Photosystem II characteristics of a constructedSynechocystis 6803 mutant lacking synthesis of the D1 polypeptide

Photosystem II (PSII) composition was studied in a mutant of the cyanobacteriumSynechosystis 6803 in which synthesis of the reaction center polypeptide D1 has been inactivated. The mutant thylakoids had lost also the other reaction center polypeptide D2 and the chlorophylla-binding protein CP47. Cytochromeb559 and the chlorophylla-binding protein CP43 accumulated to almost wild-type amounts in mutant thylakoids. Also the 33 kDa polypeptide involved in water oxidation was present and membrane-bound in mutant thylakoids. The intrinsic 22 kDa polypeptide, so far known only from plants, was detected both in wild-type and mutant thylakoids.     

Effect of exogenous glucose on the expression and activity of NADPH dehydrogenase complexes in the cyanobacterium Synechocystis sp. strain PCC 6803

Active NADPH dehydrogenase super- and medium-complexes were newly identified in cyanobacteria and are essential to cyclic photosystem I (PSI) activity and respiration and to CO₂ uptake, respectively. Synechocystis sp. strain PCC 6803 cells were treated with exogenous glucose (Glc) for different times. Active staining of NADPH–nitroblue tetrazolium oxidoreductase and western blot were conducted, and the initial rate of P700⁺ dark reduction was measured. The expression and enzyme activity of the NADPH dehydrogenase super-complex were gradually inhibited and were found to be closely associated with the decrease in cyclic PSI activity, as reflected by the initial rate of P700⁺ dark reduction. By contrast, those of the NADPH dehydrogenase medium-complex and the activity of CO₂ uptake reflected by the expression levels of NdhD3 and NdhF3 were not significantly affected by the addition of exogenous Glc to the cultures; however, the expression and enzyme activity of this medium-complex were found to be significantly influenced by the changes in CO₂ concentration. These results indicated that (1) the responses of the 2 cyanobacterial NADPH dehydrogenase complexes to exogenous Glc in terms of their expression and activity differed and that (2) these responses were closely associated with their respective physiological roles.     

集胞藻6803藻胆体别藻蓝蛋白多克隆抗体的制备

藻胆体是蓝藻细胞主要的捕光天线色素超分子复合体,主要由核心体和外围的杆两部分组成,核心体主要由别藻蓝蛋白组装而成,参与光能向光合作用反应中心的传递.该研究通过PCR扩增出集胞藻6803别藻蓝蛋白α亚基(ApcA)编码基因apcA,构建表达质粒pET-32a(+)-apcA,并将其转入大肠杆菌BL21(DE3)pLysS菌株中;通过IPTG诱导表达重组蛋白,并利用组氨酸标签将可溶性目的蛋白进行亲和纯化后,免疫日本大耳白兔,从而获得多克隆抗体.间接ELISA法揭示ApcA抗体效价可高达1∶1 025 000;蛋白免疫印迹确定该抗体具有高度特异性.表明该研究成功制备了集胞藻6803藻胆体别藻蓝蛋白多克隆抗体,为进一步研究藻胆体的核心体在光能传递过程中所承担的重要生理角色奠定了生化基础.     

集胞藻6803中ndhK基因启动子结构与功能的初步分析

ndhK是蓝藻NDH-1复合体的1个亚基编码基因,其表达受低浓度CO2和高光的诱导,对于蓝藻应对低CO2胁迫起着重要的作用。为进一步阐明ndhK基因的转录调控机制,本研究利用5′-RACE(Rapid Amplification of cDNAEnds,cDNA末端快速扩增)技术鉴定了该基因的转录起始位点,利用生物信息学预测发现ndhK基因含有4个可能的启动子,构建了含增强型黄色荧光蛋白报告基因的启动子探针型载体,并利用蛋白免疫印迹的方法进行检测。结果表明:在集胞藻6803的ndhK基因上游的-374～-274bp,-438～-374bp,-604～-543bp,+1～+52bp区域具有较高的启动子活性,而-543～-440bp区域则可能存在1个抑制ndhK基因表达的转录因子。     

Site-specific mutations in the D1 polypeptide affect the susceptibility of Synechocystis 6803 cells to photoinhibition

Photoinhibition of photosystem II in the cyanobacterium Synechocystis 6803 was followed after site-specific mutagenesis of the D1 polypeptide. Mutations were created in the stromal/cytosolic loop connecting helices D and E. Two mutations E243K and CA1, a deletion of the three glutamates 242–244 and a substitution Q241H, were made in the putative cleavage area of the D1 polypeptide. A third mutation E229D was made in the PEST-like sequence. Mutants and control cells were illuminated and F_V/F_M was recorded. Compared to the control, the mutants were less photoinhibited. Fluorescence relaxation after a single flash was delayed in CA1. Restoration of F_V/F_M after photoinhibition in the mutants was totally dependent on protein synthesis while control cells were able to recover partially also when protein synthesis was inhibited. In addition, the protein synthesis-dependent recovery of CA1 was slowed down. Our results indicate a correlation between the mutated amino acids and photoinhibition of photosystem II.     

Conjugative transfer and autonomous replication of a promiscuous IncQ plasmid in the cyanobacterium Synechocystis PCC 6803

Summary The promiscuous IncQ plasmid pKT210 (Cm^r, Sm^r) is efficiently transferred by transpecific conjugation from Escherichia coli to the facultatively heterotrophic cyanobacterium Synechocystis PCC6803 when mobilized by a helper plasmid coding for IncP transfer functions. The IncQ plasmid is stably maintained in the cyanobacterium as an autonomously replicating multicopy plasmid with no detectable structural alterations and can be recovered by transformation back to E. coli when using a mcrA mcrB host. Thus, the replicative host-range of IncQ plasmids extends beyond purple bacteria to the distinct procaryotic taxon of cyanobacteria, allowing the use of these small plasmids as convenient cloning vectors in Synechocystis PCC6803 and presumably also in cyanobacteria that are not amenable to genetic transformation. In contrast, an IncQ plasmid bearing the TRP1 gene of Saccharomyces cerevisiae failed to replicate when transferred to that yeast by transformation.     

云南不同高原湖泊噬藻体psbA基因多样性

【目的】噬藻体(cyanophage)广泛存在于自然水体生态系统中,通过侵染蓝藻进而调控蓝藻种群及群落结构,具有重要生态功能和生态地位,在控制蓝藻水华方面有巨大开发潜力。本研究旨在探究云南高原湖泊噬藻体psbA基因多样性,分析其系统进化地位,为深入了解高原湖泊生态功能、开发利用噬藻体资源奠定理论基础。【方法】以云南高原主要湖泊滇池、抚仙湖和星云湖等为研究对象,以psbA基因作为分子靶标,对湖泊水体中噬藻体遗传多样性进行研究。【结果】从不同湖泊中共获得100条环境噬藻体psbA基因序列,系统发育分析表明,湖泊的噬藻体psbA基因序列与中国东湖、中国东北稻田、日本稻田等淡水中的环境噬藻体psbA基因亲缘关系较近,与海洋环境噬藻体psbA基因亲缘关系较远;抚仙湖中的噬藻体psbA基因多样性高于滇池、星云湖和异龙湖中的噬藻体psbA基因多样性;云南高原湖泊中存在新的噬藻体类群;各湖泊秋冬季节噬藻体psbA基因遗传多样性差异不明显。【结论】云南主要高原湖泊噬藻体psbA基因遗传多样性高,与淡水环境噬藻体psbA基因亲缘关系较近,且存在独特的噬藻体类群。     

ClpP2蛋白失活对集胞藻PCC6803生长、光合作用和蛋白质组的影响

【背景】蛋白酶能够降解细胞中错误折叠或是无功能的蛋白,Clp家族蛋白就是一类重要的蛋白酶复合物。Clp蛋白酶复合物的水解核心是ClpP,集胞藻PCC6803中存在4种不同的ClpP蛋白,分别为ClpP1-ClpP4。作为重要的蛋白水解复合物的功能组分,目前对集胞藻ClpP的研究十分有限,对其生理功能与调控底物的研究甚少。【目的】选择集胞藻为研究对象探究ClpP2蛋白的功能,鉴定其潜在底物,为集胞藻ClpP2作用机制提供实验支撑。【方法】构建集胞藻ClpP2突变株(ΔClpP2),进行其生长实验和光合生理功能研究。通过标记定量蛋白质组学技术(isobaric tag for relative absolute quantitation, iTRAQ)鉴定ClpP2调控的靶标蛋白,生物信息学分析底物蛋白参与的代谢通路,最后利用平行反应监测(parallel reaction monitoring, PRM)技术对部分定量数据进行验证。【结果】ΔClpP2可以在自然条件下光合自养生长至对数生长期,但高光或高温胁迫下则无法正常生长。相较于野生型,ΔClpP2有着显著降低的PSⅡ电子传递效率及P...     

集胞藻PCC 6803中丝氨酸/苏氨酸激酶SpkC对高温胁迫的响应

丝氨酸/苏氨酸激酶是蓝藻感知和转导外界刺激的重要元件,但至今蓝藻中很多丝氨酸/苏氨酸激酶的功能尚属未知。【目的】研究集胞藻PCC6803中的丝氨酸/苏氨酸激酶Spk C是否参与对高温胁迫的响应。【方法】本研究采用同源重组的方法构建spC基因完全敲除突变株,检测突变株与野生株在高温胁迫下的生长状况、色素组成,并对高温胁迫下叶绿素荧光参数差异进行分析,比较光合系统Ⅱ活性差异。此外,通过测定生长速率来判断高温胁迫后藻株的恢复情况。【结果】经过42℃高温胁迫后,与野生株相比,突变株ΔspkC生长减缓,光合色素(叶绿素、类胡萝卜素和藻胆色素)的含量降低;45℃高温胁迫下突变株ΔspkC的光合系统Ⅱ活性下降幅度更大;经过5 d 42℃高温处理后,突变株生长几乎停滞,存活率较野生株明显降低。【结论】集胞藻PCC 6803中spkC基因的缺失导致突变株对高温胁迫响应出现缺陷,提示丝氨酸/苏氨酸激酶SpkC参与响应高温胁迫。     

Cloning, nucleotide sequence and mutational analysis of the gene encoding the Photosystem II manganese-stabilizing polypeptide of Synechocystis 6803

Summary Affinity purified, polyclonal antibodies raised against the Photosystem II 33 kDa manganese-stabilizing polypeptide of the spinach oxygen-evolving complex were used to isolate the gene encoding the homologous protein from Synechocystis 6803. Comparison of the amino acid sequence deduced from the Synechocystis psb1 nucleotide sequence with recently published sequences of spinach and pea confirms the homology indicated by antigenic crossreactivity and shows that the cyanobacterial and higher plant sequences are 43% identical and 63% conserved. Regions of identity, varying in length from 1 to 10 consecutive residues, are distributed throughout the protein. The 28 residues at the amino terminus of the psb1 gene product, characteristic of prokaryotic signal peptides, show homology with the carboxyl-terminal third of the transit sequences of pea and spinach and are most likely needed for the transport of the manganese-stabilizing protein across the thylakoid membrane to its destination of the lumen. Synechocystis mutants which contain a kanamycin resistance gene cassette inserted into the coding region for the 32 kDa polypeptide were constructed. These mutants contain no detectable 32 kDa polypeptide, do not evolve oxygen, and are incapable of photoautotrophic growth.     

Localisation and processing of the precursor form of photosystem II protein D1 inSynechocystis 6803

Pure plasma membrane and thylakoid membrane fractions from Synechocystis 6803 were isolated to study the localisation and processing of the precursor form of the D1 protein (pD1) of photosystem II (PSII). PSII core proteins (D1, D2 and cytb559) were localised both to plasma and thylakoid membrane fractions, the majority in thylakoids. pD1 was found only in the thylakoid membrane where active PSII is known to function. Membrane fatty acid unsaturation was shown to be critical in processing of pD1 into mature D1 protein. This was concluded from pulse-labelling experiments at low temperature using wild type and a mutant Synechocystis 6803 with a low level of membrane fatty acid unsaturation. Further, pD1 was identified as two distinct bands, an indication of two cleavage sites in the precursor peptide or, alternatively, two different conformations of pD1. Our results provide evidence for thylakoid membranes being a primary synthesis site for D1 protein during its light-activated turnover. The existence of the PSII core proteins in the plasma membrane, on the other hand, may be related to the biosynthesis of new PSII complexes in these membranes.     

Nucleotide sequence and homology comparison of two genes of the sulfate transport operon from the cyanobacterium Synechocystis sp. PCC 6803

The genome of Synechocystis sp. PCC 6803 contains an operon with homology to the sulfate permease of other prokaryotes. We used antibodies raised against cytoplasmic membrane protein to find three genes with strong homology to sbpA, orf81 and cysT genes of the cyanobacterium Synechococcus sp. PCC 7942, Escherichia coli, Salmonella typhymurium and Marchantia polymorpha. It is likely that the permease genes are expressed and the proteins are inserted into the cytoplasmic membrane.