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Cyanobacteria respond to light stimulation, activating localised assembly of type IV pili for motility. The resulting phototactic response is highly dependent on the nature of the incoming light stimulus, and the final motility parameters depend on the surface properties. Conventionally, phototaxis studies are carried out on hydrogel surfaces, such as agarose, with surface properties that vary in time due to experimental conditions. This study considers five substrates, widely utilized in microfluidic technology, to identify the most suitable alternative for performing reliable and repeatable phototaxis assays. The surfaces are characterised via a contact angle goniometer to determine the surface energy, white light interferometry for roughness, zeta-potentials and AFM force distance curves for charge patterns, and XPS for surface composition. Cell motility assays showed 1.25 times increment on surfaces with a water contact angle of 80° compared to a reference glass surface. To prove that motility can be enhanced, polydimethylsiloxane (PDMS) surfaces were plasma treated to alter their surface wettability. The motility on the plasma-treated PDMS showed similar performance as for glass surfaces. In contrast, untreated PDMS surfaces displayed close to zero motility. We also describe the force interactions of cells with the test surfaces using DLVO (Derjaguin-Landau-Verwey-Overbeek) and XDLVO (extended DLVO) theories. The computed DLVO/XDLVO force-distance curves are compared with those obtained using atomic force microscopy. Our findings show that twitching motility on tested surfaces can be described mainly from adhesive forces and hydrophobicity/hydrophilicity surface properties. STATEMENT OF SIGNIFICANCE: The current article focuses on unravelling the potential Micro-Electro-Mechanical System (MEMS) compatible surfaces for studying phototactic twitching motility of cyanobacteria. This is the first exhaustive surface characterization study coupled with phototaxis experiments, to understand the forces contributing to twitching motility. The methods shown in this paper can be further extended to study other surfaces and also to other bacteria exhibiting twitching motility.

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that two-step protein secretion in cyanobacteria is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further.ImportanceProtein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here we introduce a NanoLuc (NLuc)-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge on cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

Photoreceptors are proteins that sense incident light and then trigger downstream signaling events. Phytochromes are linear tetrapyrrole-binding photoreceptors present in plants, algae, fungi, and various bacteria. Most phytochromes respond to red and far-red light signals. Among the phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show much more diverse optical properties covering the entire visible region. Both phytochromes and cyanobacteriochromes share the GAF domain scaffold to cradle the chromophore as the light-sensing region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here we utilize ancestral sequence reconstruction and report that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore enables the photoreceptor to perceive green light. The ancestral cyanobacteriochromes show modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense green-rich irradiance rather than red light, which is preferentially utilized for photosynthesis. In contrast to plants and green algae, many cyanobacteria can utilize green light for photosynthesis with their special light-harvesting complexes, phycobilisomes. The evolution of green/red sensing cyanobacteriochromes may therefore have allowed ancient cyanobacteria to acclimate to different light environments by rearranging the absorption capacity of the cyanobacterial antenna complex by chromatic acclimation.Significance StatementLight serves as a crucial environmental stimulus affecting the physiology of organisms across all kingdoms of life. Photoreceptors serve as important players of light responses, absorbing light and actuating biological processes. Among a plethora of photoreceptors, cyanobacteriochromes arguably have the wealthiest palette of color sensing, largely contributing to the success of cyanobacteria in various illuminated habitats. Our ancestral sequence reconstruction and the analysis of the resurrected ancestral proteins suggest that the very first cyanobacteriochrome most probably responded to the incident green-to-red light ratio, in contrast to modern red/far-red absorbing plant phytochromes. The deprotonation of the light-absorbing pigment for green light-sensing was a crucial molecular event for the invention of the new class of photoreceptors with their huge color tuning capacity.

Synechococcus elongatus PCC 7942 is the model organism for circadian research in Cyanobacteria, and many breakthroughs in our understanding of the clock came through an early focus on a single model organism. However, Cyanobacteria are known for their diversity, and blanket generalization from a single species may be inaccurate. Bioinformatic sequence and data analyses combined with in-depth biochemical, structural, and physiological characterization revealed a range of timing systems and gave insights into shared and distinct features of the clock in different cyanobacteria. Those clock systems range from a largely reduced system in Prochlorococcus, which lacks the core factor KaiA along with central input and output factors, to an extended system in Synechocystis sp. PCC 6803, which harbors two additional Kai systems with rather unknown functions. In between, there are more cyanobacterial strains for which circadian rhythmicity was shown. Moreover, homologs of cyanobacterial clock proteins are present in some Bacteria and Archaea, opening questions about the evolution of circadian rhythmicity in prokaryotes. In this chapter, we will highlight common aspects and key differences of timing systems that are based on Kai proteins.

Photoautotrophic organisms depend on the ambient light for their growth and viability; therefore, it is not surprising that they utilize sophisticated light-regulated signaling systems to acclimate to variable light environments. Cyanobacteria are important primary producers that perform oxygenic photosynthesis in various environmental niches. Cyanobacterial genomes encode multiple and diverse photoreceptors which are often connected to second messenger signaling networks. Here, we review the current knowledge of light-regulated second messenger signaling in cyanobacteria, focusing on two examples: cyclic di-GMP signaling systems for regulation of Thermosynechococcus sessility and Synechocystis motility. We also briefly introduce the present research on various nucleotide second messenger molecules, such as cAMP, cGMP, cyclic di-GMP, cyclic di-AMP, and the alarmone (p)ppGpp in cyanobacteria. In natural conditions, incident light contains a lot of different information on wavelength, intensity, and time scales. Further understanding of second messenger signaling in cyanobacteria will uncover how cyanobacteria extract the crucial information from their light environment to regulate cellular responses of ecophysiological importance.

The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways., Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro. KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems. IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways.

Photoautotrophic cyanobacteria and microalgae offer significant potential for the renewable synthesis of high-value products. As yet, however, the productivity of phototrophic cultures is limited due to the low cell densities that are typically obtained in current pilot scale photobioreactors. Here, we explore the use of ultrahigh-density cultivation of cyanobacteria for the production of cyanophycin, a non-ribosomally synthesized biopolymer of high biotechnological interest. We demonstrate that ultrahigh-density cultivation using a two-tier vessel with membrane-mediated CO2 supply yields a cyanophycin content per cellular dry weight similar to previously reported values, while the volumetric productivity per culture volume is significantly increased. Already after 96 h of cultivation, the engineered production strain BW86 reached up to 1 g cyanophycin per liter culture, approximately a 4-fold increase over the previously reported maximal yield obtained after 12 days of cultivation. Under phosphate-limiting growth conditions, the wild-type strain Synechcocystis sp. PCC 6803 accumulates up to 0.6 g cyanophycin per L culture. Our results demonstrate that ultrahigh-density cultivation is a suitable strategy towards the development of viable phototrophic production processes for cyanophycin and possibly other products of interest.

The natural light environment is important to many prokaryotes. Most obviously, phototrophic prokaryotes need to acclimate their photosynthetic apparatus to the prevailing light conditions, and such acclimation is frequently complemented by motility to enable cells to relocate in search of more favorable illumination conditions. Non-phototrophic prokaryotes may also seek to avoid light at damaging intensities and wavelengths, and many prokaryotes with diverse lifestyles could potentially exploit light signals as a rich source of information about their surroundings and a cue for acclimation and behavior. Here we discuss our current understanding of the ways in which bacteria can perceive the intensity, wavelength and direction of illumination, and the signal transduction networks that link light perception to the control of motile behavior. We discuss the problems of light perception at the prokaryotic scale, and the challenge of directional light perception in small bacterial cells. We explain the peculiarities and the common features of light-controlled motility systems in prokaryotes as diverse as cyanobacteria, purple photosynthetic bacteria, chemoheterotrophic bacteria and haloarchaea., The authors discuss our understanding of the ways in which prokaryotes can control their motility in response to the intensity, spectral quality and direction of illumination.

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed “phototaxis,” enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJ(Se) (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis. Plate-based phototaxis assays indicate that UTEX 3055 uses PixJ(Se) to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJ(Se) controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

Significance Iron limits the growth of photosynthetic organisms, especially in marine environments. Understanding the response of photosynthetic organisms to changing iron concentrations is therefore important for agriculture and biotechnology. We have identified a protein that is essential for the correct response to changing iron concentrations in photosynthetic bacteria (cyanobacteria). This protein was previously annotated as an electron transfer component of photosynthesis, called Fed2, and contains an iron−sulfur cluster. We tested Fed2, and found that it cannot act in photosynthetic electron transport. The corresponding gene is essential, and is highly conserved between cyanobacteria, algae, and higher plants. By specifically perturbing its function, we could show that it is essential for the low-iron response at the posttranscriptional level., Iron chronically limits aquatic photosynthesis, especially in marine environments, and the correct perception and maintenance of iron homeostasis in photosynthetic bacteria, including cyanobacteria, is therefore of global significance. Multiple adaptive mechanisms, responsive promoters, and posttranscriptional regulators have been identified, which allow cyanobacteria to respond to changing iron concentrations. However, many factors remain unclear, in particular, how iron status is perceived within the cell. Here we describe a cyanobacterial ferredoxin (Fed2), with a unique C-terminal extension, that acts as a player in iron perception. Fed2 homologs are highly conserved in photosynthetic organisms from cyanobacteria to higher plants, and, although they belong to the plant type ferredoxin family of [2Fe-2S] photosynthetic electron carriers, they are not involved in photosynthetic electron transport. As deletion of fed2 appears lethal, we developed a C-terminal truncation system to attenuate protein function. Disturbed Fed2 function resulted in decreased chlorophyll accumulation, and this was exaggerated in iron-depleted medium, where different truncations led to either exaggerated or weaker responses to low iron. Despite this, iron concentrations remained the same, or were elevated in all truncation mutants. Further analysis established that, when Fed2 function was perturbed, the classical iron limitation marker IsiA failed to accumulate at transcript and protein levels. By contrast, abundance of IsiB, which shares an operon with isiA, was unaffected by loss of Fed2 function, pinpointing the site of Fed2 action in iron perception to the level of posttranscriptional regulation.

Motility in cyanobacteria is useful for purposes that range from seeking out favourable light environments to establishing symbioses with plants and fungi. No known cyanobacterium is equipped with flagella, but a diverse range of species is able to 'glide' or 'twitch' across surfaces. Cyanobacteria with this capacity range from unicellular species to complex filamentous forms, including species such as Nostoc punctiforme, which can generate specialised motile filaments called hormogonia. Recent work on the model unicellular cyanobacterium Synechocystis sp. PCC 6803 has shown that its means of propulsion has much in common with the twitching motility of heterotrophs such as Pseudomonas and Myxococcus. Movement depends on Type IV pili, which are extended, adhere to the substrate and then retract to pull the cell across the surface. Previous work on filamentous cyanobacteria suggested a very different mechanism, with movement powered by the directional extrusion of polysaccharide from pores close to the cell junctions. Now a new report by Khayatan and colleagues in this issue of Molecular Microbiology suggests that the motility of Nostoc hormogonia has much more in common with Synechocystis than was previously thought. In both cases, polysaccharide secretion is important for preparing the surface, but the directional motive force comes from Type IV pili.

Diguanylate cyclases are enzymes that use two GTP molecules to produce one molecule cyclic dimeric guanosine monophosphate (c-di-GMP). This cyclic dinucleotide is an ubiquitous prokaryotic second messenger that controls a variety of cell functions. Several proteins have been described which contain a photoreceptor domain fused to a diguanylate cyclase. The cyanobacterial light sensor Cph2 is responsible for the blue-light induced synthesis of c-di-GMP in Synechocystis sp. PCC 6803. Here, we provide a detailed protocol for an in vitro enzymatic assay with a purified photoreceptor protein using light as the crucial reaction parameter for c-di-GMP synthesis. The assay is accomplished under continuous illumination with light of different quality with inactivation of the enzyme by heat denaturation. Analytics are performed using HPLC-UV.

Phototaxis is a mechanism that allows cyanobacteria to respond to fluctuations in the quality and quantity of illumination by moving either towards or away from a light source. Phototactic movement on low concentration agar or agarose plates can be analyzed at macroscopic and microscopic scales representing group behavior and single cell motility, respectively. Here, we describe a detailed procedure for phototaxis assays on both scales using the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria are able to move directly towards or away from a light source, a process called phototaxis. Recent studies have revealed that the spherical unicellular cyanobacterium Synechocystis sp. PCC 6803 exhibits a cell polarity in response to unidirectional illumination and that micro-optic properties of cyanobacterial cells are the basis of their directional light sensing. Further functional and physiological studies highlight a very complex control of cyanobacterial phototaxis by sensory proteins, histidine kinases and response regulators. Notably, PATAN domain response regulators appear to participate in directional control of phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. In this review we explain the problem of directional light sensing at the small scale of bacteria and discuss our current understanding of signal transduction in cyanobacterial phototaxis.

The coordination of biological activities into daily cycles provides an important advantage for the fitness of diverse organisms. Most eukaryotes possess an internal clock ticking with a periodicity of about one day to anticipate sunrise and sunset. The 24-hour period of the free-running rhythm is highly robust against many changes in the natural environment. Among prokaryotes, only Cyanobacteria are known to harbor such a circadian clock. Its core oscillator consists of just three proteins, KaiA, KaiB, and KaiC that produce 24-hour oscillations of KaiC phosphorylation, even in vitro. This unique three-protein oscillator is well documented for the freshwater cyanobacterium Synechococcus elongatus PCC 7942. Several physiological studies demonstrate a circadian clock also for other Cyanobacteria including marine species. Genes for the core clock components are present in nearly all marine cyanobacterial species, though there are large differences in the specific composition of these genes. In the first section of this review we summarize data on the model circadian clock from S. elongatus PCC 7942 and compare it to the reduced clock system of the marine cyanobacterium Prochlorococcus marinus MED4. In the second part we discuss the diversity of timing mechanisms in other marine Cyanobacteria with regard to the presence or absence of different components of the clock.

Background 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0741-0) contains supplementary material, which is available to authorized users.

Cyanobacteria are blue-green bacteria that are abundant in the environment. Cyanobacteria in the oceans are among the world’s most important oxygen producers and carbon dioxide consumers. Synechocystis is a spherical single-celled cyanobacterium that measures about three thousandths of a millimetre across. Because Synechocystis needs sunlight to produce energy, it is important for it to find places where the light is neither too weak nor too strong. Unlike some bacteria, Synechocystis can’t swim, but it can crawl across surfaces. It uses this ability to move to places where the light conditions are better. It was already known that Synechocystis cells move towards a light source that is shone at them from one side, which implies that the cyanobacteria can “see” where the light is. But how can such a tiny cell accurately detect where light is coming from? Schuergers et al. tracked how Synechocystis moved in response to different light conditions, and found that the secret of “vision” in these cyanobacteria is that the cells act as tiny spherical lenses. When a light is shone at the cell, an image of the light source is focused at the opposite edge of the cell. Light-detecting molecules called photoreceptors respond to the focused image of the light source, and this provides the information needed to steer the cell towards the light. Although the details are different, and although a Synechocystis cell is in terms of volume about 500 billion times smaller than a human eyeball, vision in Synechocystis actually works by principles similar to vision in humans. Schuergers et al.’s findings open plenty of further questions, as other types of bacteria may also act as tiny lenses. More also remains to be learnt about how the cyanobacteria process visual information.

The dynamics of photosystem I assembly in cyanobacteria have been addressed using in vivo pulse-chase labeling of Synechocystis sp. PCC 6803 proteins in combination with blue native polyacrylamide gel electrophoresis. The analyses indicate the existence of three different monomeric photosystem I complexes and also the high stability of photosystem I trimers. We show that in addition to a complete photosystem I monomer, containing all 11 subunits, we detected a PsaK-less monomer and a short-lived PsaL/PsaK-less complex. The latter two monomers were missing in the ycf37 mutant of Synechocystis sp. PCC 6803 that accumulates also less trimers. Pulse-chase experiments suggest that the three monomeric complexes have different functions in the biogenesis of the trimer. Based on these findings we propose a model where PsaK is incorporated in the latest step of photosystem I assembly. The PsaK-less photosystem I monomer may represent an intermediate complex that is important for the exchange of the two PsaK variants during high light acclimation. Implications of the presented data with respect to Ycf37 function are discussed.

Cyanobacteria are well established model organisms for the study of oxygenic photosynthesis, nitrogen metabolism, toxin biosynthesis, and salt acclimation. However, in comparison to other model bacteria little is known about regulatory networks, which allow cyanobacteria to acclimate to changing environmental conditions. The current work has begun to illuminate how transcription factors modulate expression of different photosynthetic regulons. During the past few years, the research on other regulatory principles like RNA-based regulation showed the importance of non-protein regulators for bacterial lifestyle. Investigations on modulation of photosynthetic components should elucidate the contributions of all factors within the context of a larger regulatory network. Here, we focus on regulation of photosynthetic processes including transcriptional and posttranscriptional mechanisms, citing examples from a limited number of cyanobacterial species. Though, the general idea holds true for most species, important differences exist between various organisms, illustrating diversity of acclimation strategies in the very heterogeneous cyanobacterial clade. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux.

Cyanobacteria are model organisms for the analysis of photosynthetic processes, nitrogen fixation and circadian rhythm. Genetic characterization and manipulation of cyanobacteria is essential to investigate these processes and to potentially exploit their biotechnological or unique properties. Genomes of diverse cyanobacteria have been sequenced so far. This primary information bears a multitude of putative genes with completely unknown or only computational predicted functions. Such predictions might be misleading without experimental evidence. Thus, reverse genetics and molecular approaches are necessary to analyze the functions of these genes. This chapter will describe tools and strategies currently available for genetic manipulation of cyanobacteria, with a special emphasis on Synechocystis sp. PCC 6803.

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.

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.

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.

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.