The inner workings of an ancient biological clock.

Circadian clocks evolved in diverse organisms as an adaptation to the daily swings in ambient light and temperature that derive from Earth's rotation. These timing systems, based on intracellular molecular oscillations, synchronize organisms' behavior and physiology with the 24-h environmental rhythm. The cyanobacterial clock serves as a special model for understanding circadian rhythms because it can be fully reconstituted in vitro. This review summarizes recent advances that leverage new biochemical, biophysical, and mathematical approaches to shed light on the molecular mechanisms of cyanobacterial Kai proteins that support the clock, and their homologues in other bacteria. Many questions remain in circadian biology, and the tools developed for the Kai system will bring us closer to the answers.

Protocols for in vitro reconstitution of the cyanobacterial circadian clock.

Circadian clocks are intracellular systems that orchestrate metabolic processes in anticipation of sunrise and sunset by providing an internal representation of local time. Because the ~24-h metabolic rhythms they produce are important to health across diverse life forms there is growing interest in their mechanisms. However, mechanistic studies are challenging in vivo due to the complex, that is, poorly defined, milieu of live cells. Recently, we reconstituted the intact circadian clock of cyanobacteria in vitro. It oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of individual clock proteins and promoter DNA simultaneously under defined conditions without user intervention. We found that reproducibility of the reactions required strict adherence to the quality of each recombinant clock protein purified from Escherichia coli. Here, we provide protocols for preparing in vitro clock samples so that other labs can ask questions about how changing environments, like temperature, metabolites, and protein levels are reflected in the core oscillator and propagated to regulation of transcription, providing deeper mechanistic insights into clock biology.

Synechococcus elongatus Argonaute reduces natural transformation efficiency and provides immunity against exogenous plasmids.

IMPORTANCE: S. elongatus is an important cyanobacterial model organism for the study of its prokaryotic circadian clock, photosynthesis, and other biological processes. It is also widely used for genetic engineering to produce renewable biochemicals. Our findings reveal an SeAgo-based defense mechanism in S. elongatus against the horizontal transfer of genetic material. We demonstrate that deletion of the ago gene facilitates genetic studies and genetic engineering of S. elongatus.

Phenotypically complex living materials containing engineered cyanobacteria.

The field of engineered living materials lies at the intersection of materials science and synthetic biology with the aim of developing materials that can sense and respond to the environment. In this study, we use 3D printing to fabricate a cyanobacterial biocomposite material capable of producing multiple functional outputs in response to an external chemical stimulus and demonstrate the advantages of utilizing additive manufacturing techniques in controlling the shape of the fabricated photosynthetic material. As an initial proof-of-concept, a synthetic riboswitch is used to regulate the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942 within a hydrogel matrix. Subsequently, a strain of S. elongatus is engineered to produce an oxidative laccase enzyme; when printed within a hydrogel matrix the responsive biomaterial can decolorize a common textile dye pollutant, indigo carmine, potentially serving as a tool in environmental bioremediation. Finally, cells are engineered for inducible cell death to eliminate their presence once their activity is no longer required, which is an important function for biocontainment and minimizing environmental impact. By integrating genetically engineered stimuli-responsive cyanobacteria in volumetric 3D-printed designs, we demonstrate programmable photosynthetic biocomposite materials capable of producing functional outputs including, but not limited to, bioremediation.

Roles for the Synechococcus elongatus RNA-Binding Protein Rbp2 in Regulating the Circadian Clock.

The cyanobacterial circadian oscillator, consisting of KaiA, KaiB, and KaiC proteins, drives global rhythms of gene expression and compaction of the chromosome and regulates the timing of cell division and natural transformation. While the KaiABC posttranslational oscillator can be reconstituted in vitro, the Kai-based oscillator is subject to several layers of regulation in vivo. Specifically, the oscillator proteins undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localized as a focus at or near the pole of the cell at night. Here, we report that the CI domain of KaiC, when in a hexameric state, is sufficient to target KaiC to the pole. Moreover, increased ATPase activity of KaiC correlates with enhanced polar localization. We identified proteins associated with KaiC in either a localized or diffuse state. We found that loss of Rbp2, found to be associated with localized KaiC, results in decreased incidence of KaiC localization and long-period circadian phenotypes. Rbp2 is an RNA-binding protein, and it appears that RNA-binding activity of Rbp2 is required to execute clock functions. These findings uncover previously unrecognized roles for Rbp2 in regulating the circadian clock and suggest that the proper localization of KaiC is required for a fully functional clock in vivo.

Cell specialization in cyanobacterial biofilm development revealed by expression of a cell-surface and extracellular matrix protein.

Cyanobacterial biofilms are ubiquitous and play important roles in diverse environments, yet, understanding of the processes underlying the development of these aggregates is just emerging. Here we report cell specialization in formation of Synechococcus elongatus PCC 7942 biofilms-a hitherto unknown characteristic of cyanobacterial social behavior. We show that only a quarter of the cell population expresses at high levels the four-gene ebfG-operon that is required for biofilm formation. Almost all cells, however, are assembled in the biofilm. Detailed characterization of EbfG4 encoded by this operon revealed cell-surface localization as well as its presence in the biofilm matrix. Moreover, EbfG1-3 were shown to form amyloid structures such as fibrils and are thus likely to contribute to the matrix structure. These data suggest a beneficial 'division of labor' during biofilm formation where only some of the cells allocate resources to produce matrix proteins-'public goods' that support robust biofilm development by the majority of the cells. In addition, previous studies revealed the operation of a self-suppression mechanism that depends on an extracellular inhibitor, which supresses transcription of the ebfG-operon. Here we revealed inhibitor activity at an early growth stage and its gradual accumulation along the exponential growth phase in correlation with cell density. Data, however, do not support a threshold-like phenomenon known for quorum-sensing in heterotrophs. Together, data presented here demonstrate cell specialization and imply density-dependent regulation thereby providing deep insights into cyanobacterial communal behavior.

Synchronization of the circadian clock to the environment tracked in real time.

The circadian system of the cyanobacterium Synechococcus elongatus PCC 7942 relies on a three-protein nanomachine (KaiA, KaiB, and KaiC) that undergoes an oscillatory phosphorylation cycle with a period of ~24 h. This core oscillator can be reconstituted in vitro and is used to study the molecular mechanisms of circadian timekeeping and entrainment. Previous studies showed that two key metabolic changes that occur in cells during the transition into darkness, changes in the ATP/ADP ratio and redox status of the quinone pool, are cues that entrain the circadian clock. By changing the ATP/ADP ratio or adding oxidized quinone, one can shift the phase of the phosphorylation cycle of the core oscillator in vitro. However, the in vitro oscillator cannot explain gene expression patterns because the simple mixture lacks the output components that connect the clock to genes. Recently, a high-throughput in vitro system termed the in vitro clock (IVC) that contains both the core oscillator and the output components was developed. Here, we used IVC reactions and performed massively parallel experiments to study entrainment, the synchronization of the clock with the environment, in the presence of output components. Our results indicate that the IVC better explains the in vivo clock-resetting phenotypes of wild-type and mutant strains and that the output components are deeply engaged with the core oscillator, affecting the way input signals entrain the core pacemaker. These findings blur the line between input and output pathways and support our previous demonstration that key output components are fundamental parts of the clock.

Glycogen metabolism is required for optimal cyanobacterial growth in the rapid light-dark cycle of low-Earth orbit.

Some designs for bioregenerative life support systems to enable human space missions incorporate cyanobacteria for removal of carbon dioxide, generation of oxygen, and treatment of wastewater, as well as providing a source of nutrition. In this study, we examined the effects of the short light-dark (LD) cycle of low-Earth orbit on algal and cyanobacterial growth, approximating conditions on the International Space Station, which orbits Earth roughly every 90 min. We found that growth of green algae was similar in both normal 12 h light:12 h dark (12 h:12 h LD) and 45':45' LD cycles. Three diverse strains of cyanobacteria were not only capable of growth in short 45':45' LD cycles, but actually grew better than in 12 h:12 h LD cycles. We showed that 45':45' LD cycles do not affect the endogenous 24 h circadian rhythms of Synechococcus elongatus. Using a dense library of randomly barcoded transposon mutants, we identified genes whose loss is detrimental for the growth of S. elongatus under 45':45' LD cycles. These include several genes involved in glycogen metabolism and the oxidative pentose phosphate pathway. Notably, 45':45' LD cycles did not affect the fitness of strains that carry mutations in the biological circadian oscillator or the clock input and output regulatory pathways. Overall, this study shows that cultures of cyanobacteria could be grown under natural sunlight of low-Earth orbit and highlights the utility of a functional genomic study in a model organism to better understand key biological processes in conditions that are relevant to space travel.

An unexpected role for leucyl aminopeptidase in UV tolerance revealed by a genome-wide fitness assessment in a model cyanobacterium.

UV radiation (UVR) has significant physiological effects on organisms living at or near the Earth's surface, yet the full suite of genes required for fitness of a photosynthetic organism in a UVR-rich environment remains unknown. This study reports a genome-wide fitness assessment of the genes that affect UVR tolerance under environmentally relevant UVR dosages in the model cyanobacterium Synechococcus elongatus PCC 7942. Our results highlight the importance of specific genes that encode proteins involved in DNA repair, glutathione synthesis, and the assembly and maintenance of photosystem II, as well as genes that encode hypothetical proteins and others without an obvious connection to canonical methods of UVR tolerance. Disruption of a gene that encodes a leucyl aminopeptidase (LAP) conferred the greatest UVR-specific decrease in fitness. Enzymatic assays demonstrated a strong pH-dependent affinity of the LAP for the dipeptide cysteinyl-glycine, suggesting an involvement in glutathione catabolism as a function of night-time cytosolic pH level. A low differential expression of the LAP gene under acute UVR exposure suggests that its relative importance would be overlooked in transcript-dependent screens. Subsequent experiments revealed a similar UVR-sensitivity phenotype in LAP knockouts of other organisms, indicating conservation of the functional role of LAPs in UVR tolerance.

Transcriptomic and Phenomic Investigations Reveal Elements in Biofilm Repression and Formation in the Cyanobacterium Synechococcus elongatus PCC 7942.

Biofilm formation by photosynthetic organisms is a complex behavior that serves multiple functions in the environment. Biofilm formation in the unicellular cyanobacterium Synechococcus elongatus PCC 7942 is regulated in part by a set of small secreted proteins that promotes biofilm formation and a self-suppression mechanism that prevents their expression. Little is known about the regulatory and structural components of the biofilms in PCC 7942, or response to the suppressor signal(s). We performed transcriptomics (RNA-Seq) and phenomics (RB-TnSeq) screens that identified four genes involved in biofilm formation and regulation, more than 25 additional candidates that may impact biofilm formation, and revealed the transcriptomic adaptation to the biofilm state. In so doing, we compared the effectiveness of these two approaches for gene discovery.

Coupling of distant ATPase domains in the circadian clock protein KaiC.

The AAA+ family member KaiC is the central pacemaker for circadian rhythms in the cyanobacterium Synechococcus elongatus. Composed of two hexameric rings of adenosine triphosphatase (ATPase) domains with tightly coupled activities, KaiC undergoes a cycle of autophosphorylation and autodephosphorylation on its C-terminal (CII) domain that restricts binding of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryogenic-electron microscopy to investigate how daytime and nighttime states of CII regulate KaiB binding on CI. We find that the CII hexamer is destabilized during the day but takes on a rigidified C2-symmetric state at night, concomitant with ring-ring compression. Residues at the CI-CII interface are required for phospho-dependent KaiB association, coupling ATPase activity on CI to cooperative KaiB recruitment. Together, these studies clarify a key step in the regulation of cyanobacterial circadian rhythms by KaiC phosphorylation.

Comparative Genomics of Synechococcus elongatus Explains the Phenotypic Diversity of the Strains.

Strains of the freshwater cyanobacterium Synechococcus elongatus were first isolated approximately 60 years ago, and PCC 7942 is well established as a model for photosynthesis, circadian biology, and biotechnology research. The recent isolation of UTEX 3055 and subsequent discoveries in biofilm and phototaxis phenotypes suggest that lab strains of S. elongatus are highly domesticated. We performed a comprehensive genome comparison among the available genomes of S. elongatus and sequenced two additional laboratory strains to trace the loss of native phenotypes from the standard lab strains and determine the genetic basis of useful phenotypes. The genome comparison analysis provides a pangenome description of S. elongatus, as well as correction of extensive errors in the published sequence for the type strain PCC 6301. The comparison of gene sets and single nucleotide polymorphisms (SNPs) among strains clarifies strain isolation histories and, together with large-scale genome differences, supports a hypothesis of laboratory domestication. Prophage genes in laboratory strains, but not UTEX 3055, affect pigmentation, while unique genes in UTEX 3055 are necessary for phototaxis. The genomic differences identified in this study include previously reported SNPs that are, in reality, sequencing errors, as well as SNPs and genome differences that have phenotypic consequences. One SNP in the circadian response regulator rpaA that has caused confusion is clarified here as belonging to an aberrant clone of PCC 7942, used for the published genome sequence, that has confounded the interpretation of circadian fitness research. IMPORTANCE Synechococcus elongatus is a versatile and robust model cyanobacterium for photosynthetic metabolism and circadian biology research, with utility as a biological production platform. We compared the genomes of closely related S. elongatus strains to create a pangenome annotation to aid gene discovery for novel phenotypes. The comparative genomic analysis revealed the need for a new sequence of the species type strain PCC 6301 and includes two new sequences for S. elongatus strains PCC 6311 and PCC 7943. The genomic comparison revealed a pattern of early laboratory domestication of strains, clarifies the relationship between the strains PCC 6301 and UTEX 2973, and showed that differences in large prophage regions, operons, and even single nucleotides have effects on phenotypes as wide-ranging as pigmentation, phototaxis, and circadian gene expression.

Impairment of a cyanobacterial glycosyltransferase that modifies a pilin results in biofilm development.

A biofilm inhibiting mechanism operates in the cyanobacterium Synechococcus elongatus. Here, we demonstrate that the glycosyltransferase homologue, Ogt, participates in the inhibitory process - inactivation of ogt results in robust biofilm formation. Furthermore, a mutational approach shows requirement of the glycosyltransferase activity for biofilm inhibition. This enzyme is necessary for glycosylation of the pilus subunit and for adequate pilus formation. In contrast to wild-type culture in which most cells exhibit several pili, only 25% of the mutant cells are piliated, half of which possess a single pilus. In spite of this poor piliation, natural DNA competence was similar to that of wild-type; therefore, we propose that the unglycosylated pili facilitate DNA transformation. Additionally, conditioned medium from wild-type culture, which contains a biofilm inhibiting substance(s), only partially blocks biofilm development by the ogt-mutant. Thus, we suggest that inactivation of ogt affects multiple processes including production or secretion of the inhibitor as well as the ability to sense or respond to it.

Reconstitution of an intact clock reveals mechanisms of circadian timekeeping.

Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.

A Cyanobacterial Component Required for Pilus Biogenesis Affects the Exoproteome.

Protein secretion as well as the assembly of bacterial motility appendages are central processes that substantially contribute to fitness and survival. This study highlights distinctive features of the mechanism that serves these functions in cyanobacteria, which are globally prevalent photosynthetic prokaryotes that significantly contribute to primary production. Our studies of biofilm development in the cyanobacterium Synechococcus elongatus uncovered a novel component required for the biofilm self-suppression mechanism that operates in this organism. This protein, which is annotated as "hypothetical," is denoted EbsA (essential for biofilm self-suppression A) here. EbsA homologs are highly conserved and widespread in diverse cyanobacteria but are not found outside this clade. We revealed a tripartite complex of EbsA, Hfq, and the ATPase homolog PilB (formerly called T2SE) and demonstrated that each of these components is required for the assembly of the hairlike type IV pili (T4P) appendages, for DNA competence, and affects the exoproteome in addition to its role in biofilm self-suppression. These data are consistent with bioinformatics analyses that reveal only a single set of genes in S. elongatus to serve pilus assembly or protein secretion; we suggest that a single complex is involved in both processes. A phenotype resulting from the impairment of the EbsA homolog in the cyanobacterium Synechocystis sp. strain PCC 6803 implies that this feature is a general cyanobacterial trait. Moreover, comparative exoproteome analyses of wild-type and mutant strains of S. elongatus suggest that EbsA and Hfq affect the exoproteome via a process that is independent of PilB, in addition to their involvement in a T4P/secretion machinery.IMPORTANCE Cyanobacteria, environmentally prevalent photosynthetic prokaryotes, contribute ∼25% of global primary production. Cyanobacterial biofilms elicit biofouling, thus leading to substantial economic losses; however, these microbial assemblages can also be beneficial, e.g., in wastewater purification processes and for biofuel production. Mechanistic aspects of cyanobacterial biofilm development were long overlooked, and genetic and molecular information emerged only in recent years. The importance of this study is 2-fold. First, it identifies novel components of cyanobacterial biofilm regulation, thus contributing to the knowledge of these processes and paving the way for inhibiting detrimental biofilms or promoting beneficial ones. Second, the data suggest that cyanobacteria may employ the same complex for the assembly of the motility appendages, type 4 pili, and protein secretion. A shared pathway was previously shown in only a few cases of heterotrophic bacteria, whereas numerous studies demonstrated distinct systems for these functions. Thus, our study broadens the understanding of pilus assembly/secretion in diverse bacteria and furthers the aim of controlling the formation of cyanobacterial biofilms.

The circadian clock and darkness control natural competence in cyanobacteria.

The cyanobacterium Synechococcus elongatus is a model organism for the study of circadian rhythms. It is naturally competent for transformation-that is, it takes up DNA from the environment, but the underlying mechanisms are unclear. Here, we use a genome-wide screen to identify genes required for natural transformation in S. elongatus, including genes encoding a conserved Type IV pilus, genes known to be associated with competence in other bacteria, and others. Pilus biogenesis occurs daily in the morning, while natural transformation is maximal when the onset of darkness coincides with the dusk circadian peak. Thus, the competence state in cyanobacteria is regulated by the circadian clock and can adapt to seasonal changes of day length.

The international journeys and aliases of Synechococcus elongatus.

This perspective provides a historical account of the isolation and nomenclature of the cyanobacterial strains currently known as Synechococcus elongatus. The story focuses on an isolate from the San Francisco Bay area of California (Pasteur Culture Collection PCC 7942) that has, for decades, been the genetic model for this species, and its close relative isolated from Waller Creek in Texas (PCC 6301, also known as the University of Texas at Austin Culture Collection of Algae UTEX 625). Until recently, these strains have been the only representatives of the species. A new wild isolate, UTEX 3055, is distinctly different from the prior reference strains. S. elongatus strains have been widely used by labs around the world to discover fundamental cellular processes and to engineer cyanobacteria to generate useful products. The review clarifies relationships among strains that carry different names, and explains how names that appear in the literature have changed over the years.

A microcin processing peptidase-like protein of the cyanobacterium Synechococcus elongatus is essential for secretion of biofilm-promoting proteins.

Small secreted compounds, e.g. microcins, are characterized by a double-glycine (GG) secretion motif that is cleaved off upon maturation. Genomic analysis suggests that small proteins that possess a GG motif are widespread in cyanobacteria; however, the roles of these proteins are largely unknown. Using a biofilm-proficient mutant of the cyanobacterium Synechococcus elongatus PCC 7942 in which the constitutive biofilm self-suppression mechanism is inactivated, we previously demonstrated that four small proteins, Enable biofilm formation with a GG motif (EbfG1-4), each with a GG motif, enable biofilm formation. Furthermore, a peptidase belonging to the C39 family, Peptidase transporter enabling Biofilm (PteB), is required for secretion of these proteins. Here, we show that the microcin processing peptidase-like protein encoded by gene Synpcc7942_1127 is also required for biofilm development - inactivation of this gene in the biofilm-proficient mutant abrogates biofilm development. Additionally, this peptidase-like protein (denoted EbfE - enables biofilm formation peptidase) is required for secretion of the EbfG biofilm-promoting small proteins. Given their protein-domain characteristics, we suggest that PteB and EbfE take part in a maturation-secretion system, with PteB being located to the cell membrane while EbfE is directed to the periplasmic space via its secretion signal.

Predicting the metabolic capabilities of Synechococcus elongatus PCC 7942 adapted to different light regimes.

There is great interest in engineering photoautotrophic metabolism to generate bioproducts of societal importance. Despite the success in employing genome-scale modeling coupled with flux balance analysis to engineer heterotrophic metabolism, the lack of proper constraints necessary to generate biologically realistic predictions has hindered broad application of this methodology to phototrophic metabolism. Here we describe a methodology for constraining genome-scale models of photoautotrophy in the cyanobacteria Synechococcus elongatus PCC 7942. Experimental photophysiology parameters coupled to genome-scale flux balance analysis resulted in accurate predictions of growth rates and metabolic reaction fluxes at low and high light conditions. Additionally, by constraining photon uptake fluxes, we characterized the metabolic cost of excess excitation energy. The predicted energy fluxes were consistent with known light-adapted phenotypes in cyanobacteria. Finally, we leveraged the modeling framework to characterize existing photoautotrophic and photomixtotrophic engineering strategies for 2,3-butanediol production in S. elongatus. This methodology, applicable to genome-scale modeling of all phototrophic microorganisms, can facilitate the use of flux balance analysis in the engineering of light-driven metabolism.