The heterocyst regulatory protein HetP and its homologs modulate heterocyst commitment in Anabaena sp. strain PCC 7120

Abstract

The commitment of differentiating cells to a specialized fate is fundamental to the correct assembly of tissues within a multicellular organism. Because commitment is often irreversible, entry into and progression through this phase of development must be tightly regulated. Under nitrogen-limiting conditions, the multicellular cyanobacterium Anabaena sp. strain PCC 7120 terminally commits ∼10% of its cells to become specialized nitrogen-fixing heterocysts. Although commitment is known to occur 9–14 h after the induction of differentiation, the factors that regulate the initiation and duration of this phase have yet to be elucidated. Here, we report the identification of four genes that share a functional domain and modulate heterocyst commitment: hetP (alr2818), asl1930, alr2902, and alr3234. Epistatic relationships between all four genes relating to commitment were revealed by deleting them individually and in combination; asl1930 and alr3234 acted most upstream to delay commitment, alr2902 acted next in the pathway to inhibit development, and hetP acted most downstream to drive commitment forward. Possible protein–protein interactions between HetP, its homologs, and the heterocyst master regulator, HetR, were assessed, and interaction partners were defined. Finally, patterns of gene expression for each homolog, as determined by promoter fusions to gfp and reverse transcription–quantitative PCR, were distinct from that of hetP in both spatiotemporal organization and regulation. We posit that a dynamic succession of protein–protein interactions modulates the timing and efficiency of the commitment phase of development and note that this work highlights the utility of a multicellular cyanobacterium as a model for the study of developmental processes.

The hetZ gene indirectly regulates heterocyst development at the level of pattern formation in Anabaena sp. strain PCC 7120

“…It is possible to determine the relative placement of a factor involved in heterocyst differentiation by defining the point at which the cascade of genetic events governing development fails to progress. Recent work has shown that hetP governs the timing and efficacy of commitment to a differentiated cell fate in Anabaena (Videau et al ., ). Expression of genes involved in differentiation also displayed wild‐type patterning in a Δ hetP mutant (Fig.…”

“…S4) (Zhang et al ., ). Patterned hetP expression can be observed about 18–24 h after the induction of differentiation in the wild type (Videau et al ., ). In the Δ hetZ 354–762 mutant, however, none of the introduced transcriptional fusions displayed fluorescence in a periodic pattern.…”

“…To perform its functions, it could be necessary for HetZ to interact with other positive effectors of differentiation. To gain insight into the mechanism used by HetZ to enact differentiation, a network of interaction partners involved in cellular differentiation (HetR, HetZ, HetP, Asl1930, Alr2902 and Alr3234) was assessed using a bacterial two‐hybrid assay (BACTH) (Videau et al ., ). Each protein was translationally fused to a functional domain of adenylate cyclase that, when brought together by protein‐protein interaction, result in the production of β‐galactosidase (Karimova et al ., ).…”

“…The interaction of HetZ with HetR and Alr2902, a homolog of HetP and antagonist of HetR involved in defining the timing of commitment, could provide the basis for its function. Previous work has shown that a complex network of protein–protein interactions occurs between HetR, HetP, Asl1930, Alr2902 and Alr3234 and may contribute to the timing and efficacy of heterocyst formation (Videau et al ., ). We infer that protein–protein interactions may be involved in HetZ function during cellular differentiation in Anabaena .…”

“…The interaction of HetR (activator) and PatS (inhibitor) fulfill the activator–inhibitor model of de novo pattern formation to define the cells along a filament that are capable of differentiation in Anabaena . Patterned cells then irreversibly commit to a differentiated cell fate through an unknown mechanism, the timing and efficacy of which are governed by HetP, its homologs Asl1930, Alr2902 and Alr3234 and their interaction with HetR (Videau et al ., ). Following commitment, developing heterocysts vastly alter their transcriptome, cease to divide and complete morphogenesis into a mature, functional form.…”