Nixon P J, Rögner M, Diner B A
E. I. du Pont de Nemours and Company, Experimental Station, Central Research and Development Department, Wilmington, Delaware 19880-0173.
Plant Cell. 1991 Apr;3(4):383-95. doi: 10.1105/tpc.3.4.383.
The psbA gene codes for the D1 polypeptide of the photosystem II reaction center complex and is found in all photosynthetic organisms that carry out oxygenic photosynthesis. Here we describe the construction and characterization of a strain of the cyanobacterium Synechocystis sp PCC 6803 in which the three endogenous psbA genes are replaced by a single psbA gene from the chloroplast genome of the higher plant Poa annua. The resulting chimeric strain, KWPAS, grows photoautotrophically with a doubling time of 26 hours compared with 20 hours for wild-type Synechocystis 6803. The mutant oxidizes water to oxygen at light-saturated rates comparable with wild type, despite differences in 15% of the primary structure of D1 between these species. RNA gel blot analysis indicates the presence in KWPAS of a psbA transcript of approximately 1.25 kilobases, consistent with the chloroplast promoter also acting as a promoter in Synechocystis. By using antibodies specific for the carboxyl-terminal extension of the D1 polypeptide of higher plants, we showed that the D1 polypeptide synthesized by KWPAS is post-translationally modified at the carboxyl terminus, probably through processing. A detailed biophysical analysis of the chimeric photosystem II complex indicated that the rates of forward electron transfer are similar to wild type. The rates of charge recombination between the donor and acceptor sides of the reaction center are, however, accelerated by as much as a factor of nine (QA- to S2) and are the most likely explanation for the lower rate of photoautotrophic growth in the mutant. We conclude that the psbA gene from a higher plant can be expressed in cyanobacteria and its product processed and assembled into a functional chimeric photosystem II reaction center.
psbA基因编码光系统II反应中心复合物的D1多肽，存在于所有进行产氧光合作用的光合生物中。在此，我们描述了一种集胞藻属蓝藻（Synechocystis sp PCC 6803）菌株的构建与特性，该菌株中的三个内源性psbA基因被来自高等植物一年生早熟禾叶绿体基因组的单个psbA基因所取代。所得的嵌合菌株KWPAS能进行光合自养生长，其倍增时间为26小时，而野生型集胞藻6803的倍增时间为20小时。尽管这两个物种的D1一级结构有15%的差异，但该突变体在光饱和速率下将水氧化为氧气的能力与野生型相当。RNA凝胶印迹分析表明，KWPAS中存在一个约1.25千碱基的psbA转录本，这与叶绿体启动子在集胞藻中也能作为启动子的情况一致。通过使用针对高等植物D1多肽羧基末端延伸的特异性抗体，我们发现KWPAS合成的D1多肽在羧基末端进行了翻译后修饰，可能是通过加工实现的。对嵌合光系统II复合物的详细生物物理分析表明，正向电子传递速率与野生型相似。然而，反应中心供体侧和受体侧之间的电荷复合速率加快了多达9倍（QA - 到S2），这很可能是突变体光合自养生长速率较低的原因。我们得出结论，高等植物的psbA基因可以在蓝藻中表达，其产物经过加工并组装成功能性的嵌合光系统II反应中心。
