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 optimal exchange of bioavailable nitrogen and reduced carbon. The spacing between heterocysts is regulated by the activity of two developmental inhibitors: PatS and PatS. 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 encode the peptide motif ERGSGR, which is sufficient to inhibit development. While the small size of PatS makes interpretation of inhibitory domains relatively simple, HetN is a 287 amino acid protein with multiple functional regions. Previous work has 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 HetN uses two methionine start codons (M1, 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.