm之间，ZP1和ZP2菌株都可良好生长并快速降解菲；表面活性剂Brij 30和Trition 100在低浓度时对ZP2菌降解菲的速率有促进作用，但对ZP1菌株的菲降解作用有明显的抑制作用，Tween 80在实验的三个浓度梯度中对ZP1和ZP2均表现出一定的加速菲降解速度效应。
3、酶活实验证明，ZP1和ZP2菌株都有较高的1，2．邻苯二酚双加氧酶2，3-邻苯二酚双加氧酶活性，特别是2,3-邻苯二酚双加氧酶活性。在两个菌株的菲代谢过程中，都发现了邻苯二甲酸代谢途径中的关键代谢产物1-羟基-2-萘甲酸和2-羟基-1-萘甲酸及邻苯二甲酸，所以推测两个菌株都是经过邻苯二甲酸途径利用菲为碳源而实现对它的降解。
4、根据己知序列设计引物在菌株ZPl中扩增得到芳香环经化双加氧酶铁硫蛋白α大亚基编码基因，命名为pha-ZP1，其在Genbank登陆号为EU082776，该基因全长1287bp，编码383个氨基酸。利用Cn3D4．1软件和Swiss Model Workspace网络工具(http：／／swissmodel．expasy．org)推导蛋白质三维结构，结果显示其具有Rieske型[2Fe-2S]中心和单核铁原子结合域这两个在酶的催化功能上具有重要作用的保守结构。并且其氨基酸序列与来自Sphingomonas yanoikuyae B1和Sphingomonasr sp．P2的铁硫蛋白α亚基同源性高达97％。
5、根据已知序列设计引物分别在菌株ZP1和ZP2中扩增得到了各自的邻苯二酚双加氧酶基因，分别命名为phn—ZP1和phn-ZP2，它们在Genbank中的注册登陆号分别为：EU082777和EU082777。利用Cn3D4．1软件和Swiss Model Workspace网络工具(http：／／swissmodel．expasy．org)推导了基因所编码蛋白质的三维结构。通过与已知基因序列和编码氨基酸序列的比对，对两段基因进行了遗传亲缘分析。ZP1菌株C230基因phn—ZP1全长797bp，编码233个氨基酸，其二级结构主要为α螺旋和B链结构，phn—ZP2基因全长1047bp，编码331个氨基酸，其二级结构同样主要为α螺旋和β链结构。
6、通过基因重组将菌株ZP2的邻苯二酚2,3-双加氧酶编码基因phn—ZP2在Rosetta进行了成功表达，初步检测结果显示，重组菌株的酶表达活性要明显高于出发菌株。
7、卵磷脂脂质体作为模拟细胞膜被用来研究PAHs的跨膜特性和膜毒性。结果显示，此类亲脂性有机物在跨膜的过程中符合Nernst分配定律。从而可以计算出菲在卵磷脂脂质体和水之间的分配系数。研究了离子强度、温度、酸度等条件对分配作用的影响。此外，首次研究了菲在卵磷脂微囊与模拟细胞液之间的反分配作用。文中利用大肠杆菌及菲降解菌ZP1进一步研究了菲在不同细菌上的跨膜作用。实验表明相对于大肠杆菌，菲更容易进入降解菌的细胞膜内，而展开进一步的降解；在膜上的累积从而导致膜通透性的降低，是芳香环类有机物表现毒性的可能原因；亲脂性有机物菲通过分配作用富集到磷脂层，然后通过反分配作用从磷脂双分子层进入到细胞液，从而最终影响细胞的活性和功能。由以上实验得出跨膜屏障．结构效应(TBSE)理论，从而很好的解释了有机物跨膜过程的行为和膜毒性。
In this study, two phenanthrene-degrading bacterial and another bacterial with can improve the phenanthrene degradation speed when inoculated with phenanthrene degrading strains were isolated using traditional incubation method. Their identification was taken by studying its biochemical and genetic character. The factors influencing growth of phenanthrene-degrading bacteria and degradation of phenanthrene, the phenanthrene degradation pathway, the cloning and expression of phenanthrene degrading gene of two strains, the membrane toxicity mechanism of phenanthrene were also tested in this paper. The results was expected to supply useful reference for building up alert index systems in soil polluted by phenanthrene, for environmental quality evaluation and for bioremediation of PAHs pollution.
Here are presented the main results of this study:
1. Two phenanthrene-degrading bacteria strains ZP1 and ZP2 and another strain ZP5 which can improve ZP1's phenanthrene degradation speed were isolated from soil in oil refinery field in Shanghai. They were identified as belong to genus Sphingomonas, Pseudomonas and Tistrella, respectively based on Gram staining, morphology, oxydase reaction, biochemical tests, FAME analysis, G+C content and 16S rDNA gene sequence analysis. Strain ZP1 was identified as Sphingomonas paucimobilis, while strain ZP2 was identified as Pseudomonas stutzeri which is the first representative of Pseudomonas stutzeri sp., able to degrade phenanthrene very fast at high experimental concentration.
2. Both ZP1 and ZP2 has wide temperature, pH range for growth and degradation, and could tolerate high concentration of phenanthrene. The optimal growth conditions of strain ZP1 and ZP2 were determined to be at pH 7.0, 30℃and pH 8.0, 37℃, respectively. Addition of yeast extraction, peptone or glucose could promote the growth and phenanthrene degradation ability of both ZP1 and ZP2 to diferent degree. The phenanthrene degradation speed was nearly the same when (NH_4)_2SO_4, NH_4Cl, NH_4NO_3 were tested as different nitrogen resorce. Strain ZP1 can remove more than 90% of phenanthrene at any concentrations ranged from 250 to 1000ppm in 8 days while ZP2 can nearly consume them all in 6 days. Both Brij 30 and Trition 100 can inhibit the phenanthrene degradation speed of ZP2 at high concentration, but has no obvious effect on ZP1. Tween 80 can promote the degradation of phenanthrene by both ZP1 and ZP2.
3. Both ZP1 and ZP2 show high salicylate hydroxylase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activity especially catechol 2,3-dioxygenase. Two key products 1-Hydroxy-2-naphthoic acid and 2-Hydroxy-1-naphthoic acid were found in the phenanthrene degradation process, which could suggest the isolates degrade phenanthrene via phthalic acid pathway.
4. The gene pha-ZP1 encoding a subunit of aromatic hydrocarbon dioxygenase was cloned from strain ZP1, it's accession number in Genbank is EU082776. The determination and sequence analysis of the gene indicated that the DNA fragment was 1287 bp in length, encoding 383 amino acids. Two conserved regions: the [2Fe-2S] Rieske center and the mononuclear iron binding domain were found at the expected positions by deducing the protein using Cn3D4.1 software and Swiss Model Workspace web instrument. The amino acid sequence of the protein showed the highest similarity with that of Sphingomonas yanoikuyae B1 and Sphingomonas sp. P2.
5. The gene phn-ZP1 and phn-ZP2 encoding catechol 2,3-dioxygense was cloned from strain ZP1 and ZP2 respectively, their accession number in Genbank were EU082777 and EU082778. The determination and sequence analysis of the gene indicated that phn-ZP1 fragment was 797 bp in length, encoding 233 amino acids, phn-ZP2 fragment was 1047 bp in length, encoding 331 amino acids. The structure of these two enzymes deduced by using Cn3D4.1 software and Swiss Model Workspace web instrument suggested they were mostly composed by a helix and (3 strand.
6. The gene phn-ZP2 of C230 of strain ZP2 was recombined and expressed in Rosetta successfully. The activity of recombined enzyme protein expressed by recombinant obtained was much more than that by the original ZP2 strain in primary detection.
7. Lecithin liposome as the simulation membrane was used to investigate the interactions with PAHs such as phenanthrene. Results reveal that apolar compounds obeyed the Nerst partition law. The partition coefficient of phenanthrene was calculated and its' binding bonds in liposomes were clarified. Effects of electrolyte, temperature, acidity of solution were analyzed as well as effect of the molecular structure was discussed in detail. Besides, the inverse partition of apolar compounds from liposome to an analogue cytosol was first proposed. Transmembrane distribution difference of phenanthrene between Eschericha coli and phenanthrene-degrading strain Sphingomonas sp. ZP1 was investigated. The apolar compounds penetrated in membrane phospholipid bilayer by distribution effect and into cytosol by anti-distribution way. Results show that compare to E. coli, phenanthrene was easier to enter into cytoplasm of phenanthrene-degrading bacteria. The accumulation of phenanthrene in cell membrane cause the barrier effect, so it maybe the reason of toxicity of PAHs pollutants. The transmembrane barrier-structure effect (TBSE) was advanced and it will provide a very helpful experimental strategy for toxicity assessment of a lipophilic compound.
本研究所获主要结论如下：
1、从石油污染土壤中筛选分离到两株菲降解菌株，分别命名为ZP1和ZP2菌株，同时分离到一株可加速菌株ZPl对菲降解速率的菌株，命名为ZP5菌株。通过革兰氏染色、细菌形态学观察、生理生化测试、抗生素抗性实验、碳源利用实验、全细胞脂肪酸分析、G+C mol％含量分析以及16S rDNA序列同源性分析，证明ZP1、ZP2和．ZP5菌株这三株菲降解细菌应分别属于鞘氨醇单胞菌属、假单胞菌属及tistrella属。其中菌株ZP1被鉴定为少动鞘氨醇单胞菌种(Sphingomonaspaucimobilis)的又一菌株，ZP2被鉴定为施氏假单胞菌种(Pseudomonas stutzeri)，这是首次报道施氏假单胞菌种菌株具有菲降解活性。
2、ZP1和ZP2两株菲降解菌都有很好的温度、pH及菲含量适应性。少量蛋白胨、酵母膏及葡萄糖的加入可不同程度的加速两株菌对菲的降解；氯化铵、硝酸铵和硫酸铵为氮源时对这两株菌的菲降解速率几乎没有影响；菲浓度范围从250 ppm到1000 ppm之间，ZP1和ZP2菌株都可良好生长并快速降解菲；表面活性剂Brij 30和Trition 100在低浓度时对ZP2菌降解菲的速率有促进作用，但对ZP1菌株的菲降解作用有明显的抑制作用，Tween 80在实验的三个浓度梯度中对ZP1和ZP2均表现出一定的加速菲降解速度效应。
3、酶活实验证明，ZP1和ZP2菌株都有较高的1，2．邻苯二酚双加氧酶2，3-邻苯二酚双加氧酶活性，特别是2,3-邻苯二酚双加氧酶活性。在两个菌株的菲代谢过程中，都发现了邻苯二甲酸代谢途径中的关键代谢产物1-羟基-2-萘甲酸和2-羟基-1-萘甲酸及邻苯二甲酸，所以推测两个菌株都是经过邻苯二甲酸途径利用菲为碳源而实现对它的降解。
4、根据己知序列设计引物在菌株ZPl中扩增得到芳香环经化双加氧酶铁硫蛋白α大亚基编码基因，命名为pha-ZP1，其在Genbank登陆号为EU082776，该基因全长1287bp，编码383个氨基酸。利用Cn3D4．1软件和Swiss Model Workspace网络工具(http：／／swissmodel．expasy．org)推导蛋白质三维结构，结果显示其具有Rieske型[2Fe-2S]中心和单核铁原子结合域这两个在酶的催化功能上具有重要作用的保守结构。并且其氨基酸序列与来自Sphingomonas yanoikuyae B1和Sphingomonasr sp．P2的铁硫蛋白α亚基同源性高达97％。
5、根据已知序列设计引物分别在菌株ZP1和ZP2中扩增得到了各自的邻苯二酚双加氧酶基因，分别命名为phn—ZP1和phn-ZP2，它们在Genbank中的注册登陆号分别为：EU082777和EU082777。利用Cn3D4．1软件和Swiss Model Workspace网络工具(http：／／swissmodel．expasy．org)推导了基因所编码蛋白质的三维结构。通过与已知基因序列和编码氨基酸序列的比对，对两段基因进行了遗传亲缘分析。ZP1菌株C230基因phn—ZP1全长797bp，编码233个氨基酸，其二级结构主要为α螺旋和B链结构，phn—ZP2基因全长1047bp，编码331个氨基酸，其二级结构同样主要为α螺旋和β链结构。
6、通过基因重组将菌株ZP2的邻苯二酚2,3-双加氧酶编码基因phn—ZP2在Rosetta进行了成功表达，初步检测结果显示，重组菌株的酶表达活性要明显高于出发菌株。
7、卵磷脂脂质体作为模拟细胞膜被用来研究PAHs的跨膜特性和膜毒性。结果显示，此类亲脂性有机物在跨膜的过程中符合Nernst分配定律。从而可以计算出菲在卵磷脂脂质体和水之间的分配系数。研究了离子强度、温度、酸度等条件对分配作用的影响。此外，首次研究了菲在卵磷脂微囊与模拟细胞液之间的反分配作用。文中利用大肠杆菌及菲降解菌ZP1进一步研究了菲在不同细菌上的跨膜作用。实验表明相对于大肠杆菌，菲更容易进入降解菌的细胞膜内，而展开进一步的降解；在膜上的累积从而导致膜通透性的降低，是芳香环类有机物表现毒性的可能原因；亲脂性有机物菲通过分配作用富集到磷脂层，然后通过反分配作用从磷脂双分子层进入到细胞液，从而最终影响细胞的活性和功能。由以上实验得出跨膜屏障．结构效应(TBSE)理论，从而很好的解释了有机物跨膜过程的行为和膜毒性。
In this study, two phenanthrene-degrading bacterial and another bacterial with can improve the phenanthrene degradation speed when inoculated with phenanthrene degrading strains were isolated using traditional incubation method. Their identification was taken by studying its biochemical and genetic character. The factors influencing growth of phenanthrene-degrading bacteria and degradation of phenanthrene, the phenanthrene degradation pathway, the cloning and expression of phenanthrene degrading gene of two strains, the membrane toxicity mechanism of phenanthrene were also tested in this paper. The results was expected to supply useful reference for building up alert index systems in soil polluted by phenanthrene, for environmental quality evaluation and for bioremediation of PAHs pollution.
Here are presented the main results of this study:
1. Two phenanthrene-degrading bacteria strains ZP1 and ZP2 and another strain ZP5 which can improve ZP1's phenanthrene degradation speed were isolated from soil in oil refinery field in Shanghai. They were identified as belong to genus Sphingomonas, Pseudomonas and Tistrella, respectively based on Gram staining, morphology, oxydase reaction, biochemical tests, FAME analysis, G+C content and 16S rDNA gene sequence analysis. Strain ZP1 was identified as Sphingomonas paucimobilis, while strain ZP2 was identified as Pseudomonas stutzeri which is the first representative of Pseudomonas stutzeri sp., able to degrade phenanthrene very fast at high experimental concentration.
2. Both ZP1 and ZP2 has wide temperature, pH range for growth and degradation, and could tolerate high concentration of phenanthrene. The optimal growth conditions of strain ZP1 and ZP2 were determined to be at pH 7.0, 30℃and pH 8.0, 37℃, respectively. Addition of yeast extraction, peptone or glucose could promote the growth and phenanthrene degradation ability of both ZP1 and ZP2 to diferent degree. The phenanthrene degradation speed was nearly the same when (NH_4)_2SO_4, NH_4Cl, NH_4NO_3 were tested as different nitrogen resorce. Strain ZP1 can remove more than 90% of phenanthrene at any concentrations ranged from 250 to 1000ppm in 8 days while ZP2 can nearly consume them all in 6 days. Both Brij 30 and Trition 100 can inhibit the phenanthrene degradation speed of ZP2 at high concentration, but has no obvious effect on ZP1. Tween 80 can promote the degradation of phenanthrene by both ZP1 and ZP2.
3. Both ZP1 and ZP2 show high salicylate hydroxylase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activity especially catechol 2,3-dioxygenase. Two key products 1-Hydroxy-2-naphthoic acid and 2-Hydroxy-1-naphthoic acid were found in the phenanthrene degradation process, which could suggest the isolates degrade phenanthrene via phthalic acid pathway.
4. The gene pha-ZP1 encoding a subunit of aromatic hydrocarbon dioxygenase was cloned from strain ZP1, it's accession number in Genbank is EU082776. The determination and sequence analysis of the gene indicated that the DNA fragment was 1287 bp in length, encoding 383 amino acids. Two conserved regions: the [2Fe-2S] Rieske center and the mononuclear iron binding domain were found at the expected positions by deducing the protein using Cn3D4.1 software and Swiss Model Workspace web instrument. The amino acid sequence of the protein showed the highest similarity with that of Sphingomonas yanoikuyae B1 and Sphingomonas sp. P2.
5. The gene phn-ZP1 and phn-ZP2 encoding catechol 2,3-dioxygense was cloned from strain ZP1 and ZP2 respectively, their accession number in Genbank were EU082777 and EU082778. The determination and sequence analysis of the gene indicated that phn-ZP1 fragment was 797 bp in length, encoding 233 amino acids, phn-ZP2 fragment was 1047 bp in length, encoding 331 amino acids. The structure of these two enzymes deduced by using Cn3D4.1 software and Swiss Model Workspace web instrument suggested they were mostly composed by a helix and (3 strand.
6. The gene phn-ZP2 of C230 of strain ZP2 was recombined and expressed in Rosetta successfully. The activity of recombined enzyme protein expressed by recombinant obtained was much more than that by the original ZP2 strain in primary detection.
7. Lecithin liposome as the simulation membrane was used to investigate the interactions with PAHs such as phenanthrene. Results reveal that apolar compounds obeyed the Nerst partition law. The partition coefficient of phenanthrene was calculated and its' binding bonds in liposomes were clarified. Effects of electrolyte, temperature, acidity of solution were analyzed as well as effect of the molecular structure was discussed in detail. Besides, the inverse partition of apolar compounds from liposome to an analogue cytosol was first proposed. Transmembrane distribution difference of phenanthrene between Eschericha coli and phenanthrene-degrading strain Sphingomonas sp. ZP1 was investigated. The apolar compounds penetrated in membrane phospholipid bilayer by distribution effect and into cytosol by anti-distribution way. Results show that compare to E. coli, phenanthrene was easier to enter into cytoplasm of phenanthrene-degrading bacteria. The accumulation of phenanthrene in cell membrane cause the barrier effect, so it maybe the reason of toxicity of PAHs pollutants. The transmembrane barrier-structure effect (TBSE) was advanced and it will provide a very helpful experimental strategy for toxicity assessment of a lipophilic compound.
引文
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