A phosphorylation-dependent partner-switching-like module controls heterocyst polysaccharide layer formation in Anabaena sp. strain PCC 7120

Abstract Heterocysts are specialized cells formed by filamentous cyanobacteria to enable nitrogen fixation under aerobic conditions. These cells are surrounded by a multilayered envelope, including a heterocyst-specific polysaccharide (Hep) layer that restricts oxygen diffusion and protects nitrogenase activity. Although several genes required for Hep biosynthesis have been identified, the regulatory mechanisms controlling Hep layer formation remain poorly understood. Here, we show that Hep layer formation in Anabaena sp. PCC 7120 is regulated by a phosphorylation-dependent partner-switching-like system. The phosphatase HenR and the STAS domain-containing protein All4160 are both required for Hep formation and nitrogen fixation. A nonphosphorylatable All4160 variant restores Hep formation and nitrogen fixation in a henR disruptant, indicating that phosphorylation negatively regulates All4160 function. Using a bacterial two-hybrid assay, we identified two candidate kinases, Alr3423 and All2284, that interact with All4160, and both proteins phosphorylate All4160 in vitro. Genetic analysis revealed that deletion of alr3423, but not all2284, suppresses the nitrogen fixation defect of the henR disruptant, suggesting that Alr3423 is a major kinase regulating All4160 in vivo. These findings uncover a phosphorylation switch that controls heterocyst polysaccharide layer formation and expand the role of partner-switching systems beyond σ factor-dependent transcription to the direct regulation of a biosynthetic enzyme involved in exopolysaccharide (EPS) synthesis. Importance Exopolysaccharides (EPS) are involved in diverse aspects of bacterial physiology, including environmental acclimation, motility, host association, and cellular differentiation, yet their regulatory mechanisms remain poorly understood. Cyanobacteria play central roles in global carbon and nitrogen cycles through photosynthesis and nitrogen fixation, the latter requiring a micro-oxic environment provided by specialized cells called heterocysts. This study reveals a previously unrecognized mechanism for controlling polysaccharide biosynthesis, in which a partner-switching-like system directly regulates the activity of a biosynthetic enzyme. By expanding the role of partner-switching systems beyond σ factor–dependent transcription, these findings provide a framework for understanding how bacteria control EPS production in response to environmental and developmental cues. Competing Interest Statement The authors have declared no competing interest.