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The phycobilisome (PBS) is an antenna protein complex in cyanobacteria, Glaucocystophytes, and red algae. In the standard PBS, the rod-core PBS, the rods are connected to the core by the rod-core linker protein CpcG. The rod-core PBS transfers the light energy mainly to photosystem (PS) II and to a lesser extent to PSI. Cyanobacteria assemble another type of PBS, the CpcL-PBS, which consists of only one rod. This rod-type PBS is connected to the thylakoid membrane by the linker protein CpcL and is a PSI-specific antenna. In the filamentous heterocyst-forming cyanobacterium Anabaena (Nostoc) sp. PCC 7120, the CpcL-PBS forms a complex with the tetrameric PSI (PBS-PSI supercomplex). The CpcL-PBS and the rod part of the rod-core PBS are identical except for the linker proteins CpcL and CpcG. How cells control the accumulation of the two different types of PBS is unknown. Here, we analyzed two mutant strains which either lack the major rod-core linker CpcG4 or overexpress the rod-membrane linker CpcL. In both mutant strains, more and larger PBS-PSI supercomplexes accumulated compared to the wild type. Our results suggest that CpcL and CpcG4 compete for the same phycobiliprotein pool, and therefore the CpcL/CpcG4 ratio determines the levels of PBS-PSI supercomplexes. We propose that the CpcL-PBS and the rod-core PBS fulfill distinct functions in light harvesting. Graphical abstract

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The ~100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-encoded abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.

RNA degradation is crucial for many processes in pro- and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y towards 5’-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5’ sensing. Cyanobacteria, such asSynechocystissp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologs. Here, we constructed aSynechocystisstrain lacking the 5’ sensing function of RNase E and mapped on a transcriptome-wide level 292 5’-sensing-dependent cleavage sites. These included so far unknown targets such as the 5’ untranslated region of the response regulator genelsiR;trxA, apcEandatpImRNAs, encoding proteins related to energy metabolism; as well assbtBandrbcLXSencoding proteins relevant for carbon fixation. Cyanobacterial 5’ sensing is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and most prokaryotes. We found that increased RNase activities leads to a higher copy number of the majorSynechocystisplasmids pSYSA and pSYSM. The results provide a first step towards understanding the relative importance of different target mechanisms of RNase E outsideEscherichia coli.

Phytochromes are linear tetrapyrrole-binding photoreceptors in eukaryotes and bacteria, primarily responding to red and far-red light signals reversibly. Among the GAF domain-based phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show various optical properties covering the entire visible region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here, we utilize ancestral sequence reconstruction and biochemical verification to show 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 is crucial to perceive green light. The ancestral cyanobacteriochromes show only modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense the incident green/red light ratio. Many cyanobacteria can utilize green light for photosynthesis using phycobilisome light-harvesting complexes. The green/red sensing cyanobacteriochromes may have allowed better acclimation to changing light environments by rearranging the absorption capacity of the phycobilisome through chromatic acclimation. Graphical abstract

Nucleotide-derived signalling molecules control a wide range of cellular processes in all organisms. The bacteria-specific cyclic dinucleotide c-di-GMP plays a crucial role in regulating motility-to-sessility transitions, cell cycle progression, and virulence. Cyanobacteria are phototrophic prokaryotes that perform oxygenic photosynthesis and are widespread microorganisms that colonize almost all habitats on Earth. In contrast to photosynthetic processes that are well understood, the behavioural responses of cyanobacteria have rarely been studied in detail. Analyses of cyanobacterial genomes have revealed that they encode a large number of proteins that are potentially involved in the synthesis and degradation of c-di-GMP. Recent studies have demonstrated that c-di-GMP coordinates many different aspects of the cyanobacterial lifestyle, mostly in a light-dependent manner. In this review, we focus on the current knowledge of light-regulated c-di-GMP signalling systems in cyanobacteria. Specifically, we highlight the progress made in understanding the most prominent behavioural responses of the model cyanobacterial strains Thermosynechococcus vulcanus and Synechocystis sp. PCC 6803. We discuss why and how cyanobacteria extract crucial information from their light environment to regulate ecophysiologically important cellular responses. Finally, we emphasize the questions that remain to be addressed.

Many prokaryotes show complex behaviors that require the intricate spatial and temporal organization of cellular protein machineries, leading to asymmetrical protein distribution and cell polarity. One such behavior is cyanobacterial phototaxis which relies on the dynamic localization of the Type IV pilus motor proteins in response to light. In the cyanobacterium Synechocystis, various signaling systems encompassing chemotaxis-related CheY- and PatA-like response regulators are critical players in switching between positive and negative phototaxis depending on the light intensity and wavelength. In this study, we show that PatA-type regulators evolved from chemosensory systems. Using fluorescence microscopy and yeast two-hybrid analysis, we demonstrate that they localize to the inner membrane, where they interact with the N-terminal cytoplasmic domain of PilC and the pilus assembly ATPase PilB1. By separately expressing the subdomains of the response regulator PixE, we confirm that only the N-terminal PATAN domain interacts with PilB1, localizes to the membrane, and is sufficient to reverse phototactic orientation. These experiments established that the PATAN domain is the principal output domain of PatA-type regulators which we presume to modulate pilus extension by binding to the pilus motor components.

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacteriumSynechocystissp. PCC 6803, four large and three smaller plasmids exist. The ∼100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encodedssr7036transcript. Together with a cis-located abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion ofslr7037led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence ofslr7037was required for successful replication of a pSYSA-derived vector in another model cyanobacterium,Synechococcus elongatusPCC 7942. Therefore, we annotated the protein encoded byslr7037as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus inSynechocystissp. PCC 6803.

SummarySignal-sequence dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and facilitated by dedicated protein targeting factors, such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins, but can also be present within mRNAs. Byin vivoandin vitroassays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors, but is dependent on the SecYEG-translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised.

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins via their CRISPR-associated Rossmann fold (CARF) domains. Commonly, these are ribonucleases (RNases) that unspecifically degrade both invader and host RNA. To mitigate adverse effects on cell growth, ring nucleases can degrade extant cOAs to switch off ancillary nucleases. Here we show that the model organism Synechocystis sp. PCC 6803 harbors functional CARF-domain effector and ring nuclease proteins. We purified and characterized the two ancillary CARF-domain proteins from the III-D type CRISPR system of this cyanobacterium. The Csx1 homolog, SyCsx1, is a cyclic tetraadenylate(cA4)-dependent RNase with a strict specificity for cytosine nucleotides. The second CARF-domain protein with similarity to Csm6 effectors, SyCsm6, did not show RNase activity in vitro but was able to break down cOAs and attenuate SyCsx1 RNase activity. Our data suggest that the CRISPR systems in Synechocystis confer a multilayered cA4-mediated defense mechanism.

Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibers that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Three further gene products with similarity to PilX-like minor pilins have a function in flocculation of Synechocystis. The results of our study indicate that different minor pilins facilitate distinct pilus functions. Further, our microarray analysis demonstrated that the transcription levels of the minor pilin genes change in response to surface contact. A total of 122 genes were determined to have altered transcription between planktonic and surface growth, including several plasmid genes which are involved exopolysaccharide synthesis and the formation of bloom-like aggregates.

Many cyanobacteria, which use light as an energy source via photosynthesis, show directional movement towards or away from a light source. However, the molecular and cell biological mechanisms for switching the direction of movement remain unclear. Here, we visualized type IV pilus-dependent cell movement in the rod-shaped thermophilic cyanobacterium Thermosynechococcus vulcanus using optical microscopy at physiological temperature and light conditions. Positive and negative phototaxis were controlled on a short time scale of 1 min. The cells smoothly moved over solid surfaces towards green light, but the direction was switched to backward movement when we applied additional blue light illumination. The switching was mediated by three photoreceptors, SesA, SesB, and SesC, which have cyanobacteriochrome photosensory domains and synthesis/degradation activity of the bacterial second messenger cyclic dimeric GMP (c-di-GMP). Our results suggest that the decision-making process for directional switching in phototaxis involves light-dependent changes in the cellular concentration of c-di-GMP. Direct visualization of type IV pilus filaments revealed that rod-shaped cells can move perpendicular to the light vector, indicating that the polarity can be controlled not only by pole-to-pole regulation but also within-a-pole regulation. This study provides insights into previously undescribed rapid bacterial polarity regulation via second messenger signalling with high spatial resolution.

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in many bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as E. coli, have evolved tightly functioning feedback regulation of RNase E that is mediated in cis by the rne 5′-untranslated region (5′ UTR); however, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. Here, we investigated the global gene expression response in the cyanobacterium Synechocystis sp. PCC 6803 after exposure to UV light and discovered a unique response of RNase E: a rapidly increasing enzymatic activity, although the stability of the protein was decreased. In parallel, we observed an increased accumulation of full-length rne mRNA that was caused by the stabilization of its 5′ UTR and suppression of premature transcriptional termination but not by an increased transcription rate. Mapping of RNA 3′ ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5′ UTR, indicating autoregulation via its own 5′ UTR. These observations imply that RNase E in cyanobacteria contributes substantially to reshaping the transcriptome during the UV stress response and that its required activity level is maintained despite enhanced turnover of the protein by posttranscriptional feedback regulation.

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.

Many cyanobacteria, which use light as an energy source via photosynthesis, show directional movement towards or away from a light source. However, the molecular and cell biological mechanisms for switching the direction of movement remain unclear. Here, we visualized type IV pilus-dependent cell movement in the rod-shaped thermophilic cyanobacterium Thermosynechococcus vulcanus using optical microscopy at physiological temperature and light conditions. Positive and negative phototaxis were controlled on a short time scale of 1 min. The cells smoothly moved over solid surfaces towards green light, but the direction was switched to backward movement when we applied additional blue light illumination. The switching was mediated by three photoreceptors, SesA, SesB and SesC, which have cyanobacteriochrome photosensory domains and synthesis/degradation activity of the bacterial second messenger cyclic dimeric GMP (c-di-GMP). Our results suggest that the decision-making process for directional switching in phototaxis involves light-dependent changes in the cellular concentration of c-di-GMP. Furthermore, we reveal that rod-shaped cells can move perpendicular to the light vector, indicating that the polarity can be controlled not only by pole-to-pole regulation but also within-a-pole regulation. This study provides insights into previously undescribed rapid bacterial polarity regulation via second messenger signalling with high spatial resolution.

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and spontaneously occurring V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1,472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified putative RNase-E-dependent instances of operon discoordination, mRNAs likely regulated jointly by RNase E and an sRNA, potential 3’ end-derived sRNAs and a dual-acting mechanism for the glutamine riboswitch. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria.

The rotation of the Earth results in predictable environmental changes that pose challenges for organisms and force them to adapt. To address this daily rhythm, organisms from all kingdoms of life have evolved diverse timing mechanisms. In the cyanobacterium Synechococcus elongatus PCC 7942, the three proteins KaiA, KaiB, and KaiC constitute the central timing mechanism that drives circadian oscillations. In addition to the standard oscillator, named KaiAB1C1, Synechocystis sp. PCC 6803 harbors several, diverged clock homologs. The nonstandard KaiB3-KaiC3 system was suggested to impact the metabolic switch in response to darkness. Here, we demonstrate the direct interaction of KaiC3 with Sll0485, which is a potential new chimeric KaiA homolog that we named KaiA3. The existence of a functional link between these proteins is further supported by the co-occurrence of genes encoding KaiA3 with the KaiB3–KaiC3-like gene products in 10 cyanobacterial and five other bacterial species. KaiA3 is annotated as a NarL-type response regulator due to its similarity to the response regulator receiver domains. However, its similarity to canonical NarL drastically decreases in the C-terminal domain, which resembles the circadian clock protein KaiA. In line with this, we detected the stimulation of KaiC3 phosphorylation by KaiA3 in vitro. Furthermore, we showed that deletion of the kaiA3 gene led to growth defects during mixotrophic growth conditions and, like a kaiC3-deficient mutant, viability was impaired during chemoheterotrophic growth in complete darkness. In summary, we suggest KaiA3 as a novel, nonstandard KaiA homolog within the cyanobacterial phylum, extending the KaiB3-KaiC3 system in Cyanobacteria and other prokaryotes.Significance StatementMany organisms that are subjected to day and night cycles are able to predict daily changes using a circadian clock system. Cyanobacteria use a relatively simple timekeeper, a protein-based oscillator consisting of KaiA, KaiB and KaiC. Several cyanobacteria and other prokaryotes harbor additional sets of oscillator components that, however, lack the KaiA protein. Our study identified a divergent KaiA homolog that is functionally and genetically linked to potential KaiB3-KaiC3 timekeepers. The data show that the nonstandard KaiA homolog affects the phosphorylation of KaiC3 from the cyanobacterium Synechocystis, suggesting that a similar function exists in other prokaryotes.

Many prokaryotes show complex behaviors that require the intricate spatial and temporal organization of cellular protein machineries, leading to asymmetrical protein distribution and cell polarity. One such behavior is cyanobacterial phototaxis which relies on the dynamic localization of the Type IV pilus motor proteins in response to light. In the cyanobacterium Synechocystis, various signaling systems encompassing chemotaxis-related CheY- and PatA-like response regulators are critical players in switching between positive and negative phototaxis depending on the light intensity and wavelength. In this study, we show that PatA-type regulators evolved from chemosensory systems. Using fluorescence microscopy and yeast-two-hybrid analysis, we demonstrate that they localize to the inner membrane, where they interact with the N-terminal cytoplasmic domain of PilC and the pilus assembly ATPase PilB1. By separately expressing the subdomains of the response regulator PixE, we confirm that only the N-terminal PATAN domain interacts with PilB1, localizes to the membrane, and is sufficient to reverse phototactic orientation. These experiments established that the PATAN domain is the principal output domain of PatA-type regulators which we presume to modulate pilus extension by binding to the pilus motor components.

The cyanobacterium Synechocystis secretes a specific sulphated polysaccharide to form floating cell aggregates.

The putative circadian clock system of the facultative heterotrophic cyanobacterial strain Synechocystis sp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and nontargeted metabolite profiling. The strongest metabolic changes were observed in the dark in ΔrpaA and, to a lesser extent, in the ΔkaiAB1C1 mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the ΔrpaA mutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulator rpaA had a more pronounced effect on metabolism than the deletion of the kai genes. The larger impact of the rpaA mutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins in Synechocystis for balanced carbon and nitrogen metabolism during light-to-dark transitions.

This protocol could be used to analyze Kai-protein complexes in Clear-native PAGE perform Western Blot transfers of large native protein complexes with a semi dry blotting system perform the immunodetection of his-tagged proteins with a single monoclonal anti-his antibody conjugated to horseradish peroxidase (HRP) that catalyses the chemical reaction of the substrate ECL generating a chemiluminescence signal

This protocol can be used to - express his-tagged proteins in E. coli cells by using a T7-polymerase based expression system - Lyse cells with sonification - purify target protein by Ni-affinity chromatography with nickel magnetic beads

Cyanobacteria exhibit rhythmic gene expression with a period length of 24 hours to adapt to daily environmental changes. In the model organism Synechococcuselongatus PCC 7942, the central oscillator consists of the three proteins KaiA, KaiB and KaiC and utilizes the histidine kinase SasA and its response regulator RpaA as output-signaling pathway. Synechocystis sp. PCC 6803 contains in addition to the canonical kaiAB1C1 gene cluster two further homologs of the kaiB and kaiC genes. Here, we demonstrate that the SasA-RpaA system interacts with the KaiAB1C1 core oscillator only. Interaction with KaiC2 and KaiC3 proteins was not detected, suggesting different signal transduction components for the clock homologs. Inactivation of rpaA in Synechocystis sp. PCC 6803 leads to reduced viability of the mutant in light-dark cycles, especially under mixotrophic growth conditions. Chemoheterotrophic growth of the ∆rpaA strain in the dark was abolished completely. Transcriptomic data revealed that RpaA is mainly involved in the regulation of genes related to CO2 - acclimation in the light and to carbon metabolism in the dark. Further, our results indicate a link between the circadian clock and phototaxis.

The putative circadian clock system of the facultative heterotrophic cyanobacterial strain Synechocystis sp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and non-targeted metabolite profiling. The strongest metabolic changes were observed in the dark in ΔrpaA and, to a lesser extent, in the ΔkaiAB1C1 mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the ΔrpaA mutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulator rpaA had a more pronounced effect on metabolism than the deletion of the kai genes. The larger impact of the rpaA mutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins in Synechocystis for balanced carbon and nitrogen metabolism during light-to-dark transitions.

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.

Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibres that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. The transcription of the minor pilin genes pilA5, pilA6 and pilA9-pilA11 is inversely regulated in response to different conditions. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Microarray analysis demonstrated that the transcription levels of known and newly predicted minor pilin genes change in response to surface contact. A total of 120 genes were determined to have altered transcription between planktonic and surface growth. Among these genes, 13 are located on the pSYSM plasmid. The results of our study indicate that different minor pilins facilitate distinct pilus functions.

Type IV pili (T4P) are proteinaceous filaments found on the cell surface of many prokaryotic organisms and convey twitching motility through their extension/retraction cycles, moving cells across surfaces. In cyanobacteria, twitching motility is the sole mode of motility properly characterised to date and is the means by which cells perform phototaxis, the movement towards and away from directional light sources. The wavelength and intensity of the light source determine the direction of movement and, sometimes in concert with nutrient conditions, act as signals for some cyanobacteria to form mucoid multicellular assemblages. Formation of such aggregates or flocs represents an acclimation strategy to unfavourable environmental conditions and stresses, such as harmful light conditions or predation. T4P are also involved in natural transformation by exogenous DNA, secretion processes, and in cellular adaptation and survival strategies, further cementing the role of cell surface appendages. In this way, cyanobacteria are finely tuned by external stimuli to either escape unfavourable environmental conditions via phototaxis, exchange genetic material, and to modify their surroundings to fit their needs by forming multicellular assemblies.

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 cyanobacterial phytochrome Cph2 is a light-dependent diguanylate cyclase of the cyanobacterium Synechocystis 6803. Under blue light, Cph2-dependent increase in the cellular c-di-GMP concentration leads to inhibition of surface motility and enhanced flocculation of cells in liquid culture. However, the targets of second messenger signalling in this cyanobacterium and its mechanism of action remained unclear. Here, we determined the cellular concentrations of cAMP and c-di-GMP in wild-type and Δcph2 cells after exposure to blue and green light. Inactivation of cph2 completely abolished the blue-light dependent increase in c-di-GMP content. Therefore, a microarray analysis with blue-light grown wild-type and Δcph2 mutant cells was used to identify c-di-GMP dependent alterations in transcript accumulation. The increase in the c-di-GMP content alters expression of genes encoding putative cell appendages, minor pilins and components of chemotaxis systems. The mRNA encoding the minor pilins pilA5-pilA6 was negatively affected by high c-di-GMP content under blue light, whereas the minor pilin encoding operon pilA9-slr2019 accumulates under these conditions, suggesting opposing functions of the respective gene sets. Artificial overproduction of c-di-GMP leads to similar changes in minor pilin gene expression and supports previous findings that c-di-GMP is important for flocculation via the function of minor pilins. Mutational and gene expression analysis further suggest that SyCRP2, a CRP-like transcription factor, is involved in regulation of minor pilin and putative chaperone usher pili gene expression.

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.

Motile strains of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 readily aggregate into flocs, or floating multicellular assemblages, when grown in liquid culture. As described here, we used confocal imaging to probe the structure of these flocs, and we developed a quantitative assay for floc formation based on fluorescence imaging of 6-well plates. The flocs are formed from strands of linked cells, sometimes packed into dense clusters but also containing voids with very few cells. Cells within the dense clusters show signs of nutrient stress, as judged by the subcellular distribution of green fluorescent protein (GFP)-tagged Vipp1 protein. We analyzed the effects on flocculation of a series of mutations that alter piliation and motility, including Δhfq, ΔpilB1, ΔpilT1, and ΔushA mutations and deletion mutations affecting major and minor pilins. The extent of flocculation is increased in the hyperpiliated ΔpilT1 mutant, but active cycles of pilus extension and retraction are not required for flocculation. Deletion of PilA1, the major subunit of type IV pili, has no effect on flocculation; however, flocculation is lost in mutants lacking an operon coding for the minor pilins PilA9 to -11. Therefore, minor pilins appear crucial for flocculation. We show that flocculation is a tightly regulated process that is promoted by blue light perception by the cyanobacteriochrome Cph2. Floc formation also seems to be a highly cooperative process. A proportion of nonflocculating Δhfq cells can be incorporated into wild-type flocs, but the presence of a high proportion of Δhfq cells disrupts the large-scale architecture of the floc.IMPORTANCE Some bacteria form flocs, which are multicellular floating assemblages of many thousands of cells. Flocs have been relatively little studied compared to surface-adherent biofilms, but flocculation could play many physiological roles, be a crucial factor in marine carbon burial, and enable more efficient biotechnological cell harvesting. We studied floc formation and architecture in the model cyanobacterium Synechocystis sp. strain PCC 6803, using mutants to identify specific cell surface structures required for floc formation. We show that floc formation is regulated by blue and green light perceived by the photoreceptor Cph2. The flocs have a characteristic structure based on strands of linked cells aggregating into dense clusters. Cells within the dense clusters show signs of nutrient stress, pointing to a disadvantage of floc formation.

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.

Ethylene is a gaseous signal sensed by plants and bacteria. Heterologous expression of the ethylene-forming enzyme (EFE) from Pseudomonas syringae in cyanobacteria leads to the production of ethylene under photoautotrophic conditions. The recent characterization of an ethylene-responsive signalling pathway affecting phototaxis in the cyanobacterium Synechocystis sp. PCC 6803 implied that biotechnologically relevant ethylene synthesis may induce regulatory processes that are not related to changes in metabolism. Here, we provide data that indicate that endogenously produced ethylene accelerates the movement of cells towards light. Microarray analysis demonstrates that ethylene mainly deactivates transcription from the csiR1/lsiR promoter, which is under the control of the two-component system consisting of the ethylene- and UV-A-sensing histidine kinase UirS and the DNA-binding response regulator UirR. Surprisingly, ethylene production triggers a very specific transcriptional response and only a few other smaller transcriptional changes are detected in the microarray analysis.

We utilized a photoautotrophic organism to synthesize 1,2-propanediol from carbon dioxide and water fueled by light. A synthetic pathway comprising mgsA (methylglyoxal synthase), yqhD (aldehyde reductase), and adh (alcohol dehydrogenase) was inserted into Synechocystis sp. PCC6803 to convert dihydroxyacetone phosphate to methylglyoxal, which is subsequently reduced to acetol and then to 1,2-propanediol. 1,2-propanediol could be successfully produced by Synechocystis, at an approximate rate of 55 μmol h-1 gCDW-1 . Surprisingly, maximal productivity was observed in the stationary phase. The production of 1,2-propanediol was clearly coupled to the turn-over of intracellular glycogen. Upon depletion of the glycogen pool, product formation stopped. Reducing the carbon flux to glycogen significantly decreased final product titers. Optimization of cultivation conditions allowed final product titers of almost 1 g L-1 (12 mM), which belongs to the highest values published so far for photoautotrophic production of this compound.

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.

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. 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 non-standard KaiC homologs in vitro. KaiC3 displayed ATPase activity, which were lower compared to 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 crosstalk between the KaiC1 and KaiC3 based systems.

The cyanobacterium Synechocystis sp. PCC 6803 can move directionally on a moist surface toward or away from a light source to reach optimal light conditions for its photosynthetic lifestyle. This behavior, called phototaxis, is mediated by type IV pili (T4P), which can pull a single cell into a certain direction. Several photoreceptors and their downstream signal transduction elements are involved in the control of phototaxis. However, the critical steps of local pilus assembly in positive and negative phototaxis remain elusive. One of the photoreceptors controlling negative phototaxis in Synechocystis is the blue-light sensor PixD. PixD forms a complex with the CheY-like response regulator PixE that dissociates upon illumination with blue light. In this study, we investigate the phototactic behavior of pixE deletion and overexpression mutants in response to unidirectional red light with or without additional blue-light irradiation. Furthermore, we show that PixD and PixE partly localize in spots close to the cytoplasmic membrane. Interaction studies of PixE with the motor ATPase PilB1, demonstrated by in vivo colocalization, yeast two-hybrid and coimmunoprecipitation analysis, suggest that the PixD–PixE signal transduction system targets the T4P directly, thereby controlling blue-light-dependent negative phototaxis. An intriguing feature of PixE is its distinctive structure with a PATAN (PatA N-terminus) domain. This domain is found in several other regulators, which are known to control directional phototaxis. As our PilB1 coimmunoprecipitation analysis revealed an enrichment of PATAN domain response regulators in the eluate, we suggest that multiple environmental signals can be integrated via these regulators to control pilus function.

This protocol can be used for: (i) heterologous expression of GST-tagged proteins from pGEX-6P1 based expression vectors in E. coli. (ii) purification of recombinant proteins via affinity chromatography using glutathione-agarose or glutathione-sepharose (GST tagged protein can be eluted with glutathione. Alternatively, the tag can be cleaved off by prescission protease) (iii) further purification of the eluted protein via anion exchange chromatography This protocol was modified from Wiegard A, Dörrich AK, Deinzer HT, Beck C, Wilde A, Holtzendorff J, Axmann IM: Biochemical analysis of three putative KaiC clock proteins from Synechocystis sp. PCC 6803 suggests their functional divergence. Microbiology 2013, 159, 948-958 Snijder J, Schuller JM, Wiegard A, Lössel, P, Schmelling NM, Axmann IM, Plitzko JM, Förster F, Heck AJR: Structures of the cyanobacterial circadian oscillator frozen in a fully assembled state. Science 2017, 355(6330):1181-1184

This protocol describes how to detect synthesis of [α32P]ATP from [α32P]ATP by recombinant KaiC proteins. Radioactive nucleotides are separated via thin layer chromatography using TLC PEI Cellulose F plates as stationary phase and LiCl as soluble phase. The principle of this method is based on Egli et al. (Egli M, Mori T, Pattanayek R, Xu Y, Qin X, Johnson CH.2012. Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism. Biochemistry 51:1547-58.)

100x BG11 (500 ml) 74.79 g NaNO3 3.75 g MgSO4 × 7H2O 1.8 g CaCl2 × 2H2O 0.30 g Citric Acid 0.28 ml 0,5 M Na2EDTA (pH 8) Add Aqua bidest (ddH2O) to a final volume of 500 ml. Aliquot (5x100 ml) and autoclave. Store at 4°C. Trace-Metal-Mix (500 ml) 1.43 g H3BO3 900 mg MnCl2 × 4H2O 110 mg ZnSO4 × 7H2O 195 mg Na2MoO4 × 2H2O 39.5 mg CuSO4 × 5H2O 24.7 mg Co(NO3)2 × 6H2O Put some ddH2O into the bottle; dissovle the salts seperately and fill up to 500 ml. Sterile filter and aliquot the solution (10 x 50 ml). Store at 4°C. 2x BG11 (1 L) 20 ml 100x BG11 2 ml K2HPO4 × 3H2O (30 mg/ml) 2 ml Na2CO3 (20 mg/ml) 2 ml Trace-Metal-Mix Add 900 ml deionized water, afterwards add the solutions, fill up to 1 L with deionized water and autoclave. Store at RT. Before use add 2 ml of Fe-Ammonium-Citrate (6 mg/ml). 1xBG11 (1 L) 10 ml 100xBG11 10 ml TES pH 8 1 ml K2HPO4 (30 mg/ml) 1 ml Na2CO3 (20 mg/ml) 1 ml Trace-Metal-Mix Add 900 ml deionized water, afterwards add the solutions, fill up to 1 L with deionized water and autoclave. Store at RT. Before use add 1 ml of Fe-Ammonium-Citrate (6 mg/ml).

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.

This protocol can be used to investigate protein-protein interaction via yeast two-hybrid experiments. It describes the yeast-two hybrid method relying on the activity of the histidine-synthetase. If the proteins interact, the yeast cells are able to grow on selective medium without histidine.

This protocol can be used to investigate protein-protein interaction via yeast two-hybrid experiments. It describes the yeast-two hybrid method relying on a color change using β-galactosidase activity.

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.

Since cyanobacteria need to decrease PSI content to avoid absorption of excess light energy, down-regulation of PSI gene expression is one of the key characteristics of the high-light (HL) acclimation response. The transcriptional regulator RpaB and the small RNA PsrR1 (photosynthesis regulatory RNA1) 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. A 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 pre-existing PSI transcripts. By this dual repression at transcriptional and post-transcriptional levels, rapid and strict down-regulation 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.

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.

The phototactic cyanobacterium Synechocystis spec. PCC 6803 is spherical, two to three micrometer in diameter, and can directly and accurately perceive the position of a light source and consequently move into this direction. The cell body acts as a very effective spherical microlens, focusing a sharp image of the light source close to the non-illuminated side of the cell. We predict that this lensing effect leads to the inactivation of type IV pili on the far side of the light source whereas they assemble on the front side to pull the cells towards the light source.