Functional dissection and evidence for intercellular transfer of the heterocyst‐differentiation PatS morphogen

Abstract

SummaryThe formation of a diazotrophic cyanobacterial filament represents a simple example of biological development. In Anabaena, a non-random pattern of one nitrogen-fixing heterocyst separated by about 10 photosynthetic vegetative cells results from lateral inhibition elicited by the cells differentiating into heterocysts. Key to this process is the patS gene, which has been shown to produce an inhibitor of heterocyst differentiation that involves the C-terminal RGSGR pentapeptide. Complementation of a ΔpatS Anabaena mutant with different versions of PatS, including point mutations or tag fusions, showed that patS is translated into a 17-amino acid polypeptide. Alterations in the N-terminal part of PatS produced inhibition of heterocyst differentiation, thus this part of the peptide appears necessary for proper processing and selfimmunity in the producing cells. Alterations in the C-terminal part of PatS led to over-differentiation, thus supporting its role in inhibition of heterocyst differentiation. A polypeptide, produced in proheterocysts, consisting of a methionine followed by the eight, but not the five, terminal amino acids of PatS recreated the full activity of the native peptide. Immunofluorescence detection showed that an RGSGR-containing peptide accumulated in the cells adjacent to the producing proheterocysts, illustrating intercellular transfer of a morphogen in the cyanobacterial filaments.

Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120

“…This evidence is consistent with a requirement for maintained hetR expression in heterocysts to facilitate proper functioning following morphogenesis. at 24 h N2 growth that resolves to a normal pattern by roughly 96 h N2 growth (Corrales-Guerrero et al, 2013;Yoon & Golden, 2001). In contrast, a DhetN mutant produces a normal pattern of heterocysts at 24 N2 growth and Mch arises by 48 h N2 growth (Callahan & Buikema, 2001).…”

ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium

“…In addition to regulating the transcription of a large number of morphogenesis and metabolism genes, HetR positively autoregulates its own production and is necessary for induction of transcription of patS and hetN (16)(17)(18). The full-length 17-amino-acid (17-aa) patS-encoded peptides and 287-aa hetN-encoded peptides do not appear to move from cell to cell, and the presumed fragments that do are unknown (3,12). However, experimental evidence suggests a Cterminal 5-, 6-, or 8-aa peptide cleavage product in the case of patS (3,13,19).…”

“…Morphological differentiation of functional heterocysts is complete after about 24 h. There are two prominent theories on how the cells that differentiate are specified. In the first, a small diffusible peptide, PatS, acts by lateral inhibition in a Turing-like reaction-diffusion fashion to differentially regulate the activity of HetR, an activator of differentiation, to specify individual cells that will differentiate (3)(4)(5)(6)(7). Once the initial pattern is specified for the initial round of differentiation, it has been proposed that a second diffusible inhibitor, HetN, participates in the placement of heterocysts in subsequent rounds of differentiation (8,9).…”

“…The patS and hetN genes encode developmental signals that have many of the characteristics of morphogens (3,7,12). These signals move from source cells to neighboring vegetative cells to prevent their differentiation (3,7,13,14).…”

“…These signals move from source cells to neighboring vegetative cells to prevent their differentiation (3,7,13,14). Both patS and hetN encode putative peptides that contain the sequence RGSGR, which is necessary for inhibitory activity and represents the C terminus of the putative product of patS (3,7,13).…”