盐胁迫条件下古菌超氧化物歧化酶增强细菌降解硝基酚

Archaeal superoxide dismutase enhances bacterial degradation of nitrophenols under salt stress

摘要:

【背景】Burkholderia sp. SJ98利用对硝基酚和2-氯-4-硝基酚为唯一碳源和能源进行生长，通过异源表达嗜盐古菌Haloferax sp. D1227中的超氧化物歧化酶SodA，使菌株SJ98在500 mmol/L NaCl条件下仍具有降解对硝基酚的能力。然而该重组细菌在普通和高盐条件下其降解基因的转录和降解酶比活力的高低，以及该菌在高盐条件下是否还能降解对硝基酚衍生物尚未知晓。【目的】研究Burkholderia sp. SJ98的耐盐上限，观察含有sodA的细菌SJ98在普通和高盐条件下降解对硝基酚和2-氯-4-硝基酚的能力，检测重组菌中pnpA基因的转录和硝基酚单加氧酶的活力。【方法】在添加葡萄糖、对硝基酚或2-氯-4-硝基酚的无机盐培养基(分别含400?800 mmol/L NaCl)或M9培养基(含0和500 mmol/L NaCl)中培养细菌SJ98及其重组菌。通过紫外分光光度计和高效液相色谱法检测菌株生长和底物降解。通过实时荧光定量PCR分别以两种硝基酚为诱导物，检测未添加和添加500 mmol/L NaCl时，硝基酚单加氧酶编码基因pnpA的转录量变化。利用紫外分光光度计分别以两种硝基酚为底物，检测在添加500 mmol/L NaCl时，重组菌和空载体菌的粗酶液中硝基酚单加氧酶对两种底物的活力变化。【结果】野生型菌株SJ98以葡萄糖为碳源生长的NaCl耐受浓度是600 mmol/L。未添加NaCl时，重组菌SJ98[pCM-pnpR-PpnpA-sodA-rfp]生长和降解对硝基酚的能力远优于野生菌。添加500 mmol/L NaCl时，重组菌SJ98[pBBR-sodA]仍保持了利用2-氯-4-硝基苯酚底物生长和降解该底物的能力，而空载体菌SJ98[pBBR1MCS-2]的生长和降解能力完全丧失；重组菌SJ98[pBBR-sodA]粗酶液中单加氧酶对于对硝基酚和2-氯-4-硝基酚的活力均约为野生菌的1/3。分别以两种硝基酚为诱导物时，无论是否添加NaCl，重组菌SJ98[pBBR-sodA]中硝基酚单加氧酶编码基因pnpA的转录量比野生型中高出约17?25倍；但添加500 mmol/L NaCl时，pnpA的转录均受到部分抑制。【结论】本研究为利用古菌超氧化物歧化酶对细菌进行改造以提高普通环境和高盐环境中细菌降解硝基芳烃污染物能力的应用提供了潜在的可行性。

Abstract:

[Background] Burkholderia sp. SJ98 utilizes para-nitrophenol or 2-chloro-4-nitrophenol as the sole carbon and energy source for its growth. The superoxide dismutase SodA from Haloferax sp. D1227 endows strain SJ98 with the ability to degrade para-nitrophenol with 500 mmol/L NaCl. However, it is unknown whether strain SJ98 containing sodA can degrade para-nitrophenol derivatives under conditions with high salt concentration. [Objective] We are aiming to study the salt tolerance of strain SJ98, and the degradation of para-nitrophenol and 2-chloro-4-nitrophenol by strain SJ98 containing sodA under normal and high salt concentrations. We also hope to detect the transcriptions of pnpA and the activities of the nitrophenols monooxygenase in the recombinants of strain SJ98. [Methods] Strain SJ98 and its recombinants were cultured in minimal medium (containing 400 to 800 mmol/L NaCl) or M9 medium (containing 0 and 500 mmol/L NaCl, respectively) supplemented with glucose, para-nitrophenol or 2-chloro-4-nitrophenol, respectively. The strains growth and substrates degradation were detected by UV spectrophotometer and high-performance liquid chromatography, respectively. The transcriptions of pnpA (nitrophenol monooxygenases encoding gene) induced by two nitrophenols were detected by quantitative real-time PCR. The activities of nitrophenols monooxygenase in the crude enzyme solutions were detected by UV spectrophotometer using two above substrates. [Results] The NaCl tolerance concentration of strain SJ98 was 600 mmol/L with glucose as the carbon source. The growth and para-nitrophenol degradation by strain SJ98[pCM-pnpR-PpnpA-sodA-rfp] were much better than those by the wild-type strain. With 500 mmol/L NaCl, strain SJ98[pBBR-sodA] still maintained the ability of degrading and growing on 2-chloro-4-nitrophenol, while strain SJ98[pBBR1MCS-2] completely lost these abilities; the activities of the nitrophenols monooxygenase against two above substrates in the crude enzyme solutions of strain SJ98[pBBR-sodA] were both about 1/3 of those in the wild strain. When two nitrophenols were used as inducers, the transcriptions of pnpA in strain SJ98[pBBR-sodA] were about 17–25 times higher than those in the wild-type strain, regardless of the presence of NaCl. However, when 500 mmol/L NaCl was added, the transcriptions of pnpA were partially inhibited. [Conclusion] This study provides a potential possibility for the application of superoxide dismutase from archaea to improve the ability of nitroaromatics degradation under normal and high salt concentrations by bacteria.