Circadian clocks create a 24-hour temporal structure, which allows organisms to occupy a niche formed by time rather than space. They are pervasive throughout nature, yet they remain unexpectedly unexplored and uncharacterized in nonphotosynthetic bacteria. Here, we identify in Bacillus subtilis circadian rhythms sharing the canonical properties of circadian clocks: free-running period, entrainment, and temperature compensation. We show that gene expression in B. subtilis can be synchronized in 24-hour light or temperature cycles and exhibit phase-specific characteristics of entrainment. Upon release to constant dark and temperature conditions, bacterial biofilm populations have temperature-compensated free-running oscillations with a period close to 24 hours. Our work opens the field of circadian clocks in the free-living, nonphotosynthetic prokaryotes, bringing considerable potential for impact upon biomedicine, ecology, and industrial processes.

Circadian timing is a fundamental biological process, underlying cellular physiology in animals, plants, fungi, and cyanobacteria. Circadian clocks organize gene expression, metabolism, and behavior such that they occur at specific times of day. The biological clocks that orchestrate these daily changes confer a survival advantage and dominate daily behavior, for example, waking us in the morning and helping us to sleep at night. The molecular mechanism of circadian clocks has been sketched out in genetic model systems from prokaryotes to humans, revealing a combination of transcriptional and posttranscriptional pathways, but the clock mechanism is far from solved. Although Saccharomyces cerevisiae is among the most powerful genetic experimental systems and, as such, could greatly contribute to our understanding of cellular timing, it still remains absent from the repertoire of circadian model organisms. Here, we use continuous cultures of yeast, establishing conditions that reveal characteristic clock properties similar to those described in other species. Our results show that metabolism in yeast shows systematic circadian entrainment, responding to cycle length and zeitgeber (stimulus) strength, and a (heavily damped) free running rhythm. Furthermore, the clock is obvious in a standard, haploid, auxotrophic strain, opening the door for rapid progress into cellular clock mechanisms.he circadian clock is a cell-based, regulatory network that controls processes from gene expression to behavior. These daily clocks, found in diverse organisms, share a set of signature properties (1). One of these is a free-running, circa 24-h (circadian) oscillation in constant conditions. The phenomenon of selfsustained rhythm, however, has never been the "aim" of evolution. It is per se not a prerequisite for the timing system but rather a consequence of how a daily timing system has developed in an environment that is utterly predictable in its alternation of light and darkness, warmer and colder temperatures, and numerous other qualities (2). Notably, many organisms do not show obvious freerunning rhythms. For instance, the ascomycete, Neurospora crassa, suppresses daily, rhythmic circadian spore formation when CO 2 accumulates (3). The accidental discovery of a mutant strain that makes "bands" of spores once every 22 h in constant darknesswithout exchanging the air to decrease CO 2 levels-permitted development of Neurospora as a clock model system (4). Even the banding strain of Neurospora appears arrhythmic in constant light, as do many animals. Yet, in the case of Neurospora, several transcript levels and the activity of the enzyme nitrate reductase are oscillating with a circa 24-h period despite no observable rhythms in spore formation (5, 6). When animals become arrhythmic in constant light, usually a decrease in irradiance will allow rhythmicity to emerge (7). These examples suggest that the expression of a free-running clock very much depends on conditions or that it is not a universal property of circadian clocks. The...

Circadian clocks in plants, animals, fungi, and in photosynthetic bacteria have been well-described. Observations of circadian rhythms in non-photosynthetic Eubacteria have been sporadic, and the molecular basis for these potential rhythms remains unclear. Here, we present the published experimental and bioinformatical evidence for circadian rhythms in these non-photosynthetic Eubacteria. From this, we suggest that the timekeeping functions of these organisms will be best observed and studied in their appropriate complex environments. Given the rich temporal changes that exist in these environments, it is proposed that microorganisms both adapt to and contribute to these daily dynamics through the process of temporal mutualism. Understanding the timekeeping and temporal interactions within these systems will enable a deeper understanding of circadian clocks and temporal programs and provide valuable insights for medicine and agriculture.

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words)A transcriptional feedback loop is central to clock function in animals, plants and fungi. The clock genes involved in its regulation are specific to -and highly conserved within -the kingdoms of life. However, other shared clock mechanisms, such as phosphorylation, are mediated by proteins found broadly among living organisms, performing functions in many cellular sub-systems. Use of homology to directly infer involvement/association with the clock mechanism in new, developing model systems, is therefore of limited use. Here we describe the approach PREMONition, PREdicting Molecular Networks, that uses functional relationships to predict molecular circadian clock associations. PREMONition is based on the incorporation of proteins encoded by known clock genes (when available), rhythmically expressed clockcontrolled genes and non-rhythmically expressed but interacting genes into a cohesive network.After tuning PREMONition on the networks derived for human, fly and fungal circadian clocks, we deployed the approach to predict a molecular clock network for Saccharomyces cerevisiae, for which there are no readily-identifiable clock gene homologs. The predicted network was validated using gene expression data and a growth assay for sensitivity to light, a zeitgeber of circadian clocks of most organisms. PREMONition may be used to identify candidate clockregulated processes and thus candidate clock genes in other organisms.Keywords: circadian clock / interactome / S. cerevisiae / network / circadian rhythm the absence of identified clock genes. For instance, Neurospora crassa shows free-running rhythms and circadian entrainment without the clock gene frequency 3,4 and rhythms in the redox state of peroxiredoxin persist in many model genetic systems in the absence of transcription or of clock genes 5 . Paracrine signaling can sustain circadian rhythms in pacemaker (neuronal) tissue that lacks clock genes and displays no endogenous rhythm 6 . One interpretation of this is that feedback integral to circadian clocks spans multiple molecular 'levels', from transcription to metabolism. We conjecture that this multi-level response can be captured through functional relationships, referred to as functional interactions. We know of no models that explicitly attempt to integrate these different levels to more fully describe the mechanisms of the circadian clock. Here, we describe the method PREMONition, PREdicting MOlecular Networks, to construct multi-level (functional) molecular clock networks using publicly available datasets and bioinformatics tools. After tuning the method on clock genetic model systems, we deployed PREMONition to predict a molecular clock network in S. cerevisiae, an experimental system with a circadian phenotype that is not suited to genetic screens 7 . We validated the results of the yeast experiment in silico and in vivo. Methods like PREMONition, that span multiple levels in the cell, are essential to developing integrative models of complex biological processes. Results PREMONition applied to mod...