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Organization of Photosystem I Polypeptides (A Structural Interaction between the PsaD and PsaL Subunits).

The wild-type, PsaD-less, and PsaL-less strains of the cyanobacterium Synechocystis sp. PCC 6803 were used to study subunit interactions in photosystem I (PSI). When the membranes of a PsaD-less strain were solubilized with Triton X-100 and PSI was purified using ion-exchange chromatography and sucrose-gradient ultracentrifugation, the PsaL subunit was substantially removed from the core of PSI, whereas other subunits, such as PsaE and PsaF, were quantitatively retained during purification. When the wild-type PSI was exposed to increasing concentrations of NaI, the PsaE, PsaD, and PsaC subunits were gradually removed, whereas PsaF, PsaL, PsaK, and PsaJ resisted removal by up to 3 M NaI. The absence of PsaL enhanced the accessibility of PsaD to removal by NaI. Treatment of the wild-type PSI complexes with glutaraldehyde at 4[deg] C resulted in a 29-kD cross-linked product between PsaD and PsaL. The formation of such cross-linked species was independent of PSI concentrations, suggesting an intracomplex cross-linking between PsaD and PsaL. Taken together, these results demonstrate a structural interaction between PsaD and PsaL that plays a role in their association with the PSI core.

Journal Article↗

Organization of photosystem I polypeptides. Identification of PsaB domains that may interact with PsaD.

PsaA and PsaB are homologous integral membrane-proteins that form the heterodimeric core of photosystem i (PSI). We used subunit-deficient PSI complexes from the mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 to examine interactions between PsaB and other PSI subunits. Incubation of the wild-type PSI with thermolysin yielded 22-kD C-terminal fragments of PsaB that were resistant to further proteolysis. Modification of the wild-type PSI with N-hydroxysuccinimidobiotin and subsequent cleavage by thermolysin showed that the lysyl residues in the 22-kD C-terminal domain were inaccessible to modification by N-hydroxysuccinimidobiotin. The absence of PsaE, PsaF, PsaI, PsaJ, or PsaL facilitated accumulation of 22-kD C-terminal fragments of PsaB but did not alter their resistance to further proteolysis. When the PsaD-less PSI was treated with thermolysin, the 22-kD C-terminal fragments of PsaB were rapidly cleaved, with concomitant accumulation of a 16-kD fragment and then a 3.4-kD one. We mapped the N termini of these fragments by N-terminal amino acid sequencing and the C termini from their positive reaction with an antibody against the C-terminal peptide of PsaB. The cleavage sites were proposed to be in the extramembrane loops on the cytoplasmic side. Western blot analyses showed resistance of PsaC and PsaI to proteolysis prior to cleavage of the 22-kD fragments. Therefore, we propose that PsaD shields two extramembrane loops of PsaB and protects the C-terminal domain of PsaB from in vitro proteolysis.

Amino Acid Sequence↗

Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I.

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent N-ethyl-3-(3-dimethylaminopropyl) carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes. The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP(+) reductase.

Journal Article↗

A two-component system that controls the expression of pneumococcal surface antigen A (PsaA) and regulates virulence and resistance to oxidative stress in Streptococcus pneumoniae.

Recent genomic-based studies have identified 13 two-component signal transduction systems (TCS) in Streptococcus pneumoniae. Bacterial TCSs are important for regulating expression of bacterial genes, including those which are important to the virulence of pathogenic bacteria. We have used virulence assays together with microarray analysis to investigate the importance of pneumococcal TCS04 in the virulence and gene regulation of this pathogen. Deletion mutants of the response regulator of TCS04, rr04, were examined in three independent pneumococcal strains representing three different pneumococcal serotypes. Analysis of the virulence of the three strains enabled us to identify a serotype-specific attenuation of virulence due to deletion of rr04. Microarray comparison of the transcriptional profiles of the wild-type strains with the rr04 mutants allowed us to determine which transcriptional changes were occurring in the rr04 mutants. Virulence-associated changes were demonstrated in the attenuated strain with significant downregulation of a previously determined virulence locus, psaB, psaC and psaA.

ATP-Binding Cassette Transporters↗

Molecular analysis of the psa permease complex of Streptococcus pneumoniae.

The psaBCA locus of Streptococcus pneumoniae encodes a putative ABC Mn2+-permease complex. Downstream of the operon is psaD, which may be co-transcribed and encodes a thiol peroxidase. Previously, there has been discordance concerning the phenotypic impact of mutations in the psa locus, resolution of which has been complicated by differences in mutant construction and the possibility of polar effects. Here, we constructed unmarked, in frame deletion mutants DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaD, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD in S. pneumoniae D39 to examine the role of each gene within the locus in Mn2+ uptake, susceptibility to oxidative stress, virulence, nasopharyngeal colonization and chain morphology. The requirement for Mn2+ for growth and transformation was also investigated for all mutants. Inductively coupled plasma mass spectrometry (ICP-MS) analysis provided the first direct evidence that PsaBCA is indeed a Mn2+ transporter. However, this study did not substantiate previous reports that the locus plays a role in choline-binding protein pro-duction or chain morphology. We also confirmed the importance of the Psa permease in systemic virulence and resistance to superoxide and hydrogen peroxide, as well as demonstrating a role in nasopharyngeal colonization for the first time. Further evi-dence is provided to support the requirement for Mn2+ supplementation for growth and transformation of DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD mutants. However, transformation, as well as growth, of the DeltapsaD mutant was not dependent upon Mn2+ supplementation. We also show that, apart from sensitivity to hydrogen peroxide, the DeltapsaD mutant exhibited essentially similar phenotypes to those of the wild type. Western blot analysis with a PsaD antiserum showed that deleting any of the genes upstream of psaD did not affect its expression. However, we found that deleting psaB resulted in decreased expression of PsaA relative to that in D39, whereas deleting both psaB and psaC resulted in at least wild-type levels of PsaA.

Animals↗

Brassica rapa plants adapted to microgravity with reduced photosystem I and its photochemical activity.

The photosynthetic apparatus contains several protein complexes, many of which are regulated by environmental conditions. In this study, the influences of microgravity on PSI and PSII in Brassica rapa plants grown aboard the space shuttle were examined. We found that Brassica plants grown in space had a normal level of growth relative to controls under similar conditions on Earth. Upon return to Earth, cotyledons were harvested and thylakoid membranes were isolated. Analysis of chlorophyll contents showed that the Chl a/b ratio (3.5) in flight cotyledons was much higher than a ratio of 2.42 in the ground controls. The flight samples also had a reduction of PSI complexes and a corresponding 30% decrease of PSI photochemical activity. Immunoblotting showed that the reaction centre polypeptides of PSI were more apparently decreased (e.g. by 24-33% for PsaA and PsaB, and 57% for PsaC) than the light-harvesting complexes. In comparison, the accumulation of PSII complex was less affected in microgravity, thus only a slight reduction in D1, D2 and LHCII was observed in protein blots. However, there was a 32% decrease of OEC1 in the flight samples, indicating a defective OEC subcomplex. In addition, an average 54% increase of the 54 kDa CF1-beta isoform was found in the flight samples, suggesting that space-grown plants suffered from certain stresses, consistent with implications of the increased Chl a/b ratio. Taken together, the results demonstrated that Brassica plants can adapt to spaceflight microgravity, but with significant alterations in chloroplast structures and photosynthetic complexes, and especially reduction of PSI and its activity.

Biomass↗

Isolation and structural characterization of trimeric cyanobacterial photosystem I complex with the help of recombinant antibody fragments.

A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb. A one-step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N-terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/Fv was 139 +/- 9 chlorophyll a/primary electron donor. Flash-induced absorption-change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe-S center, FA/FB. The position of the bound Fv fragment on the trimeric PSI surface was determined by high-resolution electron microscopy and digital image processing.

Amino Acid Sequence↗

Protective effect of active oxygen scavengers on protein degradation and photochemical function in photosystem I submembrane fractions during light stress.

The protective role of reactive oxygen scavengers against photodamage was studied in isolated photosystem (PS) I submembrane fractions illuminated (2000 microE x m(-2) x s(-1)) for various periods at 4 degrees C. The photochemical activity of the submembrane fractions measured as P700 photooxidation was significantly protected in the presence of histidine or n-propyl gallate. Chlorophyll photobleaching resulting in a decrease of absorbance and fluorescence, and a blue-shift of both absorbance and fluorescence maximum in the red region, was also greatly delayed in the presence of these scavengers. Western blot analysis revealed the light harvesting antenna complexes of PSI, Lhca2 and Lhca1, were more susceptible to strong light when compared to Lhca3 and Lhca4. The reaction-center proteins PsaB, PsaC, and PsaE were most sensitive to strong illumination while other polypeptides were less affected. Addition of histidine or n-propyl gallate lead to significant protection of reaction-center proteins as well as Lhca against strong illumination. Circular dichroism (CD) spectra revealed that the alpha-helix content decreased with increasing period of light exposure, whereas beta-strands, turns, and unordered structure increased. This unfolding was prevented with the addition of histidine or n-propyl gallate even after 10 h of strong illumination. Catalase or superoxide dismutase could not minimize the alteration of PSI photochemical activity and structure due to photodamage. The specific action of histidine and n-propyl gallate indicates that 1O2 was the main form of reactive oxygen species responsible for strong light-induced damage in PSI submembrane fractions.

Circular Dichroism↗

Bundle sheath cell-specific expression of chloroplast genes encoding subunits of the NADH dehydrogenase-like complex in maize.

C4 photosynthesis alleviates the limitation caused by the oxygenase activity of Rubisco by partitioning photosynthetic functions between two distinct cell types: bundle sheath cells (BSCs) and mesophyll cells (MCs). These cell types perform different steps of photosynthesis using specialized machinery, accompanied by differential expression of chloroplast genes. To uncover the underlying molecular mechanisms for this differentiation, we isolated BSCs and MCs and compared their chloroplast transcriptomes, focusing on the chloroplast NADH dehydrogenase-like (NDH) complex, which is enriched in BSCs. To investigate whether RNA stabilization contributes to differential gene expression, we analyzed RNA footprints that reflect the binding of pentatricopeptide repeat (PPR) proteins to their RNA targets. We could not detect cell-type-specific accumulation of footprint RNAs. We then focused on transcriptional regulation, specifically on an operon that starts with the rps15 gene. The operon includes six ndh genes and the psaC gene encoding a photosystem I subunit. Transcript levels of all genes in this operon were higher in BSCs than in MCs, suggesting coordinated regulation as a transcriptional unit. Based on the genomic location of the rps15 gene within inverted repeats near the junctions on both sides of the small single copy region, we demonstrated that rps15, through two distinct promoters, is sufficient to drive preferential accumulation of downstream transcripts in BSCs.

Zea mays↗

Energy needs versus environmental pollution: a reconciliation?

In this article I have presented, for discussion, a proposed system for energy generation by which the principal sources of environmental pollution by power plants could be eliminated. For stationary power plants the concept appears feasible technically and, according to my " horseback estimates," perhaps economically as well, depending upon the economic value of the by-products of sulfur, CO(2), water, and possibly nitrogen, and upon the price we are willing to pay for a clean environment .Thus, a more thorough engineering and economic analysis to explore these and other factors in greater depth seems warranted. In the case of turbine-driven vehicles, the technical and economic feasibility of widespread distribution and handling of the fuel constitutes a serious question, but one which deserves equally serious consideration before the possibility is discounted. The reports of the cited study panels notwithstanding, the technology required for the proposed system exists today, with one exception. This exception (which is not essential for trial of the system but will be required for its complete fruition) is the development of a nuclear reactor for the prime purpose of delivering process heat for the steam reforming of natural gas and, ultimately, for gas production from coal in a continuous process, such as those discussed by Pieroni et al. (16). Today's intermittent processes of coking and gas production are both archaic and themselves large sources of atmospheric pollution, and a development program aimed at advancing the technology of the coal industry in this regard would seem long overdue. The report of the PSAC Environmental Pollution Panel recommended "demonstration of the feasibility and economy of new developments for abating or controlling pollution through their use at Federal installations" and suggested the coalburning TVA power plants as a likely place for such demonstration. This suggestion is doubly appropriate since the TVA is in a region of subnormal " atmospheric ventilation" (8). By design these plants are adjacent to the AEC's Oak Ridge National Laboratory, and such a location would seem ideal for an experiment on the wedding of nuclear and fossil sources of energy. In comments on a preliminary draft of this article, proponents of "conventional " nuclear power pointed out that such power is hard to beat on the basis of cost, and that dissipation of heat to the air by way of cooling towers can also be accomplished in conventional plants (17). These observations are individually correct but not compatible: the low power costs cited are for very large plants [of the order of 1000 mega-watts lectrical) and larger], and the costs of cooling towers and associated equipment needed to dissipate such large amounts of heat [of the order of 2000 megawatts (thermal)] to air from a closed cycle would offset the power cost advantage of the large plant. In regard to the proposed use of nuclear process heat, Weinberg (20) has expressed doubt that much advantage can be derived from this approach because the temperatures involved are too high for low-cost reactors, and heat transfer from surfaces could involve materials problems. In the case of gas production, this is indeed an anticipated problem-not a technologically insuperable one, but a problem of reducing the cost of the materials required (16). Indeed, Weinberg himself has mentioned this possible use of nuclear heat in a recent publication discussing the steam reforming of coal to liquid fuel(21). Also, an improved process for synthesizing methane from lignitec has recently been reported (22). Since the earlier studies date back a decade, a new look at the problems and costs involved relative to the benefits to be derived (not the least of which could be new vigor for the coal industry) would seem to be in order. In the case of steam reforming of natural gas, the temperature level (about 1500 degrees F) is such that the technology is available today, and a process-heat-reactor design study could be initiated without awaiting further developments.

Air Pollution↗

The sufR gene (sll0088 in Synechocystis sp. strain PCC 6803) functions as a repressor of the sufBCDS operon in iron-sulfur cluster biogenesis in cyanobacteria.

The suf operon is composed of four genes (sufB, sufC, sufD, and sufS) and is highly conserved in the genomes of cyanobacteria. Open reading frame sll0088 in Synechocystis sp. strain PCC 6803 is located near the 5' end of the suf operon but is transcribed in the direction opposite that of the suf operon. We previously reported the isolation of two independent suppressor strains of C14S(PsaC) that mapped to sll0088 and restored photoautotrophic growth. The protein encoded by sll0088 has two significant features: (i) a DNA-binding domain near the N terminus and (ii) four highly conserved cysteine residues near the C terminus. The protein has high sequence similarity to transcription regulatory proteins with a conserved DNA-binding domain and can be classified in the DeoR family of helix-loop-helix proteins. The protein falls into a further subclass that contains a C-X(12)-C-X(13)-C-X(14)-C motif near the C terminus, which may represent a metal-binding site. The expressed Sll0088 protein harbored an iron-sulfur cluster as shown by optical and electron paramagnetic resonance spectroscopy. Compared to the wild type, expression levels of the sufBCDS genes were elevated when cells were grown under conditions of oxidative and iron stress and were even higher in a null mutant of Synechococcus sp. strain PCC 7002 in which the sll0088 homolog was insertionally inactivated. In agreement with the proposed role of the sufBCDS genes in iron metabolism, the growth rate of the null mutant was significantly higher than that of the wild type under iron-limiting conditions. We propose that the protein encoded by sll0088 is a transcriptional repressor of the suf operon, and we name the gene sufR.

Amino Acid Sequence↗

Evolutionary dynamics of the chloroplast genome in Abutilon (Malvoideae, Malvaceae).

The genus Abutilon Mill. (Malvaceae) comprises approximately 178 species distributed across tropical and subtropical regions, many of which hold significant ornamental, economic, and medicinal value; yet its taxonomic classification remains challenging. In this study, six species were sequenced from herbarium specimens, and the chloroplast (cp.) genomes of ten additional species were assembled de novo from publicly available raw data. Three previously reported cp. genomes were also incorporated to characterise cp. genome structure, identify polymorphic loci, and perform phylogenetic analyses. The cp. genomes ranged from 159,458 to 160,454 bp and exhibited the typical quadripartite structure, with each genome containing 112 unique genes (78 protein-coding, 30 tRNA, and 4 rRNA) that showed conserved content and organisation. These genomes exhibited high similarity in GC content, inverted repeat boundaries, relative synonymous codon usage, amino acid frequencies, and substitution patterns. However, notable variation was observed in the total number of simple sequence repeats, ranging from 70 to 97 per genome. Selection analyses indicated predominant purifying selection, with evidence of episodic positive selection detected in rpoC2, rbcL, and ycf1. Two codons in rbcL were clade-specific and provided phylogenetic signal distinguishing Australian and Old World pantropical species. Nucleotide diversity analysis identified six highly polymorphic intergenic spacers (trnH-psbA, rps19-rpl2, psbT-pbf1, psaC-ndhD, trnR-atpA, and ndhJ-ndhK) that may be suitable for taxonomic studies. The phylogeny from maximum likelihood (ML) and Bayesian inference (BI) resolved two major clades: one comprising an exclusively Australian lineage occurring predominantly in arid and semi-arid environments, and the other a pantropical lineage spanning multiple continents. Abutilon grandifolium was recovered as sister to the remaining sampled Abutilon taxa in both ML and BI analyses, although no biogeographic origin inference can be drawn from this placement pending broader taxon sampling and integration of nuclear genomic data. These findings provide insights into the evolutionary dynamics of the cp. genome in Abutilon and offer a foundational genomic framework for refining Abutilon taxonomy.

Genome, Chloroplast↗

Isolation of photosystem I particles from spinach.

A method to prepare photosystem I (PSI) particles is described. Spinach leaves are used to prepare broken chloroplasts that are then solubilized by using a detergent (Triton X-100). Solubilized chloroplasts are then applied on an ion-exchange column. Eluted by a linear concentration gradient of NaCl, fractions enriched in PSI particles are collected and applied on a small hydroxyapatite column. By eluting with phosphate buffer, a concentrated preparation of PSI particles is obtained. The particles consist of PsaA, PsaB, PsaC, PsaD, PsaE, and PsaY. Assay methods that involve SDS-PAGE and P700 determination are also presented.

Chloroplasts↗

The amino-acid sequence of three proteins of photosystem I of the cyanobacterium Fremyella diplosiphon (Calothrix sp PCC 7601).

Three proteins containing 138 amino acids (psaD protein), 80 amino acids (psaC protein) and 66 amino acids (psaE protein) of the photosystem I (PS I) complex of the cyanobacterium Fremyella diplosiphon (Calothrix sp PCC 7601) were isolated and sequenced. Comparison with previously known sequences showed a close relationship to homologous proteins of Nostoc, another filamentous cyanobacterium.

Amino Acid Sequence↗

Ultrastructural observation of altered chloroplast morphology in space-grown Brassica rapa cotyledons.

Photosynthesis will be indispensable in a bioregenerative life-support systems for long space missions. It is critical understand the effects of space on this complex process, especially the loss of gravity. Past has noted changes in plant growth and development; differences about cell size, shape, division, and differentiation; and plastid distribution and structure alterations. The amyloplast-containing columelar cells in root tips were carefully examined since they are likely gravity-sensing sites. Changes on photosynthetic physiology and chloroplast structure have been reported. Both increases and decreases of chlorophyll and carotenoid contents were reported. Structural changes of thylakoid membranes in chloroplasts were observed in pea and Arabidopsis grown in space or clinorotation. Recently, a decrease of CO2 assimilation rate and of electron transport rate of both PSI and PSII on thylakoid membranes were reported in space-grown wheat. These imply an overall decrease of photosynthetic activities, and implicate thylakoid-old structural changes. For example, PSI activity, and its reaction center subunits (PsaA, PsaB, and PsaC) and the LHCIs, were decreased under microgravity. Here, we further examined cellular morphology and ultrastructural features of the chloroplast and its thylakoid membranes by electron microscopy and in situ immunolocalization.

Brassica↗

[Bioinformatics studies on photosynthetic system genes in cyanobacteria and chloroplasts].

This study compared homology of base sequences in genes encoding photosynthetic system proteins of cyanobacteria (Synechocystics sp. PCC6803, Nostoc sp. PCC7120) with these of chloroplasts (from Marchantia Polymorpha, Nicotiana tobacum, Oryza sativ, Euglena gracilis, Pinus thunbergii, Zea mays, Odentella sinesis, Cyanophora paradoxa, Porphyra purpurea and Arabidopsis thaliana) by BLAST method. While the gene sequence of Synechocystics sp. PCC6803 was considered as the criterion (100%) the homology of others were compared with it. Among the genes for photosystem I, psaC homology was the highest (90.14%) and the lowest was psaJ (52.24%). The highest ones were psbD (83.71%) for photosystem II, atpB (79.58%) for ATP synthase and petB (81.66%) for cytochrome b6/f complex. The lowest ones were psbN (49.70%) for photosystem II, atpF (26.69%) for ATP synthase and petA (55.27%) for cytochrome b6/f complex. Also, this paper discussed why the homology of gene sequences was the highest or the lowest. No report has been published and this bioinformatics research may provide some evidences for the origin and evolution of chloroplasts.

Chloroplast Proton-Translocating ATPases↗

Purification and characterization of the photosystem I complex from the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

A photoactive photosystem I complex has been purified from the filamentous, nitrogen-fixing cyanobacterium Anabaena variabilis ATCC 29413. Cells were broken using glass beads, and the membrane fraction was solubilized with beta-dodecyl maltoside followed by two rounds of fast protein liquid chromatography on anion exchange columns. The polypeptide composition of the isolated complex was determined by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis and N-terminal amino acid sequencing of the fractionated proteins. The purified complex consists of at least 11 proteins, identified as the PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaI, PsaJ, PsaK, PsaL, and PsaN proteins. The spectrum of the flash-induced absorbance change measured between 670 and 830 nm shows that the purified complex contains 99 +/- 11 chlorophyll a molecules per P700, the primary donor in photosystem I. The kinetics of the rereduction of oxidized P700 following an actinic flash indicate that forward electron transfer from P700 to the FA/FB iron-sulfur center acceptors is functional in the isolated complex.

Amino Acid Sequence↗

Science, government, and information: 1988 perspective.

Twenty-five years have passed since the appearance of Science, Government, and Information (SGI), the 1963 report by the President's Science Advisory Committee (PSAC). The Information Analysis Center, which was prominently recommended in SGI, has not become a central element of the information system. Instead automation, which was only beginning in 1963, has become dominant. Though extreme automation may be appropriate for those activities that are time constrained, it may lead to clogged information channels for those scientific activities for which time is less important than depth of understanding.

Government↗