Bacteria can anticipate the seasons: photoperiodism in cyanobacteria.

Photoperiodic Time Measurement is the ability of plants and animals to measure differences in day/night-length (photoperiod) and use that information to anticipate critical seasonal transformations such as annual temperature cycles. This timekeeping phenomenon triggers adaptive responses in higher organisms such as gonadal growth/regression, flowering, and hibernation. Unexpectedly, we discovered this capability in cyanobacteria, unicellular prokaryotes with generation times of only 5-6 h. Cyanobacteria in short winter-like days develop enhanced resistance to cold that involves desaturation of membrane lipids and differential programs of gene transcription, including stress response pathways. As in eukaryotes, this photoperiodic timekeeping requires an intact circadian clockwork and develops over multiple cycles. Therefore, photoperiodic timekeeping evolved in much simpler organisms than previously appreciated, and involved genetic responses to stresses that recur seasonally.

Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Evaluating the Adaptive Fitness of Circadian Clocks and their Evolution.

Surely most chronobiologists believe circadian clocks are an adaptation of organisms that enhances fitness, but are we certain that this focus of our research effort really confers a fitness advantage? What is the evidence, and how do we evaluate it? What are the best criteria? These questions are the topic of this review. In addition, we will discuss selective pressures that might have led to the historical evolution of circadian systems while considering the intriguing question of whether the ongoing climate change is modulating these selective pressures so that the clock is still evolving.

Host circadian behaviors exert only weak selective pressure on the gut microbiome under stable conditions but are critical for recovery from antibiotic treatment.

The circadian rhythms of hosts dictate an approximately 24 h transformation in the environment experienced by their gut microbiome. The consequences of this cyclic environment on the intestinal microbiota are barely understood and are likely to have medical ramifications. Can daily rhythmicity in the gut act as a selective pressure that shapes the microbial community? Moreover, given that several bacterial species have been reported to exhibit circadian rhythms themselves, we test here whether a rhythmic environment is a selective pressure that favors clock-harboring bacteria that can anticipate and prepare for consistent daily changes in the environment. We observed that the daily rhythmicity of the mouse gut environment is a stabilizing influence that facilitates microbiotal recovery from antibiotic perturbation. The composition of the microbiome recovers to pretreatment conditions when exposed to consistent daily rhythmicity, whereas in hosts whose feeding and activity patterns are temporally disrupted, microbiotal recovery is incomplete and allows potentially unhealthy opportunists to exploit the temporal disarray. Unexpectedly, we found that in the absence of antibiotic perturbation, the gut microbiome is stable to rhythmic versus disrupted feeding and activity patterns. Comparison of our results with those of other studies reveals an intriguing correlation that a stable microbiome may be resilient to one perturbation alone (e.g., disruption of the daily timing of host behavior and feeding), but not to multiple perturbations in combination. However, after a perturbation of the stable microbiome, a regular daily pattern of host behavior/feeding appears to be essential for the microbiome to recover to the original steady state. Given the inconsistency of daily rhythms in modern human life (e.g., shiftwork, social jet-lag, irregular eating habits), these results emphasize the importance of consistent daily rhythmicity to optimal health not only directly to the host, but also indirectly by preserving the host's microbiome in the face of perturbations.

A BRET Ca2+ sensor enables high-throughput screening in the presence of background fluorescence.

The intrinsic fluorescence of samples confounds the use of fluorescence-based sensors. This is of particular concern in high-throughput screening (HTS) applications using large chemical libraries containing intrinsically fluorescent compounds. To overcome this problem, we developed a bioluminescence resonance energy transfer (BRET) Ca2+ sensor, CalfluxCTN. We demonstrated that it reliably reported changes in intracellular Ca2+ concentrations evoked by an agonist and an antagonist of the human muscarinic acetylcholine receptor M1 (hM1R) even in the presence of the fluorescent compound fluorescein, which interfered with a standard fluorescent HTS sensor (Fluo-8). In an HTS using a chemical library containing fluorescent compounds, CalfluxCTN accurately identified agonists and antagonists that were missed or miscategorized using Fluo-8. Moreover, we showed that a luciferase substrate that becomes activated only when inside cells generated long-lasting BRET signals in HTS, enabling results to be reliably compared among replicate samples for hours. Thus, the use of a self-luminescent sensor instead of a fluorescent sensor could facilitate the complete screening of chemical libraries in a high-throughput context and enable analysis of autofluorescent samples in many different applications.

Screen for Small-Molecule Modulators of Circadian Rhythms Reveals Phenazine as a Redox-State Modifying Clockwork Tuner.

A high-throughput cell-based screen identified redox-active small molecules that produce a period lengthening of the circadian rhythm. The strongest period lengthening phenotype was induced by a phenazine carboxamide (VU661). Comparison to two isomeric benzquinoline carboxamides (VU673 and VU164) shows the activity is associated with the redox modulating phenazine functionality. Furthermore, ex vivo cell analysis using optical redox ratio measurements shows the period lengthening phenotype to be associated with a shift to the NAD/FAD oxidation state of nicotinamide and flavine coenzymes.

Circadian Rhythms and Sleep Are Dependent Upon Expression Levels of Key Ubiquitin Ligase Ube3a.

Normal neurodevelopment requires precise expression of the key ubiquitin ligase gene Ube3a. Comparing newly generated mouse models for Ube3a downregulation (models of Angelman syndrome) vs. Ube3a upregulation (models for autism), we find reciprocal effects of Ube3a gene dosage on phenotypes associated with circadian rhythmicity, including the amount of locomotor activity. Consistent with results from neurons in general, we find that Ube3a is imprinted in neurons of the suprachiasmatic nuclei (SCN), the pacemaking circadian brain locus, despite other claims that SCN neurons were somehow exceptional to these imprinting rules. In addition, Ube3a-deficient mice lack the typical drop in wake late in the dark period and have blunted responses to sleep deprivation. Suppression of physical activity by light in Ube3a-deficient mice is not due to anxiety as measured by behavioral tests and stress hormones; quantification of stress hormones may provide a mechanistic link to sleep alteration and memory deficits caused by Ube3a deficiency, and serve as an easily measurable biomarker for evaluating potential therapeutic treatments for Angelman syndrome. We conclude that reduced Ube3a gene dosage affects not only neurodevelopment but also sleep patterns and circadian rhythms.

Synechocystis: A model system for expanding the study of cyanobacterial circadian rhythms.

The study of circadian rhythms in bacteria was transformed by studies of the cyanobacterium Synechococcus elongatus. However, in a number of respects S. elongatus is atypical, and while those unusual characteristics were helpful for rapid progress in the past, another commonly used cyanobacterial species, Synechocystis sp. PCC 6803, may be more representative and therefore more productive for future insights into bacterial clock mechanisms. In the past, circadian studies of Synechocystis have suffered from not having an excellent reporter of circadian gene expression, but we introduce here a new luminescence reporter that rivals the reporters that have been used so successfully in S. elongatus. Using this new system, we generate for the first time in Synechocystis circadian period mutants resulting from point mutations. The temperature compensation and dark-pulse resetting that mediates entrainment to the environment is characterized. Moreover, we analyse the complex organization of clock genes in Synechocystis and identify which genes are essential for circadian rhythmicity and adaptive fitness for entrainment and optimal phase alignment to environmental cycles (and which genes are not). These developments will provide impetus for new approaches towards understanding daily timekeeping mechanisms in bacteria.

Bioluminescent Sensors for Ca++ Flux Imaging and the Introduction of a New Intensity-Based Ca++ Sensor.

Sensitive detection of biological events is a goal for the design and characterization of sensors that can be used in vitro and in vivo. One important second messenger is Ca++ which has been a focus of using genetically encoded Ca++ indicators (GECIs) within living cells or intact organisms in vivo. An ideal GECI would exhibit high signal intensity, excellent signal-to-noise ratio (SNR), rapid kinetics, a large dynamic range within relevant physiological conditions, and red-shifted emission. Most available GECIs are based on fluorescence, but bioluminescent GECIs have potential advantages in terms of avoiding tissue autofluorescence, phototoxicity, photobleaching, and spectral overlap, as well as enhancing SNR. Here, we summarize current progress in the development of bioluminescent GECIs and introduce a new and previously unpublished biosensor. Because these biosensors require a substrate, we also describe the pros and cons of various substrates used with these sensors. The novel GECI that is introduced here is called CalBiT, and it is a Ca++ indicator based on the functional complementation of NanoBiT which shows a high dynamic change in response to Ca++ fluxes. Here, we use CalBiT for the detection of Ca++ fluctuations in cultured cells, including its ability for real-time imaging in living cells.

Time-optimized feeding is beneficial without enforced fasting.

Time-restricted feeding (TRF) studies underscore that when food is consumed during the daily cycle is important for weight gain/loss because the circadian clock rhythmically modulates metabolism. However, the interpretation of previous TRF studies has been confounded by study designs that introduced an extended period of enforced fasting. We introduce a novel time-optimized feeding (TOF) regimen that disentangles the effects of phase-dependent feeding from the effects of enforced fasting in mice, as well as providing a laboratory feeding protocol that more closely reflects the eating patterns of humans who usually have 24 hour access to food. Moreover, we test whether a sudden switch from ad libitum food access to TRF evokes a corticosterone (stress) response. Our data indicate that the timing of high-fat feeding under TOF allows most of the benefit of TRF without obligatory fasting or evoking a stress response. This benefit occurs through stable temporal coupling of carbohydrate/lipid oxidation with feeding. These results highlight that timing the ingestion of calorically dense foods to optimized daily phases will enhance lipid oxidation and thereby limit fat accumulation.

Spectres of Clock Evolution: Past, Present, and Yet to Come.

Circadian clocks are phylogenetically widespread biological oscillators that allow organisms to entrain to environmental cycles and use their steady-state phase relationship to anticipate predictable daily phenomena - such as the light-dark transitions of a day - and prepare accordingly. Present from cyanobacteria to mammals, circadian clocks are evolutionarily ancient and are thought to increase the fitness of the organisms that possess them by allowing for better resource usage and/or proper internal temporal order. Here, we review literature with respect to the ecology and evolution of circadian clocks, with a special focus on cyanobacteria as model organisms. We first discuss what can be inferred about future clock evolution in response to climate change, based on data from latitudinal clines and domestication. We then address our current understanding of the role that circadian clocks might be contributing to the adaptive fitness of cyanobacteria at the present time. Lastly, we discuss what is currently known about the oldest known circadian clock, and the early Earth conditions that could have led to its evolution.

Publisher Correction: Methylation deficiency disrupts biological rhythms from bacteria to humans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Methylation deficiency disrupts biological rhythms from bacteria to humans.

The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies.

Systematic identification and elimination of flux bottlenecks in the aldehyde production pathway of Synechococcus elongatus PCC 7942.

Isotopically nonstationary metabolic flux analysis (INST-MFA) provides a versatile platform to quantitatively assess in vivo metabolic activities of autotrophic systems. By applying INST-MFA to recombinant aldehyde-producing cyanobacteria, we identified metabolic alterations that correlated with increased strain performance in order to guide rational metabolic engineering. We identified four reactions adjacent to the pyruvate node that varied significantly with increasing aldehyde production: pyruvate kinase (PK) and acetolactate synthase (ALS) fluxes were directly correlated with product formation, while pyruvate dehydrogenase (PDH) and phosphoenolpyruvate carboxylase (PPC) fluxes were inversely correlated. Overexpression of enzymes for PK or ALS did not result in further improvements to the previous best-performing strain, while downregulation of PDH expression (through antisense RNA expression) or PPC flux (through expression of the reverse reaction, phosphoenolpyruvate carboxykinase) provided significant improvements. These results illustrate the potential of INST-MFA to enable a systematic approach for iterative identification and removal of pathway bottlenecks in autotrophic host cells.

Eating breakfast and avoiding late-evening snacking sustains lipid oxidation.

Circadian (daily) regulation of metabolic pathways implies that food may be metabolized differentially over the daily cycle. To test that hypothesis, we monitored the metabolism of older subjects in a whole-room respiratory chamber over two separate 56-h sessions in a random crossover design. In one session, one of the 3 daily meals was presented as breakfast, whereas in the other session, a nutritionally equivalent meal was presented as a late-evening snack. The duration of the overnight fast was the same for both sessions. Whereas the two sessions did not differ in overall energy expenditure, the respiratory exchange ratio (RER) was different during sleep between the two sessions. Unexpectedly, this difference in RER due to daily meal timing was not due to daily differences in physical activity, sleep disruption, or core body temperature (CBT). Rather, we found that the daily timing of nutrient availability coupled with daily/circadian control of metabolism drives a switch in substrate preference such that the late-evening Snack Session resulted in significantly lower lipid oxidation (LO) compared to the Breakfast Session. Therefore, the timing of meals during the day/night cycle affects how ingested food is oxidized or stored in humans, with important implications for optimal eating habits.

Circadian clock helps cyanobacteria manage energy in coastal and high latitude ocean.

The circadian clock coordinates cellular functions over the diel cycle in many organisms. The molecular mechanisms of the cyanobacterial clock are well characterized, but its ecological role remains a mystery. We present an agent-based model of Synechococcus (harboring a self-sustained, bona fide circadian clock) that explicitly represents genes (e.g., kaiABC), transcripts, proteins, and metabolites. The model is calibrated to data from laboratory experiments with wild type and no-clock mutant strains, and it successfully reproduces the main observed patterns of glycogen metabolism. Comparison of wild type and no-clock mutant strains suggests a main benefit of the clock is due to energy management. For example, it inhibits glycogen synthesis early in the day when it is not needed and energy is better used for making the photosynthesis apparatus. To explore the ecological role of the clock, we integrate the model into a dynamic, three-dimensional global circulation model that includes light variability due to seasonal and diel incident radiation and vertical extinction. Model output is compared with field data, including in situ gene transcript levels. We simulate cyanobaceria with and without a circadian clock, which allows us to quantify the fitness benefit of the clock. Interestingly, the benefit is weakest in the low latitude open ocean, where Prochlorococcus (lacking a self-sustained clock) dominates. However, our attempt to experimentally validate this testable prediction failed. Our study provides insights into the role of the clock and an example for how models can be used to integrate across multiple levels of biological organization.

Autokinase Activity of Casein Kinase 1 δ/ε Governs the Period of Mammalian Circadian Rhythms.

Circadian rhythms exist in nearly all organisms. In mammals, transcriptional and translational feedback loops (TTFLs) are believed to underlie the mechanism of the circadian clock. Casein kinase 1δ/ε (CK1δ/ε) are key kinases that phosphorylate clock components such as PER proteins, determining the pace of the clock. Most previous studies of the biochemical properties of the key kinases CK1ε and CK1δ in vitro have focused on the properties of the catalytic domains from which the autoinhibitory C-terminus has been deleted (ΔC); those studies ignored the significance of self-inhibition by autophosphorylation. By comparing the properties of the catalytic domain of CK1δ/ε with the full-length kinase that can undergo autoinhibition, we found that recombinant full-length CK1 showed a sequential autophosphorylation process that induces conformational changes to affect the overall kinase activity. Furthermore, a direct relationship between the period change and the autokinase activity among CK1δ, CK1ε, and CK1ε-R178C was observed. These data implicate the autophosphorylation activity of CK1δ and CK1ε kinases in setting the pace of mammalian circadian rhythms and indicate that the circadian period can be modulated by tuning the autophosphorylation rates of CK1δ/ε.

Circadian biology and sleep in monogenic neurological disorders and its potential application in drug discovery.

Sleep disturbances are common in people with monogenic neurological disorders and they dramatically affect the life of individuals with the disorders and their families. The associated sleep problems are probably caused by multiple factors that have not been elucidated. Study of the underlying molecular cause, behavioral phenotypes, and reciprocal interactions in several single-gene disorders (Angelman Syndrome, Fragile X Syndrome, Rett Syndrome, and Huntington's Disease) leads to the suggestion that sleep disruption and other symptoms may directly result from abnormal operation of circadian systems due to genetic alteration and/or conflicting environmental cues for clock entrainment. Therefore, because circadian patterns modify the symptoms of neurological disorders, treatments that modulate our daily rhythms may identify heretofore unappreciated therapies for the underlying disorders.

Revealing circadian mechanisms of integration and resilience by visualizing clock proteins working in real time.

The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a remarkable circa-24 h oscillation of KaiC phosphorylation that persists for many days in vitro. Here we use high-speed atomic force microscopy (HS-AFM) to visualize in real time and quantify the dynamic interactions of KaiA with KaiC on sub-second timescales. KaiA transiently interacts with KaiC, thereby stimulating KaiC autokinase activity. As KaiC becomes progressively more phosphorylated, KaiA's affinity for KaiC weakens, revealing a feedback of KaiC phosphostatus back onto the KaiA-binding events. These non-equilibrium interactions integrate high-frequency binding and unbinding events, thereby refining the period of the longer term oscillations. Moreover, this differential affinity phenomenon broadens the range of Kai protein stoichiometries that allow rhythmicity, explaining how the oscillation is resilient in an in vivo milieu that includes noise. Therefore, robustness of rhythmicity on a 24-h scale is explainable by molecular events occurring on a scale of sub-seconds.

Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production.

We applied isotopically nonstationary 13C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity. Single-gene overexpression of the three enzymes in the proposed PK bypass pathway also led to improvements in IBA production, although to a lesser extent than pk overexpression. Combinatorial overexpression of two of the three genes in the proposed PK bypass pathway (mdh and me) led to improvements in specific productivity that were similar to those achieved by single-gene pk overexpression. Our work demonstrates how 13C flux analysis can be used to identify potential metabolic bottlenecks and novel metabolic routes, and how these findings can guide rational metabolic engineering of cyanobacteria for increased production of desired molecules.