Rachel A. Foster

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.

Photosynthetic organelles (plastids), which originated via primary endosymbiosis between an Archaeplastida ancestor and cyanobacteria, have shaped the Earth's oxygen-rich atmosphere and spread across the eukaryotic tree of life through multiple secondary and higher order endosymbioses. Yet, the mechanisms driving the transition from endosymbiont to organelle remain poorly understood, highlighting the need for novel systems to elucidate the stages of endosymbiont integration and test the generality of plastid evolution models. Here, we employ single-cell genomics and catalyzed reporter deposition fluorescence in-situ hybridization (CARD-FISH) to investigate the dictyochophyte plastids of the poorly known marine centrohelid Meringosphaera across diverse geographic locations. Our analyses detected novel microdiversity of cells both containing and lacking plastids. Using phylogenomics we show that host and plastid data are perfectly congruent across a large plastid-containing clade (named MER-2), strongly indicating co-evolution. Extensive environmental screening using double CARD-FISH simultaneously targeting host and plastid further confirms that MER-2 cells almost always harbor plastids, supporting the hypothesis of permanent plastid integration and vertical transmission. Additionally, we find that both MER-2 hosts and other Meringosphaera lineages encode multiple plastid-associated genes from diverse phylogenetic origins, with many of their products predicted to be plastid-targeted. Collectively, our findings represent the first report of algae in centrohelids, a large eukaryotic group of otherwise heterotrophic predators. The discovery of new plastids is rare and underscores the importance of exploring uncultured algae to provide new insights into plastid origin and evolution.

Picocyanobacteria contribute significantly to carbon export but the mechanism by which their biomass is transported to depth is unclear. By analyzing 16S rRNA gene sequences from sediment trap material and size-fractionated water samples from different depths in the Baltic Sea, we show that picocyanobacterial contribution to phytoplankton biomass export was largely proportional (70%–90%) to their surface water abundances. However, we also found that both cell aggregation (enrichment in >10 μm fraction) and sedimentation (trap enrichment) varied among Synechococcus ASVs (amplicon sequence variants). A robust linear relationship was identified between ASV enrichment in traps and ASV enrichment in the >10 μm fraction, suggesting that cell aggregation is a direct determinant of Synechococcus sedimentation. Seasonal abundance patterns were similar among the dominant aggregate-forming ASVs but distinct from those of single-cell ASVs, suggesting aggregate/colony formation being an important trait in determining distribution of Synechococcus strains and their response to environmental conditions. Correlating microscopy- and sequence-based abundances further allowed us to propose genotypes for the dominant Baltic Sea colonial picocyanobacterial morphotypes. Our results provide in situ data linking phenotypic characteristics of picocyanobacteria with sedimentation properties and several lines of evidence for a quantitative dependency between surface water picocyanobacterial aggregate/colony formation and sinking. These findings are important for our understanding of mechanisms involved in export of pico-sized phytoplankton biomass from the photic zone to depth and emphasize the need to investigate occurrence and environmental regulation of cell aggregation in other picophytoplankton-dominated waters (e.g. open ocean gyres).

Introduction: Decades of research has been devoted to understanding the occurrence, distribution, and activity of different N₂-fixers in the oceans. Marine N₂-fixing diatom symbioses involving the heterocyst-forming cyanobacterium Richelia are widespread, yet often go undetected. Some of the diatom-Richelia symbioses have the advantage to be identified by microscopy, however, observations alone cannot inform on the metabolic state of a cell. Methods: Here, we developed nine new specific quantitative PCR assays that detect three common symbiotic Richelia strains (ReuHH01, RintRC01, RrhiSC01) of diatoms based on molecular markers for: a high affinity phosphate transporter (pstS), an iron transporter (exbB), and a constitutively expressed gene (rnpA, protein component of ribonuclease P). We first tested the new assays in the lab, including a diel experiment to elucidate the temporal dynamics of gene expression for each molecular marker, then applied the new assays to field collected samples. In the field we also made microscopy observations of the diatom-Richelia symbioses and measured bulk N₂ and Carbon (C) fixation rates with stable isotope labelling incubations. Results: The number of genes encoding the molecular markers varied; typically, fewer in the endobiont strains. Detection in field samples based on qPCR was consistent with microscopy observations. The environmental gene expression for all the new targets, and additionally nifH for N₂ fixation (nitrogenase) were low, and highest expression was detected in the upper water column (0-40 m) consistent with higher densities of the Richelia by qPCR and microscopy observations. Low in situ bulk rates of N₂ and C fixation corroborated the aforementioned low nifH expression. Expression of both nifH and pstS was temporally regulated in the lab experiment of the facultative symbiotic Richelia strain RrhiSC01 with higher expression in the early and late photoperiods, respectively. Discussion: The nine new assays are an improvement over the previous assays based on the nifH gene as cross-reactivity between Richelia strains is minimum, expression can be normalized to rnpA, and expression is informative of the symbiont nutrient status.

A few genera of diatoms are widespread in the oceans and form stable partnerships with N2-fixing filamentous cyanobacteria Richelia spp. A unique feature of the diatom-Richelia symbioses is the symbiont cellular location spans a continuum of integration (epibiont, periplasmic, endobiont) that is reflected in the symbiont genome size and content. In this study we analyzed genomes derived from cultures and environmental metagenome-assembled genomes focusing on characters indicative of genome evolution. Our results show an enrichment of short length transposable elements (TEs) and pseudogenes in the periplasmic endosymbiont genomes, suggesting an active and transitionary period in genome evolution. In contrast, genomes of endobionts exhibited fewer TEs and pseudogenes, reflecting advanced stages of genome reduction and increased host dependency in the endobionts. Pangenome analyses identified that endobionts streamline their genomes and conserve a majority of their genes in the core, whereas periplasmic endobionts and epibionts maintain larger flexible genomes, suggesting increased genomic plasticity. Functional gene comparisons with other N2-fixing cyanobacteria revealed that Richelia endobionts have similar patterns of metabolic loss but are distinguished by absence of specific pathways (e.g. cytochrome bd ubiquinol oxidase, lipid-A) that increase dependency and direct interaction with their respective hosts. In conclusion, our findings underscore the dynamic nature of genome reduction in N2-fixing cyanobacterial symbionts and demonstrate the diatom-Richelia symbioses as a valuable and rare model to study genome evolution in the transitional stages from a free-living facultative symbiont to a host-dependent endobiont.

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.