The hetF Gene Product Is Essential to Heterocyst Differentiation and Affects HetR Function in the Cyanobacterium Nostoc punctiforme

Abstract

A novel gene, hetF, was identified as essential for heterocyst development in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133. In the absence of combined nitrogen, hetF mutants were unable to differentiate heterocysts, whereas extra copies of hetF in trans induced the formation of clusters of heterocysts. Sequences hybridizing to a hetF probe were detected only in heterocyst-forming cyanobacteria. The inactivation and multicopy effects of hetF were similar to those of hetR, which encodes a self-degrading serine protease thought to be a central regulator of heterocyst development. Increased transcription of hetR begins in developing cells 3 to 6 h after deprivation for combined nitrogen (N step-down), and the HetR protein specifically accumulates in heterocysts. In the hetF mutant, this increase in hetR transcription was delayed, and a hetR promoter::green fluorescent protein (GFP) transcriptional reporter indicated that increased transcription of hetR occurred in all cells rather than only in developing heterocysts. When a fully functional HetR-GFP fusion protein was expressed in the hetF mutant from a multicopy plasmid, HetR-GFP accumulated nonspecifically in all cells under nitrogen-replete conditions; when expressed in the wild type, HetR-GFP was observed only in heterocysts after N step-down. HetF therefore appears to cooperate with HetR in a positive regulatory pathway and may be required for the increased transcription of hetR and localization of the HetR protein in differentiating heterocysts.

Establishment of a functional symbiosis between the cyanobacterium Nostoc punctiforme and the bryophyte Anthoceros punctatus requires genes involved in nitrogen control and initiation of heterocyst differentiation The GenBank accession numbers for the coding regions of ntcA, hetR and hetF are AY038370, AF318069 and AF288130, respectively.

“…Complementation in trans and reconstruction of the hetF mutant was previously reported (Wong & Meeks, 2001). The hetR mutation in strain UCD 415 was complemented both with the wild-type gene in trans, and with a full-length HetR-green fluorescent protein fusion (data not shown).…”

“…1 and Wong & Meeks, 2001). Both mutants grew normally as vegetative cell filaments when supplemented with nitrate or ammonium.…”

“…HetR exhibits a serine-dependent autoprotease activity (Zhou et al, 1998), regulates its own transcription (Black et al, 1993 ;Buikema & Haselkorn, 2001), and is sufficient to induce heterocyst differentiation when ectopically expressed under nitrogenreplete conditions (Buikema & Haselkorn, 2001). Transcription of hetR is enhanced in specific cells following combined nitrogen deprivation (N step-down), and HetR accumulates in heterocysts (Black et al, 1993 ;Wong & Meeks, 2001). HetF is an ancillary positive activator of heterocyst differentiation that functions in the enhanced transcription of hetR and the localization of HetR to differentiating cells (Wong & Meeks, 2001).…”

“…Transcription of hetR is enhanced in specific cells following combined nitrogen deprivation (N step-down), and HetR accumulates in heterocysts (Black et al, 1993 ;Wong & Meeks, 2001). HetF is an ancillary positive activator of heterocyst differentiation that functions in the enhanced transcription of hetR and the localization of HetR to differentiating cells (Wong & Meeks, 2001). …”

“…Het − mutants were distinguished from the Fox − mutants by microscopically examining strains during combined-nitrogen starvation (N stepdown). Characterization of the hetF mutant strain UCD 416 was reported elsewhere (Wong & Meeks, 2001). To verify that the phenotype of hetR mutant strain UCD 415 was due to the transposition event, the mutation was reconstructed as follows.…”