沼泽红假单胞菌重组LH2表达体系的构建及功能评价

Expression system construction and activity evaluation of reconstituted LH2 of Rhodopseudomonas palustris

摘要:

【背景】外周捕光色素蛋白复合体(peripheral light-harvesting complex, LH2)在光合作用中执行光能捕获和传递功能，对于不产氧光合细菌(anoxygenic phototrophic bacteria, APB)的光合生长至关重要。在APB中，LH2的αβ亚基通常是由多拷贝pucBA基因编码，尤其是红假单胞菌属(Rhodopseudomonas)菌株，可高达5−7拷贝。由于不同pucBA基因合成的异质性LH2彼此之间难以分离纯化，极大限制了LH2结构与功能的深入研究。【目的】构建以沼泽红假单胞菌为宿主菌的重组LH2表达体系，利用该重组表达体系在体内和体外评价LH2的光能传递活性。【方法】以5对pucBA基因全部缺失的沼泽红假单胞菌(Rhodopseudomonaspalustris) CGA009 (ΔpucBA)为宿主菌，采用基因重组方法，以pucBAd和mrfp(红色荧光蛋白基因)基因共表达产物为检测指标，对表达载体进行遗传改造和启动子筛选；采用光谱法测定重组菌的生物量和基因表达产物光谱特征，以及纯化LH2的光谱特征、光合色素分析和能量传递活性。【结果】T7启动子(P_T7)和T7 RNA聚合酶基因联合使用，可使pucBAd和mrfp在ΔpucBA宿主菌中共表达。用启动子P_badR、P_pckA和P_ars替换P_T7，启动子活性从高到低依次为P_pckA、P_ars、P_badR和P_T7，并与重组菌mrfp基因表达量、pucBAd表达产物(d-LH2)合成量和光合生长速率呈正相关关系，其中以P_pckA启动子构建的重组LH2表达体系ΔpucBA(pucBAd)表达活性最高。在此基础上，以CGA009菌株的pucBAa基因(编码a-LH2)替换ΔpucBA(pucBAd)菌株的pucBAd基因，获得ΔpucBA(pucBAa)重组菌。相较于ΔpucBA(pucBAa)重组菌，低光时ΔpucBA(pucBAd)重组菌生长速率显著升高，高光时有升高趋势，但未呈显著差异。纯化的d-LH2和a-LH2分别呈现典型的B800-only和B800-850特征光谱，d-LH2产生~863 nm荧光的量子效率高于a-LH2，但采用经典法测定的类胡萝卜素(carotenoids, Car)到细菌叶绿素(bacteriochlorophylls, BChl)的光能传递效率却低于a-LH2，2种结果不一致的原因是“经典法”未考虑不同光谱类型LH2对~863 nm光子吸收的差异。【结论】构建沼泽红假单胞菌的重组LH2表达系统，从基因纯LH2水平和重组菌光合生长水平评价了多拷贝pucBA基因菌株中2种不同光谱类型的LH2能量传递活性，证明异常光谱LH2 (d-LH2)能量传递活性高于典型光谱LH2 (a-LH2)，本研究为多拷贝pucBA菌株光适应过程中LH2形成和组装的分子调控机制的全面理解奠定了基础。

Abstract:

[Background] The peripheral light-harvesting complex (LH2) performs the function of light energy capture and transfer in photosynthesis and is essential for the phototrophic growth of anoxygenic phototrophic bacteria (APB). The α and β subunits of LH2 are usually encoded by multiple copies of pucBA genes, which reach 5–7 copies inRhodopseudomonas. As a result, the heterogeneous synthesis of LH2 by multiple genes makes LH2 difficult to be separated from each other, which greatly limits the full understanding about the contributions of different pucBAgenes to the structures and physiological roles of LH2. [Objective] To develop an expression system and evaluate the light energy transfer activity in vivo andin vitro for the reconstituted LH2 of R. palustris. [Methods] Using Rhodopseudomonas palustris CGA009 (ΔpucBA) with all five pucBA genes deleted as the host bacterium and the co-expression products of pucBAd and mrfp (red fluorescent protein gene) as the markers, we conducted genetic modification of the expression vector and promoter screening. The spectroscopic method was employed to determine the biomass of recombinant bacteria, the spectral characteristics of gene expression products, and the spectral characteristics, photosynthetic pigments and light energy transfer activity of purified LH2. [Results] The T7 promoter (P_T7) combined with T7 RNA polymerase gene promoted the co-expression of pucBAd and mrfpin ΔpucBA. We replaced P_T7 with P_badR, P_pckA, and P_ars, respectively, and the activities of the promoters followed a descending order of P_pckA, P_ars, P_badR, and P_T7. This strategy increased the mrfp gene expression, the synthesis product of pucBAd (d-LH2), and the phototrophic growth rate in the host bacteria. The expression system ΔpucBA(pucBAd) harboring P_pckA promoter had the highest expression activity of reconstituted LH2. Then, we used pucBAa from strain CGA009 to replace pucBAd in ΔpucBA(pucBAd) and successfully obtained the recombinant strain ΔpucBA(pucBAa). Compared with the pucBAaconstruct, the recombinant strain with pucBAd showed significantly accelerated growth under low light and slightly accelerated growth under high light. The purified d-LH2 and a-LH2 showed typical characteristic spectra of B800-only and B800-850, respectively. The fluorescence quantum efficiency of d-LH2 at ~863 nm was higher than that of a-LH2. However, the light energy transfer efficiency from carotenoids (Car) to bacteriochlorophylls (BChl) of d-LH2 measured by the classical method was lower than that of a-LH2. The inconsistency between the two results was due to the fact that the classical method did not consider the photon absorption difference at ~863 nm by different spectral types of LH2. [Conclusion] The reconstituted LH2 expression system ofR.palustris was successfully constructed. The light energy transfer activities of two different spectral types of LH2 in strains with multiple copies of pucBA were evaluated on the levels of gene-pure LH2 and photosynthetic growth rate of the recombinant bacteria. The results showed that the energy transfer activity of abnormal spectral LH2 (d-LH2) was higher than that of typical spectral LH2 (a-LH2). The present work laid a foundation for comprehensively understanding the molecular regulatory mechanisms of the formation and assembly of LH2 in the bacteria with multiple copies of pucBA.