ApcD is required for state transition but not involved in blue-light induced quenching in the cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

Pbycobilisomes （PBS） are able to transfer absorbed energy to photosystem Ⅰ and Ⅱ, and the distribution of light energy between two photosystems is regulated by state transitions. In this study we show that energy transfer from PBS to photosystem Ⅰ （PSI） requires ApcD. Cells were unable to perform state transitions in the absence of ApcD. The apcD mutant grows more slowly in light mainly absorbed by PBS, indicating that ApcD-dependent energy transfer to PSI is required for optimal growth under this condition. The apcD mutant showed normal blue-light induced quenching, suggesting that ApcD is not required for this process and state transitions are independent of blue-light induced quenching. Under nitrogen fixing condition, the growth rates of the wild type and the mutant were the same, indicating that energy transfer from PBS to PSI in heterocysts was not required for nitrogen fixation.     

Transient Effects of Salt Shock on the Photosynthetic Machinery in Synechocystis sp. PCC 6803

IntroductionSalt stress is one of the main detrimental factors inthe environment that limit the growth andproductivity of plants.Salt stress causessignificant decreases in photosynthetic activity,such as the electron transport[1,2 ] ,but themechanisms by which salt stress inhibitsphotosynthesis remain poorly understood[3] .　Cyanobacteria are prokaryotes that performoxygenic photosynthesis using a photosyntheticapparatus similar to that in the chloroplasts ofgreen algae and higher plants[4 ] .…     

New mechanism revealed for light-state transition in cyanobacterium Arthrospira platensis according to 77-K fluorescence kinetics

The mechanisms of oxygen evolution and carbon fixation in oxygenic organisms depend on the equal distribution of excitation energy to photosystems Ⅰ and Ⅱ, which is regulated by a mechanism referred to as light-state transition. In this work, a novel mechanism, energy spillover from PS Ⅰ to PS Ⅱ referred to as "inverse spillover", was revealed besides "mobile phycobilisome (PBS)" and the "spillover" of energy from PS Ⅱ to PS Ⅰ in cyanobacteria. Under continuous illumination with blue light, time-dependent 77-K fluorescence spectra demonstrated heterogeneous kinetics for the PBS and photosystem components, indicating that inverse spillover and mobile PBS work successively to regulate the excitation to a balanced distribution in cyanobacterial cells under blue light. Inverse spillover and mobile PBS occur under both 100 and 300 μmol m-2 s-1 blue-light conditions but they are accelerated under the latter.     

鱼腥藻PCC 7120染色体上relBE同源基因的克隆及表达

为研究鱼腥藻PCC7120染色体上与relBE同源的基因asl4561和asl4562表达产物的毒素-抗毒素作用,从鱼腥藻细胞中提取总基因组DNA,设计特异引物PCR扩增目的基因asl4561和asl4562,与T载体连接后经XhoI和EcoR I双酶切并与表达载体pET28a（＋）连接,由IPTG诱导表达,并在IPTG浓度和诱导时间上对asl4561基因的表达条件进行了优化.琼脂糖凝胶电泳显示扩增出了264bp的asl4561基因和213bp的asl4562基因,SDS-PAGE检测表明成功表达出15.65kD和13.55kD的两蛋白,当诱导温度28℃、IPTG浓度0.4 mmol/L、诱导时间6h时,asl4561基因表达蛋白在细菌裂解液上清中表达量最大.     

鱼腥藻铁吸收调节蛋白基因(alr0957)的克隆和表达

依据CyanoBase提供的鱼腥藻PCC7120 furC基因(alr0957)的序列信息设计了一对特异性引物,用Touch-down PCR的方法从基因组DNA中扩增得到大小约450bp的目的片段.通过TA克隆的方法将该片段连接到pMD18-T载体上筛选出重组质粒pMD18-T-fur,然后进行双酶切,纯化furC基因,再连接到原核表达载体pET-28a(+)上,转化表达菌株BL21(DE3).经PCR、双酶切和测序鉴定,对阳性菌株进行IPTG诱导表达,SDS-PAGE检测重组蛋白.结果表明:在25℃条件下经1mmol/L IPTG诱导20h,融合蛋白被成功表达,其分子量约为19 000,为进一步纯化蛋白和对基因的调控功能方面研究奠定了基础.     

Effect of Iron Deficiency on Heterocyst Differentiation and Physiology of the Filamentous Cyanobacterium Anabaena sp. PCC 7120

0　IntroductionIron playsimportantrolesinthemetabolismofcyanobacteria,andisin volvedinavarietyofbiologicalactivities,bothasacofactorandcatalyst.Thesein cludephotosynthesis,pigmentsynthesisandfunction,respiration ,nitrateandnitritere duction ,hydrogenasereactions,dinitrogenfixation,andotherbiologicaloxidations.Numerousstudieshavebeencarriedoutontheeffectofnutritionaldeficiencyoncyanobacteria .Thephysiologicalandmolec ulareffectsofirondeficiencyoncyanobacte riahavebeenstudiedfromavarietyofper sp…     

Cloning and Characterization of the fecC Gene Necessary for Optimal Growth under Iron-Deficiency Conditions in the Cyanobacterium Anabaena sp. PCC7120

0　IntroductionIronisthefourthmostabundantelementbyweightintheearth’scrust.Itisrequiredfornearlyallorganisms.Itinvolvedinawidevarietyofbiochemicalprocessesbothasacofactorandcatalyst.Theseprocessesincludephotosynthesis,chlorophyllproduction ,respiration,nitrateandnitritereduction,nitrogenfixation ,andotherbi ologicaloxidations.However,itisanelementwithalowbiologicalavailabilityduetoitspoorsolubilityinoxygenicaqueoussolution .Livingor ganismsareconstantlyfacedtotheproblemofhowtoovercomethelows…     

丝状蓝藻Calothrix sp.PCC7601原生质球的分离和再生

采用溶菌酶、纤维素酶和果胶酶混合处理法，首次较好地分离具活力的丝状蓝藻的原生质球。通过再生培养，跟踪显微观察了原生质球的再生过程，并且获得了再生藻株。为丝状蓝藻的转基因工作提供了一种潜在的受体细胞。     

Specific degradation of photosystem II D1 protein by a protease （Air3815） in heterocysts of the cyanobacterium Anabaena sp. PCC7120

Some filamentous cyanobacteria form heterocysts under conditions lacking combined nitrogen for nitrogen fixation.Photosystem II is removed from heterocyst during the process of cell differentiation.Here,we demonstrate that Alr3815 is a protease that is capable of degrading D1 protein of photosystem II.Strain-322,which lacks alr3815,is impaired in nitrogen fixation in air because some oxygen evolving activity is retained in its heterocysts.Our results also suggest that calcium may play a regulatory role in D1 degradation during heterocyst differentiation.     

水稻cFBA和菠菜cpTPI串联基因在鱼腥藻7120中的表达及其对光合作用的影响

利用转基因技术,将水稻cFBA和菠菜cpTPI串联基因转入到鱼腥藻7120中进行表达．结果发现：与野生藻相比,转基因藻蛋白粗提液中两个酶的比活力提高了33．3％;转基因鱼腥藻的生长明显优于野生型鱼腥藻,当在培养基中添加适量NaHCO3时,这一优势更加明显;转基因鱼腥藻的净光合速率和真实光合速率都有明显提高,光饱和点和光补偿点也有所提高．以上这些结果表明,在鱼腥藻7120中特异的提高“非限速酶”——FBA和TPI的水平,能在一定程度上提高转基因鱼腥藻的光合作用速率和生长速度．     

鱼腥藻PCC7120质粒上毒素-抗毒素基因对alr9029/asr9028的初步研究

指出了大肠杆菌的毒素-抗毒素系统(TAs)能介导解离后致死机制维持细菌质粒的稳定性和参与环境胁迫诱导细菌生长抑制或死亡,在鱼腥藻PCC7120质粒上的基因对alr9029/asr9028具有与TA系统较高的同源性.为了证实该基因对属于TA系统,通过生物信息学分析了alr9029/asr9028的遗传结构,设计特异性引物,扩增得到大小为198 bp的asr9028和387 bp的alr9029.PCR产物经Bam H I和HindШ双酶切后被插入p MD18-T和p ET-30a中,依次构建克隆载体和表达载体.经SDS-PAGE电泳检测,含有His6标签的表达蛋白相对分子量分别为12.4k Da和19.2 k Da,且为可溶性蛋白.故初步认定asr9028为抗毒素基因,alr9029为毒素基因,二者共同构成一个TA系统.     

Kinetics of blue-green light-induced state transition and fluorescence quenching in the wild-type cyanobacterium Synechocystis PCC 6803 and apcD − and apcF − mutants

State transition and blue-green light-induced fluorescence quenching are two short-term processes in cyanobacteria. The details of their kinetics and the relationship between these processes have not been elucidated. In this work, these two processes were studied in the wildtype cyanobacterium Synechocystis PCC 6803 cells as well as in apcD^- and apcF^- mutants by monitoring their time- dependent 77 K fluorescence responses to blue-green light （430-540 nm） at a series of intensities ranging from 20-800 μE m^-2 s^-1. The lowest light intensity to induce fluorescence quenching in wild-type cells was 160 μE m^-2 s^-1 under the selected experimental conditions, while state transition took place at the intensities lower than 160 μE m^-2 s^-1 at a conservative level, but at variable rates. The quenching level increased at intensities higher than 160 μE m^-2 s^-1, reaching the maximum level at intensities equal to or higher than 200 μEm^-2 s^-1. Fluorescence kinetics indicated that both the length of the induction period and time required to reach the maximum level were functions of light intensity. State transitions as well as fluorescence quenching took place in both wildtype and mutant cells, but might involve different mechanisms.     

all 1691基因表达及铁元素对鱼腥藻生长的影响

为实现FurA的表达,探究铁元素对藻生长的影响,首先运用Primer5.0设计引物,克隆出鱼腥藻PCC7120染色体上all1691(furA)基因片段;构建含组氨酸融合表达标签和T7启动子标签的表达载体.IPTG诱导FurA蛋白表达.然后设计不同Fe3+浓度梯度培养基,利用SDS-PAGE检测高、中、低Fe3+浓度培养液的藻细胞总蛋白,考马斯亮蓝g-250法测定蛋白含量,并用Trizol法提取不同Fe3+浓度培养的藻细胞总RNA.结果表明,获得纯化的all 1691克隆片段,大小为456bp,pET-1691重组蛋白在1mmol/L的IPTG诱导下,于37℃下摇床培养15h得到成功表达;低浓度Fe3+促进藻生长,高浓度Fe3+抑制藻生长;Fe3+浓度为2.0mg/L时rRNA位置与亮度清晰可见,而缺铁培养的藻生长几乎停滞,转录和翻译水平都很低.     

Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp. PCC 6803

From a random insertion mutant library of Synechocystis sp. PCC 6803, a mutant defective in photoautotrophic growth was obtained. The interrupted gene was identified to be slr2094 （fbpl）, which encodes the fructose-1,6-biphosphatase （FBPase）/sedoheptulose-1,7-biphosphatase （SBPase） bifunctional enzyme （F-I）. Two other independently constructed slr2094 mutants showed an identical phenotype. The FBPase activity was found to be virtually lacking in an slr2094 mutant, which was sensitive to light under mixotrophic growth conditions. These results indicate that slr2094 is the only active FBPase-encoding gene in this cyanobacterium. Inactivation of photosystem II by interrupting psbB in slr2094 mutant alleviated the sensitiveness to light. This report provides the direct genetic evidence for the essential role of F-I in the photosynthesis of Synechocystis sp. PCC 6803.     

Changes in the transthylakoid proton gradient are caused by the movement of phycobilisomes in the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. strain PCC 6803

Phycobilisomes (PBSs) are the main accessory light-harvesting complexes in cyanobacteria and their movement between photosystems (PSs) affects cyclic and respiratory electron transport. However, it remains unclear whether the movement of PBSs between PSs also affects the transthylakoid proton gradient (ΔpH). We investigated the effect of PBS movement on ΔpH levels in a unicellular cyanobacterium Synechocystis sp. strain PCC 6803, using glycinebetaine to immobilize and couple PBSs to photosystem II (PSII) or photosystem I (PSI) by applying under far-red or green light, respectively. The immobilization of PBSs at PSII inhibited decreases in ΔpH, as reflected by the slow phase of millisecond-delayed light emission (ms-DLE) that occurs during the movement of PBSs from PSII to PSI. By contrast, the immobilization of PBSs at PSI inhibited the increase in ΔpH that occurs when PBSs move from PSI to PSII. Comparison of the changes in ΔpH and electron transport caused by the movement of PBSs between PSs indicated that the changes in ΔpH were most likely caused by respiratory electron transport. This will further improve our understanding of the physiological role of PBS movement in cyanobacteria.     

Kinetics of blue-green light-induced state transition and fluorescence quenching in the wild-type cyanobacterium Synechocystis PCC 6803 and apc D2 and apc F2 mutants

State transition and blue-green light-induced fluorescence quenching are two short-term processes in cyanobacteria. The details of their kinetics and the relationship between these processes have not been elucidated.In this work, these two processes were studied in the wildtype cyanobacterium Synechocystis PCC 6803 cells as well as in apc D-and apc F-mutants by monitoring their timedependent 77 K fluorescence responses to blue-green light(430–540 nm) at a series of intensities ranging from20–800 l E m-2s-1. The lowest light intensity to induce fluorescence quenching in wild-type cells was160 l E m-2s-1under the selected experimental conditions, while state transition took place at the intensities lower than 160 l E m-2s-1at a conservative level, but at variable rates. The quenching level increased at intensities higher than 160 l E m-2s-1, reaching the maximum level at intensities equal to or higher than 200 l E m-2s-1.Fluorescence kinetics indicated that both the length of the induction period and time required to reach the maximum level were functions of light intensity. State transitions as well as fluorescence quenching took place in both wildtype and mutant cells, but might involve different mechanisms.     

Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp. PCC 6803

From a random insertion mutant library of Synechocystis sp. PCC 6803, a mutant defective in photoautotrophic growth was obtained. The interrupted gene was identified to be slr2094 (fbp1), which encodes the fructose-1,6-biphosphatase (FBPase) / sedoheptulose-1,7-biphosphatase (SBPase) bifunctional enzyme (F-I). Two other independently constructed slr2094 mutants showed an identical phenotype. The FBPase activity was found to be virtually lacked in slr2094 mutant, which was sensitive to light under mixotrophic growth conditions. These results indicate that slr2094 is the only active FBPase-encoding gene in this cyanobacterium. Inactivation of photosystem II by interrupting psbB in slr2094 mutant alleviated the sensitiveness to light. This report provides the direct genetic evidence for the essential role of F-I in the photosynthesis of Synechocystis sp. PCC 6803.     

Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp. PCC 6803

From a random insertion mutant library of Synechocystis sp. PCC 6803, a mutant defective in photoautotrophic growth was obtained. The interrupted gene was identified to be slr2094 (fbp1), which encodes the fructose-1,6-biphosphatase (FBPase) / sedoheptulose-1,7-biphosphatase (SBPase) bifunctional enzyme (F-I). Two other independently constructed slr2094 mutants showed an identical phenotype. The FBPase activity was found to be virtually lacked in slr2094 mutant, which was sensitive to light under mixotrophic growth conditions. These results indicate that slr2094 is the only active FBPase-encoding gene in this cyanobacterium. Inactivation of photosystem II by interrupting psbB in slr2094 mutant alleviated the sensitiveness to light. This report provides the direct genetic evidence for the essential role of F-I in the photosynthesis of Synechocystis sp. PCC 6803.