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Synthetic biology conceptualizes biological complexity as a network of biological parts, devices, and systems with predetermined functionalities and has had a revolutionary impact on fundamental and applied research. With the unprecedented ability to synthesize and transfer any DNA and RNA across organisms, the scope of synthetic biology is expanding and being recreated in previously unimaginable ways. The field has matured to a level where highly complex networks, such as artificial communities of synthetic organisms, can be constructed. In parallel, computational biology became an integral part of biological studies, with computational models aiding the unravelling of the escalating complexity and emerging properties of biological phenomena. However, there is still a vast untapped potential for the complete integration of modelling into the synthetic design process, presenting exciting opportunities for scientific advancements. Here, we first highlight the most recent advances in computer-aided design of microbial communities. Next, we propose that such a design can benefit from an organism-free modular modelling approach that places its emphasis on modules of organismal function towards the design of multispecies communities. We argue for a shift in perspective from single organism-centred approaches to emphasizing the functional contributions of organisms within the community. By assembling synthetic biological systems using modular computational models with mathematical descriptions of parts and circuits, we can tailor organisms to fulfil specific functional roles within the community. This approach aligns with synthetic biology strategies and presents exciting possibilities for the design of artificial communities. Graphical Abstract.
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Mismanaged plastics, upon entering the environment, undergo degradation through physicochemical and/or biological processes. This process often results in the formation of microplastics (MPs), the most prevalent form of plastic debris (<1 mm). MPs pose severe threats to aquatic and terrestrial ecosystems, necessitating innovative strategies for effective remediation. Some photosynthetic microorganisms can degrade MPs but there lacks a comprehensive review. Here we examine the specific role of photoautotrophic microorganisms in water and soil environments for the biodegradation of plastics, focussing on their unique ability to grow persistently on diverse polymers under sunlight. Notably, these cells utilise light and CO2 to produce valuable compounds such as carbohydrates, lipids, and proteins, showcasing their multifaceted environmental benefits. We address key scientific questions surrounding the utilisation of photosynthetic microorganisms for MPs and nanoplastics (NPs) bioremediation, discussing potential engineering strategies for enhanced efficacy. Our review highlights the significance of alternative biomaterials and the exploration of strains expressing enzymes, such as polyethylene terephthalate (PET) hydrolases, in conjunction with microalgal and/or cyanobacterial metabolisms. Furthermore, we delve into the promising potential of photo-biocatalytic approaches, emphasising the coupling of plastic debris degradation with sunlight exposure. The integration of microalgal-bacterial consortia is explored for biotechnological applications against MPs and NPs pollution, showcasing the synergistic effects in wastewater treatment through the absorption of nitrogen, heavy metals, phosphorous, and carbon. In conclusion, this review provides a comprehensive overview of the current state of research on the use of photoautotrophic cells for plastic bioremediation. It underscores the need for continued investigation into the engineering of these microorganisms and the development of innovative approaches to tackle the global issue of plastic pollution in aquatic and terrestrial ecosystems.
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Cyanobacteria are a promising platform for the production of the triterpene squalene (C30), a precursor for all plant and animal sterols, and a highly attractive intermediate towards triterpenoids, a large group of secondary plant metabolites. Synechocystis sp. PCC 6803 natively produces squalene from CO2 through the MEP pathway. Based on the predictions of a constraint-based metabolic model, we took a systematic overexpression approach to quantify native Synechocystis gene's impact on squalene production in a squalene-hopene cyclase gene knock-out strain (Δshc). Our in silico analysis revealed an increased flux through the Calvin-Benson-Bassham cycle in the Δshc mutant compared to the wildtype, including the pentose phosphate pathway, as well as lower glycolysis, while the tricarboxylic acid cycle predicted to be downregulated. Further, all enzymes of the MEP pathway and terpenoid synthesis, as well as enzymes from the central carbon metabolism, Gap2, Tpi and PyrK, were predicted to positively contribute to squalene production upon their overexpression. Each identified target gene was integrated into the genome of Synechocystis Δshc under the control of the rhamnose-inducible promoter Prha. Squalene production was increased in an inducer concentration dependent manner through the overexpression of most predicted genes, which are genes of the MEP pathway, ispH, ispE, and idi, leading to the greatest improvements. Moreover, we were able to overexpress the native squalene synthase gene (sqs) in Synechocystis Δshc, which reached the highest production titer of 13.72 mg l-1 reported for squalene in Synechocystis sp. PCC 6803 so far, thereby providing a promising and sustainable platform for triterpene production.
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The growing use of photosynthetic microorganisms for food and food-related applications is driving related biotechnology research forward. Increasing consumer acceptance, high sustainability, demand of eco-friendly sources for food, and considerable global economic concern are among the main factors to enhance the focus on the novel foods. In the cases of not toxic strains, photosynthetic microorganisms not only provide a source of sustainable nutrients but are also potentially healthy. Several published studies showed that microalgae are sources of accessible protein and fatty acids. More than 400 manuscripts were published per year in the last 4 years. Furthermore, industrial approaches utilizing these microorganisms are resulting in new jobs and services. This is in line with the global strategy for bioeconomy that aims to support sustainable development of bio-based sectors. Despite the recognized potential of the microalgal biomass value chain, significant knowledge gaps still exist especially regarding their optimized production and utilization. This review highlights the potential of microalgae and cyanobacteria for food and food-related applications as well as their market size. The chosen topics also include advanced production as mixed microbial communities, production of high-value biomolecules, photoproduction of terpenoid flavoring compounds, their utilization for sustainable agriculture, application as source of nutrients in space, and a comparison with heterotrophic microorganisms like yeast to better evaluate their advantages over existing nutrient sources. This comprehensive assessment should stimulate further interest in this highly relevant research topic.
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Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis, a solar-driven process which allows them to obtain electrons from water to reduce and finally assimilate carbon dioxide. Consequently, they are in the spotlight of biotechnology as photoautotrophic cell factories to generate a large variety of chemicals and biofuels in a sustainable way. Recent progress in synthetic biology has enlarged the molecular toolset to genetically engineer the metabolism of cyanobacteria, mainly targeting common model strains, such as Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, Synechococcus sp. PCC 7002, or Anabaena sp. PCC 7120. Nevertheless, the accessibility and flexibility of engineering cyanobacteria is still somewhat limited and less predictable compared to other biotechnologically employed microorganisms.This chapter gives a broad overview of currently available methods for the genetic modification of cyanobacterial model strains as well as more recently discovered and promising species, such as Synechococcus elongatus PCC 11801. It comprises approaches based on homologous recombination, replicative broad-host-range or strain-specific plasmids, CRISPR/Cas, as well as markerless selection. Furthermore, common and newly introduced molecular tools for gene expression regulation are presented, comprising promoters, regulatory RNAs, genetic insulators like transcription terminators, ribosome binding sites, CRISPR interference, and the utilization of heterologous RNA polymerases. Additionally, potential DNA assembly strategies, like modular cloning, are described. Finally, considerations about post-translational control via protein degradation tags and heterologous proteases, as well as small proteins working as enzyme effectors are briefly discussed.
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Synechococcus , Synechocystis , Synechococcus/genética , Synechococcus/metabolismo , Fotosíntesis , Synechocystis/genética , Synechocystis/metabolismo , BiotecnologíaRESUMEN
In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5'/3' gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.
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ADN-Topoisomerasas , Regiones Promotoras Genéticas , Synechocystis , División Celular/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Synechocystis/enzimología , Synechocystis/genética , Activación Transcripcional , ADN-Topoisomerasas/genética , ADN-Topoisomerasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
With more than 80 000 compounds, terpenoids represent one of the largest classes of secondary metabolites naturally produced by various plants and other organisms. Owing to the tremendous structural diversity, they offer a wide range of properties relevant for biotechnological and pharmaceutical applications. In this context, heterologous terpenoid production in engineered microbial hosts represents an often cost-effective and eco-friendly way to make these valuable compounds industrially available. This review provides an overview of current strategies to employ and engineer oxygenic and anoxygenic phototrophic bacteria as alternative cell factories for sustainable terpenoid production. Besides terpenoid pathway engineering, the effects of different illumination strategies on terpenoid photoproduction are key elements in the latest studies.
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Ingeniería Metabólica , Terpenos , Bacterias/metabolismo , Plantas/metabolismo , Terpenos/metabolismoRESUMEN
Genetic engineering of cyanobacteria is currently limited to genomic integration via homologous recombination and RSF1010-based conjugative vector systems. Here, we introduce a rationally designed conjugative vector with two BioBrick-based cloning sites which enables facilitated and modular cloning. This streamlined vector is suitable for a variety of synthetic biology applications, such as expression of multiple enzymes from metabolic pathways for the production of biofuels or secondary metabolites, or screening of modular parts such as promoters, further facilitating applications to improve crop plants using synthetic biology. Finally, we present a general approach to cloning of constructs, as well as detailed protocols for conjugation and culturing of strains carrying said constructs.
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Cianobacterias , Vectores Genéticos , Cianobacterias/genética , Ingeniería Genética/métodos , Vectores Genéticos/genética , Ingeniería Metabólica/métodos , Plásmidos , Biología Sintética/métodosRESUMEN
Cyanobacteria are extremely adaptable, fast-growing, solar-powered cell factories that, like plants, are able to convert carbon dioxide into sugar and oxygen and thereby produce a large number of important compounds. Due to their unique phototrophy-associated physiological properties, i.e. naturally occurring isoprenoid metabolic pathway, they represent a highly promising platform for terpenoid biosynthesis. Here, we implemented a carefully devised engineering strategy to boost the biosynthesis of commercially attractive plant sequiterpenes, in particular valencene. Sesquiterpenes are a diverse group of bioactive metabolites, mainly produced in higher plants, but with often low concentrations and expensive downstream extraction. In this work we successfully demonstrate a multi-component engineering approach towards the photosynthetic production of valencene in the cyanobacterium Synechocystis sp. PCC 6803. First, we improved the flux towards valencene by markerless genomic deletions of shc and sqs. Secondly, we downregulated the formation of carotenoids, which are essential for viability of the cell, using CRISPRi on crtE. Finally, we intended to increase the spatial proximity of the two enzymes, ispA and CnVS, involved in valencene formation by creating an operon construct, as well as a fusion protein. Combining the most successful strategies resulted in a valencene production of 19 mg/g DCW in Synechocystis. In this work, we have devised a useful platform for future engineering steps.
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Design and implementation of synthetic biological circuits highly depends on well-characterized, robust promoters with predictable input-output responses. While great progress has been made with heterotrophic model organisms such as Escherichia coli, the available variety of tunable promoter parts for phototrophic cyanobacteria is still limited. Commonly used synthetic and semisynthetic promoters show weak dynamic ranges or no regulation at all in cyanobacterial models. Well-controlled alternatives such as native metal-responsive promoters, however, pose the problems of inducer toxicity and lacking orthogonality. Here, we present the comparative assessment of dose-response functions of four different inducible promoter systems in the model cyanobacterium Synechocystis sp. PCC 6803. Using the novel bimodular reporter plasmid pSHDY, dose-response dynamics of the re-established vanillate-inducible promoter PvanCC was compared to the previously described rhamnose-inducible Prha, the anhydrotetracycline-inducible PL03, and the Co2+-inducible PcoaT. We estimate individual advantages and disadvantages regarding dynamic range and strength of each promoter, also in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in cyanobacteria, facilitating the scalability and robustness of synthetic regulatory network designs and of complex metabolic pathway engineering strategies.
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Regulación Bacteriana de la Expresión Génica/genética , Ingeniería Metabólica/métodos , Regiones Promotoras Genéticas/genética , Synechocystis/genética , Biología Sintética/métodos , Plásmidos/genética , Synechocystis/metabolismo , Ácido Vanílico/metabolismoRESUMEN
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.
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Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/fisiología , Péptidos y Proteínas de Señalización del Ritmo Circadiano/fisiología , Synechocystis/crecimiento & desarrollo , Relojes Circadianos/fisiología , Oscuridad , Synechocystis/enzimología , TemperaturaRESUMEN
Plant cells are surrounded by an extracellular matrix that consists mainly of polysaccharides. Many molecular components involved in plant cell wall polymer synthesis have been identified, but it remains largely unknown how these molecular players function together to define the length and decoration pattern of a polysaccharide. Synthetic biology can be applied to answer questions beyond individual glycosyltransferases by reconstructing entire biosynthetic machineries required to produce a complete wall polysaccharide. Recently, this approach was successful in establishing the production of heteromannan from several plant species in an orthogonal host-a yeast-illuminating the role of an auxiliary protein in the biosynthetic process. In this review we evaluate to what extent a selection of organisms from three kingdoms of life (Bacteria, Fungi and Animalia) might be suitable for the synthesis of plant cell wall polysaccharides. By identifying their key attributes for glycoengineering as well as analyzing the glycosidic linkages of their native polymers, we present a valuable comparison of their key advantages and limitations for the production of different classes of plant polysaccharides.
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Residual phenol, carried over from RNA purification, can alter RNA concentration measurements and is assumed to inhibit PCR. Here, we demonstrate that Impurities A260 values of spectral content profiling (SCP) UV/Vis measurements correlated with phenol concentration, whereas absorbance ratios of classical UV/Vis systems failed to reliably detect phenol in RNA samples. Phenol contamination led to over- or underestimation of RNA concentration on UV/Vis systems, whereas it had no influence on fluorometry quantification. Wrong RNA concentration results led to altered template input in qRT-PCR and consequently caused quantification cycle (Cq) shifts, althoughâ¯≤â¯0.01% phenol had no direct influence.
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Fenol/análisis , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN/aislamiento & purificaciónRESUMEN
Precisely timing the regulation of gene expression by anticipating recurring environmental changes is a fundamental part of global gene regulation. Circadian clocks are one form of this regulation, which is found in both eukaryotes and prokaryotes, providing a fitness advantage for these organisms. Whereas many different eukaryotic groups harbour circadian clocks, cyanobacteria are the only known oxygenic phototrophic prokaryotes to regulate large parts of their genes in a circadian fashion. A decade of intensive research on the mechanisms and functionality using computational and mathematical approaches in addition to the detailed biochemical and biophysical understanding make this the best understood circadian clock. Here, we summarize the findings and insights into various parts of the cyanobacterial circadian clock made by mathematical modelling. These findings have implications for eukaryotic circadian research as well as synthetic biology harnessing the power and efficiency of global gene regulation.
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Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo. However, riboregulators are generally designed in silico and tested in vivo, which provides little quantitative information about their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable information about the performances of in silico designed riboregulators. We first propose a simple model that provides a quantitative definition of the dynamic range of a riboregulator. We further characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off, in agreement with our model, and that relative dynamic ranges of riboregulators determined in vitro are in agreement with published in vivo data. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.
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Regulación de la Expresión Génica , Técnicas Genéticas , ARN/química , ARN/genética , Regiones no Traducidas 5' , Sistema Libre de Células , ADN/química , ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Fluorescencia , Modelos Genéticos , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN de Transferencia , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase), LUP1 (lupeol synthase), THAS1 (thalianol synthase) and MRN1 (marneral synthase) derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates for providing new bacterial access to the broad class of triterpenes for biotechnological applications.
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Rhodobacter capsulatus/metabolismo , Synechocystis/metabolismo , Triterpenos/metabolismo , Ciclización , Regulación Bacteriana de la Expresión GénicaRESUMEN
BACKGROUND: The 6S RNA is a global transcriptional riboregulator, which is exceptionally widespread among most bacterial phyla. While its role is well-characterized in some heterotrophic bacteria, we subjected a cyanobacterial homolog to functional analysis, thereby extending the scope of 6S RNA action to the special challenges of photoautotrophic lifestyles. RESULTS: Physiological characterization of a 6S RNA deletion strain (ΔssaA) demonstrates a delay in the recovery from nitrogen starvation. Significantly decelerated phycobilisome reassembly and glycogen degradation are accompanied with reduced photosynthetic activity compared to the wild type. Transcriptome profiling further revealed that predominantly genes encoding photosystem components, ATP synthase, phycobilisomes and ribosomal proteins were negatively affected in ΔssaA. In vivo pull-down studies of the RNA polymerase complex indicated that the presence of 6S RNA promotes the recruitment of the cyanobacterial housekeeping σ factor SigA, concurrently supporting dissociation of group 2 σ factors during recovery from nitrogen starvation. CONCLUSIONS: The combination of genetic, physiological and biochemical studies reveals the homologue of 6S RNA as an integral part of the cellular response of Synechocystis sp. PCC 6803 to changing nitrogen availability. According to these results, 6S RNA supports a rapid acclimation to changing nitrogen supply by accelerating the switch from group 2 σ factors SigB, SigC and SigE to SigA-dependent transcription. We therefore introduce the cyanobacterial 6S RNA as a novel candidate regulator of RNA polymerase sigma factor recruitment in Synechocystis sp. PCC 6803. Further studies on mechanistic features of the postulated interaction should shed additional light on the complexity of transcriptional regulation in cyanobacteria.
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Aclimatación/genética , Regulación Bacteriana de la Expresión Génica , Nitrógeno/deficiencia , ARN Bacteriano/metabolismo , ARN no Traducido/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Perfilación de la Expresión Génica , Fotosíntesis/genética , Ficobilisomas/genética , ARN Bacteriano/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , Factor sigma/metabolismo , Transactivadores/genéticaRESUMEN
BACKGROUND: Circadian clocks are found in organisms of almost all domains including photosynthetic Cyanobacteria, whereby large diversity exists within the protein components involved. In the model cyanobacterium Synechococcus elongatus PCC 7942 circadian rhythms are driven by a unique KaiABC protein clock, which is embedded in a network of input and output factors. Homologous proteins to the KaiABC clock have been observed in Bacteria and Archaea, where evidence for circadian behavior in these domains is accumulating. However, interaction and function of non-cyanobacterial Kai-proteins as well as homologous input and output components remain mainly unclear. RESULTS: Using a universal BLAST analyses, we identified putative KaiC-based timing systems in organisms outside as well as variations within Cyanobacteria. A systematic analyses of publicly available microarray data elucidated interesting variations in circadian gene expression between different cyanobacterial strains, which might be correlated to the diversity of genome encoded clock components. Based on statistical analyses of co-occurrences of the clock components homologous to Synechococcus elongatus PCC 7942, we propose putative networks of reduced and fully functional clock systems. Further, we studied KaiC sequence conservation to determine functionally important regions of diverged KaiC homologs. Biochemical characterization of exemplary cyanobacterial KaiC proteins as well as homologs from two thermophilic Archaea demonstrated that kinase activity is always present. However, a KaiA-mediated phosphorylation is only detectable in KaiC1 orthologs. CONCLUSION: Our analysis of 11,264 genomes clearly demonstrates that components of the Synechococcus elongatus PCC 7942 circadian clock are present in Bacteria and Archaea. However, all components are less abundant in other organisms than Cyanobacteria and KaiA, Pex, LdpA, and CdpA are only present in the latter. Thus, only reduced KaiBC-based or even simpler, solely KaiC-based timing systems might exist outside of the cyanobacterial phylum, which might be capable of driving diurnal oscillations.
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Relojes Circadianos/genética , Synechococcus/genética , Synechococcus/fisiología , Secuencias de Aminoácidos , Archaea/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Ritmo Circadiano/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/química , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Secuencia Conservada , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Fosforilación , Filogenia , Homología de Secuencia de Ácido Nucleico , Transcriptoma/genéticaRESUMEN
Cyanobacteria have a robust circadian oscillator, known as the Kai system. Reconstituted from the purified protein components KaiC, KaiB, and KaiA, it can tick autonomously in the presence of adenosine 5'-triphosphate (ATP). The KaiC hexamers enter a natural 24-hour reaction cycle of autophosphorylation and assembly with KaiB and KaiA in numerous diverse forms. We describe the preparation of stoichiometrically well-defined assemblies of KaiCB and KaiCBA, as monitored by native mass spectrometry, allowing for a structural characterization by single-particle cryo-electron microscopy and mass spectrometry. Our data reveal details of the interactions between the Kai proteins and provide a structural basis to understand periodic assembly of the protein oscillator.
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Proteínas Bacterianas/química , Relojes Circadianos , Péptidos y Proteínas de Señalización del Ritmo Circadiano/química , Ritmo Circadiano , Cianobacterias/fisiología , Adenosina Trifosfato/química , Proteínas Bacterianas/ultraestructura , Péptidos y Proteínas de Señalización del Ritmo Circadiano/ultraestructura , Microscopía por Crioelectrón , Espectrometría de Masas , Modelos Moleculares , Multimerización de ProteínaRESUMEN
Many organisms harbor circadian clocks with periods close to 24 h. These cellular clocks allow organisms to anticipate the environmental cycles of day and night by synchronizing circadian rhythms with the rising and setting of the sun. These rhythms originate from the oscillator components of circadian clocks and control global gene expression and various cellular processes. The oscillator of photosynthetic cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, linked to a complex regulatory network. Synechocystis sp. strain PCC 6803 possesses the standard cyanobacterial kaiABC gene cluster plus multiple kaiB and kaiC gene copies and antisense RNAs for almost every kai transcript. However, there is no clear evidence of circadian rhythms in Synechocystis sp. PCC 6803 under various experimental conditions. It is also still unknown if and to what extent the multiple kai gene copies and kai antisense RNAs affect circadian timing. Moreover, a large number of small noncoding RNAs whose accumulation dynamics over time have not yet been monitored are known for Synechocystis sp. PCC 6803. Here we performed a 48-h time series transcriptome analysis of Synechocystis sp. PCC 6803, taking into account periodic light-dark phases, continuous light, and continuous darkness. We found that expression of functionally related genes occurred in different phases of day and night. Moreover, we found day-peaking and night-peaking transcripts among the small RNAs; in particular, the amounts of kai antisense RNAs correlated or anticorrelated with those of their respective kai target mRNAs, pointing toward the regulatory relevance of these antisense RNAs. Surprisingly, we observed that the amounts of 16S and 23S rRNAs in this cyanobacterium fluctuated in light-dark periods, showing maximum accumulation in the dark phase. Importantly, the amounts of all transcripts, including small noncoding RNAs, did not show any rhythm under continuous light or darkness, indicating the absence of circadian rhythms in Synechocystis.