Circadian clocks are pervasive throughout nature, yet only recently has this adaptive regulatory program been described in non-photosynthetic bacteria. Here, we describe an inherent complexity in the Bacillus subtilis circadian clock. We find that B. subtilis entrains to blue and red light and that circadian entrainment is separable from masking through fluence titration and frequency demultiplication protocols. We identify circadian rhythmicity in constant light, consistent with Aschoff’s Rule, and entrainment aftereffects, both of which are properties described for eukaryotic circadian clocks. We report that circadian rhythms occur in wild isolates of this prokaryote, thus establishing them as a general property of this species, and that its circadian system responds to the environment in a complex fashion that is consistent with multicellular eukaryotic circadian systems.

Luminometry datasets

White, 96-well plates (Nunclon Delta, Thermo Fisher Scientific) were used, with each well inoculated with 5 × 10⁵ cells. Plates were sealed with a transparent, evaporation-free cover (Optical Adhesive Covers, Applied Biosystems, Life Technologies). Cultures were exposed to light-darkness cycles as indicated in the manuscript, after which the cultures were released to constant conditions (either constant darkness or light). The temperature was kept constant at 27 °C. We measured bioluminescence (Berthold Centro LB960 XS3 or Berthold Mithras LB 940 Multimode Plate Reader) for 1 s each hour. All experiments were carried out in temperature-controlled incubators (MIR-154, Panasonic, Japan or Percival Intellus, Percival, USA). In entrainment experiments, the plates were ejected from the machine between readings for exposure to light.

Datasets uploaded correspond to unprocessed, raw data. Luminometry data have been collected every hour and data analysis has been performed on detrended, normalised data as described in the manuscript. 

Data uploaded are in XLSX format.