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Multicellular development requires the careful orchestration of gene expression to correctly create and position specialized cells. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are differentiated from vegetative cells in a reproducibly periodic and physiologically relevant pattern. While many genetic factors required for heterocyst development have been identified, the role of HetZ has remained unclear. Here, we present evidence to clarify the requirement of hetZ for heterocyst production and support a model where HetZ functions in the patterning stage of differentiation. We show that a clean, nonpolar deletion of hetZ fails to express the developmental genes hetR, patS, hetP and hetZ correctly and fails to produce heterocysts. Complementation and overexpression of hetZ in a hetP mutant revealed that hetZ was incapable of bypassing hetP, suggesting that it acts upstream of hetP. Complementation and overexpression of hetZ in a hetR mutant, however, demonstrated bypass of hetR, suggesting that it acts downstream of hetR and is capable of bypassing the need for hetR for differentiation irrespective of nitrogen status. Finally, protein-protein interactions were observed between HetZ and HetR, Alr2902 and HetZ itself. Collectively, this work suggests a regulatory role for HetZ in the patterning phase of cellular differentiation in Anabaena.

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.