Inactivation of the petE gene for plastocyanin lowers photosynthetic capacity and exacerbates chilling-induced photoinhibition in the cyanobacterium Synechococcus.

We describe the identification and expression of a petE gene in Synechococcus sp. PCC 7942, a cyanobacterium previously thought to lack plastocyanin. The petE gene is a 420-bp open reading frame that encodes a protein 70 to 75% similar to plastocyanins from other cyanobacteria. Synechococcus possesses a single genomic copy of petE located immediately upstream of the clpB gene. It is transcribed as a single mRNA (550 bases) and, in contrast to most other photobionts, the level of petE expression in Synechococcus is unaffected by variable copper concentrations during acclimated growth. Inactivation of petE does not prevent photoautotrophic growth, but does induce a dramatic increase in mRNA for the alternative electron carrier cytochrome C6. Despite this adjustment, loss of plastocyanin results in slower growth, lower photosystem I content, and a decreased maximum capacity for photosynthetic electron transport. The mutant is also more susceptible to chilling-induced photoinhibition during a shift from 37 to 25 degrees C, at which temperature its inherently lower photosynthetic capacity exacerbates the normal slowing of electron transfer reactions at low temperatures. Under similar conditions, the amount of petE message in the wild type decreases by 50% in the 1st h, but then increases dramatically to almost three times the 37 degrees C level by 9 h.     

集胞藻PCC6803铜离子诱导表达平台的构建

在集胞藻PCC6803中,基因敲除是研究基因功能的最直接有效的方法,但是对于某些生存必需的基因则无法通过这种方法获得突变株。为研究集胞藻PCC6803中此类基因的功能,在其基因组中构建了一个petE基因启动子（PpetE）控制的铜离子诱导表达的平台。将集胞藻PpetE装配在lacZ报告基因的上游,通过同源双交换整合到这种蓝藻的基因组中。通过调节培养基中铜离子的浓度发现,lacZ的表达能够人为控制。特别是当铜离子浓度在6－400nmoL／L范围时,LacZ活力随铜离子浓度增加呈S型增长关系。利用这个铜离子诱导表达平台,可以控制某些必需基因的表达：提供铜离子维持细胞生存;而撤去铜离子时则关闭基因的表达,可以观察其对生命活动的影响。     

Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin.

Synechococcus sp. PCC7942 recipient strains were constructed for the chromosomal integration of DNA fragments cloned in any pBR322-derived vector, which carries the ampicillin resistance (ApR) marker. The construction was based on the incorporation of specific recombination targets, the so-called 'integration platforms', into the chromosomal metF gene. These platforms consist of an incomplete bla gene (ApS) and the pBR322 ori separated from each other by a gene encoding an antibiotic (streptomycin or kanamycin) resistance (SmR or KmR). Recombination between a pBR322-derived donor plasmid and such a chromosomal platform results with high frequency in restoration of the bla gene and replacement of the chromosomal marker (SmR or KmR) by the insert of the donor plasmid. The integration into the platform depends on recombination between pBR322 ori and bla sequences only and is therefore independent of the DNA insert to be transferred. The desired recombinants are found by selection for a functional bla gene (ApR) and subsequent screening for absence of the chromosomal antibiotic marker. Gene transfer with this integration system was found to occur efficiently and reliably. Furthermore, the presence of the pBR322 ori in the platform allowed for 'plasmid rescue' of integrated sequences. The system was applied successfully for the transfer of the gene encoding plastocyanin (petE1) from Anabaena sp. PCC7937 and for the integration of an extra copy of the gene encoding ferredoxin I (petF1) from Synechococcus sp. PCC7942 itself.     

Depletion of Vipp1 in Synechocystis sp. PCC 6803 affects photosynthetic activity before the loss of thylakoid membranes

A vipp1 mutant of Synechocystis sp. PCC 6803 could not be completely segregated under either mixotrophic or heterotrophic conditions. A vipp1 gene with a copper-regulated promoter (P _petE -vipp1 ) was integrated into a neutral platform in the genome of the merodiploid mutant. The copper-induced expression of P _petE -vipp1 allowed a complete segregation of the vipp1 mutant and observation of the phenotype of Synechocystis 6803 with different levels of vesicle-inducing protein in plastids 1 (Vipp1). When P _petE -vipp1 was turned off by copper deprivation, Synechocystis lost Vipp1 and photosynthetic activity almost simultaneously, and at a later stage, thylakoid membranes and cell viability. The photosystem II (PSII)-mediated electron transfer was much more rapidly reduced than the PSI-mediated electron transfer. By testing a series of concentrations, we found that P _petE -vipp1 cells grown in medium with 0.025 μM Cu²⁺ showed no reduction of thylakoid membranes, but greatly reduced photosynthetic activity and viability. These results suggested that in contrast to a previous report, the loss of photosynthetic activity may not have been due to the loss of thylakoid membranes, but may have been caused more directly by the loss of Vipp1 in Synechocystis 6803.     

Restricted capacity for PSI-dependent cyclic electron flow in ΔpetE mutant compromises the ability for acclimation to iron stress in Synechococcus sp. PCC 7942 cells

Exposure of wild type (WT) and plastocyanin coding petE gene deficient mutant (ΔpetE) of Synechococcus cells to low iron growth conditions was accompanied by similar iron-stress induced blue-shift of the main red Chl a absorption peak and a gradual decrease of the Phc/Chl ratio, although ΔpetE mutant was more sensitive when exposed to iron deficient conditions. Despite comparable iron stress induced phenotypic changes, the inactivation of petE gene expression was accompanied with a significant reduction of the growth rates compared to WT cells. To examine the photosynthetic electron fluxes in vivo, far-red light induced P700 redox state transients at 820nm of WT and ΔpetE mutant cells grown under iron sufficient and iron deficient conditions were compared. The extent of the absorbance change (ΔA(820)/A(820)) used for quantitative estimation of photooxidizable P700(+) indicated a 2-fold lower level of P700(+) in ΔpetE compared to WT cells under control conditions. This was accompanied by a 2-fold slower re-reduction rate of P700(+) in the ΔpetE indicating a lower capacity for cyclic electron flow around PSI in the cells lacking plastocyanin. Thermoluminescence (TL) measurements did not reveal significant differences in PSII photochemistry between control WT and ΔpetE cells. However, exposure to iron stress induced a 4.5 times lower level of P700(+), 2-fold faster re-reduction rate of P700(+) and a temperature shift of the TL peak corresponding to S(2)/S(3)Q(B)(-) charge recombination in WT cells. In contrast, the iron-stressed ΔpetE mutant exhibited only a 40% decrease of P700(+) and no significant temperature shift in S(2)/S(3)Q(B)(-) charge recombination. The role of mobile electron carriers in modulating the photosynthetic electron fluxes and physiological acclimation of cyanobacteria to low iron conditions is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

The efficient functioning of photosynthesis and respiration in Synechocystis sp. PCC 6803 strictly requires the presence of either cytochrome c6 or plastocyanin

In cyanobacteria, cytochrome c6 and plastocyanin are able to replace each other as redox carriers in the photosynthetic and respiratory electron transport chains with the synthesis of one or another protein being regulated by the copper concentration in the culture medium. However, the presence of a third unidentified electron carrier has been suggested. To address this point, we have constructed two deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803, each variant lacking either the petE or petJ gene, which respectively codes for the copper or heme protein. The photoautotrophic and heterotrophic growth rate of the two mutants in copper-free and copper-supplemented medium as well as their photosystem I reduction kinetics in vivo were compared with those of wild-type cells. The two mutant strains grow at equivalent rates and show similar in vivo photosystem I reduction kinetics as wild-type cells when cultured in media that allow the expression of just one of the two electron donor proteins, but their ability to grow and reduce photosystem I is much lower when neither cytochrome c6 nor plastocyanin is expressed. These findings indicate that the normal functioning of the cyanobacterial photosynthetic and respiratory chains obligatorily depends on the presence of either cytochrome c6 or plastocyanin.     

Transient binding of plastocyanin to its physiological redox partners modifies the copper site geometry

The transient complexes of plastocyanin with cytochrome f and photosystem I are herein used as excellent model systems to investigate how the metal sites adapt to the changes in the protein matrix in transient complexes that are involved in redox reactions. Thus, both complexes from the cyanobacterium Nostoc sp. PCC 7119 (former Anabaena sp. PCC 7119) have been analysed by X-ray absorption spectroscopy. Our data are consistent with a significant distortion of the trigonal pyramidal geometry of the Cu coordination sphere when plastocyanin binds to cytochrome f, no matter their redox states are. The resulting tetrahedral geometry shows a shortening of the distance between Cu and the S(delta) atom of its ligand Met-97, with respect to the crystallographic structure of free plastocyanin. On the other hand, when plastocyanin binds to photosystem I instead of cytochrome f, the geometric changes are not significant but a displacement in charge distribution around the metal centre can be observed. Noteworthy, the electronic density around the Cu atom increases or decreases when oxidised plastocyanin binds to cytochrome f or photosystem I, respectively, thus indicating that the protein matrix affects the electron transfer between the two partners during their transient interaction.     

Amino-terminal and hydrophobic regions of the Chlamydomonas reinhardtii plastocyanin transit peptide are required for efficient protein accumulation in vivo

Nucleus-encoded chloroplast proteins of vascular plants are synthesized as precursors and targeted to the chloroplast by stroma-targeting domains in N-terminal transit peptides. Transit peptides in Chlamydomonas reinhardtii are considerably shorter than those in vascular plants, and their stroma-targeting domains have similarities to both mitochondrial and chloroplast targeting sequences. To examine Chlamydomonas transit peptide function in vivo, deletions were introduced into the transit peptide coding region of the petE gene, which encodes the thylakoid lumen protein plastocyanin (PC). The mutant petE genes were introduced into a plastocyanin-deficient Chlamydomonas strain, and transformants that accumulated petE mRNA were analyzed for PC accumulation. The most profound defects were observed with deletions at the N-terminus and those that extended into the hydrophobic region in the C-terminal half of the transit peptide. PC precursors were detected among pulse-labeled proteins in transformants with N-terminal deletions, suggesting that these precursors cannot be imported and are degraded in the cytosol. Intermediate PC species were observed in a transformant deleted for part of the hydrophobic region, suggesting that this protein is defective in lumen translocation and/or processing. Thus, despite its shorter length, the bipartite nature of the Chlamydomonas PC transit peptide appears similar to that of lumen-targeted proteins in vascular plants. Analysis of the synthesis, stability, and accumulation of PC species in transformants bearing deletions in the stroma-targeting domain suggests that specific regions probably have distinct roles in vivo. Abbreviations: cyt, cytochrome; ECL, enhanced chemiluminescence; LSU, large subunit; PC, plastocyanin; TP, transit peptide     

Balanced regulation of expression of the gene for cytochrome cM and that of genes for plastocyanin and cytochrome c6 in Synechocystis

The cytM gene for cytochrome cM was previously found in Synechocystis sp. PCC 6803. Northern blotting analysis revealed that the cytM gene was scarcely expressed under normal growth conditions but its expression was enhanced when cells were exposed to low temperature or high-intensity light. By contrast, the expression of the genes for cytochrome c6 and plastocyanin was suppressed at low temperature or under high-intensity light. These observations suggest that plastocyanin and/or cytochrome c6, which are dominant under non-stressed conditions, are replaced by cytochrome cM under the stress conditions.     

Structural and functional properties of the cyanobacterial photosystem I complex

Photosystem I (PSI) complexes have been isolated from two cyanobacterial strains, Synechococcus sp. PCC 7002 and 6301. These complexes contain six to seven low molecular mass subunits in addition to the two high molecular mass subunits previously shown to bind the primary reaction center components. Chemical cross-linking of ferredoxin to the complex identified a 17.5-kDa subunit as the ferredoxin-binding protein in the Synechococcus sp. PCC 6301-PSI complex. The amino acid sequence of this subunit, deduced from the DNA sequence of the gene, confirmed its identity as the psaD gene product. A 17-kDa subunit cross-links to the electron donor, cytochrome c-553, in a manner analogous to the cross-linking of plastocyanin to the higher plant PSI complex. Using antibodies raised against the spinach psaC gene product (a 9-kDa subunit which binds Fe-S centers A and B), we identified an analogous protein in the cyanobacterial PSI complex.     

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.     

重组鱼腥藻脂肪氧合酶基因的克隆表达、分离纯化及活性分析

克隆鱼腥藻PCC7120基因组中脂肪氧合酶(ana-LOX)基因,对该功能基因进行了定点突变研究,确定了ana-LOX的最短功能基因长度,构建原核重组表达载体,对重组ana-LOX进行了分离纯化和性质研究。从GenBank中搜索到鱼腥藻PCC7120基因组中含有LOX基因,通过序列分析和比对,发现LOX功能基因位于双功能酶AOS(单加氧酶)-LOX的C端,通过定点突变研究,证实了ana-LOX活性中心位点为His197、His202、His369、Asn373和Ile455。通过逐步缩短基因长度的策略,获得ana-LOX基因的最短功能基因长度为1 254 bp。构建的表达载体pET-32a/ana-LOX转化入BL21(DE3)宿主内,在低温16℃条件下的诱导表达,重组脂肪氧合酶活力可达6 750 U/mL。表达产物通过Ni-NTA亲和柱进行分离纯化,比活达到11.4×104U/mg蛋白,酶活回收率为60.89%。重组ana-LOX最适反应温度45℃,最适反应pH 6.0,在常温下具有较好的稳定性,金属离子Fe2+、Mg2+、Ca2+对该酶存在明显的激活作用,而Fe3+和Cu2+对该酶有强烈的抑制作用。重组ana-LOX能够改善面团的显微结构。该研究获得了高效表达重组ana-LOX,为实现其在食品加工中的应用提供了参考。     

Uptake of Pb2+ by a cyanobacterium belonging to the genus Synechocystis, isolated from East Kolkata Wetlands

East Kolkata Wetlands is a conserved wetland utilizing sewage and garbage, generated by Kolkata Municipal Corporation area for cultivation purpose. Cyanobacteria are the photosynthetic prokaryotes having bioremedial capacity. We have isolated a cyanobacterium from the sewage recycling fish-pond of East Kolkata Wetlands. Partial sequence of 16S rDNA gene of the isolated strain showed 100% similarity with that of genus Synechocystis. Isolated strain and Synechocystis sp. PCC6803 survived up to 300 mug ml(-1) Pb(2+ )and growth was completely inhibited at 400 mug ml(-1) Pb(2+). All experiments were carried out with 100 mug ml(-1) Pb(2+) in which growth was the maximum. 91.67% of the total Pb(2+) got adsorbed to the outer surface of the cell and 1% of the total Pb(2+) entered the cell of the isolated strain as estimated by atomic absorption spectrometry, but in Synechocystis sp. PCC6803 72.72% adsorbed and 0.96% penetrated. Intracellular and periplasmic depositions of Pb(2+) were observed in both the strain. A filamentous structure developed outside the cell wall of the isolated cyanobacterium, but very little change was observed in Synechocystis sp. PCC6803. ZiaR-SmtB like regulator gene was expressed in both the strains after Pb(2+) induction. The cDNA sequence of ZiaR of the isolated cyanobacterium shows 100% homology with that of Synechocystis sp. PCC6803. Upon Pb(2+) induction, expression of SOD gene increased. cDNA sequence of the SOD gene from the isolated strain showed 98% homology with that of Synechocystis sp. PCC6803. Enzymatic activity of catalase and SOD was also increased. No DNA damage was monitored upon induction with Pb(2+).     

A Brownian dynamics study of the interaction of Phormidium cytochrome f with various cyanobacterial plastocyanins

Brownian dynamics simulations were used to study the role of electrostatic forces in the interactions of cytochrome f from the cyanobacterium Phormidium laminosum with various cyanobacterial plastocyanins. Both the net charge on the plastocyanin molecule and the charge configuration around H92 (H87 in higher plants) are important in determining the interactions. Those plastocyanins (PCs) with a net charge more negative than -2.0, including those from Synechococcus sp. PCC7942, Synechocystis sp. 6803, and P. laminosum showed very little complex formation. On the other hand, complex formation for those with a net charge more positive than -2.0 (including Nostoc sp. PCC7119 and Prochlorothrix hollandica) as well as Nostoc plastocyanin mutants showed a linear dependence of complex formation upon the net charge on the plastocyanin molecule. Mutation of charged residues on the surface of the PC molecules also affected complex formation. Simulations involving plastocyanin mutants K35A, R93A, and K11A (when present) showed inhibition of complex formation. In contrast, D10A and E17A mutants showed an increase in complex formation. All of these residues surround the H92 (H87 in higher plant plastocyanins) ligand to the copper. An examination of the closest electrostatic contacts shows that these residues interact with D63, E123, R157, D188, and the heme on Phormidium cytochrome f. In the complexes formed, the long axis of the PC molecule lies perpendicular to the long axis of cytochrome f. There is considerable heterogeneity in the orientation of plastocyanin in the complexes formed.     

patS minigenes inhibit heterocyst development of Anabaena sp. strain PCC 7120

The patS gene encodes a small peptide that is required for normal heterocyst pattern formation in the cyanobacterium Anabaena sp. strain PCC 7120. PatS is proposed to control the heterocyst pattern by lateral inhibition. patS minigenes were constructed and expressed by different developmentally regulated promoters to gain further insight into PatS signaling. patS minigenes patS4 to patS8 encode PatS C-terminal 4 (GSGR) to 8 (CDERGSGR) oligopeptides. When expressed by P(petE), P(patS), or P(rbcL) promoters, patS5 to patS8 inhibited heterocyst formation but patS4 did not. In contrast to the full-length patS gene, P(hepA)-patS5 failed to restore a wild-type pattern in a patS null mutant, indicating that PatS-5 cannot function in cell-to-cell signaling if it is expressed in proheterocysts. To establish the location of the PatS receptor, PatS-5 was confined within the cytoplasm as a gfp-patS5 fusion. The green fluorescent protein GFP-PatS-5 fusion protein inhibited heterocyst formation. Similarly, full-length PatS with a C-terminal hexahistidine tag inhibited heterocyst formation. These data indicate that the PatS receptor is located in the cytoplasm, which is consistent with recently published data indicating that HetR is a PatS target. We speculated that overexpression of other Anabaena strain PCC 7120 RGSGR-encoding genes might show heterocyst inhibition activity. In addition to patS and hetN, open reading frame (ORF) all3290 and an unannotated ORF, orf77, encode an RGSGR motif. Overexpression of all3290 and orf77 under the control of the petE promoter inhibited heterocyst formation, indicating that the RGSGR motif can inhibit heterocyst development in a variety of contexts.