Characterization of transcription regulation of HetR and investigation of functional significance of different regions of HetN in Anabaena sp PCC 7120

The multicellular cyanobacterium Anabaena sp. PCC 7120 fixes atmospheric nitrogen in specially differentiated cells called heterocysts. In a filament of vegetative cells, approximately every tenth cell differentiates in response to nitrogen-starvation, and the resulting pattern is maintained as the filament elongates. This simple one-dimensional pattern serves as a paradigm to study developmental regulation in prokaryotes. HetR is a DNA-binding protein that positively upregulates its own transcription. Its expression is necessary and sufficient for differentiation even under non-inducing conditions. Four hetR transcripts are made from transcription start points (tsp) originating at nucleotides --728, --696, --271 and --184 with respect to the translational start site. The tsp --271 is implicated in auto-regulation. We investigated the spatiotemporal regulation of transcription from the four transcriptional start sites of hetR and found that the tsp at --271 alone is regulated spatially. We also analyzed the role played by the heterocyst regulators PatA, PatS and HetN in controlling transcription and found that each affects transcription from one or more tsps. We examined the binding of HetR to different regions in the hetR promoter using mobility shift assays to identify HetR-binding sites and found that HetR binds to several regions in its promoter with different binding affinities. HetN is a putative short-chain alcohol dehydrogenase, and it also contains an RGSGR motif known to inhibit heterocyst differentiation. To evaluate whether HetN function is due to the presence of the RGSGR motif or predicted dehydrogenase activity, site-directed mutagenesis was used to make conservative substitutions in the five amino acid residues of the RGSGR motif and three residues known to be essential to the catalytic function of short-chain dehydrogenases. We found that HetN function in heterocyst differentiation is caused by the presence of the RGSGR motif. To investigate the role of different regions of HetN in protein localization, the full length HetN protein and three different truncated versions were translationally fused to yellow fluorescent protein (YFP) and introduced into Anabaena. YFP fluorescence was observed as a ring around the cells in the filament only for the full length HetN transcriptionally fused to YFP.