Temporal regulation of metabolic processes in the marine diazotroph Crocosphaera watsonii WH8501.

Marine diazotrophic cyanobacteria play a crucial role in oceanic nitrogen cycling, supporting primary production and ecosystem balance. Crocosphaera watsonii WH8501 exemplifies this capability by temporally separating photosynthesis and diazotrophy. To investigate the regulatory mechanisms underlying this process, we employed mass spectrometry-based proteomics in a high-resolution diel culturing experiment, revealing tightly coordinated protein abundance patterns. Unsupervised clustering of diel protein abundance profiles identified six groups of co-regulated proteins characterized by distinct temporal expression patterns and functional categories. Cluster 1 contained nitrogen fixation and amino acid biosynthesis proteins that peaked at night, while cluster 2 included glycogen metabolism and photosynthetic dark reaction proteins that were most abundant during the night and day-night transition, supporting carbon consumption and energy production. Clusters 3 and 4 were enriched in proteins involved in photosynthesis, cell division, and lipid synthesis, with maximal abundance around midday. Cluster 5 comprised peptide biosynthesis proteins that peaked later in the day and may facilitate nitrogenase complex formation. Cluster 6, expressed during the day-night transition, revealed coordinated nitrogenase assembly, with Fe-S cluster proteins preceding peak nitrogenase iron protein abundance, suggesting a temporally ordered sequence of enzyme maturation. Distinct temporal patterns in iron trafficking to heme and Fe-S cluster biosynthetic systems further indicated tightly regulated allocation of iron to metalloproteins across the diel cycle. Together, these results reveal the intricate temporal coordination that enables Crocosphaera to balance nitrogen fixation and photosynthesis within a single cell and underscore the central role of diel regulation in sustaining these globally important processes.IMPORTANCEMarine diazotrophic cyanobacteria play an essential role in global nitrogen cycling and ocean productivity. In this study, we investigate the temporal regulation of Crocosphaera watsonii metabolism using high-resolution proteomics under diel culturing conditions. Our analysis resolves six distinct clusters of protein abundance patterns across the light/dark cycle, reflecting intricate coordination of nitrogen fixation, photosynthesis, carbon allocation, and iron homeostasis. Notably, we identify temporally ordered assembly of nitrogenase subunits, including precursory expression of Fe-S cluster proteins and indications of tightly controlled iron trafficking systems. These findings provide new mechanistic insight into how unicellular diazotrophs synchronize key metabolic processes under natural diel cycles.