Nitrogen-Responsive Extracellular Proteomics Reveals Evidence for a Novel Heterocyst-Specific Protein Secretion Pathway in Anabaena

Nitrogen availability is a major factor governing the physiology, ecology, and metabolism of cyanobacteria. Here, we performed a comparative extracellular proteomic analysis of Anabaena sp. PCC 7120 grown under nitrate-replete and diazotrophic conditions. Using LC-MS/MS, we identified 115 extracellular proteins in nitrate-grown cultures and 113 proteins under N2-fixing conditions. Remarkably, SignalP 6.0 predicted canonical signal peptides in only [~]22% of the identified proteins, suggesting that extracellular protein export in Anabaena predominantly occurs through non-classical secretion mechanisms, potentially involving extracellular vesicles or other unrecognized pathways. Six highly abundant extracellular proteins (Alr2938, Alr4550, Alr2328, All4121, Alr0528, and Alr0529) were detected under both nitrogen regimes. In contrast, All4337, Alr0608, and All3093 were preferentially enriched under nitrate-replete conditions, whereas Alr0267 and Alr1050 emerged among the most abundant extracellular proteins during diazotrophic growth. Notably, an Alr0267-GFP fusion protein was detected exclusively in heterocysts, the specialized N2-fixing cells of Anabaena, with GFP fluorescence concentrated at the cell periphery. The extracellular localization of Alr0267 is particularly intriguing because heterocysts are surrounded by specialized polysaccharide and glycolipid envelope layers that establish the microoxic environment required for nitrogenase activity. The apparent export of Alr0267 across these barriers provides evidence for a previously unrecognized heterocyst-associated protein secretion pathway. Together, these findings reveal a nitrogen-responsive extracellular proteome and provide the first evidence for heterocyst-specific extracellular protein secretion. This work advances our understanding of heterocyst biology and protein trafficking while laying a foundation for engineering Anabaena as a sustainable photosynthetic platform for secreting high-value proteins using sunlight, CO2, N2, and mineralized water.