Mitsunori Katayama

Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein

Cyanobacteriochromes are a newly recognized group of photoreceptors that are distinct relatives of phytochromes but are found only in cyanobacteria. A putative cyanobacteriochrome, CcaS, is known to chromatically regulate the expression of the phycobilisome linker gene (cpcG2) in Synechocystis sp. PCC 6803. In this study, we isolated the chromophore-binding domain of CcaS from Synechocystis as well as from phycocyanobilin-producing Escherichia coli. Both preparations showed the same reversible photoconversion between a green-absorbing form (Pg, λmax = 535 nm) and a red-absorbing form (Pr, λmax = 672 nm). Mass spectrometry and denaturation analyses suggested that Pg and Pr bind phycocyanobilin in a double-bond configuration of C15-Z and C15-E, respectively. Autophosphorylation activity of the histidine kinase domain in nearly full-length CcaS was up-regulated by preirradiation with green light. Similarly, phosphotransfer to the cognate response regulator, CcaR, was higher in Pr than in Pg. From these results, we conclude that CcaS phosphorylates CcaR under green light and induces expression of cpcG2, leading to accumulation of CpcG2-phycobilisome as a chromatic acclimation system. CcaS is the first recognized green light receptor in the expanded phytochrome superfamily, which includes phytochromes and cyanobacteriochromes.

The Membrane-Associated CpcG2-Phycobilisome in Synechocystis : A New Photosystem I Antenna

The phycobilisome (PBS) is a supramolecular antenna complex required for photosynthesis in cyanobacteria and bilin-containing red algae. While the basic architecture of PBS is widely conserved, the phycobiliproteins, core structure and linker polypeptides, show significant diversity across different species. By contrast, we recently reported that the unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses two types of PBSs that differ in their interconnecting “rod-core linker” proteins (CpcG1 and CpcG2). CpcG1-PBS was found to be equivalent to conventional PBS, whereas CpcG2-PBS retains phycocyanin rods but is devoid of the central core. This study describes the functional analysis of CpcG1-PBS and CpcG2-PBS. Specific energy transfer from PBS to photosystems that was estimated for cells and thylakoid membranes based on low-temperature fluorescence showed that CpcG2-PBS transfers light energy preferentially to photosystem I (PSI) compared to CpcG1-PBS, although they are able to transfer to both photosystems. The preferential energy transfer was also supported by the increased photosystem stoichiometry (PSI/PSII) in the cpcG2 disruptant. The cpcG2 disruptant consistently showed retarded growth under weak PSII light, in which excitation of PSI is limited. Isolation of thylakoid membranes with high salt showed that CpcG2-PBS is tightly associated with the membrane, while CpcG1-PBS is partly released. CpcG2 is characterized by its C-terminal hydrophobic segment, which may anchor CpcG2-PBS to the thylakoid membrane or PSI complex. Further sequence analysis revealed that CpcG2-like proteins containing a C-terminal hydrophobic segment are widely distributed in many cyanobacteria.

Cyanobacteriochrome CcaS regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic adapter

Responding to green and red light, certain cyanobacteria change the composition of their light-harvesting pigments, phycoerythrin (PE) and phycocyanin (PC). Although this phenomenon—complementary chromatic adaptation—is well known, the green light–sensing mechanism for PE accumulation is unclear. The filamentous cyanobacterium Nostoc punctiforme ATCC 29133 (N. punctiforme) regulates PE synthesis in response to green and red light (group II chromatic adaptation). We disrupted the green/red-perceiving histidine-kinase gene (ccaS) or the cognate response regulator gene (ccaR), which are clustered with several PE and PC genes (cpeC-cpcG2-cpeR1 operon) in N. punctiforme. Under green light, wild-type cells accumulated a significant amount of PE upon induction of cpeC-cpcG2-cpeR1 expression, whereas they accumulated little PE with suppression of cpeC-cpcG2-cpeR1 expression under red light. Under both green and red light, the ccaS mutant constitutively accumulated some PE with constitutively low cpeC-cpcG2-cpeR1 expression, whereas the ccaR mutant accumulated little PE with suppression of cpeC-cpcG2-cpeR1 expression. The results of an electrophoretic mobility shift assay suggest that CcaR binds to the promoter region of cpeC-cpcG2-cpeR1, which contains a conserved direct-repeat motif. Taken together, the results suggest that CcaS phosphorylates CcaR under green light and that phosphorylated CcaR then induces cpeC-cpcG2-cpeR1 expression, leading to PE accumulation. In contrast, CcaS probably represses cpeC-cpcG2-cpeR1 expression by dephosphorylation of CcaR under red light. We also found that the cpeB-cpeA operon is partially regulated by green and red light, suggesting that the green light-induced regulatory protein CpeR1 activates cpeB-cpeA expression together with constitutively induced CpeR2.

ldpA Encodes an Iron-Sulfur Protein Involved in Light-Dependent Modulation of the Circadian Period in the Cyanobacterium Synechococcus elongatus PCC 7942

We generated random transposon insertion mutants to identify genes involved in light input pathways to the circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942. Two mutants, AMC408-M1 and AMC408-M2, were isolated that responded to a 5-h dark pulse differently from the wild-type strain. The two mutants carried independent transposon insertions in an open reading frame here named ldpA (for light-dependent period). Although the mutants were isolated by a phase shift screening protocol, the actual defect is a conditional alteration in the circadian period. The mutants retain the wild-type ability to phase shift the circadian gene expression (bioluminescent reporter) rhythm if the timing of administration of the dark pulse is corrected for a 1-h shortening of the circadian period in the mutant. Further analysis indicated that the conditional short-period mutant phenotype results from insensitivity to light gradients that normally modulate the circadian period in S. elongatus, lengthening the period at low light intensities. The ldpA gene encodes a polypeptide that predicts a 7Fe-8S cluster-binding motif expected to be involved in redox reactions. We suggest that the LdpA protein modulates the circadian clock as an indirect function of light intensity by sensing changes in cellular physiology.

Three major output pathways from the KaiABC-based oscillator cooperate to generate robust circadian kaiBC expression in cyanobacteria

Circadian kaiBC expression in the cyanobacterium Synechococcus elongatus PCC 7942 is generated by temporal information transmission from the KaiABC-based circadian oscillator to RpaA, a putative transcriptional factor, via the SasA-dependent positive pathway and the LabA-dependent negative pathway which is responsible for feedback regulation of KaiC. However, the labA/sasA double mutant has a circadian kaiBC expression rhythm, suggesting that there is an additional circadian output pathway. Here we describe a third circadian output pathway, which is CikA-dependent. The cikA mutation attenuates KaiC overexpression-induced kaiBC repression and exacerbates the low-amplitude phenotype of the labA mutant, suggesting that cikA acts as a negative regulator of kaiBC expression independent of the LabA-dependent pathway. In the labA/sasA/cikA triple mutant, kaiBC promoter activity becomes almost arrhythmic, despite preservation of the circadian KaiC phosphorylation rhythm, suggesting that CikA largely accounts for the residual kaiBC expression rhythm observed in the labA/sasA double mutant. These results also strongly suggest that transcriptional regulation in the labA/sasA/cikA triple mutant is insulated from the circadian signals of the KaiABC-based oscillator. Based on these observations, we propose a model in which temporal information from the KaiABC-based circadian oscillator is transmitted to gene expression through three separate output pathways.

Distinct roles of CpcG1 and CpcG2 in phycobilisome assembly in the cyanobacterium Synechocystis sp. PCC 6803.

Structural role of the second copy of the rod–core linker CpcG, which was found by genome analysis, was studied in Synechocystis sp. PCC 6803 by gene disruption and fractionation of phycobilisome (sub)complexes. Disruption of cpcG2 (sll1471) resulted in a marked decrease in phycocyanin content both in the background of wild-type and cpcG1 (slr2051)-disruptant. The unique phycocyanin rod–CpcG2 complex without the major allophycocyanin components was isolated from the cpcG1-disruptant. By fluorescence analysis, it was proposed that CpcG2 protein connects the rods with a minor allophycocyanin component, to support energy transfer to Photosystem I.

A Novel Mutation in kaiC Affects Resetting of the Cyanobacterial Circadian Clock

Light is the most important factor controlling circadian systems in response to day-night cycles. In order to better understand the regulation of circadian rhythms by light in Synechococcus elongatus PCC 7942, we screened for mutants with defective phase shifting in response to dark pulses. Using a 5-h dark-pulse protocol, we identified a mutation in kaiC that we termed pr1, for phase response 1. In the pr1 mutant, a 5-h dark pulse failed to shift the phase of the circadian rhythm, while the same pulse caused a 10-h phase shift in wild-type cells. The rhythm in accumulation of KaiC was abolished in the pr1 mutant, and the rhythmicity of KaiC phosphorylation was reduced. Additionally, the pr1 mutant was defective in mediating the feedback inhibition of kaiBC. Finally, overexpression of mutant KaiC led to a reduced phase shift compared to that for wild-type KaiC. Thus, KaiC appears to play a role in resetting the cellular clock in addition to its documented role in the feedback regulation of circadian rhythms.

Cellulose Accumulation and a Cellulose Synthase Gene are Responsible for Cell Aggregation in the Cyanobacterium Thermosynechococcus vulcanus RKN

A thermophilic cyanobacterium, Thermosynechococcus vulcanus RKN, exhibits cell aggregation under low temperature illuminated conditions as a means of physiological acclimation to avoid excess light stress. The cell aggregation was dispersed with cellulase treatment. We developed a method to quantify small amounts of cellulose by partial cellulose purification followed by quantitation of liberated glucose by cellulase. Under low temperature illuminated light conditions, cellulose accumulation was induced approximately 2-fold, to 10 μg (4 × 10(9) cells)(-1), and slightly preceded aggregation. Based on sequence similarity, three candidate genes for cellulose synthase (Tvtll0007, Tvtlr1795 and Tvtlr1930-33) were cloned from T. vulcanus. Gene disruption analysis showed that only Tvtll0007 was responsible for both the light- and low temperature-induced cell aggregation and the induction of cellulose accumulation. Gene expression analysis suggested that the low temperature illuminated conditions quickly induced expression of Tvtlr1795 and Tvtlr1930-33, while the induction of Tvtll0007 was slow. These results suggest that Tvtll0007 encodes a functional cellulose synthase whose activity may not be regulated at the transcriptional level.

The Circadian Clock-Related Gene pex Regulates a Negative cis Element in the kaiA Promoter Region

In the cyanobacterium Synechococcus sp. strain PCC 7942, a circadian clock-related gene, pex, was identified as the gene prolonging the period of the clock. A PadR domain, which is a newly classified transcription factor domain, and the X-ray crystal structure of the Pex protein suggest a role for Pex in transcriptional regulation in the circadian system. However, the regulatory target of the Pex protein is unknown. To determine the role of Pex, we monitored bioluminescence rhythms that reported the expression activity of the kaiA gene or the kaiBC operon in pex deficiency, pex constitutive expression, and the wild-type genotype. The expression of kaiA in the pex-deficient or constitutive expression genotype was 7 or 1/7 times that of the wild type, respectively, suggesting that kaiA is the target of negative regulation by Pex. In contrast, the expression of the kaiBC gene in the two pex-related genotypes was the same as that in the wild type, suggesting that Pex specifically regulates kaiA expression. We used primer extension analysis to map the transcription start site for the kaiA gene 66 bp upstream of the translation start codon. Mapping with deletion and base pair substitution of the kaiA upstream region revealed that a 5-bp sequence in this region was essential for the regulation of kaiA. The repression or constitutive expression of the kaiA transgene caused the prolongation or shortening of the circadian period, respectively, suggesting that the Pex protein changes the period via the negative regulation of kaiA.

Transcriptional regulation of the circadian clock operon kaiBC by upstream regions in cyanobacteria

In the cyanobacterium, Synechococcus elongatus PCC 7942, the kaiBC operon is upregulated by the KaiA protein and downregulated by the KaiC protein to generate circadian oscillation. We investigated the regulation of kaiBC transcription. A primer extension and deletion analyses of the upstream region mapped the sufficient promoter region (SPR) to base pairs −55 to +1 (the transcription start site, TSS) and identified a constitutive negative regulatory region upstream of the SPR (base pairs −897 to −56) that extended into the coding sequence of kaiA. Base‐pair substitution within the SPR identified a sequence from −52 to −28 that was the essential element for transcription. Most of the examined sequences drove rhythmic expression of a luxAB reporter that was similar to the expression driven by the kaiBC promoter (PkaiBC) and responded to the overexpression of kaiA or kaiC, even in a promoter activity range of 1–8000%. These results indicate that circadian feedback regulation by KaiA and KaiC is addressed to a  global step preceding transcription driven by PkaiBC. However, increasing or decreasing the intrinsic activity of PkaiBC greatly affected the rhythm, suggesting that constitutive adjustment of PkaiBC activity by the sequences identified here is essential for the oscillator.