Yukako Hihara

Cyanobacteria are monophyletic organisms that perform oxygenic photosynthesis. While they exhibit great diversity, they have a common set of genes. However, the essentiality of them for viability has hampered the elucidation of their functions. One example of the genes is cyabrB1 encoding a transcriptional regulator. In the present study, we investigated the function of cyabrB1 in heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 through CRISPR interference, a method we recently utilized for the photosynthetic production of a useful chemical in the strain. Conditional knockdown of cyabrB1 in the presence of nitrate resulted in formation of heterocysts. Two genes, hetP and hepA, which are required for heterocyst formation, were up-regulated by cyabrB1 knockdown in the presence of combined nitrogen sources. The genes are known to be induced by HetR, a master regulator of heterocyst formation. hetR was not induced by cyabrB1 knockdown. hetP and hepA were repressed by direct binding of cyAbrB1 to their promoter regions in a HetR-independent manner. In addition, the over-expression of cyabrB1 abolished heterocyst formation upon nitrogen depletion. Also, knockout of cyabrB2, a paralogue gene of cyabrB1, in addition to cyabrB1 knockdown, enhanced heterocyst formation in the presence of nitrate, suggesting functional redundancy of cyAbrB proteins. We propose that a balance between amounts of HetR and cyAbrB1 is a key factor influencing heterocyst differentiation during nitrogen step-down. cyAbrB proteins are essential safety devices inhibiting heterocyst differentiation.Importance: Spore formation in Bacillus subtilis and Streptomyces represents non-terminal differentiation and has been extensively studied as models of prokaryotic cell differentiation. In the two organisms, many cells differentiate simultaneously, and the differentiation is governed by a network in which one regulator stands at the top. Differentiation of heterocysts in Anabaena sp. PCC 7120 has also been extensively studied. The differentiation is unique because it is terminal and only 5-10% vegetative cells differentiate into heterocysts. In the present study, we identified cyAbrB1 as a repressor of two genes that are essential for heterocyst formation, hetP and hepA, independent of HetR, which is a master activator for heterocyst differentiation. The finding is reasonable for unique cell differentiation of Anabaena because cyAbrB1 could suppress heterocyst differentiation tightly in vegetative cells, while only cells in which HetR is over-expressed could differentiate into heterocysts.

The transcription factor RpaB regulates the expression of genes encoding photosynthesis-associated proteins during light acclimation. The binding site of RpaB is the HLR1 motif, a pair of imperfect octameric direct repeats, separated by two random nucleotides. Here, we used high-resolution mapping data of transcriptional start sites (TSSs) in the model Synechocystis sp. PCC 6803 in conjunction with the positional distribution of HLR1 sites for the global prediction of the RpaB regulon. The results demonstrate that RpaB regulates the expression of more than 150 promoters, driving the transcription of protein-coding and non-coding genes and antisense transcripts under low light and upon the shift to high light when DNA binding activity is lost. Transcriptional activation by RpaB is achieved when the HLR1 motif is located 66 to 45 nt upstream, repression occurs when it is close to or overlapping the TSS. Selected examples were validated by multiple experimental approaches, including chromatin affinity purification, reporter gene, northern hybridization and electrophoretic mobility shift assays. We found that RpaB controls ssr2016/pgr5 , which is involved in cyclic electron flow and state transitions; six out of nine ferredoxins; three of four FtsH proteases; gcvP/slr0293 , encoding a crucial photorespiratory protein; and nirA and isiA for which we suggest cross-regulation with the transcription factors NtcA or FurA, respectively. In addition to photosynthetic gene functions, RpaB contributes to the control of genes affiliated with nitrogen assimilation, cofactor biosyntheses, the CRISPR system and the circadian clock, making it one of the most versatile regulators in cyanobacteria. Significance Statement: RpaB is a transcription factor in cyanobacteria and in the chloroplasts of several lineages of eukaryotic algae. Like other important transcription factors, the gene encoding RpaB cannot be deleted, making the study of deletion mutants impossible. Based on a bioinformatic approach, we increased the number of known genes controlled by RpaB by a factor of 5. Depending on the distance to the TSS, RpaB mediates transcriptional activation or repression. The high number and functional diversity among its target genes and co-regulation with other transcriptional regulators characterize RpaB as a regulatory hub.

LexA is a well-established transcriptional repressor of SOS genes induced by DNA damage in E. coli and other bacterial species. However, LexA in the cyanobacterium Synechocystis sp. PCC 6803 has been suggested not to be involved in SOS response. In this study, we performed RNA-seq analysis of the wild-type strain and the lexA-disrupted mutant to obtain the comprehensive view of LexA-regulated genes in Synechocystis. Disruption of lexA positively or negatively affected expression of genes related to various cellular functions such as phototactic motility, accumulation of the major compatible solute glucosylglycerol and subunits of bidirectional hydrogenase, photosystem I and phycobilisome complexes. We also observed increase in the expression level of genes related to iron and manganese uptake in the mutant at the later stage of cultivation. However, none of the genes related to DNA metabolism were affected by disruption of lexA. DNA gel mobility shift assay using the recombinant LexA protein suggested that LexA binds to the upstream region of pilA7, pilA9, ggpS and slr1670 to directly regulate their expression, but changes in the expression level of photosystem I genes by disruption of lexA is likely a secondary effect.

Since cyanobacteria need to decrease photosystem I (PSI) content to avoid absorption of excess light energy, downregulation of PSI gene expression is one of the key characteristics of high light (HL) acclimation response. The transcriptional regulator RpaB and the small RNA PsrR1 have been suggested to be the two most critical factors for this response in Synechocystis sp. PCC 6803. In this study, we found that the HLR1 DNA-binding motif, the recognition sequence for RpaB, is highly conserved in the core promoter region of the psrR1 gene among cyanobacterial species. Gel mobility shift assay revealed that RpaB binds to the HLR1 sequence of psrR1 in vitro. RNA gel blot analysis together with chromatin affinity purification (ChAP) analysis suggested that PSI genes are activated and the psrR1 gene is repressed by the binding of RpaB under low-light (LL) conditions. Decrease in DNA binding affinity of RpaB occurs within 5 min after the shift from LL to HL conditions, leading to the prompt decrease in PSI promoter activity together with derepression of psrR1 gene expression. Accumulating PsrR1 molecules then prevent translation from preexisting PSI transcripts. By this dual repression at transcriptional and post-transcriptional levels, rapid and strict downregulation of PSI expression under HL is secured. Our findings suggest that RpaB and PsrR1 constitute a feed-forward loop for the regulation of PSI gene expression to achieve a rapid acclimation response to the damaging HL conditions.

The redox state of the photosynthetic electron transport chain is known to act as a signal to regulate the transcription of key genes involved in the acclimation responses to environmental changes. We hypothesized that the protein thioredoxin (Trx) acts as a mediator connecting the redox state of the photosynthetic electron transport chain and transcriptional regulation, and established a screening system to identify transcription factors (TFs) that interact with Trx. His-tagged TFs and S-tagged mutated form of Trx, TrxM_C35S, whose active site cysteine 35 was substituted with serine to trap the target interacting protein, were co-expressed in E. coli cells and Trx-TF complexes were detected by immuno-blotting analysis. We examined the interaction between Trx and ten OmpR family TFs encoded in the chromosome of the cyanobacterium Synechocystis sp. PCC 6803 (S.6803). Although there is a highly conserved cysteine residue in the receiver domain of all OmpR family TFs, only three, RpaA (Slr0115), RpaB (Slr0946) and ManR (Slr1837), were identified as putative Trx targets. The recombinant forms of wild-type TrxM, RpaA, RpaB and ManR proteins from S.6803 were purified following over-expression in E. coli and their interaction was further assessed by monitoring changes in the number of cysteine residues with free thiol groups. An increase in the number of free thiols was observed after incubation of the oxidized TFs with Trx, indicating the reduction of cysteine residues as a consequence of interaction with Trx. Our results suggest, for the first time, the possible regulation of OmpR family TFs through the supply of reducing equivalents from Trx, as well as through the phospho-transfer from its cognate sensor histidine kinase.

The cyAbrB2 transcriptional regulator is essential for active sugar catabolism in Synechocystis sp. PCC 6803 grown under light conditions. In the light-grown cyabrB2-disrupted mutant, glycogen granules and sugar phosphates corresponding to early steps in the glycolytic pathway accumulated to higher levels than those in the wild-type (WT) strain, whereas the amounts of 3-phosphoglycerate, phosphoenolpyruvate and ribulose 1,5-bisphosphate were significantly lower. We further determined that accumulated glycogen granules in the mutant could be actively catabolized under dark conditions. Differences in metabolite levels between WT and the mutant became less substantial during dark incubation due to a general quantitative decrease in metabolite levels. Notable exceptions, however, were increases in 2-oxoglutarate, histidine, ornithine and citrulline in the WT but not in the mutant. The amounts of cyAbrBs were highly responsive to the availability of light both in transcript and protein levels. When grown under light-dark cycle conditions, diurnal oscillatory pattern of glycogen content of the mutant was lost after the second dark period. These observations indicate that cyAbrB2 is dispensable for activation of sugar catabolism under dark conditions but involved in the proper switching between day and night metabolisms.

CyAbrB is a transcriptional regulator unique to and highly conserved among cyanobacterial species. A gene-disrupted mutant of cyabrB2 (sll0822) in Synechocystis sp. PCC 6803 exhibited severe growth inhibition and abnormal accumulation of glycogen granules within cells under photomixotrophic conditions. Within 6 h after the shift to photomixotrophic conditions, sodium bicarbonate-dependent oxygen evolution activity markedly declined in the ΔcyabrB2 mutant, but the decrease in methyl viologen-dependent electron transport activity was much smaller, indicating inhibition in carbon dioxide fixation. Decreases in the transcript levels of several genes related to sugar catabolism, carbon dioxide fixation, and nitrogen metabolism were also observed within 6 h. Metabolome analysis by capillary electrophoresis mass spectrometry revealed that several metabolites accumulated differently in the wild-type and mutant strains. For example, the amounts of pyruvate and 2-oxoglutarate (2OG) were significantly lower in the mutant than in the wild type, irrespective of trophic conditions. The growth rate of the ΔcyabrB2 mutant was restored to a level comparable to that under photoautotrophic conditions by addition of 2OG to the growth medium under photomixotrophic conditions. Activities of various metabolic processes, including carbon dioxide fixation, respiration, and nitrogen assimilation, seemed to be enhanced by 2OG addition. These observations suggest that cyAbrB2 is essential for the active transcription of genes related to carbon and nitrogen metabolism upon a shift to photomixotrophic conditions. Deletion of cyAbrB2 is likely to deregulate the partition of carbon between storage forms and soluble forms used for biosynthetic purposes. This disorder may cause inactivation of cellular metabolism, excess accumulation of reducing equivalents, and subsequent loss of viability under photomixotrophic conditions.

Photosynthetic organisms have evolved various acclimatory responses to high-light (HL) conditions to maintain a balance between energy supply (light harvesting and electron transport) and consumption (cellular metabolism) and to protect the photosynthetic apparatus from photodamage. The molecular mechanism of HL acclimation has been extensively studied in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Whole genome DNA microarray analyses have revealed that the change in gene expression profile under HL is closely correlated with subsequent acclimatory responses such as (1) acceleration in the rate of photosystem II turnover, (2) downregulation of light harvesting capacity, (3) development of a protection mechanism for the photosystems against excess light energy, (4) upregulation of general protection mechanism components, and (5) regulation of carbon and nitrogen assimilation. In this review article, we survey recent progress in the understanding of the molecular mechanisms of these acclimatory responses in Synechocystis sp. PCC 6803. We also briefly describe attempts to understand HL acclimation in various cyanobacterial species in their natural environments.

All known cyanobacterial genomes possess multiple copies of genes encoding AbrB-like transcriptional regulators, known as cyAbrBs, which are distinct from those conserved among other bacterial species. In this study, we addressed the physiological roles of Sll0822 and Sll0359, the two cyAbrBs in Synechocystis sp. strain PCC 6803, under nonstress conditions (20 μmol of photons m ⁻² s ⁻¹ in ambient CO ₂ ). When the sll0822 gene was disrupted, the expression levels of nitrogen-related genes such as urtA , amt1 , and glnB significantly decreased compared with those in the wild-type cells. Possibly due to the increase of the cellular carbon/nitrogen ratio in the sll0822-disrupted cells, a decrease in pigment contents, downregulation of carbon-uptake related genes, and aberrant accumulation of glycogen took place. Moreover, the mutant exhibited the decrease in the expression level of cytokinesis-related genes such as ftsZ and ftsQ , resulting in the defect in cell division and significant increase in cell size. The pleiotrophic phenotype of the mutant was efficiently suppressed by the introduction of Sll0822 and also partially suppressed by the introduction of Sll0359. When His-tagged cyAbrBs were purified from overexpression strains, Sll0359 and Sll0822 were copurified with each other. The cyAbrBs in Synechocystis sp. strain PCC 6803 seem to interact with each other and regulate carbon and nitrogen metabolism as well as the cell division process under nonstress conditions.