Cell-type-specific glutamine synthetase inhibition reveals the intercellular nitrogen economy in Anabaena sp. PCC...

Nitrogen homeostasis in heterocyst-forming filamentous diazotrophic cyanobacteria depends on the coordination between two cell types: heterocysts, which fix atmospheric N2, and vegetative cells, which carry out oxygenic photosynthesis. Yet the cell-type-specific contribution of glutamine synthetase (GS), the central enzyme of nitrogen assimilation, to this coordination remains poorly understood. We used cell-type-specific overexpression of IF7A, the GS post-translational inhibitor, in both wild-type Anabaena sp. PCC 7120 and Δamt genetic backgrounds, the latter lacking the high-affinity ammonium transporters (Amt) responsible for NH4+ uptake, to dissect the contribution of each cell type to the nitrogen economy of the filament. Whole-filament GS inhibition caused photosynthetic impairment, disruption of the NtcA-PII-PipX signalling axis and growth arrest, establishing GS in vegetative cells as rate-limiting for filament growth. Heterocyst-targeted GS inhibition triggered accumulation of the nitrogen storage polymer cyanophycin, revealing that heterocyst GS governs the routing of fixed nitrogen either towards distribution or storage. Both phenotypes were amplified in the Δamt background. Using pharmacological inhibition of GS with ammonium excretion measurements, we demonstrate that Amt transporters recover nitrogen lost by passive NH3 diffusion under diazotrophic conditions, and that their absence renders the internal nitrogen economy chronically more vulnerable to GS inhibition. In natural nitrogen-limited environments, where diazotrophic cyanobacteria cannot compensate for passive nitrogen loss through external uptake, this recycling function is critical for nitrogen fixation efficiency. Overall, our findings redefine the architecture of intercellular nitrogen homeostasis in filamentous diazotrophic cyanobacteria, establishing GS and Amt transporters as interdependent determinants of intercellular nitrogen retention, distribution and storage.