Temporal regulation of metabolic processes in the marine diazotroph Crocosphaera watsonii WH 8501.

Marine diazotrophic cyanobacteria play a crucial role in oceanic nitrogen cycling, supporting primary production and ecosystem balance. Crocosphaera watsonii WH8501 exemplifies this ability by temporally separating photosynthesis and diazotrophy to sustain metabolism. To investigate the regulatory mechanisms underlying this process, we employed LC/MS-MS proteomics in a diel culturing experiment, revealing tightly coordinated protein abundance patterns. Our findings showed a sophisticated temporal regulation of metabolic processes categorized within six distinct protein abundance clusters: (1) nitrogen fixation and amino acid biosynthesis proteins peaked during the night, while (2) glycogen metabolism and dark reactions of photosynthesis were most abundant during the night and day-night transition, likely supporting carbon consumption and energy production. Midday (3 and 4) was dominated by proteins related to photosynthesis, cellular division, and lipid synthesis, whereas late-day peaks (5) in peptide biosynthesis may facilitate nitrogenase complex formation. Notably, the day-night transition (6) exhibited fine-tuned coordination of nitrogenase assembly, with FeS cluster proteins preceding peak nitrogenase iron protein abundance, implying a temporally ordered sequence for functional enzyme formation. Within these categories, sharp temporal patterns emerged in iron trafficking to heme and iron cluster biosynthetic systems, consistent with the need to maintain tight control of iron distribution to metalloproteins at each temporal transition. These results highlight the intricate diel regulation that enables Crocosphaera to balance nitrogen fixation and photosynthesis within a single cell. The observed coordination supports the existence of a complex regulatory system ensuring optimal metabolic performance, reinforcing the critical role of temporal control in sustaining these globally significant biological processes.