Pseudomonas putida DLL-E4对硝基苯酚代谢基因簇的克隆、功能分析和表达调控的研究
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摘要
对硝基苯酚(p-Nitrophenol, PNP)是重要的环境污染物,进入环境的PNP会长期残留在深层土壤和地下水中,对动植物和人类的健康造成严重的危害。大量能够降解PNP的微生物已被分离,同时在微生物许多种属中PNP代谢途径已经得到相应的研究,但与PNP降解基因和调控机制相关的报道甚少,特别是在革兰氏阴性菌中。
本研究以本实验室分离的PNP降解菌株Pseudomonas putida DLL-E4为材料,通过SEFA PCR扩增偏三苯酚1,2-双加氧酶基因(pnpC)的侧翼序列,获得12kb的片段。序列分析发现该片段中存在10个参与PNP代谢的ORF,并预测了每个ORF的功能。这10个ORF可以分为三个部分,第一部分是催化PNP脱硝基形成对苯二酚的相关基因,包括pnpA和pnpB,这一部分是整个PNP降解基因簇的关键部分；第二部分是与对苯二酚开环及催化开环产物进入TCA循环的相关基因,包括pnpC1、pnpC2、pnpD、 pnpE、pnpC、pnpX1和pnpX2;第三部分是调控PNP降解基因簇表达的基因,只有属于LysR家族的调控基因pnpR。因此我们认为克隆到的片段包含了P. putida DLL-E4代谢PNP的所有必需的功能基因,这些基因在基因组中成簇排列,因此把克隆得到的12kb的片段命名为对硝基苯酚基因簇(pnp基因簇)。
与革兰氏阳性菌中双组分脱硝基单加氧酶不同,P. putida DLL-E4中的PNP4-单加氧酶(PnpA)是单组分酶,由基因pnpA编码。我们利用表达载体pET-29a将pnpA在大肠杆菌BL21中进行了表达。通过Ni-NTA亲和层析以及分子筛纯化了PnpA,在此基础上对PnpA6勺酶学特性进行了研究。PnpA催化PNP生成苯醌和亚硝酸根。PnpA的活性依赖于FAD和NADH。 PnpA催化PNP的最佳反应条件为pH8.0,温度20℃。PnpA对PNP的活力是2.57±0.04U·-mg-1, Km值为302μM。过渡金属离子(Cu2+、Ni2+、 Zn2+、Fe2+、Fe3+和Cr3+)对PnpA活性有明显抑制作用,金属螯合剂(EDTA, EGTA,1,10-phenanthrolin)对PnpA活性没有影响,结合文献报道,我们认为PnpA是非金属酶。构建了PnpA的系统发育树,PnpA在进化上和其它黄素单加氧酶相距较远,可能有不同的起源。通过同源重组,敲除了P. putida DLL-E4中pnpA,敲除菌株丧失了降解PNP能力,说明DLL-E4中只存在一个PNP单加氧酶即PnpA。
P. putida DLL-E4中对苯二酚双加氧酶复合体是由a亚基(PnpC1)和β亚基(PnpC2)组成的。分别表达PnpCl和PnpC2,纯化后在体外重聚试验中未能获得具有对苯二酚开环活性的酶复合体。通过多顺反子表达载体成功获得了有活性的对苯二酚双加氧酶复合体(PnpC1C2BL21)。PnpC1C2BL21能够催化对苯二酚生成4-羟基粘康酸半醛(4-hydroxymuconic semialdehyde),但对其它酚类化合物没有催化能力。与来源于原始菌株DLL-E4的对苯二酚双加氧酶复合体(PnPC1C2DLL-E4)相比,PnpC1C2BL21存在活性不稳定,瞬间催化的问题。在排除大量因素后,结合PnpC1C2DLL-E4的催化对苯二酚不同的动力学行为和活性特性,我们推测造成该现象的原因是不同宿主表达的对苯二酚双加氧酶复合体在结构上存在差异。P. putida DLL-E4中的PnpC1C2DLL.E4催化对苯二酚的活性存在产物(4-羟基粘康酸半醛)抑制,该抑制能够在高浓度底物条件下被解除,此时催化反应的Vmax不变。因此认为该抑制为竞争性抑制。
4-硝基儿茶酚只有在PNP存在的条件下,形成混合底物才能够被DLL-E4代谢。通过酶活分析和突变子降解试验,我们认为P. putida DLL-E4中pnp基因簇不仅参与PNP的代谢,同时也参与菌株降解和利用4-硝基儿茶酚的过程。P. putida DLL-E4中的pnp基因簇混合了菌株代谢不同物质(PNP和4-硝基儿茶酚)的基因,是一个杂合的代谢基因簇。
pnp基因簇中的调控基因pnpR敲除突变子DLL-△pnpR丧失了对苯二酚降解能力,但是仍可以降解PNP,并积累对苯二酚。通过RT-PCR证明Pnp(?)(?)是在转录水平上调控pnp基因簇中的对苯二酚代谢相关基因(pnpC1C2DE),对pnp基因簇中的脱硝基相关基因(onpBA)无调控作用,PnpR调控方式为正调控。对PnpR氨基酸进行序列分析,发现PnpR在N端存在DNA结合保守域(HTH),在C端存在诱导物作用保守域。结合PnpR的序列分析信息和在P. putida DLL-E4中的生物学功能,把PnpR归类为LysR家族调控蛋白。
pnpA替换为氨基糖苷磷酸转移酶Ⅱ基因(aph2II,卡那霉素抗性基因)的突变菌株DLL-A-aph,必须在PNP诱导条件下才能够在含有10mg/L的卡那霉素的无机盐培养基中生长,说明了pnpA的表达需要PNP的诱导。经RT-PCI(?)验证,该诱导是在转录水平上控制pnpA的表达。RT-PCR结果揭示了DLL-E4不能够单独降解4-硝基儿茶酚的原因是其不能够诱导pnpA的转录。
p-Nitrophenol (PNP) is widely used in the manufacture of pesticides,dyes, explosives, and drug intermediates. PNP can also accumulate in soil as a result of the hydrolysis of several organophosphorous insecticides, such as parathion or methyl parathion. Due to its potential toxicity and persistence in the environment, PNP was listed on the "Priority Pollutants List"by US EPA.
Pseudomonas putida DLL-E4has the ability to grow on PNP as the sole source of carbon, nitrogen, and energy, and hydroquinone was detected as the key intermediate in PNP degradation. In this study, we cloned a pnp gene cluster involved in the catabolism of PNP from DLL-E4. The functions of five genes in the cluster were verified by expression in E. coli and gene deletion in DLL-E4.
A12,540bp DNA fragment around the conserved region was obtained after several rounds of SEFA PCR. Ten open reading frames (ORFs) were found in the fragment and they were annotated on the basis of BLAST analysis. The10ORFs can be divided into three groups by functional analysis. The pnpC1C2DE genes were proposed to be involved in hydroquinone catabolism by DLL-E4. pnpA and pnpB were determined to encode PNP4-monooxygenase and p-benzoquinone reductase, they could oxidatively remove the nitro group from PNP. pnpR was located at the beginning of the hydroquinone degradation gene cluster of DLL-E4and proposed to be encode a LysR regulatory protein. The12kb fragment named as pnp gene cluster, contained all essential genes involved in PNP degradation.
PnpA was overexpressed in E. coli and purified to apparent homogeneity by Ni-NTA affinity chromatography and size exclusion Sephadex G-200column. PnpA is a flavin adenine dinucleotide-dependent single-component PNP4-monooxygenase that converts PNP to para-benzoquinone. The optimal reaction temperature of PnpA is20℃The optimal reaction pH of PnpA is8.0. Purified PnpA had a specific activity of2.57±0.04U mg-1for PNP. The Km value of PnpA for PNP was302μM. PnpA activity is NADH and FAD dependent. NADH can be replaced by NADPH with the lower efficiency. PnpA transformed4-NC with a lower specific activity (1.17±0.032U·mg-1) than that of PNP transformation. No activity was observed by PnpA on o-nitrophenol,m-nitrophenoland2,4-Dinitrophenol. Transition metal (Cu2+、Ni2+、Zn2+、Fe2+、Fe3+and Cr3+)inhibit the activity of PnpA and metal chelators(EDTA, EGTA,1,10-phenanthrolin)has no effect on PnpA activity, these results indicate that PnpA is non-metallic enzyme. To elucidate the phylogenetic relationships among the functionally identified nitroarene monooxygenases, PnpA and other flavoprotein monooxygenases were used to construct the distance neighbor-joining tree. The results indicated that PnpA belongs to a different group of flavin monooxygenases.
PnpCl and PnpC2from the pnp gene cluster in DLL-E4are the oc-(PnpCl) and P-subunits (PnpC2) of hydroquinone dioxygenase. Purified PnpC1and PnpC2failed to reconstitute into active complex in vitro. Most of separately expressed PnpC2existed as inclusion bodies, and a mixture of separately expressed and purified PnpC1and PnpC2showed no activity on hydroquinone. A poly-cistronic plasmid pET-pnpC1C2containing pnpCl and pnpC2was constructed to express the dual component hydroquinone dioxygenase complex (PnpC1C2BL21)-Size-exclusion spectrometry revealed that hydroquinone dioxygenase is a α2and β2heterotetramer of112.4kDa. The reconstitution of a hydroquinone dioxygenase complex in vitro failed. Most of separately expressed PnpC2existed as inclusion bodies, and a mixture of separately expressed and purified PnpC1and PnpC2showed no activity on hydroquinone. A poly-cistronic plasmid pET-pnpC1C2containing pnpCl and pnpC2was constructed to express the dual component hydroquinone dioxygenase complex (PnpC1C2BL21).Hydroquinone is transformed to4-hydroxymuconic semialdehyde by PnpC1C2BL21under aerobic conditions. The expressed PnpC1C2BL21seemed to be unstable when acting on hydroquinone since complete conversion of hydroquinone was only possible by repeated addition of fresh PnpC1C2BL21.This phenomenon had not been observed from PnpC1C2DLL-E4of P. putida DLL-E4. Since a large number of factors have been studied, we believe that different behavior of the two complexes is caused by differences in protein structure. The activity of PnpC1C2DLL-E4is greatly inhibited by the product (4-hydroxymuconic semialdehyde). The inhibition can be overcome by increasing the substrate (hydroquinone) concentration, and the maximum velocity (Vmax) of the reaction is unchanged. These results suggest that the product inhibit the activity of PnpC1C2DLL-E4by competitive inhibition.
In an attempt to identify the function of pnpR, a pnpR deletion mutant, strain DLL-ApnpR was constructed by homologous recombination in DLL-E4. Activity assay showed that DLL-△pnpR could still degradate PNP with nitrite release, but lost the ability to degrade hydroquinone. The accumulated product was determined by HPLC analysis to be hydroquinone. RT-PCR analysis showed that the pnpCl can be transcripted under the presence of PnpR. These results indicated that PnpR positively controls the transcription of hydroquinone catabolic genes but not pnpA in pnp gene cluster. The analysis of amino acid sequence shows that PnpR contains a conserved helix-turn-helix DNA binding motif, as well as a LysR substrate-binding domain.
Since PnpA can transform4-nitrocatechol to1,2,4-trihydroxybenzene, and PnpC converted1,2,4-trihydroxybenzene to maleylacetate, the pnp gene cluster from DLL-E4must contain the essential genes to catalyze the degradation of4-nitrocatechol; however, DLL-E4did not degrade4-nitrocatechol when it was used as the single substrate, but degraded up to92%of4-nitrocatechol when mixed with PNP. The RT-PCR analysis showed that4-nitrocatechol cannot act as an inductor for inducible transcription of the pnp gene cluster in DLL-E4.
本研究以本实验室分离的PNP降解菌株Pseudomonas putida DLL-E4为材料,通过SEFA PCR扩增偏三苯酚1,2-双加氧酶基因(pnpC)的侧翼序列,获得12kb的片段。序列分析发现该片段中存在10个参与PNP代谢的ORF,并预测了每个ORF的功能。这10个ORF可以分为三个部分,第一部分是催化PNP脱硝基形成对苯二酚的相关基因,包括pnpA和pnpB,这一部分是整个PNP降解基因簇的关键部分；第二部分是与对苯二酚开环及催化开环产物进入TCA循环的相关基因,包括pnpC1、pnpC2、pnpD、 pnpE、pnpC、pnpX1和pnpX2;第三部分是调控PNP降解基因簇表达的基因,只有属于LysR家族的调控基因pnpR。因此我们认为克隆到的片段包含了P. putida DLL-E4代谢PNP的所有必需的功能基因,这些基因在基因组中成簇排列,因此把克隆得到的12kb的片段命名为对硝基苯酚基因簇(pnp基因簇)。
与革兰氏阳性菌中双组分脱硝基单加氧酶不同,P. putida DLL-E4中的PNP4-单加氧酶(PnpA)是单组分酶,由基因pnpA编码。我们利用表达载体pET-29a将pnpA在大肠杆菌BL21中进行了表达。通过Ni-NTA亲和层析以及分子筛纯化了PnpA,在此基础上对PnpA6勺酶学特性进行了研究。PnpA催化PNP生成苯醌和亚硝酸根。PnpA的活性依赖于FAD和NADH。 PnpA催化PNP的最佳反应条件为pH8.0,温度20℃。PnpA对PNP的活力是2.57±0.04U·-mg-1, Km值为302μM。过渡金属离子(Cu2+、Ni2+、 Zn2+、Fe2+、Fe3+和Cr3+)对PnpA活性有明显抑制作用,金属螯合剂(EDTA, EGTA,1,10-phenanthrolin)对PnpA活性没有影响,结合文献报道,我们认为PnpA是非金属酶。构建了PnpA的系统发育树,PnpA在进化上和其它黄素单加氧酶相距较远,可能有不同的起源。通过同源重组,敲除了P. putida DLL-E4中pnpA,敲除菌株丧失了降解PNP能力,说明DLL-E4中只存在一个PNP单加氧酶即PnpA。
P. putida DLL-E4中对苯二酚双加氧酶复合体是由a亚基(PnpC1)和β亚基(PnpC2)组成的。分别表达PnpCl和PnpC2,纯化后在体外重聚试验中未能获得具有对苯二酚开环活性的酶复合体。通过多顺反子表达载体成功获得了有活性的对苯二酚双加氧酶复合体(PnpC1C2BL21)。PnpC1C2BL21能够催化对苯二酚生成4-羟基粘康酸半醛(4-hydroxymuconic semialdehyde),但对其它酚类化合物没有催化能力。与来源于原始菌株DLL-E4的对苯二酚双加氧酶复合体(PnPC1C2DLL-E4)相比,PnpC1C2BL21存在活性不稳定,瞬间催化的问题。在排除大量因素后,结合PnpC1C2DLL-E4的催化对苯二酚不同的动力学行为和活性特性,我们推测造成该现象的原因是不同宿主表达的对苯二酚双加氧酶复合体在结构上存在差异。P. putida DLL-E4中的PnpC1C2DLL.E4催化对苯二酚的活性存在产物(4-羟基粘康酸半醛)抑制,该抑制能够在高浓度底物条件下被解除,此时催化反应的Vmax不变。因此认为该抑制为竞争性抑制。
4-硝基儿茶酚只有在PNP存在的条件下,形成混合底物才能够被DLL-E4代谢。通过酶活分析和突变子降解试验,我们认为P. putida DLL-E4中pnp基因簇不仅参与PNP的代谢,同时也参与菌株降解和利用4-硝基儿茶酚的过程。P. putida DLL-E4中的pnp基因簇混合了菌株代谢不同物质(PNP和4-硝基儿茶酚)的基因,是一个杂合的代谢基因簇。
pnp基因簇中的调控基因pnpR敲除突变子DLL-△pnpR丧失了对苯二酚降解能力,但是仍可以降解PNP,并积累对苯二酚。通过RT-PCR证明Pnp(?)(?)是在转录水平上调控pnp基因簇中的对苯二酚代谢相关基因(pnpC1C2DE),对pnp基因簇中的脱硝基相关基因(onpBA)无调控作用,PnpR调控方式为正调控。对PnpR氨基酸进行序列分析,发现PnpR在N端存在DNA结合保守域(HTH),在C端存在诱导物作用保守域。结合PnpR的序列分析信息和在P. putida DLL-E4中的生物学功能,把PnpR归类为LysR家族调控蛋白。
pnpA替换为氨基糖苷磷酸转移酶Ⅱ基因(aph2II,卡那霉素抗性基因)的突变菌株DLL-A-aph,必须在PNP诱导条件下才能够在含有10mg/L的卡那霉素的无机盐培养基中生长,说明了pnpA的表达需要PNP的诱导。经RT-PCI(?)验证,该诱导是在转录水平上控制pnpA的表达。RT-PCR结果揭示了DLL-E4不能够单独降解4-硝基儿茶酚的原因是其不能够诱导pnpA的转录。
p-Nitrophenol (PNP) is widely used in the manufacture of pesticides,dyes, explosives, and drug intermediates. PNP can also accumulate in soil as a result of the hydrolysis of several organophosphorous insecticides, such as parathion or methyl parathion. Due to its potential toxicity and persistence in the environment, PNP was listed on the "Priority Pollutants List"by US EPA.
Pseudomonas putida DLL-E4has the ability to grow on PNP as the sole source of carbon, nitrogen, and energy, and hydroquinone was detected as the key intermediate in PNP degradation. In this study, we cloned a pnp gene cluster involved in the catabolism of PNP from DLL-E4. The functions of five genes in the cluster were verified by expression in E. coli and gene deletion in DLL-E4.
A12,540bp DNA fragment around the conserved region was obtained after several rounds of SEFA PCR. Ten open reading frames (ORFs) were found in the fragment and they were annotated on the basis of BLAST analysis. The10ORFs can be divided into three groups by functional analysis. The pnpC1C2DE genes were proposed to be involved in hydroquinone catabolism by DLL-E4. pnpA and pnpB were determined to encode PNP4-monooxygenase and p-benzoquinone reductase, they could oxidatively remove the nitro group from PNP. pnpR was located at the beginning of the hydroquinone degradation gene cluster of DLL-E4and proposed to be encode a LysR regulatory protein. The12kb fragment named as pnp gene cluster, contained all essential genes involved in PNP degradation.
PnpA was overexpressed in E. coli and purified to apparent homogeneity by Ni-NTA affinity chromatography and size exclusion Sephadex G-200column. PnpA is a flavin adenine dinucleotide-dependent single-component PNP4-monooxygenase that converts PNP to para-benzoquinone. The optimal reaction temperature of PnpA is20℃The optimal reaction pH of PnpA is8.0. Purified PnpA had a specific activity of2.57±0.04U mg-1for PNP. The Km value of PnpA for PNP was302μM. PnpA activity is NADH and FAD dependent. NADH can be replaced by NADPH with the lower efficiency. PnpA transformed4-NC with a lower specific activity (1.17±0.032U·mg-1) than that of PNP transformation. No activity was observed by PnpA on o-nitrophenol,m-nitrophenoland2,4-Dinitrophenol. Transition metal (Cu2+、Ni2+、Zn2+、Fe2+、Fe3+and Cr3+)inhibit the activity of PnpA and metal chelators(EDTA, EGTA,1,10-phenanthrolin)has no effect on PnpA activity, these results indicate that PnpA is non-metallic enzyme. To elucidate the phylogenetic relationships among the functionally identified nitroarene monooxygenases, PnpA and other flavoprotein monooxygenases were used to construct the distance neighbor-joining tree. The results indicated that PnpA belongs to a different group of flavin monooxygenases.
PnpCl and PnpC2from the pnp gene cluster in DLL-E4are the oc-(PnpCl) and P-subunits (PnpC2) of hydroquinone dioxygenase. Purified PnpC1and PnpC2failed to reconstitute into active complex in vitro. Most of separately expressed PnpC2existed as inclusion bodies, and a mixture of separately expressed and purified PnpC1and PnpC2showed no activity on hydroquinone. A poly-cistronic plasmid pET-pnpC1C2containing pnpCl and pnpC2was constructed to express the dual component hydroquinone dioxygenase complex (PnpC1C2BL21)-Size-exclusion spectrometry revealed that hydroquinone dioxygenase is a α2and β2heterotetramer of112.4kDa. The reconstitution of a hydroquinone dioxygenase complex in vitro failed. Most of separately expressed PnpC2existed as inclusion bodies, and a mixture of separately expressed and purified PnpC1and PnpC2showed no activity on hydroquinone. A poly-cistronic plasmid pET-pnpC1C2containing pnpCl and pnpC2was constructed to express the dual component hydroquinone dioxygenase complex (PnpC1C2BL21).Hydroquinone is transformed to4-hydroxymuconic semialdehyde by PnpC1C2BL21under aerobic conditions. The expressed PnpC1C2BL21seemed to be unstable when acting on hydroquinone since complete conversion of hydroquinone was only possible by repeated addition of fresh PnpC1C2BL21.This phenomenon had not been observed from PnpC1C2DLL-E4of P. putida DLL-E4. Since a large number of factors have been studied, we believe that different behavior of the two complexes is caused by differences in protein structure. The activity of PnpC1C2DLL-E4is greatly inhibited by the product (4-hydroxymuconic semialdehyde). The inhibition can be overcome by increasing the substrate (hydroquinone) concentration, and the maximum velocity (Vmax) of the reaction is unchanged. These results suggest that the product inhibit the activity of PnpC1C2DLL-E4by competitive inhibition.
In an attempt to identify the function of pnpR, a pnpR deletion mutant, strain DLL-ApnpR was constructed by homologous recombination in DLL-E4. Activity assay showed that DLL-△pnpR could still degradate PNP with nitrite release, but lost the ability to degrade hydroquinone. The accumulated product was determined by HPLC analysis to be hydroquinone. RT-PCR analysis showed that the pnpCl can be transcripted under the presence of PnpR. These results indicated that PnpR positively controls the transcription of hydroquinone catabolic genes but not pnpA in pnp gene cluster. The analysis of amino acid sequence shows that PnpR contains a conserved helix-turn-helix DNA binding motif, as well as a LysR substrate-binding domain.
Since PnpA can transform4-nitrocatechol to1,2,4-trihydroxybenzene, and PnpC converted1,2,4-trihydroxybenzene to maleylacetate, the pnp gene cluster from DLL-E4must contain the essential genes to catalyze the degradation of4-nitrocatechol; however, DLL-E4did not degrade4-nitrocatechol when it was used as the single substrate, but degraded up to92%of4-nitrocatechol when mixed with PNP. The RT-PCR analysis showed that4-nitrocatechol cannot act as an inductor for inducible transcription of the pnp gene cluster in DLL-E4.
引文
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