Tatsuo Omata

Stoichiometry of components in the photosynthetic oxygen evolution system of Photosystem II particles prepared with Triton X-100 from spinach chloroplasts

Abstract The stoichiometry of the proteins of the photosynthetic oxygen evolution system and of the electron transport components in Photosystem II particles prepared with Triton X-100 from spinach chloroplasts were determined. Per about 220 chlorophyll molecules, there were one reaction center II, one molecule each of the 33, 24 and 18 kDa proteins, four Mn atoms, two cytochromes b -559 (one high-potential, the other low-potential), and 3.5 plastoquinone-9 molecules, but practically no cytochrome b -563, cytochrome f , phylloquinone, α-tocopherol or α-tocopherylquinone.

Transcriptional activation of NtcA-dependent promoters of Synechococcus sp. PCC 7942 by 2-oxoglutarate in vitro

The transcription factor NtcA is a global regulator of nitrogen homeostasis in cyanobacteria. It thus positively regulates the expression of genes related to nitrogen assimilation such as glnA (which encodes glutamine synthetase) and ntcA itself in response to nitrogen shortage or depletion. The binding of NtcA to the glnA and ntcA promoters of Synechococcus sp. PCC 7942 in vitro now has been shown to be enhanced by 2-oxoglutarate. In vitro analysis of gene transcription also revealed that the interaction of NtcA with its promoter element was not sufficient for activation of transcription, and 2-oxoglutarate was required for transcriptional initiation by NtcA. Given that the intracellular concentration of 2-oxoglutarate is inversely related to nitrogen availability, it is proposed that this metabolite functions as a signaling molecule that transmits information on cellular nitrogen status to NtcA and thereby regulates the transcription of genes related to nitrogen assimilation in cyanobacteria.

A RAPID AND EFFICIENT METHOD TO PREPARE CHLOROPHYLL A AND B FROM LEAVES

Abstract— Highly purified Chi a and b were prepared from spinach leaves in a short time by a combined use of the column chromatography with DEAE‐Sepharose CL‐6B and Sepharose CL‐6B. The former chromatography eliminated carotenoids, phaeophytin and chlorophyllide, and the latter chromatography efficiently separated Chi a and b.

Modes of active inorganic carbon uptake in the cyanobacterium, Synechococcus sp. PCC7942

Cyanobacteria (blue-green algae) have evolved a remarkable environmental adaptation for survival at limiting CO2 concentrations. The adaptation is known as a CO2 concentrating mechanism, and functions to actively transport and accumulate inorganic carbon (Ci; HCO3- and CO2) within the cell. Thereafter, this Ci pool is utilised to provide elevated CO2 concentrations around the primary CO2 fixing enzyme, Rubisco, which is encapsulated in a unique micro-compartment known as the carboxysome. Recently, significant progress has been gained in understanding the different types of Ci transport in cyanobacteria. This semi-review centres on the model cyanobacterium, Synechococcus sp. PCC7942, which possesses at least four distinct modes of Ci uptake when grown under Ci limitation, each possessing a high degree of functional redundancy. The four modes so far identified are: (i) BCT1, an inducible, high affinity HCO3- transporter of the bacterial ATP binding cassette transporter family, encoded by cmpABCD; (ii) a constitutive, Na+-dependent HCO3- transport system that can be allosterically activated (possibly by phosphorylation) in as little as 10 min; (iii) and (iv) two CO2 uptake systems, one constitutive and the other inducible, based on specialised forms of thylakoid-based, type 1, NAD(P)H dehydrogenase complexes (NDH-1). Here, we forward a speculative model that proposes that two unique proteins, ChpX and ChpY, possess CO2 hydration activity in the light, and when coupled to photosynthetic electron transport through the two specialised NDH-1 complexes, result in net hydration of CO2 to HCO3- as a crucial component of the CO2 uptake process.

Involvement of a CbbR Homolog in Low CO2-Induced Activation of the Bicarbonate Transporter Operon in Cyanobacteria

The cmpABCD operon of Synechococcus sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter, is transcribed only under CO2-limited conditions. In Synechocystis sp. strain PCC 6803, the slr0040, slr0041, slr0043, and slr0044 genes, forming an operon with a putative porin gene (slr0042), were identified as the cmpA, cmpB, cmpC, and cmpD genes, respectively, on the basis of their strong similarities to the corresponding Synechococcus cmp genes and their induction under low CO2 conditions. Immediately upstream of and transcribed divergently from the Synechocystis cmp operon is a gene (sll0030) encoding a homolog of CbbR, a LysR family transcriptional regulator of the CO2 fixation operons of chemoautotrophic and purple photosynthetic bacteria. Inactivation of sll0030, but not of another closely related cbbR homolog (sll1594), abolished low CO2 induction of cmp operon expression. Gel retardation assays showed specific binding of the Sll0030 protein to the sll0030-cmpA intergenic region, suggesting that the protein activates transcription of the cmp operon by interacting with its regulatory region. A cbbR homolog similar to sll0030 and sll1594 was cloned from Synechococcus sp. strain PCC 7942 and shown to be involved in the low CO2-induced activation of the cmp operon. We hence designated the Synechocystis sll0030 gene and the Synechococcus cbbR homolog cmpR. In the mutants of the cbbR homologs, upregulation of ribulose-1,5-bisphosphate carboxylase/oxygenase operon expression by CO2 limitation was either unaffected (strain PCC 6803) or enhanced (strain PCC 7942), suggesting existence of other low CO2-responsive transcriptional regulator(s) in cyanobacteria.

Substrate-binding Lipoprotein of the Cyanobacterium Synechococcus sp. Strain PCC 7942 Involved in the Transport of Nitrate and Nitrite

Of the four genes (nrtABCD) required for active transport of nitrate in the cyanobacterium Synechococcus sp. strain PCC 7942, nrtBCD encode membrane components of an ATP-binding cassette transporter involved in the transport of nitrite as well as of nitrate, whereas nrtA encodes a 45-kDa cytoplasmic membrane protein, the biochemical function of which remains unclear. Characterization of the nrtA deletional mutants showed that the 45-kDa protein is essential for the functioning of the nitrate/nitrite transporter. A truncated NrtA protein lacking the N-terminal 81 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, was shown to bind nitrate and nitrite with high affinity (Kd = 0.3 μM). Immunoblotting analysis using the antibody against the 45-kDa protein revealed a 48-kDa precursor of the protein, which accumulated in the cyanobacterial cells treated with globomycin, an antibiotic that specifically inhibits cleavage of the signal peptide of lipoprotein precursors. These findings indicated that the nrtA gene product is a nitrate- and nitrite-binding lipoprotein. The N-terminal sequences of putative cyanobacterial substrate-binding proteins suggested that lipoprotein modification of substrate-binding proteins of ATP-binding cassette transporters is common in cyanobacteria.

[23] Isolation of cyanobacterial plasma membranes

Publisher Summary This chapter focuses on the isolation of cyanobacterial plasma membranes. Cyanobacteria are classified as gram-negative bacteria, in which the cell envelope is composed of outer and inner (plasma or cytoplasmic) membranes with a peptidoglycan layer in between. In the cytoplasm are the thylakoid membranes, which are the site of light capturing, electron transport, and ATP synthesis. The thylakoid membranes represent most of the total cellular membranes, and this situation made it difficult for a long time to purify plasma membranes from the cyanobacteria. Subsequently developed methods to isolate plasma membranes are essentially the same as their method, although some modifications are employed such as using a French pressure cell for disruption of the cells and flotation centrifugation for isolation of plasma membranes. Plasma membranes thus prepared can be identified by electron microscopy and also by labeling the intact cells prior to disruption with a membrane-impermeant protein marker such as diazobenzene [ 35 S]sulfonate.

Transcriptional and Posttranscriptional Regulation of Nitrogen-Responding Expression of Phosphoenolpyruvate Carboxylase Gene in Maize

To study the regulation of gene expression for enzymes in the C4 photosynthetic pathway of maize (Zea mays L.) in response to changing N status in developing photosynthetic cells, we have studied in vitro transcription of the phosphoenolpyruvate carboxylase (PEPC) gene in leaf nuclei isolated from plants during recovery from N starvation. The induction was specific for the C4-type PEPC gene (C4Ppc1), and its transcription was N dependent and increased markedly by supply of an N source, but there was a discrepancy between the steady-state levels of mRNA and the stimulation of in vitro transcription. The results suggest that the N-inducible expression of C4Ppc1 is regulated both transcriptionally and posttranscriptionally by N availability. The in vitro transcription rate of C4Ppc1 was greatly stimulated by incubating detached leaves with zeatin alone, whereas the rate remained essentially unchanged by incubating with an exogenous N source alone. The results, taken together, imply that cytokinins up-regulate the transcription of C4Ppc1 in response to N status, whereas glutamine and/or its metabolite(s) up-regulate the level of the transcript. The transcription was totally inhibited by cycloheximide, indicating that the cytokinin-dependent transcription of C4Ppc1 requires the synthesis of protein.

Regulation of nitrate assimilation in cyanobacteria

Nitrate assimilation by cyanobacteria is inhibited by the presence of ammonium in the growth medium. Both nitrate uptake and transcription of the nitrate assimilatory genes are regulated. The major intracellular signal for the regulation is, however, not ammonium or glutamine, but 2-oxoglutarate (2-OG), whose concentration changes according to the change in cellular C/N balance. When nitrogen is limiting growth, accumulation of 2-OG activates the transcription factor NtcA to induce transcription of the nitrate assimilation genes. Ammonium inhibits transcription by quickly depleting the 2-OG pool through its metabolism via the glutamine synthetase/glutamate synthase cycle. The P(II) protein inhibits the ABC-type nitrate transporter, and also nitrate reductase in some strains, by an unknown mechanism(s) when the cellular 2-OG level is low. Upon nitrogen limitation, 2-OG binds to P(II) to prevent the protein from inhibiting nitrate assimilation. A pathway-specific transcriptional regulator NtcB activates the nitrate assimilation genes in response to nitrite, either added to the medium or generated intracellularly by nitrate reduction. It plays an important role in selective activation of the nitrate assimilation pathway during growth under a limited supply of nitrate. P(II) was recently shown to regulate the activity of NtcA negatively by binding to PipX, a small coactivator protein of NtcA. On the basis of accumulating genome information from a variety of cyanobacteria and the molecular genetic data obtained from the representative strains, common features and group- or species-specific characteristics of the response of cyanobacteria to nitrogen is summarized and discussed in terms of ecophysiological significance.

Role of NtcB in Activation of Nitrate Assimilation Genes in the Cyanobacterium Synechocystis sp. Strain PCC 6803

In Synechocystis sp. strain PCC 6803, the genes encoding the proteins involved in nitrate assimilation are organized into two transcription units, nrtABCD-narB and nirA, the expression of which was repressed by ammonium and induced by inhibition of ammonium assimilation, suggesting involvement of NtcA in the transcriptional regulation. Under inducing conditions, expression of the two transcription units was enhanced by nitrite, suggesting regulation by NtcB, the nitrite-responsive transcriptional enhancer we previously identified in Synechococcus sp. strain PCC 7942. The slr0395 gene, which encodes a protein 47% identical to Synechococcus NtcB, was identified as the Synechocystis ntcB gene, on the basis of the inability of an slr0395 mutant to rapidly accumulate the transcripts of the nitrate assimilation genes upon induction and to respond to nitrite. While Synechococcus NtcB strictly requires nitrite for its action, Synechocystis NtcB enhanced transcription significantly even in the absence of nitrite. Whereas the Synechococcus ntcB mutant expresses the nitrate assimilation genes to a significant level in an NtcA-dependent manner, the Synechocystis ntcB mutant showed only low-level expression of the nitrate assimilation genes, indicating that NtcA by itself cannot efficiently promote expression of these genes in Synechocystis. Activities of the nitrate assimilation enzymes in the Synechocystis ntcB mutant were consequently low, being 40 to 50% of the wild-type level, and the cells grew on nitrate at a rate approximately threefold lower than that of the wild-type strain. These results showed that the contribution of NtcB to the expression of nitrate assimilation capability varies considerably among different strains of cyanobacteria.