Abstract
Circadian rhythms are self-sustained and adjustable cycles, typically entrained with light/dark and/or temperature cycles. These rhythms are present in animals, plants, fungi, and several bacteria. The central mechanism behind these “pacemakers” and the connection to the circadian regulated pathways are still poorly understood. The circadian rhythm of the cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) is highly robust and controlled by only three proteins, KaiA, KaiB, and KaiC. This central clock system has been extensively studied functionally and structurally and can be reconstituted in vitro. These characteristics, together with a relatively small genome (2.7 Mbp), make S. elongatus an ideal model system for the study of circadian rhythms.Different approaches have been used to reveal the influence of the central S. elongatus clock on rhythmic gene expression, rhythmic mRNA abundance, rhythmic DNA topology changes, and cell division. However, a global analysis of its proteome dynamics has not been reported yet.To uncover the variation in protein abundances during 48 h under light and dark cycles (12:12 h), we used quantitative proteomics, with TMT 6-plex isobaric labeling. We queried the S. elongatus proteome at 10 different time points spanning a single 24-h period, leading to 20 time points over the full 48-h period.Employing multidimensional separation and high-resolution mass spectrometry, we were able to find evidence for a total of 82% of the S. elongatus proteome. Of the 1537 proteins quantified over the time course of the experiment, only 77 underwent significant cyclic variations. Interestingly, our data provide evidence for in- and out-of-phase correlation between mRNA and protein levels for a set of specific genes and proteins. As a range of cyclic proteins are functionally not well annotated, this work provides a resource for further studies to explore the role of these proteins in the cyanobacterial circadian rhythm.
Highlights
Different approaches have been used to reveal the influence of the central S. elongatus clock on rhythmic gene expression, rhythmic mRNA abundance, rhythmic DNA topology changes, and cell division
Global Proteome Analysis of the Cyanobacterium S. elongatus—Here we present the first in-depth quantitative proteome analysis of the cyanobacterium S. elongatus, conducted over a 48-h time span under LD conditions
Protein digestion, and TMT labeling, the peptides were fractionated by means of strong cation exchange (SCX); this was done in order to decrease the complexity of the proteome and, at the same time, reduce precursor ion interference upon MS/MS analysis, which is inherent to isobaric quantification
Summary
Cyanobacteria Cell Culture—The wild-type strain of S. elongatus PCC 7942 was routinely grown photoautotrophically in BG11 medium (29) at 30 °C under continuous illumination with white light at 80 M photons/m2s (Versatile Environmental Test Chamber, SANYO, Bensenville, IL) and a continuous stream of air. Significance Analysis and Clustering—Quantitative data containing protein intensities from 20 time points, as obtained from the experiments described above, was analyzed as follows. Based on the figure of merit, a method of determining the optimal number of k-means clusters, we chose to perform k-means clustering with six clusters All clusters with their containing proteins were exported and used for further analysis. Cyclic Profile Validation—The proteins considered significant and observed across all different time points were subjected to Pearson correlation analysis between the first 24-h ratios and the second 24-h ratios. For this purpose the statistical software IBM® SPSS® Statistics Data Editor was used. The “raw” data (GSE14225) were extracted from GEO (Gene Expression Omnibus) with R (35), and an average of the two replicates was performed
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