The role of FraI in cell-cell communication and differentiation in the hormogonia-forming cyanobacterium Nostoc punctiforme.

Multicellular cyanobacteria, like Nostoc punctiforme, rely on septal junctions for cell-cell communication, which is crucial for coordinating various physiological processes including differentiation of N2-fixing heterocysts, spore-like akinetes, and hormogonia-short, motile filaments important for dispersal. In this study, we functionally characterize a protein, encoded by gene Npun_F4142, which in a random mutagenesis approach, initially showed a motility-related function. The reconstructed Npun_F4142 knockout mutant exhibits further distinct phenotypic traits, including altered hormogonia formation with significant reduced motility, inability to differentiate heterocysts and filament fragmentation. For that reason, we named the protein FraI (fragmentation phenotype). The mutant displays severely impaired cell-cell communication, due to almost complete absence of the nanopore array in the septal cell wall, which is an essential part of the septal junctions. Despite lack of communication, hormogonia in the ΔfraI mutant maintain motility and phototactic behavior, even though less pronounced than the wild type (WT). This suggests an alternative mechanism for coordinated movement beyond septal junctions. Our study underscores the significance of FraI in nanopore formation and cell differentiation, and provides additional evidence for the importance of septal junction formation and communication in various differentiation traits of cyanobacteria. The findings contribute to a deeper understanding of the regulatory networks governing multicellular cyanobacterial behavior, with implications for broader insights into microbial multicellularity. IMPORTANCE: The filament-forming cyanobacterium Nostoc punctiforme serves as a valuable model for studying cell differentiation, including the formation of nitrogen-fixing heterocysts and hormogonia. Hormogonia filaments play a crucial role in dispersal and plant colonization, providing a nitrogen source through atmospheric nitrogen fixation, thus holding promise for fertilizer-free agriculture. The coordination among the hormogonia cells enabling uniform movement toward the positive signal remains poorly understood. This study investigates the role of septal junction-mediated communication in hormogonia differentiation and motility, by studying a ΔfraI mutant with significantly impaired communication. Surprisingly, impaired communication does not abolish synchronized filament movement, suggesting an alternative coordination mechanism. These findings deepen our understanding of cyanobacterial biology and have broader implications for multicellular behavior in prokaryotes.

SepN is a septal junction component required for gated cell-cell communication in the filamentous cyanobacterium Nostoc.

Multicellular organisms require controlled intercellular communication for their survival. Strains of the filamentous cyanobacterium Nostoc regulate cell-cell communication between sister cells via a conformational change in septal junctions. These multi-protein cell junctions consist of a septum spanning tube with a membrane-embedded plug at both ends, and a cap covering the plug on the cytoplasmic side. The identities of septal junction components are unknown, with exception of the protein FraD. Here, we identify and characterize a FraD-interacting protein, SepN, as the second component of septal junctions in Nostoc. We use cryo-electron tomography of cryo-focused ion beam-thinned cyanobacterial filaments to show that septal junctions in a sepN mutant lack a plug module and display an aberrant cap. The sepN mutant exhibits highly reduced cell-cell communication rates, as shown by fluorescence recovery after photobleaching experiments. Furthermore, the mutant is unable to gate molecule exchange through septal junctions and displays reduced filament survival after stress. Our data demonstrate the importance of controlling molecular diffusion between cells to ensure the survival of a multicellular organism.