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Hypothetical chloroplast open reading frames (ycfs) are highly conserved and interspecifically occurring genes in plastomes of plants and algae with significant functions in gene expression and photosynthesis. However, the function of many ycfs is still in vain so that attention is directed to other chloroplast functions such as metabolism of co-factors, protein translocation and protection against abiotic stress. We provide a comprehensive functional description of ycf53 and ycf59, two genes involved in chlorophyll biosynthesis. While ycf59 encodes an essential enzymatic component of Mg protoporphyrin monomethylester cyclase, ycf53 encodes a posttranslational regulator of chlorophyll biosynthesis. Their roles in tetrapyrrole biosynthesis were compared by using cyanobacterial and plant mutants with modulated expression of these two genes. Our work provides indications for diverse effects of these homologous gene products in plants and cyanobacteria on tetrapyrrole biosynthesis and photosynthesis.

Organisms coordinate biological activities into daily cycles using an internal circadian clock. The circadian oscillator proteins KaiA, KaiB, and KaiC are widely believed to underlie 24-h oscillations of gene expression in cyanobacteria. However, a group of very abundant cyanobacteria, namely, marine Prochlorococcus species, lost the third oscillator component, KaiA, during evolution. We demonstrate here that the remaining Kai proteins fulfill their known biochemical functions, although KaiC is hyperphosphorylated by default in this system. These data provide biochemical support for the observed evolutionary reduction of the clock locus in Prochlorococcus and are consistent with a model in which a mechanism that is less robust than the well-characterized KaiABC protein clock of Synechococcus is sufficient for biological timing in the very stable environment that Prochlorococcus inhabits.

Gun4 is a porphyrin-binding protein that activates magnesium chelatase, a multimeric enzyme catalyzing the first committed step in chlorophyll biosynthesis. In plants, GUN4 has been implicated in plastid-to-nucleus retrograde signaling processes that coordinate both photosystem II and photosystem I nuclear gene expression with chloroplast function. In this work we present the functional analysis of Gun4 from the cyanobacterium Synechocystis sp. PCC 6803. Affinity co-purification of the FLAG-tagged Gun4 with the ChlH subunit of the magnesium chelatase confirmed the association of Gun4 with the enzyme in cyanobacteria. Inactivation of the gun4 gene abolished photoautotrophic growth of the resulting gun4 mutant strain that exhibited a decreased activity of magnesium chelatase. Consequently, the cellular content of chlorophyll-binding proteins was highly inadequate, especially that of proteins of photosystem II. Immunoblot analyses, blue native polyacrylamide gel electrophoresis, and radiolabeling of the membrane protein complexes suggested that the availability of the photosystem II antenna protein CP47 is a limiting factor for the photosystem II assembly in the gun4 mutant.

The Ycf37 protein has been suggested to be involved in the biogenesis and/or stability of the cyanobacterial photosystem I (PSI) [A. Wilde, K. Lünser, F. Ossenbühl, J. Nickelsen, T. Börner, Characterization of the cyanobacterial ycf37: mutation decreases the photosystem I content, Biochem. J. 357 (2001) 211–216]. With Ycf37 specific antibodies, we analyzed the localization of Ycf37 within the thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Inspection of a sucrose gradient profile indicated that small amounts of Ycf37 co-fractionated with monomeric photosynthetic complexes, but not with trimeric PSI. Isolating 3xFLAG epitope-tagged Ycf37 by affinity-tag purification rendered several PSI subunits that specifically co-precipitated with this protein. Blue-native PAGE newly revealed two monomeric PSI complexes (PSI and PSI*) in wild-type thylakoids. The lower amount of PsaK present in PSI* may explain its higher electrophoretic mobility. PSI* was more prominent in high-light grown cells and interestingly proved absent in the Δycf37 mutant. PSI* appeared again when the mutant was complemented in trans with the wild-type ycf37 gene. In the Δycf37 mutant the amount of trimeric PSI complexes was reduced to about 70% of the wild-type level with no significant changes in photochemical activity and subunit composition of the remaining photosystems. Our results indicate that Ycf37 plays a specific role in the preservation of PSI* and the biogenesis of PSI trimers.

Inactivation of the genes for the cyanobacterial phytochromes cph1 and cph2 in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 affected the growth of the cells under certain light conditions. Differences in growth were detected by recording growth curves and in competition experiments. Mutation of cph1 and cph2 resulted in different effects. The cph1-mutant strains exhibited a reduced growth rate under far-red light (FRL), whereas the growth of the cph2-mutant strains was inhibited by red light (RL). The growth rate of a cph1- / cph2- double mutant was reduced under both RL and FRL. Furthermore, cph1-, cph2- as well as double-mutant strains showed impaired growth under high-light (HL) conditions. Acclimation of the photosynthetic apparatus of the mutants to RL, FRL and HL, as determined by pigment analysis, was similar to that of the wild type.

We analysed the effects of inactivation of the phytochrome genes cph1 and cph2 on the phototactic migration of the cyanobacterium Synechocystis sp. PCC 6803 under different light qualities. Motility towards white, green, red and far red light was not altered in several independently raised cph1, cph2 and cph1/cph2 double mutants. Blue light (lambda = 400-500 nm) did not induce motility in wild-type and cph1 mutant cells, whereas cph2 and cph1/cph2 double mutants moved towards the blue light. Inhibition of the photosynthetic electron transport by DCMU influenced the motility of cph2 mutants under blue light, but not under white, red and far red light. RNA blot hybridizations did not indicate an altered transcript accumulation of the pilin-encoding pilA1 gene under blue light. We propose that the Cph2 protein is part of a light-stimulated signal transduction chain inhibiting the movement of Synechocystis sp. PCC 6803 cells towards blue light.

Phytochromes are photoreceptors common to lower and higher plants. Plant phytochrome apoproteins autocatalytically attach phytochromobilin, a linear tetrapyrrole chromophore [1]. They regulate a wide variety of photomorphogenetic responses of plants to light [2]. The total sequence of the Synechocystis sp. PCC 6803 chromosome revealed the existence of a putative phytochrome gene (phy, ORF slr0473) [3]. The phy gene potentially encodes a protein (PhyS) that exhibits homology to phytochromes throughout its length, in particular in the N-terminal region including the chromophore binding site [4]. Moreover, the putative Synechocystis phytochrome shows distinct homology to sensory histidine kinases. Sensory histidine kinases and the so-called response regulators form the “two component systems” of signal transduction in bacteria [5]. Two component systems have been discovered more recently also in eukaryotes including components involved in the ethylene-induced signal transduction in Arabidopsis [6]. The Synechocystis chromosome encodes a number of putative response regulators and histidine kinases [3]. Several of these kinases contain stretches of amino acids with limited similarity to the N-terminal domain of phytochromes and to domains of the ethylene receptor protein of Arabidopsis (summarized in Table 1). We named these putative histidine kinases “phytochrome like proteins” (abbreviated: plp) [7]. We disrupted the gene of one of these plp’s (plpA, encoded by ORF sll1124 [3]) by directed insertional mutagenesis. The obtained mutant was found to be unable to grow under blue light [7].

A gene that may encode a novel light sensing histidine protein kinase, designated plpA (phytochrome-like protein), was isolated from the cyanobacterium Synechocystis sp. PCC 6803. The 200 COOH-terminal amino acids of the gene product show homology with conserved domains of several bacterial histidine kinases and the ethylene response gene etr1 of Arabidopsis, whereas its central region is similar to the chromophore attachment site of plant phytochromes. Interruption or partial deletion of plpA yielded mutants unable to grow under blue light.

Cyanobacteria are the simplest known cellular systems that regulate their biological activities in daily cycles. For the cyanobacterium Synechococcus elongatus, it has been shown by in vitro and in vivo experiments that the basic circadian timing process is based on rhythmic phosphorylation of KaiC hexamers. Despite the excellent experimental work, a full systems level understanding of the in vitro clock is still lacking. In this work, we provide a mathematical approach to scan different hypothetical mechanisms for the primary circadian oscillator, starting from experimentally established molecular properties of the clock proteins. Although optimised for highest performance, only one of the in silico-generated reaction networks was able to reproduce the experimentally found high amplitude and robustness against perturbations. In this reaction network, a negative feedback synchronises the phosphorylation level of the individual hexamers and has indeed been realised in S. elongatus by KaiA sequestration as confirmed by experiments.

A gene of the unicellular cyanobacterium Synechocystis sp strain PCC 6803 that is homologous to the conserved chloroplast open reading frame orf184 has been cloned and sequenced. The nucleotide sequence of the gene predicts a protein of 184 amino acids with a calculated molecular mass of 21.5 kD and two membrane-spanning regions. Amino acid sequence analysis showed 46 to 37% homology of the cyanobacterial orf184 with tobacco orf184, rice orf185, liverwort orf184, and Euglena gracilis orf206 sequences. Two orf184-specific mutants of Synechocystis sp PCC 6803 were constructed by insertion mutagenesis. Cells of mutants showed growth characteristics similar to those of the wild type. Their pigment composition was distinctly different from the wild type, as indicated by an increase in the phycocyanin-to-chlorophyll ratio. In addition, mutants also had a two- to threefold increase in photosynthetic electron transfer rates as well as in photosystem II-to-photosystem I ratio-a phenomenon hitherto not reported for mutants with altered photosynthetic characteristics. The observed alterations in the orf184-specific mutants provide strong evidence for a functional role of the orf184 gene product in photosynthetic processes.

Ycf53 is a hypothetical chloroplast open reading frame with similarity to the Arabidopsis nuclear gene GUN4. In plants, GUN4 is involved in tetrapyrrole biosynthesis. We demonstrate that one of the two Synechocystis sp. PCC 6803 ycf53 genes with similarity to GUN4 functions in chlorophyll (Chl) biosynthesis as well: cyanobacterial gun4 mutant cells exhibit lower Chl contents, accumulate protoporphyrin IX and show less activity not only of Mg chelatase but also of Fe chelatase. The possible role of Gun4 for the Mg as well as Fe porphyrin biosynthesis branches in Synechocystis sp. PCC 6803 is discussed.

Noncoding RNAs are crucial regulators of gene expression in prokaryotes and eukaryotes, but how they affect the dynamics of transcriptional networks remains poorly understood. We analyzed the temporal characteristics of the cyanobacterial iron stress response by mathematical modeling and quantitative experimental analyses and focused on the role of a recently discovered small noncoding RNA, IsrR. We found that IsrR is responsible for a pronounced delay in the accumulation of isiA mRNA encoding the late-phase stress protein, IsiA, and that it ensures a rapid decline in isiA levels once external stress triggers are removed. These kinetic properties allow the system to selectively respond to sustained (as opposed to transient) stimuli and thus establish a temporal threshold, which prevents energetically costly IsiA accumulation under short-term stress conditions. Biological information is frequently encoded in the quantitative aspects of intracellular signals (e.g., amplitude and duration). Our simulations reveal that competitive inhibition and regulated degradation allow intracellular regulatory networks to efficiently discriminate between transient and sustained inputs.

The cyanobacterial wild-type strain Synechocystis sp. PCC 6803 and its engineered strain BW86 were cultivated under defined conditions in photobioreactors to investigate the effect of phosphate availability on cyanophycin accumulation. Cyanophycin, multi- l -arginyl-poly- l -aspartate, can be deployed as amino acid source or can be chemically converted into polyaspartic acid, a biodegradable polymer. In previous studies it was demonstrated that a single point mutation in the PII signaling protein from the Synechocystis wild type is sufficient to unlock the arginine pathway causing an over accumulation of the biopolymer cyanophycin in BW86. One process strategy to evoke cyanophycin synthesis in Synechocystis is nutrient starvation. Therefore, different phosphate concentrations from 17.5 to 175 μM were tested. Progressive phosphate starvation resulted in an increased cyanophycin accumulation. The highest obtained cyanophycin amounts in g cyanophycin per g cell dry mass were 18% and 40% for Synechocystis sp. PCC 6803 wild type and BW86 respectively, demonstrating that phosphate starvation is an effective route for biotechnological cyanophycin production. By evaluating cyanophycin and phosphor quotas per cell dry mass, it was possible to determine the specific required amount of phosphor to accumulate cyanophycin and to initialize stationary growth phase. Phosphor quotas in the range of 4 to 1 mg phosphor per g cell dry mass triggered cyanophycin biosynthesis while Synechocystis entered the stationary phase at phosphor quotas of 1 mg phosphor per g cell dry mass or less. Additionally, light kinetics was determined. Photon flux densities exceeding 46 μmol photons m − 2 s − 1 result in a maximum growth rate of 1.32 d − 1 .

Future sustainable energy production can be achieved using mass cultures of photoautotrophic microorganisms, which are engineered to synthesize valuable products directly from CO2 and sunlight. As cyanobacteria can be cultivated in large scale on non-arable land, these phototrophic bacteria have become attractive organisms for production of biofuels. Synechococcus sp. PCC 7002, one of the cyanobacterial model organisms, provides many attractive properties for biofuel production such as tolerance of seawater and high light intensities. Here, we performed a systems analysis of an engineered ethanol-producing strain of the cyanobacterium Synechococcus sp. PCC 7002, which was grown in artificial seawater medium over 30 days applying a 12:12 h day–night cycle. Biosynthesis of ethanol resulted in a final accumulation of 0.25% (v/v) ethanol, including ethanol lost due to evaporation. The cultivation experiment revealed three production phases. The highest production rate was observed in the initial phase when cells were actively growing. In phase II growth of the producer strain stopped, but ethanol production rate was still high. Phase III was characterized by a decrease of both ethanol production and optical density of the culture. Metabolomics revealed that the carbon drain due to ethanol diffusion from the cell resulted in the expected reduction of pyruvate-based intermediates. Carbon-saving strategies successfully compensated the decrease of central intermediates of carbon metabolism during the first phase of fermentation. However, during long-term ethanol production the producer strain showed clear indications of intracellular carbon limitation. Despite the decreased levels of glycolytic and tricarboxylic acid cycle intermediates, soluble sugars and even glycogen accumulated in the producer strain. The changes in carbon assimilation patterns are partly supported by proteome analysis, which detected decreased levels of many enzymes and also revealed the stress phenotype of ethanol-producing cells. Strategies towards improved ethanol production are discussed. Systems analysis of ethanol production in Synechococcus sp. PCC 7002 revealed initial compensation followed by increasing metabolic limitation due to excessive carbon drain from primary metabolism.