A study of genes involved in the formation of the heterocyst spacing pattern in the cyanobacterium Nostoc punctiforme and their roles in the symbiotic growth state

Nostoc punctiforme strain ATCC 29133 is a filamentous, diazotrophic cyanobacterium capable of entering into symbiotic associations with a selected members of the four major groups of plant kingdom, including the bryophyte Anthoceros punctatus. Nitrogen fixation takes place in specialized cells called heterocysts, which differentiate in a one-dimensional pattern along the filament to 8% of the cell population upon combined nitrogen limitation. By characterizing mutants with altered heterocyst spacing pattern, two novel genes essential to the development of a normal heterocyst pattern, hetF and patN, were identified. Further, results were obtained demonstrating the essential role genes that involved in nitrogen control and heterocyst pattern formation play in the establishment of a functional symbiosis with A. punctatus.;In the absence of combined nitrogen, hetF mutants are unable to differentiate heterocysts (Het−), whereas extra copies of hetF in trans induced the formation of clusters of heterocysts. These characteristics are similar to those of hetR , a central regulator of heterocyst development. hetR is an autoregulatory gene whose transcription enhances early after nitrogen step-down in developing cells. The HetR protein specifically accumulates in heterocysts. The hetF mutation was shown to affect the autoregulated transcriptional induction of hetR. Further, HetR-GFP protein was observed to accumulate in all cells in a hetF mutant. HetF therefore appears to cooperate with HetR in a positive regulatory pathway, and may be required for the autoregultory function of hetR and localization of the HetR protein in differentiating heterocysts.;Under nitrogen limitation, the patN mutant differentiates heterocysts intervened by shorter intervals of vegetative cells, leading to a 2.5-fold increase in heterocyst frequency. This phenotype is previously unobserved in any mutant but is similar to heterocyst frequency and spacing pattern observed in the symbiotic growth state. The patN mutant phenotype was complemented with a multi-copy plasmid carrying the patN gene, and the recombinant strain exhibits the wild-type heterocyst frequency and pattern. Thus, while patN appears to be involved in negative regulation of heterocyst differentiation, it does not affect development when in excess, in contrast to patS. Further preliminary evidences suggests that PatN may belong to a new regulatory pathway of heterocyst pattern formation.