Yukako Hihara

PmgA and Ssr1600, homologs of the anti-sigma factor and the anti-sigma antagonist of the bacterial partner-switching system, respectively, are involved in coordination of the repression of photosystem I content and chlorophyll biosynthesis under prolonged high-light (HL) conditions in the cyanobacterium Synechocystis sp. PCC 6803. To characterize the transcriptional responses associated with PmgA and Ssr1600 during HL acclimation, we compared global gene expression profiles of the wild-type (WT) and knockout strains of pmgA (ΔpmgA) and ssr1600 (Δ1600) under low-light conditions and at 15 min, 1 h and 12 h after the shift to HL. The knockout of pmgA or ssr1600 induced temporally distinct transcriptomic alterations between short-term and prolonged HL exposure. At 15 min, Δ1600, but not ΔpmgA, showed enhanced induction of stress-responsive genes, including sigB and heat-shock genes, together with derepression of pmgA. At 12 h, both mutants showed significant upregulation of genes related to chlorophyll biosynthesis, photosynthetic electron transport, nitrogen assimilation, and amino acid biosynthesis. Considering that Ssr1600 does not accumulate in ΔpmgA, these observations can be explained by a shift in the role of Ssr1600, from the suppressor of the amplification of the stress response by PmgA under short-term HL conditions to a factor mediating signal output from the PmgA-Ssr1600 module under prolonged HL conditions, although the contribution of secondary effects cannot be excluded. Our transcriptomic data do not support the hypothesis that PmgA acts as a canonical anti-sigma factor for any of the sigma factors examined in this study, SigB, SigD and SigE, under either condition.

The SOS response is a widespread bacterial mechanism for coping with DNA-damaging stress. The key regulator of the process is the transcriptional repressor LexA, which represses expression of DNA-repair–related genes under nonstress conditions and undergoes self-cleavage to induce them under DNA-damaging conditions. Here, we gained insights into the SOS response and functional diversification of LexA in cyanobacteria by characterizing 2 model species, Synechocystis sp. PCC 6803 (S.6803) and Anabaena sp. PCC 7120 (A.7120), together with an early branching species Gloeobacter violaceus PCC 7421 (G.7421) whose LexA belongs to the same subclade as those of Candidatus Melainabacteria. After 3 h of treatment with UV-C or UV-B, the amount of LexA in A.7120 and G.7421 decreased to 30% to 40% of pre-stress levels concomitant with the induction of DNA repair–related genes, suggesting that LexA may function as a typical SOS repressor. In S.6803, on the other hand, we did not observe a decrease in LexA after treatment with UV radiation. This observation was consistent with previous studies reporting that LexA with substituted self-cleavage residues regulates expression of genes related to various cellular functions but not the SOS response in S.6803. The changes in gene expression profile of S.6803 after exposure to UV-C seemed to depend on transcription factors other than LexA, including the master regulator of photosynthetic genes, RpaB. In phototrophic cyanobacteria, the role of LexA as an SOS repressor may have become less important over the course of evolution, enabling LexA to diversify its functions in some species.

Under high-light conditions, the dissipation of excess energy as heat in the light-harvesting antenna is essential for photosynthetic organisms to protect the photosynthetic machinery. In the case of cyanobacteria, however, the induction of the thermal dissipation in the antennae is insufficient to dissipate all excess energy, which is manifested as the increase in the steady-state level of chlorophyll fluorescence (Fs) under high light. To elucidate the underlying cause of the incomplete dissipation of excess light in the antenna, we investigated the impact of depletion and overexpression of orange carotenoid protein (OCP), which is essential to induce thermal dissipation in the antenna, on photosynthesis in Synechocystis sp. PCC 6803. The suppression of the OCP-dependent thermal dissipation resulted in elevated Fs with a constant yield of photosynthesis, suggesting that the light-induced increase in Fs might function as an acclimation mechanism to high light, which compensated for the lower OCP-dependent thermal dissipation. By contrast, over-induction of the OCP-dependent thermal dissipation decreased not only Fs but also the yield of photosynthesis under high light, due to the reduced energy transfer from the antenna to photosystem II. These results indicate that the complete removal of excess energy via the OCP-dependent mechanism has a drawback in photosynthetic efficiency under high-light conditions, and the strategy independent of OCP is employed to cope with excess light without lowering the yield of photosynthesis in cyanobacteria.

Recently, acyl plastoquinol (APQ) and plastoquinone-B (PQ-B), which are fatty acid esters of plastoquinol and plastoquinone-C respectively, have been identified as the major neutral lipids in cyanobacteria. In Synechocystis sp. PCC 6803, Slr2103 having homology with the eukaryotic enzyme for triacylglycerol (TAG) synthesis, diacylglycerol acyltransferase 2 (DGAT2), was identified as responsible for the synthesis of these plastoquinone-related lipids. On the other hand, TAG synthesis in cyanobacteria remains controversial due to the low accumulation level within cyanobacterial cells together with the high contamination level from the environment. In this study, to quantify more precisely and elucidate the relationship between the accumulation of neutral lipids and the presence or absence of DGAT2-like genes, plastoquinone-related lipids and TAG were analyzed directly from total lipids of six cyanobacterial species with different sets of genes encoding DGAT2-like proteins belonging to two distinct subclades. The results showed that the synthesis of these neutral lipids is highly dependent on clade A DGAT2-like proteins under the culture conditions used in this study, although accumulation level of TAG was quite low. In contrast to APQ highly abundant in saturated fatty acids, the fatty acid composition of TAG was species-specific and partly reflected the total lipid composition. Gloeobacter violaceus PCC 7421, which lacks a DGAT2-like gene, accumulated APQ with a high proportion of C18:0, suggesting APQ synthesis by an unidentified acyltransferase.

Photomixotrophic growth A (PmgA) is a pleiotropic regulator essential for growth under photomixotrophic and prolonged high-light (HL) conditions in the cyanobacterium Synechocystis sp. PCC 6803. The overall similarity with the anti-sigma factor of the bacterial partner-switching system indicates that PmgA exerts a regulatory function via phosphorylation of its target proteins. In this study, we performed an in vitro phosphorylation assay and protein-protein interaction analysis and found that PmgA interacts with four anti-sigma antagonist homologs, Ssr1600, Slr1856, Slr1859, and Slr1912, but specifically phosphorylates Ssr1600. Phenotypic analyses using the set of gene disruption and overexpression strains of pmgA and ssr1600 revealed that phosphorylation by PmgA is essential for the accumulation of Ssr1600 protein in vivo. The ssr1600-disrupted mutant showed similar phenotypes as those previously reported for the pmgA-disrupted mutant, namely, no obvious phenotype just after the shift to HL, but higher chlorophyll content, 5-aminolevulinic acid synthesis activity, and psaAB transcript levels than those in the wild-type after 6 hours. These findings indicate that the phosphorylated form of Ssr1600 works as the output of the partner-switching system to coordinately repress chlorophyll biosynthesis and accumulation of photosystem I during HL acclimation.

Most cyanobacterial genomes possess more than two copies of genes encoding cyAbrBs (cyanobacterial AbrB-like proteins) having an AbrB-like DNA-binding domain at their C-terminal region. Accumulating data suggest that a wide variety of metabolic and physiologic processes are regulated by cyAbrBs. In this study, we investigated the function of the essential gene cyabrB1 (sll0359) in Synechocystis sp. PCC 6803 by using CRISPR interference technology. The conditional knockdown of cyabrB1 caused increases of cyAbrB2 transcript and protein levels. However, the effect of cyabrB1 knockdown on global gene expression profile was quite limited compared to the previously reported profound effect of knockout of cyabrB2. Among 24 up-regulated genes, 16 genes were members of the divergently transcribed icfG and sll1783 operons related to carbon metabolism. The results of this and previous studies indicate the different contributions of two cyAbrBs to transcriptional regulation of genes related to carbon, hydrogen and nitrogen metabolism. Possession of a pair of cyAbrBs has been highly conserved during the course of evolution of the cyanobacterial phylum, suggesting physiological significance of transcriptional regulation attained by their interaction.

Although cyanobacteria do not possess bacterial triacylglycerol (TAG) synthesizing enzymes, accumulation of TAGs and/or lipid droplets has been repeatedly reported in a wide range of species. In most cases, identification of TAG has been based on the detection of the spot showing the mobility similar to the TAG standard in thin-layer chromatography of neutral lipids. In this study, we identified monoacyl plastoquinol (acyl PQH) as the predominant molecular species in the TAG-like spot from the unicellular Synechocystis sp. PCC 6803 (S.6803) as well as the filamentous Nostocales species, Nostoc punctiforme PCC 73102 and Anabaena sp. PCC 7120. In S.6803, the accumulation level of acyl PQH but not TAG was affected by deletion or overexpression of slr2103, indicating that acyl PQH is the physiological product of Slr2103 having homology with the eukaryotic diacylglycerol acyltransferase-2 (DGAT2). Electron microscopy revealed that cyanobacterial strains used in this study do not accumulate lipid droplet structures such as those observed in oleaginous microorganisms. Instead, they accumulate polyhydroxybutyrate (PHB) granules and/or aggregates of alkane, free C16 and C18 saturated fatty acids and low amounts of TAG in the cytoplasmic area, which can be detected by staining with a fluorescent dye specific to neutral lipids. Unlike these lipophilic materials, acyl PQH is exclusively localized in the membrane fraction. There must be DGAT2-like enzymatic activity esterifying de novo-synthesized C16 and C18 fatty acids to PQH₂ in the thylakoid membranes.

The response regulator RpaB plays a central role in transcriptional regulation of photosynthesis-related genes in cyanobacteria. RpaB is phosphorylated by its cognate histidine kinase Hik33 and functions as both an activator and a repressor under low-light conditions, whereas its phosphorylation level and DNA-binding activity promptly decrease upon the upshift of photon flux density, causing changes in the gene expression profile. In this study, we assessed the possibility of redox regulation of the DNA-binding activity of RpaB in Synechocystis sp. PCC 6803 by the addition of inhibitors of photosynthetic electron transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, or the reducing agent dithiothreitol under different photon flux densities. Analysis of the phosphorylation level of RpaB revealed that reduction of Q_A and increase in the availability of reducing equivalents at the acceptor side of photosystem I (PSI) can independently trigger dephosphorylation. The redox-state-dependent regulation by an unidentified thiol other than Cys59 of RpaB is prerequisite for the phosphorylation-dependent regulation of the DNA-binding activity. Environmental signals, recognized by Hik33, and metabolic signals recognized as the availability of reducing equivalents, must be integrated at the master regulator RpaB, in order to attain the flexible regulation of acclimatory responses.

Different from typical LexA repressors in heterotrophic bacteria exerting SOS response by auto-cleavage, cyanobacterial LexAs, especially that of Synechocystis sp. PCC 6803 (S.6803), have been suggested be involved in regulation of a number of genes related to various cellular processes, rather than the typical SOS regulon. When and how cyanobacterial LexAs are triggered to regulate its target genes have remained unknown. In this study, we found the profound repressing effect of LexA on salt-stress inducible genes in S.6803. The repressing activity of LexA was likely to persist during salt stress and the salt response of these genes was mainly achieved by other regulators than LexA, suggesting that the physiological role of LexA is fine-tuning of gene expression in response to environmental changes. Although the amount and oligomeric state of LexA were unchanged upon salt stress, two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry analyses detected a change in posttranslational modification in a small fraction of LexA molecules, possibly dephosphorylation of Ser¹⁷³, after 30 min upon the upshift in salt concentration. Activity of LexA in S.6803 may be under gradual control by posttranslational modification to fine-tune gene expression, which is contrasted with the digital switching-off regulation by auto-cleavage in heterotrophic bacteria.