EbsA is essential for both motility and biofilm formation in the filamentous cyanobacterium Nostoc punctiforme.

Many cyanobacteria, both unicellular and filamentous, exhibit surface motility driven by type IV pili (T4P). While the component parts of the T4P machinery described in other prokaryotes are largely conserved in cyanobacteria, there are also several T4P proteins that appear to be unique to this phylum. One recently discovered component is EbsA, which has been characterized in two unicellular cyanobacteria. EbsA was found to form a complex with other T4P proteins and is essential for motility. Additionally, deletion of ebsA in one of these strains promoted the formation of biofilms. To expand the understanding of ebsA in cyanobacteria, its role in motility and biofilm formation were investigated in the model filamentous cyanobacterium Nostoc punctiforme. Expression of ebsA was strictly limited to hormogonia, the motile filaments of N. punctiforme. Deletion of ebsA did not affect hormogonium development but resulted in the loss of motility and the failure to accumulate surface pili or produce hormogonium polysaccharide (HPS), consistent with pervious observations in unicellular cyanobacteria. Protein-protein interaction studies indicated that EbsA directly interacts with PilB, and the localization of EbsA-GFP resembled that previously shown for both PilB and Hfq. Collectively, these results support the hypothesis that EbsA forms a complex along with PilB and Hfq that is essential for T4P extension. In contrast, rather than enhancing biofilm formation, deletion of both ebsA and pilB abolish biofilm formation in N. punctiforme, implying that distinct modalities for the relationship between motility, T4P function and biofilm formation may exist in different cyanobacteria.

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.

Identification of a morphogene required for tapered filament termini in filamentous cyanobacteria.

Although the photosynthetic cyanobacteria are monophyletic, they exhibit substantial morphological diversity across species, and even within an individual species due to phenotypic plasticity in response to life cycles and environmental signals. This is particularly prominent among the multicellular filamentous cyanobacteria. One example of this is the appearance of tapering at the filament termini. However, the morphogenes controlling this phenotype and the adaptive function of this morphology are not well defined. Here, using the model filamentous cyanobacterium Nostoc punctiforme ATCC29133 (PCC73102), we identify tftA, a morphogene required for the development of tapered filament termini. The tftA gene is specifically expressed in developing hormogonia, motile trichomes where the tapered filament morphology is observed, and encodes a protein containing putative amidase_3 and glucosaminidase domains, implying a function in peptidoglycan hydrolysis. Deletion of tftA abolished filament tapering inidcating that TftA plays a role in remodelling the cell wall to produce tapered filaments. Genomic conservation of tftA specifically in filamentous cyanobacteria indicates this is likely to be a conserved mechanism among these organisms. Finally, motility assays indicate that filaments with tapered termini migrate more efficiently through dense substratum, providing a plausible biological role for this morphology.