Shin-ichi Maeda

Novel gene products associated with NdhD3/D4‐containing NDH‐1 complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp. PCC7942

Cyanobacteria possess light‐dependent CO2 uptake activity that results in the net hydration of CO2 to HCO3– and may involve a protein‐mediated carbonic anhydrase (CA)‐like activity. This process is vital for the survival of cyanobacteria and may be a contributing factor in the ecological success of this group of organisms. Here, via isolation of mutants of Synechococcus sp. PCC7942 that cannot grow under low‐CO2 conditions, we have identified two novel genes, chpX and chpY, that are involved in light‐dependent CO2 hydration and CO2 uptake reactions; co‐inactivation of both these genes abolished both activities. The function and mechanism of the CO2 uptake systems supported by each chp gene product differs, with each associated with functionally distinct NAD(P)H dehydrogenase (NDH‐1) complexes. The ChpX system has a low affinity for CO2 and is de‐pendent on photosystem I cyclic electron transport, whereas the inducible ChpY system has a high affinity for CO2 and is dependent on linear electron transport. We believe that ChpX and ChpY are involved in a unique, net hydration of CO2 to HCO3–, that is coupled electron flow within the NDH‐1 complex on the thylakoid membrane.

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.

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.

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.

Bicarbonate Binding Activity of the CmpA Protein of the Cyanobacterium Synechococcus sp. strain PCC 7942 Involved in Active Transport of Bicarbonate

The cmpABCD operon of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an ATP-binding cassette transporter involved in HCO3 − uptake. The three genes, cmpBCD, encode membrane components of an ATP-binding cassette transporter, whereas cmpA encodes a 42-kDa cytoplasmic membrane protein, which is 46.5% identical to the membrane-anchored substrate-binding protein of the nitrate/nitrite transporter. Equilibrium dialysis analysis using H14CO3 −showed that a truncated CmpA protein lacking the N-terminal 31 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, specifically binds inorganic carbon (CO2 or HCO3 −). The addition of the recombinant CmpA protein to a buffer caused a decrease in the concentration of dissolved CO2 because of the binding of inorganic carbon to the protein. The decrease in CO2 concentration was accelerated by the addition of carbonic anhydrase, indicating that HCO3 −, but not CO2, binds to the protein. Mass spectrometric measurements of the amounts of unbound and bound HCO3 − in CmpA solutions containing low concentrations of inorganic carbon revealed that CmpA binds HCO3 − with high affinity (K d = 5 μm). A similar dissociation constant was obtained by analysis of the competitive inhibition of the CmpA protein on the carboxylation of phosphoenolpyruvate by phosphoenolpyruvate carboxylase at limiting concentrations of HCO3 −. These findings showed that the cmpA gene encodes the substrate-binding protein of the HCO3 −transporter.

Mechanism of low CO2‐induced activation of the cmp bicarbonate transporter operon by a LysR family protein in the cyanobacterium Synechococcus elongatus strain PCC 7942

The cmp operon of the cyanobacterium Synechococcus elongatus strain PCC 7942, encoding the subunits of the ABC‐type bicarbonate transporter, is activated under CO2‐limited growth conditions in a manner dependent on CmpR, a LysR family transcription factor of CbbR subfamily. The 0.7 kb long regulatory region of the operon carried a single promoter, which responded to CO2 limitation. Using the luxAB reporter system, three cis‐acting elements involved in the low‐CO2 activation of transcription, each consisting of a pair of LysR recognition signatures overlapping at their ends, were identified in the regulatory region. CmpR was shown to bind to the regulatory region, yielding several DNA–protein complexes in gel shift assays. Addition of ribulose‐1,5‐bisphosphate (> 1 mM) or 2‐phosphoglycolate (> 10 μM) enhanced the binding of CmpR in a concentration‐dependent manner, promoting formation of large DNA–protein complexes. Given the involvement of O2 in adaptive responses of cyanobacteria to low‐CO2 conditions, our results suggest that 2‐phosphoglycolate, which is produced by oxygenation by ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) of ribulose‐1,5‐bisphosphate under CO2‐limited conditions, acts as the co‐inducer in the activation of the cmp operon by CmpR.

Effects of High CO2 on Growth and Metabolism of Arabidopsis Seedlings During Growth with a Constantly Limited Supply of Nitrogen

Elevated CO2 has been reported to stimulate plant growth under nitrogen-sufficient conditions, but the effects of CO2 on growth in a constantly nitrogen-limited state, which is relevant to most natural habitats of plants, remain unclear. Here, we maintained Arabidopsis seedlings under such conditions by growing a mutant with reduced nitrate uptake activity on a medium containing nitrate as the sole nitrogen source. Under nitrogen-sufficient conditions (i.e. in the presence of ammonium), growth of shoots and roots of both the wild type (WT) and the mutant was increased approximately 2-fold by elevated CO2. Growth stimulation of shoots and roots by elevated CO2 was observed in the WT growing with nitrate as the sole nitrogen source, but in the mutant grown with nitrate, the high-CO2 conditions stimulated only the growth of roots. In the mutant, elevated CO2 caused well-known symptoms of nitrogen-starved plants, including decreased shoot/root ratio, reduced nitrate content and accumulation of anthocyanin, but also had an increased Chl content in the shoot, which was contradictory to the known effect of nitrogen depletion. A high-CO2-responsive change specific to the mutant was not observed in the levels of the major metabolites, although CO2 responses were observed in the WT and the mutant. These results indicated that elevated CO2 causes nitrogen limitation in the seedlings grown with a constantly limited supply of nitrogen, but the Chl content and the root biomass of the plant increase to enhance the activities of both photosynthesis and nitrogen uptake, while maintaining normal metabolism and response to high CO2.

CyanoBase: a large-scale update on its 20th anniversary

The first ever cyanobacterial genome sequence was determined two decades ago and CyanoBase (http://genome.microbedb.jp/cyanobase), the first database for cyanobacteria was simultaneously developed to allow this genomic information to be used more efficiently. Since then, CyanoBase has constantly been extended and has received several updates. Here, we describe a new large-scale update of the database, which coincides with its 20th anniversary. We have expanded the number of cyanobacterial genomic sequences from 39 to 376 species, which consists of 86 complete and 290 draft genomes. We have also optimized the user interface for large genomic data to include the use of semantic web technologies and JBrowse and have extended community-based reannotation resources through the re-annotation of Synechocystis sp. PCC 6803 by the cyanobacterial research community. These updates have markedly improved CyanoBase, providing cyanobacterial genome annotations as references for cyanobacterial research.