Phenotypic Assessment Suggests Multiple Start Codons for HetN, an Inhibitor of Heterocyst Differentiation, in Anabaena sp Strain PCC 7120

Multicellular organisms must carefully regulate the timing, number, and location of specialized cellular development. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are interspersed between vegetative cells in a periodic pattern to achieve an optimal exchange of bioavailable nitrogen and reduced carbon. The spacing between heterocysts is regulated by the activity of two developmental inhibitors, PatS and HetN. PatS functions to create a de novo pattern from a homogenous field of undifferentiated cells, while HetN maintains the pattern throughout subsequent growth. Both PatS and HetN harbor the peptide motif ERGSGR, which is sufficient to inhibit development. While the small size of PatS makes the interpretation of inhibitory domains relatively simple, HetN is a 287-amino-acid protein with multiple functional regions. Previous work suggested the possibility of a truncated form of HetN containing the ERGSGR motif as the source of the HetN-derived inhibitory signal. In this work, we present evidence that the glutamate of the ERGSGR motif is required for proper HetN inhibition of heterocysts. Mutational analysis and subcellular localization indicate that the gene encoding HetN uses two methionine start codons (M1 and M119) to encode two protein forms: M1 is required for protein localization, while M119 is primarily responsible for inhibitory function. Finally, we demonstrate that patS and hetN are not functionally equivalent when expressed from the other gene's regulatory sequences. Taken together, these results help clarify the functional forms of HetN and will help refine future work defining a HetN-derived inhibitory signal in this model of one-dimensional periodic patterning. IMPORTANCE The proper placement of different cell types during a developmental program requires the creation and maintenance of a biological pattern to define the cells that will differentiate. Here we show that the HetN inhibitor, responsible for pattern maintenance of specialized nitrogen-fixing heterocyst cells in the filamentous cyanobacterium Anabaena, may be produced from two different start methionine codons. This work demonstrates that the two start sites are individually involved in a different HetN function, either membrane localization or inhibition of cellular differentiation.