Proteomic analysis of metabolic adaptation in a unicellular cyanobacterium during light-dark cycles and nitrogen fixation.

Cyanobacteria have evolved specialized proteins and pathways to efficiently carry out photosynthesis and biological nitrogen (N2)-fixation (BNF). To deepen our understanding of these processes, we performed proteomic analysis of the unicellular cyanobacterium Crocosphaera subtropica ATCC 51142 grown with and without nitrate under 12-hour light-dark cycles. Using cells collected at six hours into the light or dark cycles for proteomic analysis, our data revealed significant shift in metabolic activities related to photosynthesis, respiration, biological nitrogen fixation (BNF), and proteostasis. Nitrogenase complexes, including NifHDK, NifT, and NifW, were highly expressed in the dark under nitrogen-fixing conditions, underscoring their central role in BNF and regulation based on nitrogen availability. These results amplify previous studies and show that key respiratory enzymes, such as CoxB1, and uptake hydrogenase HupSL, were co-expressed with nitrogenase, suggesting a tightly coordinated regulation of respiration, nitrogen fixation, and redox balance. The synchronized expression of HesA and HesB, conserved in diazotrophic cyanobacteria, further supports their roles in nitrogenase activities. Results also suggest that cells exploit alternative pathways, including oxidative pentose phosphate (OPP) and 2-oxoglutarate (2-OG) to produce excess ATP and support bioenergetic BNF. Our study provides new insights into the proteomic responses of Crocosphaera 51142 to nitrogen availability and light-dark cycles.