Sepehr Bardi

Intercellular communication is essential for metabolite exchange and cellular coordination in filamentous cyanobacteria, yet how filament organization influences this process remains poorly understood, particularly in terminal heterocyst-forming strains. Here, we compared the facultative symbiont Richelia rhizosoleniae SC01 (hereafter Richelia ; terminal heterocysts) with the free-living Anabaena sp. PCC 7120 (hereafter Anabaena sp.; intercalary heterocysts) and a septal junction mutant of Anabaena with impaired intercellular communication (CSVT22, ∆ fraC -∆ fraD ), using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). Richelia SC01 and Anabaena showed similar recovery ( R ) rates, indicating comparable average molecular exchange through septal junctions, with no significant correlation between R and filament length. By contrast, CSVT22 showed reduced recovery and accelerated, irregular decay, indicative of both disrupted septal connectivity and altered regulation of septal junctions, resulting in heterogeneous and less efficient intercellular exchange. Differences in fluorescence decay speed across cell types and strains suggest that cell identity and filament organization, including filament taper, shape patterns of intercellular exchange. FLIP analyses showed that Anabaena consistently exhibited greater exchange asymmetry, whereas Richelia SC01 vegetative cells displayed comparatively more symmetric exchange, despite pronounced filament tapering and terminal heterocyst organization. Transmission electron microscopy further showed that septal nanopores in Richelia SC01 are similar in size to those of Anabaena but exhibited marked variation in number per septal disk, suggesting a potential structural basis for variation in intercellular exchange patterns. Collectively, our findings show that combining FRAP and FLIP provides a quantitative framework to dissect the kinetics and spatial organization of intercellular exchange in filamentous cyanobacteria. IMPORTANCE: Filamentous cyanobacteria rely on intercellular exchange through septal junctions to coordinate metabolism and development, yet the principles governing multicellular communication in bacterial filaments remain poorly understood. Here, we show that intercellular exchange emerges from the interplay between septal junction properties, cell identity, and filament architecture, generating distinct communication patterns across cyanobacterial lifestyles. Notably, despite its pronounced filament tapering and terminal heterocyst organization, Richelia SC01 exhibited comparatively symmetric exchange, demonstrating that filament polarity alone does not determine communication patterns. Our findings reveal that cyanobacterial multicellularity arises from the integration of structural organization and cell-specific properties, providing a framework for understanding coordinated physiology in filamentous cyanobacteria, including symbiotic and nitrogen-fixing systems.

Symbioses between diatoms and the N2-fixing, heterocyst-forming cyanobacteria Richelia spp. are widespread and contribute to primary production. Unique to these symbioses is a variation in the symbiont location: one lives in the host cytoplasm (endobiont) vs. residing between the host frustule and plasmalemma (periplasmic endobiont). Both partners are photosynthetic, yet how the partners acquire, share, or compete for bicarbonate necessary for their photosynthesis is unknown. The genomes of both endobionts (ReuHH01 and RintRC01, respectively) contain genes encoding SulP-family proteins, which are oxyanion transporters. To study the possible involvement of these transporters in bicarbonate uptake, we used complementation in a Synechocystis sp. PCC 6803 mutant that is unable to grow at air levels of CO2 because all five of its inorganic carbon uptake systems have been inactivated. Of the five genes tested, only one (RintRC_3892) from the periplasmic endobiont complemented the mutant to grow with air levels of CO2 or at low bicarbonate concentrations. The complemented strain showed strong sodium-dependent and low-affinity bicarbonate uptake that was consistent with bicarbonate concentrations expected in the diatom periplasm. Additionally, all the amino acids involved in the bicarbonate binding site of BicA from Synechocystis sp. PCC 6803 are conserved in RintRC_3892. Finally, the importance of the RintRC_3892 protein was confirmed by the consistent detection of its transcripts in wild Richelia populations from three different oceans. Combined our results showed no evidence for a bicarbonate transporter in the cytoplasmic endobiont, whereas the periplasmic endobiont has retained a SulP-type bicarbonate transporter for its own photosynthesis.

Diatom-Diazotrophic Associations (DDAs) contribute significantly to new and primary production in the world’s oceans, yet the understanding of how production is sustained is poorly resolved. These symbioses involve diatoms and N ₂ -fixing, heterocyst-forming cyanobacteria of the genus Richelia , both partners being photosynthetic. Richelia euintracellularis resides in the cytoplasm of Hemiaulus hauckii , whereas Richelia intracellularis is periplasmic in Rhizosolenia clevei . In the ocean, bicarbonate is taken up by phytoplankton to provide CO ₂ for photosynthesis. The genomes of both Richelia endobionts (ReuHH01 and RintRC01, respectively) contain genes encoding SulP-family proteins, which are oxyanion transporters. To study the possible involvement of these transporters in bicarbonate uptake, we used complementation of a Synechocystis sp. PCC 6803 mutant with its five CO ₂ uptake systems inactivated, which is unable to grow in air levels of CO ₂ . Three genes from RintRC01 and one gene and a DNA fragment containing four partial gene sequences from ReuHH01 were chemically synthesized, cloned under the control of a strong gene promoter and incorporated in the chromosome of the Synechocystis mutant. One gene from RintRC01, RintRC_3892, complemented the Synechocystis mutant to grow with air levels of CO ₂ or with low bicarbonate concentrations. The complemented strain showed strong sodium-dependent, low affinity bicarbonate uptake, which, together with phylogenetic analyses, identified RintRC_3892 as a BicA protein. Additionally, RintRC_3892 transcripts were consistently detected in environmental samples from three ocean basins. No evidence for a bicarbonate transporter was found, however, for ReuHH01, suggesting different strategies for inorganic carbon uptake in the periplasmic and cytoplasmic endobionts.

A few genera of diatoms are widespread and thrive in low-nutrient waters of the open ocean due to their close association with N₂-fixing, filamentous heterocyst-forming cyanobacteria. In one of these symbioses, the symbiont, Richelia euintracellularis, has penetrated the cell envelope of the host, Hemiaulus hauckii, and lives inside the host cytoplasm. How the partners interact, including how the symbiont sustains high rates of N₂ fixation, is unstudied. Since R. euintracellularis has evaded isolation, heterologous expression of genes in model laboratory organisms was performed to identify the function of proteins from the endosymbiont. Gene complementation of a cyanobacterial invertase mutant and expression of the protein in Escherichia coli showed that R. euintracellularis HH01 possesses a neutral invertase that splits sucrose producing glucose and fructose. Several solute-binding proteins (SBPs) of ABC transporters encoded in the genome of R. euintracellularis HH01 were expressed in E. coli, and their substrates were characterized. The selected SBPs directly linked the host as the source of several substrates, e.g. sugars (sucrose and galactose), amino acids (glutamate and phenylalanine), and a polyamine (spermidine), to support the cyanobacterial symbiont. Finally, transcripts of genes encoding the invertase and SBPs were consistently detected in wild populations of H. hauckii collected from multiple stations and depths in the western tropical North Atlantic. Our results support the idea that the diatom host provides the endosymbiotic cyanobacterium with organic carbon to fuel N₂ fixation. This knowledge is key to understanding the physiology of the globally significant H. hauckii–R. euintracellularis symbiosis.

A few genera of diatoms are widespread and thrive in low nutrient waters of the open ocean due to their close association with N ₂ -fixing, filamentous heterocyst-forming cyanobacteria. In one of these symbioses, the symbiont, Richelia euintracellularis , has penetrated the cell envelope of the host, Hemiaulus hauckii , and lives inside the host cytoplasm. How the partners interact, including how the symbiont sustains high rates of N ₂ fixation is unstudied. Since R. euintracellularis has evaded isolation, heterologous expression of genes in model laboratory organisms was performed to identify the function of proteins from the endosymbiont. Gene complementation of a cyanobacterial invertase mutant and expression of the protein in Escherichia coli showed that R. euintracellularis HH01 possesses a neutral invertase that splits sucrose producing glucose and fructose. Several solute binding proteins (SBPs) of ABC transporters encoded in the genome of R. euintracellularis HH01 were expressed in E. coli and their substrates were characterized. The selected SBPs directly linked the host as the source of several substrates, e.g., sugars (sucrose, galactose), amino acids (glutamate, phenylalanine) and a polyamine (spermidine), to support the cyanobacterial symbiont. Finally, transcripts of genes encoding the invertase and SBPs were consistently detected in wild populations of H. hauckii collected from multiple stations and depths in the western tropical North Atlantic. Our results support the idea that the diatom host provides the endosymbiotic cyanobacterium with organic carbon to fuel N ₂ fixation. This knowledge is key to understand the physiology of the globally significant H. hauckii-R. euintracellularis symbiosis. SIGNIFICANCE: Diatom diazotroph associations (DDAs) between diatoms and N ₂ -fixing bacteria (diazotrophs) have a relevant impact on N ₂ fixation-based production, but the mechanisms underlying their integrated N ₂ and CO ₂ fixation remain unstudied. In the association between the diatom Hemiaulus hauckii (host) and the N ₂ -fixing, heterocyst-forming cyanobacterium Richelia euintracellularis (endosymbiont), the cyanobacterium is uncultivable. Here we used heterologous expression of genes from the endosymbiont to identify the function of proteins involved in the utilization of organic carbon from the host. The importance of these proteins was also confirmed by estimating gene expression in environmental samples. Our results show that the metabolisms of the symbiotic partners are integrated allowing the host to sustain the physiology of the endosymbiont for an important ecological role.