a, H. salinarum NRC-1 cells were entrained with 3 days of 12∶12 LD and subsequently released into constant darkness. Total RNA was prepared from samples collected immediately post-entrainment (t = 0 hrs), every four hours until t = 60 hours. Two additional samples were collected at t = 64.5 hrs and t = 68.5 hrs. Culture conditions during sampling were frequently monitored and controlled (Supplementary Table S1). b, Frequency histogram of genes detected with periodic transcriptional changes (binned at intervals of 0.001 hr⁻¹, p<0.2) using Lomb-Scargle analysis. c, Spectral density (black line) of the histogram in (a) shows two dominant frequencies of ∼12.5 and ∼21 hours; the blue swath shows data distribution of normally distributed gene expression changes. d, Five k-means clusters of periodic transcriptional changes of the 290 genes (from Experiment A) in (a, b) are visualized as a heatmap [average period is shown in parentheses and overrepresented GO or KEGG physiological functions (p<0.01) are also indicated]. The phasing of the diurnal L∶D cycle is indicated at the top of the heatmap with alternating white and shaded rectangles, respectively. e, Phase alignment of periodic gene expression changes shows co-induction of related cellular functions according to the diurnal cycle. DNA replication, tyrosine metabolism and ion-coupled transporters were upregulated during the middle of the light and dark phase with a period of 13.6 hours. Transcription of genes encoding components of NADH dehydrogenase (ndhG3 and ndhG4), cytochrome oxidase (coxB), the urea cycle and glutamine-glutamate metabolism peaked at the transitions from one phase to the next. Finally, nucleotide sugar metabolism, general sugar metabolism, and DNA integration were maximally induced during the latter half of the dark phase.

Three classes of average mRNA profiles for 180 of the 290 genes detected as cyclers in Experiment A. Expression profiles in all three panels are color-matched to indicate transcript profiles for the same three sets of genes over the LD cycle (A), in response to oxygen (B) and during growth in a batch culture (C). In panel A The period of oscillations in transcription upon entrainments is indicated as is the L∶D cycle (open∶grey boxes). Average transcript level changes in the same three groups of genes are plotted over the course of the growth curve for H. salinarum NRC-1 (data from Facciotii et al. submitted). (C) The transcriptional response of these genes to sudden inflow of O₂ after >6 hrs of anoxia [O2 levels are shown as a magenta dotted line (see secondary y-axis)] (Schmid et al. 2007).

Integrated analysis of transcriptional changes from a physiological context identified periodic expression of genes encoding key steps in energy production (TCA cycle and arginine metabolism), C- and N- assimilation (glutamate and arginine metabolism) and nucleotide biosynthesis. The inset graphs show transcriptional profiles (log₁₀ ratios) of gene with a specific period.