Yukako Hihara

DNA Microarray Analysis of Cyanobacterial Gene Expression during Acclimation to High Light

DNA microarrays bearing nearly all of the genes of the unicellular cyanobacterium Synechocystis sp PCC 6803 were used to examine the temporal program of gene expression during acclimation from low to high light intensity. A complete pattern is provided of gene expression during acclimation of a photosynthetic organism to changing light intensity. More than 160 responsive genes were identified and classified into distinct sets. Genes involved in light absorption and photochemical reactions were downregulated within 15 min of exposure to high light intensity, whereas those associated with CO2 fixation and protection from photoinhibition were upregulated. Changes in the expression of genes involved in replication, transcription, and translation, which were induced to support cellular proliferation, occurred later. Several unidentified open reading frames were induced or repressed. The possible involvement of these genes in the acclimation to high light conditions is discussed.

DNA Microarray Analysis of Redox-Responsive Genes in the Genome of the Cyanobacterium Synechocystis sp. Strain PCC 6803

Whole-genome DNA microarrays were used to evaluate the effect of the redox state of the photosynthetic electron transport chain on gene expression in Synechocystis sp. strain PCC 6803. Two specific inhibitors of electron transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), were added to the cultures, and changes in accumulation of transcripts were examined. About 140 genes were highlighted as reproducibly affected by the change in the redox state of the photosynthetic electron transport chain. It was shown that some stress-responsive genes but not photosynthetic genes were under the control of the redox state of the plastoquinone pool in Synechocystis sp. strain PCC 6803.

Difference in metabolite levels between photoautotrophic and photomixotrophic cultures of Synechocystis sp. PCC 6803 examined by capillary electrophoresis electrospray ionization mass spectrometry

Capillary electrophoresis mass spectrometry (CE/MS) was applied for the comprehensive survey of changes in the amounts of metabolites upon the shift from photoautotrophic to photomixotrophic conditions in Synechocystis sp. PCC 6803. When glucose was added to the photoautotrophically grown culture, the increase in the metabolites for the oxidative pentose phosphate (OPP) pathway and glycolysis, together with the decrease in those for the Calvin cycle, was observed. Concomitantly, the increase in respiratory activity and the decrease in photosynthetic activity took place in the wild-type cells. In the pmgA-disrupted mutant that shows growth inhibition under photomixotrophic conditions, lower enzymatic activities of the OPP pathway and higher photosynthetic activity were observed, irrespective of trophic conditions. These defects brought about metabolic disorders such as a decrease in ATP and NADPH contents, a failure in the activation of respiratory activity, and the aberrant accumulation of isocitrate under photomixotrophic but not under photoautotrophic conditions. A delicate balancing of the carbon flow between the Calvin cycle and the OPP pathway seems indispensable for growth specifically under photomixotrophic conditions and PmgA is likely to be involved in the regulation.

The mechanism of the degradation of psaB gene product, one of the photosynthetic reaction center subunits of photosystem I, upon photoinhibition

The psaB gene product (PsaB protein), one of the reaction center subunits of Photosystem I (PS I), was specifically degraded by light illumination of spinach thylakoid membranes. The degradation of the protein yielded N-terminal fragments of molecular mass 51 kDa and 45 kDa. The formation of the 51 kDa fragment was i) partially suppressed by the addition of phenylmethylsulfonyl fluoride or 3,4-dichloroisocoumarin, which are inhibitors of serine proteases, and ii) enhanced in the presence of hydrogen peroxide during photoinhibitory treatment, but iii) not detected following hydrogen peroxide treatment in the dark. These results suggest that the hydroxyl radical produced at the reduced iron-sulfur centers in PS I triggers the conformational change of the PS I complex, which allows access of a serine-type protease to PsaB. This results in the formation of the 51 kDa N-terminal fragment, presumably by cleavage on the loop exposed to the stromal side, between putative helices 8 and 9. On the other hand, the formation of the 45 kDa fragment, which was enhanced in the presence of methyl viologen but did not accompany the photoinhibition of PS I, was not affected by the addition of hydrogen peroxide or protease inhibitors. Another fragment of 18 kDa was identified as a C-terminal counterpart of the 45 kDa fragment. N-terminal sequence analysis of the 18 kDa fragment revealed that the cleavage occurred between Ala500 and Val501 on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.

An AbrB-Like Transcriptional Regulator, Sll0822, Is Essential for the Activation of Nitrogen-Regulated Genes in Synechocystis sp. PCC 6803

Every cyanobacterial species possesses multiple genes encoding AbrB-like transcriptional regulators (cyAbrBs) distinct from those conserved among other bacterial species. In this study, two genes encoding cyAbrBs in Synechocystis sp. PCC 6803, sll0359 and sll0822, were insertionally disrupted in order to examine their physiological roles. A fully segregated disrupted mutant of sll0822 (Δsll0822 mutant) but not of sll0359 was obtained, although both mutants exhibited similar phenotypes (i.e. decreases in growth rate and pigment content). The growth rate of the Δsll0822 mutant was low under any condition, but the low pigment content could be partially recovered by nitrate supplementation of the medium. DNA microarray and RNA-blot analyses revealed that the level of expression of a part of the NtcA regulon, such as urtA, amt1, glnB, sigE, and the nrt operon, was significantly decreased in the Δsll0822 mutant, although the induction of these genes upon nitrogen depletion was still observed to some extent. Sll0822 seems to work in parallel with NtcA to achieve flexible regulation of the nitrogen uptake system. The Sll0822 protein exists mainly in a dimeric form in vivo, and the amount of the protein was not affected by nitrogen availability. This observation, together with the low binding specificity of the purified histidine-tagged Sll0822 protein, implies that the activity of Sll0822 may be posttranslationally modulated in Synechocystis cells.

Isolation and characterization of two cDNA clones for mRNAs that are abundantly expressed in immature anthers of rice (Oryza sativa L.)

The relationship between the length of anthers and the stage of development of microspores was examined in rice (Oryza sativa L. cv. Hayayuki). Anthers of ≤2 mm and 2.1–2.2 mm in length and those ready to dehiscence were determined to be at the uninucleate, binucleate and trinucleate microspore stage, respectively.Two cDNAs (YY1 and YY2), representing genes that are specifically expressed in anthers at the uninucleate microspore stage, were isolated and characterized. YY1 cDNA encoded an open reading frame of 95 amino acids. Eight cysteine residues with the potential to form disulfide bridges were present in the amino acid sequence. There was a hydrophobic region at the N-terminus of the putative protein, suggesting that the YY1 protein might be secreted. This cysteine motif and the hydrophobic N-terminus are conserved among products of several anther-specific genes or cDNAs isolated from various plant species. These proteins are thought to form a superfamily of proteins that are confined to anthers. The YY1 transcript was localized in the tapetal cells and the peripheral cells of the vascular bundle. YY2 cDNA encoded an open reading frame of 389 amino acids and the deduced amino acid sequence exhibited substantial homology to that of chalcone synthase. Expression of YY2 mRNA was confined to the tapetal cells. The genes correspond to YY1 and YY2 cDNAs were shown to exist as single copies in the rice genome.

A cyanobacterial AbrB-like protein affects the apparent photosynthetic affinity for CO2 by modulating low-CO2-induced gene expression

In Synechocystis sp. strain PCC 6803, over 450 genes are upregulated following transfer of the cells from a high (1-5% CO(2) in air, HC) to a low level of CO(2) (as in air or lower, LC). This includes sbtA, ndhF3 and cmpA involved in inorganic carbon (Ci) uptake. Earlier studies implicated NdhR in the regulation of LC-induced genes but there are indications that additional components are involved. Following extraction of proteins from cells grown under HC and (NH4)(2)SO(4) fractionation, we have identified LexA and two AbrB-like proteins, Sll0359 and Sll0822, which bind to a fragment of the sbtA promoter. Using extracts prepared from LC-grown cells, Sll0822 did not bind to the sbtA promoter despite its presence in the cells, suggesting that it may serve as a repressor of LC-induced genes. This is supported by the fact that sbtA, ndhF3 and cmpA normally expressed only under LC in the wild-type are transcribed under both HC and LC in a Deltasll0822 mutant. When grown under HC this mutant exhibits an elevated apparent photosynthetic affinity to Ci, typically observed in the wild-type only under LC. Clearly, expression of genes essential for Ci uptake was sufficient to raise the apparent photosynthetic affinity for external Ci.

Mutation in a novel gene required for photomixotrophic growth leads to enhanced photoautotrophic growth of Synechocystis sp. PCC 6803

In the glucose-tolerant strain of Synechocystis sp. PCC 6803, we found two types of cells with distinct growth properties. Under photoautotrophic conditions at any light intensity, one type gave larger colonies (designated WL) than the other (designated WS). Notably, the WL cells produced much larger colonies than the WS cells at higher light intensity. In contrast, growth of the WL cells was severely suppressed under mixotrophic conditions with glucose and light, while the WS cells grew normally. A gene which could complement the WL phenotype was obtained from a wild-type genomic library. The gene, designated pmgA, coded for a 23 kDa polypeptide of 204 amino acid residues with no apparent homology to known genes. In the WL genome, the base substitution of T for C at position 193 of pmgA caused replacement of Leu with Phe at position 65 of the product. The phenotype of pmgA disruption mutants was similar to that of the WL cells, indicating that the WS cells expressed a functional pmgA product. By direct sequencing of polymerase chain reaction-amplified pmgA from genomic DNA, it was revealed as an example of microevolution that WL had expelled WS from the photoautotrophic culture of wild-type in our laboratory for a year or so. Mixed culture in liquid also demonstrated that the WL cells increased gradually under photoautotrophic conditions, while they decreased rapidly under photomixotrophic conditions. These results suggest that pmgA product is essential for photomixotrophic growth, whereas it represses photoautotrophic growth. To our knowledge, the WL cells and pmgA-disrupted mutants are the first in cyanobacteria, which shows much improved photosynthetic growth than wild-type especially at high light intensity.

The Mutant of sll1961, Which Encodes a Putative Transcriptional Regulator, Has a Defect in Regulation of Photosystem Stoichiometry in the Cyanobacterium Synechocystis sp. PCC 6803

In acclimation to changing light environments, photosynthetic organisms modulate the ratio of two photosynthetic reaction centers (photosystem I [PSI] and photosystem II). One mutant, which could not modulate photosystem stoichiometry upon the shift to high light, was isolated from mutants created by random transposon mutagenesis. Measurements of chlorophyll fluorescence and analysis of the reaction center subunits of PSI through western blotting in this mutant revealed that the content of PSI could not be suppressed under high-light condition. In the mutant, transposon was inserted to the sll1961 gene encoding a putative transcriptional regulator. DNA microarray analysis revealed that the expression of sll1773 was drastically induced in the sll1961 mutant upon exposure to high light for 3 h. Our results demonstrate that a transcriptional regulator, Sll1961, and its possible target proteins, including Sll1773, may be responsible for the regulation of photosystem stoichiometry in response to high light.

The Response Regulator RpaB Binds to the Upstream Element of Photosystem I Genes To Work for Positive Regulation under Low-Light Conditions in Synechocystis sp. Strain PCC 6803

The coordinated high-light response of genes encoding subunits of photosystem I (PSI) is achieved by the AT-rich region located just upstream of the core promoter in Synechocystis sp. strain PCC 6803. The upstream element enhances the basal promoter activity under low-light conditions, whereas this positive regulation is lost immediately after the shift to high-light conditions. In this study, we focused on a high-light regulatory 1 (HLR1) sequence included in the upstream element of every PSI gene examined. A gel mobility shift assay revealed that a response regulator RpaB binds to the HLR1 sequence in PSI promoters. Base substitution in the HLR1 sequence or decrease in copy number of the rpaB gene resulted in decrease in the promoter activity of PSI genes under low-light conditions. These observations suggest that RpaB acts as a transcriptional activator for PSI genes. It is likely that RpaB binds to the HLR1 sequence under low-light conditions and works for positive regulation of PSI genes and for negative regulation of high-light-inducible genes depending on the location of the HLR1 sequence within target promoters.