Cellular response of Parachlorella kessleri to a solid surface culture environment.

Attached culture allows high biomass productivity and is a promising biomass cultivating system because neither a huge facility area nor a large volume of culture medium are needed. This study investigates photosynthetic and transcriptomic behaviors in Parachlorella kessleri cells on a solid surface after their transfer from liquid culture to elucidate the physiological and gene-expression regulatory mechanisms that underlie their vigorous proliferation. The chlorophyll content shows a decrease at 12 h after the transfer; however, it has fully recovered at 24 h, suggesting temporary decreases in the amounts of light harvesting complexes. On PAM analysis, it is demonstrated that the effective quantum yield of PSII decreases at 0 h right after the transfer, followed by its recovery in the next 24 h. A similar changing pattern is observed for the photochemical quenching, with the PSII maximum quantum yield remaining at an almost unaltered level. Non-photochemical quenching was increased at both 0 h and 12 h after the transfer. These observations suggest that electron transfer downstream of PSII but not PSII itself is only temporarily damaged in solid-surface cells just after the transfer, with light energy in excess being dissipated as heat for PSII protection. It thus seems that the photosynthetic machinery acclimates to high-light and/or dehydration stresses through its temporal size-down and functional regulation that start right after the transfer. Meanwhile, transcriptomic analysis by RNA-Seq demonstrates temporary upregulation at 12 h after the transfer as to the expression levels of many genes for photosynthesis, amino acid synthesis, general stress response, and ribosomal subunit proteins. These findings suggest that cells transferred to a solid surface become stressed immediately after transfer but can recover their high photosynthetic activity through adaptation of photosynthetic machinery and metabolic flow as well as induction of general stress response mechanisms within 24 h.

Energy conservation in photosynthetic microorganisms.

Photosynthesis is a biological process of energy conversion from solar radiation to useful organic compounds for the photosynthetic organisms themselves. It, thereby, also plays a role of food production for almost all animals on the Earth. The utilization of photosynthesis as an artificial carbon cycle is also attracting a lot of attention regarding its benefits for human life. Hydrogen and biofuels, obtained from photosynthetic microorganisms, such as microalgae and cyanobacteria, will be promising products as energy and material resources. Considering that the efficiency of bioenergy production is insufficient to replace fossil fuels at present, techniques for the industrial utilization of photosynthesis processes need to be developed intensively. Increase in the efficiency of photosynthesis, the yields of target substances, and the growth rates of algae and cyanobacteria must be subjects for efficient industrialization. Here, we overview the whole aspect of the energy production from photosynthesis to biomass production of various photosynthetic microorganisms.

Physiological Properties of Photoautotrophic Microalgae and Cyanobacteria Relevant to Industrial Biomass Production.

Photoautotrophic mass culture of microalgae is currently under investigation for social implementation, since such organisms are anticipated to be resources of alternative fuels and materials for reducing global warming. Production scale-up of culture systems and economy balance are great barriers for practical usage. In order to develop new culture systems such as attachment on solid surfaces or biofilms, we investigated various characteristics of photosynthesis in Chlorella, not only in liquid but also on filter membranes. In aquatic cultures, the photosynthetic rate was almost the same as the specific exponential growth rate at over 32 °C, suggesting that highly efficient cell growth was achieved at that temperature. The algal cells could fix about 50 mmol carbons per mole photons, at cloudy-day-level light intensities, which result to produce 1.2 g dry cell weight in calculation. Moreover, Chlorella could grow on a membrane surface at almost the same rate as in liquid. Similar tolerance to water deficiency was observed in a cyanobacterium, Synechocystis, in which gene expression responded in 30 min after the stress. Such a tolerance was also observed in other species of microalgae and cyanobacteria in photosynthesis.

Genes for a series of proteins that are involved in glucose catabolism are upregulated by the Hik8-cascade in Synechocystis sp. PCC 6803.

MAIN CONCLUSION: In summary, we could show the involvement of a Hik8-cascade in the expression of genes involved in the glycolytic and OPP pathways induced by GPL, and another signal pathway under photosynthetic conditions in Synechocystis . The Hik8-cascade under GPL conditions may regulate glucose degradation to produce some energy and carbon compounds. This cascade might be important for the supply of organic materials such as amino acids and nucleotides through enhancement of the rates of the glycolysis and OPP pathways. Histidine kinase Hik8 upregulates the expression of one of the important glycolytic genes, fbaA, via sll1330 under heterotrophic growth conditions (i.e., in the presence of glucose with an indispensable short period of light) in Synechocystis sp. PCC 6803. In this study, expression of the genes for the glycolytic and OPP pathways was investigated using the wild type, and disruption mutants of Hik8 and sll1330, to determine whether or not the Hik8-involving signal transduction system generally regulates glucose catabolism. In the wild type, all the genes for the glycolytic and OPP pathways were upregulated under the same conditions as for fbaA. Analyses of the disruption mutants suggested that the signal transduction system involving Hik8 and Sll1330 plays a key role in the upregulation of genes such as pfkA, pgmB, and glk, and also that Hik8 induces genes including gap1 and pgk independently of Sll1330. This complicated signal transduction cascade, designated as the Hik8-cascade, occurs under heterotrophic growth with light pulses. In addition, a disruption mutant of a putative histidine kinase, sll1334, exhibited growth and gene expression patterns that suggested it to be a negative regulator in the cascade. Possible histidine kinases and response regulators as candidates for other components in the cascade are discussed.

Two regulatory networks mediated by light and glucose involved in glycolytic gene expression in cyanobacteria.

Fructose 1,6-bisphosphate aldolase (FBA) is an enzyme involved in both glycolytic and photosynthetic reactions in photosynthetic organisms. In prokaryotes, the bidirectional reaction proceeds in the same cellular compartment, i.e. the cytoplasm. Expression of the FBA gene, fbaA, is induced through two independent pathways, stimulated by continuous light and by glucose plus pulsed light (GPL), in a cyanobactrium, Synechocystis sp. PCC 6803. Under GPL conditions, glucose can be replaced by glucose analogs that are not even metabolized in a cell. Analyses of transcripts in deletion mutants suggested that both a histidine kinase, Hik8, and a response regulator, Sll1330, played important roles as signal components in fbaA expression under GPL conditions, but not under photosynthetic conditions. Analysis of a transformant in which sll1330 expression was enhanced demonstrated that fbaA expression was induced at least partially even without glucose, but for its further induction a pulsed light stimulus was required. These results substantiated that there are two light-dependent regulatory pathways for aldolase gene expression in this cyanobacterium.

Light-induced gene expression of fructose 1,6-bisphosphate aldolase during heterotrophic growth in a cyanobacterium, Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 exhibits light-activated heterotrophic growth (LAHG) under dark conditions with glucose as a carbon source. The light activation is remarkable at a late period of photoautotrophic preculture, such as the late-linear and stationary growth phases. To understand the physiological effects of light irradiation and glucose under LAHG conditions, their effects on the expression of soluble proteins were analyzed by means of 2D-PAGE. Various soluble proteins, which were minimal under photoautotrophic preculture conditions, were observed clearly under LAHG conditions, suggesting that proteins were synthesized actively under these conditions. Fructose 1,6-bisphosphate aldolase, one of the glycolytic enzymes, was found to be induced under LAHG conditions on 2D-PAGE. The activity of fructose 1,6-bisphosphate aldolase, which had decreased during photoautotrophic preculture, also increased under LAHG conditions, similar to the mRNA level of the encoding gene, fbaA. In addition, we found that a deletion mutant of sll1330, a putative gene containing a helix-turn-helix DNA-binding motif, could not grow under LAHG conditions, whereas it could grow photoautotrophically. The increases in the protein level of FbaA and fbaA gene expression observed in wild-type cells under LAHG conditions were greatly inhibited in the deletion mutant. These results suggest that the regulation of fbaA gene expression by way of sll1330 is one of the important processes in Synechocystis sp. PCC 6803 under light pulse LAHG conditions.

Sll1330 controls the expression of glycolytic genes in Synechocystis sp. PCC 6803.

In the complete annotated genome sequences of cyanobacterium Synechocystis sp. PCC 6803, one can find many putative genes for two-component response regulators that include a helix-turn-helix DNA-binding domain. The mRNA level of one of the putative genes, sll1330, was increased by glucose, especially in the presence of light. We successfully disrupted the sll1330 gene by targeted mutagenesis with a spectinomycin resistance cassette. Deltasll1330 could not grow well under light-activated heterotrophic growth conditions. Analyses of the expression of glycolytic genes revealed that the mRNA levels of five glycolytic genes, that is, glk (sll0593), pfkA (sll1196), fbaA (sll0018), gpmB (slr1124), and pk (sll0587), were decreased, and were regulated by Sll1330 under light and glucose-supplemented conditions. The Synechocystis sp. PCC 6803 genome each encodes two isozymes for these five glycolytic genes, suggesting that each of the two isozymes is regulated by Sll1330 at the mRNA level.

A suppressor mutation in the alpha-phycocyanin gene in the light/glucose-sensitive phenotype of the psbK-disruptant of the cyanobacterium Synechocystis sp. PCC 6803.

psbK encodes a small transmembrane component of PSII. Here we report that the psbK-disruptant of Synechocystis sp. PCC 6803 cannot survive under photomixotrophic conditions of light and glucose after transient growth, while the wild type is able to grow. A spontaneous yellow-green mutant that recovered the sustained growth under the same conditions was isolated from the psbK-disruptant. Instead of recovery, the mutant largely lost photoautotrophic growth. By phenotype complementation, the mutation was identified in cpcA as a sequence replacement with a close downstream segment, generating an inverted repeat of 23 bp. The mutant phenotype was characterized by (i) the complete loss of alpha- and beta-phycocyanin; (ii) increased accumulation of PSII; and (iii) greatly reduced transcripts harboring cpcA in abundance and in size. The inverted repeat generated in cpcA probably led to the early termination of transcription. A possible mechanism for such a mutation is discussed.

Use of segment-based microarray in the analysis of global gene expression in response to various environmental stresses in the cyanobacterium Anabaena sp. PCC 7120.

We prepared microarrays that contain genomic sequences of a heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120. The complete genome of this cyanobacterium codes for about 5,368 protein-coding genes in the main chromosome of 6.4 Mbp. In total, 2,407 DNA segments were selected from the sequencing clones, and amplified by PCR, then spotted on glass slides in duplicate. These microarrays differ from the widely used commercial or custom-made ones for other microorganisms in that each DNA segment was 3-4 kbp long, and contained about 3-4 predicted genes on average. This feature, however, did not decrease the usefulness of the microarrays, since we were able to detect a number of potentially novel genes that are induced in response to nitrogen deprivation, low temperature and drought. In addition, we found some genomic regions in which dozens of contiguous genes are simultaneously regulated. These results suggest that these segment-based microarrays are useful especially for such large genomes as Anabaena, for which the number of genes exceeds either technical or practical limitations.

Decrease in the efficiency of the electron donation to tyrosine Z of photosystem II in an SQDG-deficient mutant of Chlamydomonas.

Photosystem (PS) II activity of a sulfoquinovosyl diacylglycerol (SQDG)-deficient mutant (hf-2) of Chlamydomonas was partially decreased compared with that of wild-type. The susceptibility to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modified in the mutant. Photometric measurements in the isolated thylakoid membranes of hf-2 revealed that the lowered activity in the mutant was derived from a decrease in the efficiency of the electron donation from water to tyrosine Z, not from the efficiency of the electron transport from Q(A) to Q(B). This result was confirmed by the decay kinetics of chlorophyll fluorescence determined in vivo. We conclude that SQDG contributes to maintaining the conformation of PSII complexes, particularly that of D1 polypeptides, which are necessary for maximum activities in Chlamydomonas.

Role of sulfoquinovosyl diacylglycerol for the maintenance of photosystem II in Chlamydomonas reinhardtii.

The physiological role of sulfoquinovosyl diacylglycerol (SQDG) in photosynthesis was investigated with a SQDG defective mutant (hf-2) of Chlamydomonas reinhardtii that did not have any detectable amount of SQDG. The mutant showed a lower rate of photosystem II (PSII) activity by approximately 40% and also a lower growth rate than those of the wild-type. Results of genetical analysis of hf-2 strongly suggest that the SQDG defect and the lowered PSII activity are due to a single gene mutation. The supplementation of SQDG to hf-2 cells restored the lowered PSII activity to the same level as that of wild-type cells, and also enabled the mutant to grow even in the presence of 135 nm 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Moreover, the incubation of isolated thylakoid membranes of hf-2 with SQDG raised the lowered PSII activity. Chemical modifications of SQDG impaired the recovery of PSII activity. The results suggest that SQDG is indispensable for PSII activity in Chlamydomonas by maintaining PSII complexes in their proper state.