The HetR regulon of Anabaena sp. strain Pcc 7120

Abstract

The process of cellular differentiation relies on the action of transcriptional regulators to enact the developmental fate of the cell. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is a model system for studying pattern formation and cellular differentiation. When combined nitrogen is limiting, Anabaena forms a periodic pattern of nitrogen-fixing heterocyst cells separated by 10-20 photosynthetic vegetative cells. The pattern of cells that can differentiate is defined by the interaction of the master regulator of differentiation, HetR, and the inhibitors of differentiation, PatS and HetN. The crystal structure of HetR bound to DNA determined the amino acids that interact with DNA and mutations in these amino acids yielded non-functional hetR alleles. In this work, a 17 bp inverted repeat in the promoter of the hepA gene was bound by HetR in vitro and found to be necessary for heterocyst-specific transcription in vivo. A search of the genome identified 166 potential hepA-like HetR sites and investigation of a subset of these sites found that HetR could act as either a transcriptional activator or repressor. This study also identified a HetR-binding site in the promoter region of the trpE gene, which encodes an anthranilate synthase involved in tryptophan biosynthesis. Mutation trpE yielded a strain that differentiated heterocysts in the presence of nitrate, a nitrogen source that normally represses differentiation. Analysis of the intracellular concentration of the Kreb's cycle intermediate 2-oxoglutarate (2-OG) showed a roughly 2.5 fold increase within one hour after the transition from growth on ammonia to nitrate. This spike in 2-OG is characteristic of a nitrogen starvation response and suggests a role for amino acid metabolism in the perception of nitrogen starvation. The final study utilized an FMN-dependent fluorphore, EcFbFP, to determine the transcriptional profiles of hetR, patS, and hetN in mature heterocysts. The transcription of hetR and hetN persisted in mature heterocysts while patS expression ceased. This is consistent with roles for PatS in pattern formation, HetN in pattern maintenance, and HetR in all stages of development. This work expands the direct HetR regulon and suggest a role for amino acid metabolism in the induction of differentiation.