Nostoc punctiforme is a phenotypically complex, filamentous, nitrogen-fixing cyanobacterium, whose vegetative cells can mature in four developmental directions. The particular developmental direction is determined by environmental signals. The vegetative cell cycle is maintained when nutrients are sufficient. Limitation for combined nitrogen induces the terminal differentiation of heterocysts, cells specialized for nitrogen fixation in an oxic environment. A number of unique regulatory events and genes have been identified and integrated into a working model of heterocyst differentiation. Phosphate limitation induces the transient differentiation of akinetes, spore-like cells resistant to cold and desiccation. A variety of environmental changes, both positive and negative for growth, induce the transient differentiation of hormogonia, motile filaments that function in dispersal. Initiation of the differentiation of heterocysts, akinetes and hormogonia are hypothesized to depart from the vegetative cell cycle, following separate and distinct events. N. punctiforme also forms nitrogen-fixing symbiotic associations; its plant partners influence the differentiation and behavior of hormogonia and heterocysts. N. punctiforme is genetically tractable and its genome sequence is nearly complete. Thus, the regulatory circuits of three cellular differentiation events and symbiotic interactions of N. punctiforme can be experimentally analyzed by functional genomics.

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.

Heterocysts, cells specialized for nitrogen fixation in certain filamentous cyanobacteria, appear singly in a nonrandom spacing pattern along the chain of vegetative cells. A two-stage, biased initiation and competitive resolution model has been proposed to explain the establishment of this spacing pattern. There is substantial evidence that competitive resolution of a subset of cells initiating differentiation occurs by interactions between a selfenhancing activator protein, HetR, and a diffusible pentapeptide inhibitor PatS-5 (RGSGR). Results presented here show that the absence of a unique membrane protein, PatN, in Nostoc punctiforme strain ATCC 29133 leads to a threefold increase in heterocyst frequency and a fourfold decrease in the vegetative cell interval between heterocysts. A PatN-GFP translational fusion shows a pattern of biased inheritance in daughter vegetative cells of ammonium-grown cultures. Inactivation of another heterocyst patterning gene, patA, is epistatic to inactivation of patN, and transcription of patA increases in a patN-deletion strain, implying that patN may function by modulating levels of patA. The presence of PatN is hypothesized to decrease the competency of a vegetative cell to initiate heterocyst differentiation, and the cellular concentration of PatN is dependent on cell division that results in cells transiently depleted of PatN. We suggest that biased inheritance of cell-fate determinants is a phylogenetic domain-spanning paradigm in the development of biological patterns.activator-inhibitor | pattern differentiation T wo broad strategies used in the development of biological patterns are activator-inhibitor systems, a refinement of Turing-type reaction-diffusion systems (1, 2), and differential inheritance of cell fate determinants (3, 4). These strategies have been analyzed in multicellular eukaryotes ranging in developmental complexity from hydra to vertebrates (1, 4). Some filamentous cyanobacteria show spaced patterns of differentiated cells, such as nitrogen-fixing heterocysts and spore-like akinetes, thereby representing the simplest and oldest evolutionary model for study of the development of biological patterns and multicellular interactions. There is strong experimental evidence that an activatorinhibitor system regulates heterocyst patterning in filamentous cyanobacteria (5-7). We report here the characteristics of a gene (patN) that is required for normal heterocyst patterning, and the phenotype and protein localization of which is consistent with a role in the biased initiation of heterocyst differentiation via biased inheritance.Heterocysts terminally differentiate from vegetative cells in response to limitation of combined nitrogen. The heterocysts appear in a nonrandom spacing of approximately 1 heterocyst to every 10-15 vegetative cells. The activator-inhibitor system governing this patterning consists of a self-enhancing activator protein, HetR (8, 9), and its antagonist, proteins containing a pentapeptide, PatS-5 (RGSGR) (10, 11). These two primary positi...

Three mutant strains (ntcA, hetR, hetF) of the cyanobacterium Nostoc punctiforme unable to differentiate heterocysts were characterized and examined for their ability to form a symbiotic association with the bryophyte Anthoceros punctatus. Previously unknown characteristics of the N. punctiforme hetR mutant include differentiation of chilling-resistant akinetes, while vegetative cells of the ntcA mutant randomly lysed, yielding short filaments, following ammonium deprivation. Strains with mutations in hetF and hetR infected A. punctatus with similar frequency to that of wild-type N. punctiforme but did not support growth of the plant partner. These results confirm that the infection of A. punctatus by hormogonia leading to the establishment of an association is physiologically uncoupled from the development of a functional diazotrophic association. They also indicate that heterocyst regulatory elements downstream from HetR and HetF are required in both free-living and symbiotic heterocyst differentiation and nitrogenase expression. A strain with a mutation in the global nitrogen regulator ntcA did not infect A. punctatus despite its ability to differentiate hormogonia at a low frequency. When complemented with one or more copies of ntcA, the mutant strain infected A. punctatus at a similar frequency as the wild-type and supported growth of the plant partner in the absence of combined nitrogen. These results established a connection between the presence of a functional copy of ntcA and the magnitude of hormogonium differentiation, and the behaviour of the formed hormogonia.

SummaryNostoc punctiforme is an example of a filamentous cyanobacterium that is capable of differentiating nongrowing cells that constitute gliding filaments termed hormogonia. These gliding filaments serve in short distance dispersal and as infective units in establishing a symbiosis with plants, such as the bryophyte Anthoceros punctatus . Mutants of N . punctiforme exist which show elevated levels of initial infection of A . punctatus as a consequence of repeated cycles of hormogonium differentiation. Such mutations occur within the hrmA and hrmU genes. Further characterization of the hrm locus revealed several genes with an organizational and predicted protein sequence similarity to genes of heterotrophic bacteria that are involved in hexuronic acid metabolism. Genes in the N. punctiforme locus are transcribed in response to the presence of a plant extract containing hormogonium-repressing factors. A predicted transcriptional repressor encoded in the locus, HrmR , was shown herein to be a specific DNA binding protein that regulates the transcription of its own gene and that of hrmE , a nearby gene. The ability of HrmR to bind DNA was abolished upon addition of either galacturonate or lysate from specifically induced N . punctiforme cells, implying that the in vivo HrmR binding activity is modulated via an internal compound, most likely a sugar molecule.