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.

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.

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.

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.

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.

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.

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.

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.