Algal Research

The increasing world population necessitates new sustainable nutrient sources, making microalgae like Chlorella sorokiniana interesting due to its rich nutrient profile and sustainable cultivation methods. With genetic optimization tools like CRISPR/Cas9, microalgae as a nutrient source can be improved even further. However, degradation of the rigid cell wall of microalgae, and thereby developing protoplasts, is often necessary prior to transformation, but monitoring protoplast development in spherical, single-celled organisms like C. sorokiniana is challenging using bright-field microscopy. Carbotrace 480 and 630 were tested as fluorescent markers of the cell wall of a C. sorokiniana mutant for protoplast detection, and Carbotrace 480 was successfully used to distinguish protoplast from normal cells in a cell suspension. The enzymes Driselase, Glucanex, Snailase, and Saczyme were tested in different combinations to degrade the cell wall of the mutant, with Snailase as the most effective yielding ~60 % protoplasts. This study provides a quick and easy tool for monitoring protoplast development in the microalgae C. sorokiniana, the first step to improve C. sorokiniana as a sustainable nutrient source using genetic optimization tools like CRISPR/Cas9.

The unicellular algae cell cycle can be divided into several phases, including the commitment point (CP), a point-of-no-return where the cell decides to divide, presumably based on reaching a critical cell size. Light plays a crucial role in the fitness of photosynthetic algal cells, affecting both CP timing and the number of daughter cells produced. So far, only few genes involved in CP have been described, and the presumed sizer and its signal(s) remain unidentified. Using synchronized cells and varying light intensities as a proxy, we explored the effects of light intensity in Chlamydomonas reinhardtii and observed both physiological and transcriptional changes occurring before and after CP under low light (LL) (100 {micro}mol m-{superscript 2} s-{superscript 1}) and optimal light (OL) (500 {micro}mol m-{superscript 2} s-{superscript 1}) conditions. Although CP was delayed by approximately 6 hours in LL, resulting in smaller mother cells and fewer daughter cells, the cells divided at the same time in both conditions. Overall, nucleic acid, protein, and energy reserve levels were lower in LL, with almost no starch produced. RNA-seq analysis identified several core genes shared between both conditions, with 201 genes expressed only in pre-CP1, 161 genes specific to post-CP1, and 582 shared across different phases. In LL, RNA-seq analysis showed an increase in differentially expressed genes (DEGs) in pre-CP1 compared to post-CP1, with an emphasis on photosynthesis, RNA metabolism, and organelle production before commitment, and cell division-related pathways (microtubules, DNA recombination) after CP1. In OL, the number of DEGs increased in post-CP1 by approximately 41% compared to pre-CP1, with a strong emphasis on protein production throughout the cell cycle.

Studying algae requires specialized equipment to characterize culture growth and physiological performance and non-invasive monitoring of these cultures typically relies on optical density (OD) measurements. Currently, OD is often measured using standard spectrometers that suffer from detector saturation at high cell densities, which requires time consuming sample dilutions and lacks compatibility with flat-walled cell culture flasks like Roux bottles. Here we describe a 3D-printable device to measure optically dense algal cultures directly within these cultivation vessels. This instrument combines light attenuation and scattering, resulting in a dual-sensor system that utilizes near-infrared light (950 nm) to precisely quantify biomass without interference from changing photosynthetic pigments. Cultures that reach high cell densities can be assumed to be accurately quantified in situ using the scattered signal, preserving the integrity of the culture and eliminating the need for subsampling. This method is adaptable to the most common geometries of Roux bottles (T-25, T-75, and T-175) and could be used to monitor small-scale batch cultures without contamination risks. A cyanobacteria algal species (Limnospira platensis) and three flask sizes were analysed on light attenuation linearity and scattering detection limits at different cell concentration, demonstrating that the light attenuation module can reliably measure biomass up to an OD of 3.1 (equivalent to >1.5 g/L) without requiring dilution.

Intertidal mudflats are low hydrodynamic energy environments hosting microphytobenthic communities that experience strong spatiotemporal variability in light regimes, including changes in spectral quality and light intensity that can lead to cellular photooxidative stress. To cope with these fluctuations, autotrophs exhibit diverse and highly plastic adaptations that are often species-dependent and shaped by their ecological niches. This study investigates photophysiological responses and metabolic remodeling in a diatom assemblage originating from a natural winter microphytobenthic biofilm under contrasting red and blue light intensities. To this end, photosynthetic parameters were monitored alongside changes in lipophilic metabolites, including untargeted lipids and lipophilic pigments. While few metabolites showed temporal remodeling, rapid and contrasting changes were observed within 30 minutes in response to both spectral quality and light intensity. Red light treatments induced broader remodeling of lipophilic metabolites than blue light, whereas blue light appeared to have a greater impact on photosynthetic parameters. Moreover, red light induced xanthophyll-cycle responses comparable to those observed under blue light at equivalent incident intensity. We discuss these metabolic responses in relation to diatom photoadaptive strategies, placing these findings within the intertidal environmental framework. This work further underlines the importance of understanding rapid metabolic plasticity in coping with light fluctuations, providing new insights into the photoregulatory strategies of natural microphytobenthic communities.

O_LIVitamin B12, an essential micronutrient for many microalgae and humans, is synthesised only by certain prokaryotes. B12 is a complex tetrapyrrole that can exist in many forms (vitamers), some more bioavailable than others. Some microalgae are able to interconvert, or remodel, different B12 vitamers. As microalgae are important primary producers, it is crucial to understand how diverse microalgae acquire, utilise, and remodel this micronutrient. C_LIO_LIThrough the development of a novel algal bioassay for B12 quantification that distinguishes between B12 vitamers with different lower axial ligands, and the generation of targeted knock-out lines, we characterised the role of proteins involved in algal B12 uptake and remodelling. C_LIO_LIWe found that the previously characterised protein CoBalamin-Acquisition protein 1 (CBA1) is also necessary for the acquisition of pseudocobalamin, a less bioavailable form of B12. In addition, we provide the first experimental evidence that COBT is required for Chlamydomonas reinhardtii to remodel B12. C_LIO_LIWe apply the algal B12 bioassay to show that the edible alga Chlorella vulgaris can accumulate pseudocobalamin but is unable to remodel it, highlighting the need for thorough investigation of the metabolic requirements and capabilities of microalgae, especially given the growing interest in microalgae-based food additives. C_LI

Microalgae and cyanobacteria have emerged as promising resources for sustainable agriculture; however, integrated methodologies for the rational design of crop-specific agricultural formulations remain scarce. This study proposes an integrated framework that combines biomass production, species characterization, nutrient characterization, mixture design, nutrient profile estimation, biological validation, and statistical optimization for the rational development of agricultural formulations based on microalgae and cyanobacteria. As a proof of concept, the proposed framework was applied to formulate consortia composed of C. vulgaris, Scenedesmus sp., and A. platensis using common bean (Phaseolus vulgaris L., ecotype Sangre Toro) and rainfed rice (Oryza sativa L., cv. Fedearroz 2020) as model crops. The experimentally determined nutrient composition of the individual biomasses was integrated into a simplex-lattice mixture design coupled with response surface methodology and desirability analysis to identify crop-specific optimal formulations and estimate their nutrient profiles. The cubic model provided the best fit (P < 0.05), showing high predictive performance and a non-significant lack of fit. The optimal bean formulation consisted of 31.6% C. vulgaris and 68.4% Scenedesmus sp., whereas the optimal rice formulation comprised 62.3% A. platensis and 37.7% C. vulgaris, demonstrating distinct crop- specific responses. The optimized bean formulation exhibited higher estimated concentrations of calcium, phosphorus, iron, and zinc, whereas the rice formulation showed higher estimated potassium and Kjeldahl nitrogen contents. These findings demonstrate the feasibility of developing crop-specific microalgal formulations and highlight that different crops may require distinct formulations rather than a universal approach. The proposed approach offers a reproducible, integrated framework for the development and optimization of next-generation agricultural formulations for sustainable crop production.

A single colony of colonial coral often demonstrates conspicuous color variation. However, the molecular basis of this variation within a single, clonal individual is poorly understood. While coral tissues within a single colony have distinct colors, they share the same genetic background, suggesting differences between them are regulatory rather than genetic. This pilot study aims to characterize the transcriptional mechanisms of color regions in a green-and-orange chalice coral colony through an end-to-end transcriptomic analysis. First, we sampled the green body, an orange oral region, and a transition region between them. Then, a holobiont transcriptome was assembled de novo from paired-end RNA-seq of each color region. In the holobiont transcriptome, coral host transcripts were separated from dinoflagellate symbiont transcripts. Green fluorescent protein (GFP)-like pigment genes were classified phylogenetically. Their expression was then quantified using host-only normalization, which corrects for a large regional difference in symbiont read fraction, and compared across color regions. Relative to the green body, the orange oral region showed higher expression of two sets of host pigment genes: distinct orange fluorescent-protein and chromoprotein transcripts, and green-to-red photoconvertible EosFP-family proteins whose unconverted state is green, implicating photoconversion as a contributing mechanism. This study reveals that distinct color regions within a single colony are associated with region-specific up-regulation of different pigment transcripts, including both directly colored proteins and photoconvertible proteins. Further research is needed to confirm spectrally that photoconversion contributes to the coloration and test whether the pattern holds across multiple colonies.

The Retaria are an abundant planktonic protist lineage that play a substantial role in biogeochemical cycling, primary production, and food web dynamics in open ocean ecosystems. Their impact is, in large part, due to their capacity to host photosynthetic endosymbionts. Despite the importance of these relationships in the global oceans, our knowledge about the diversity of photosymbionts associated with the planktonic Retaria is still limited due to the difficulty of collecting and analyzing these cells. Here we apply a new protocol to assess retarian photosymbiont diversity: Fluorescence Lifetime Imaging Microscopy (FLIM) is an easy-to-use method to distinguish between different sources of fluorescence during confocal microscopy. We applied FISH probes that hybridize with the most commonly reported retarian photosymbiont lineages to environmental plankton samples and used FLIM to confirm the presence or absence of our FISH probes in retarian cells. By analyzing the fluorescence lifetime of 92 photosymbionts, we found that the retarian photosymbiosis is far more complex than originally described, where Retaria commonly host multiple algal lineages per cell, and regularly host algal lineages outside of the two most common lineages. These efforts demonstrate that FLIM is an overlooked tool in the study of host-microbe associations, with significant potential to be used across diverse host lineages and environmental samples.

A cnidarian photosymbiont alga, Breviolum minutum, is an emerging model to study symbiosis due its ability to colonise host in absence of light, and amenability to genetic and physiological manipulations. This alga undergoes subcellular reorganisation in response to stress conditions such as elevated temperature and nutrient deprivation. However, subcellular visualisation of this alga is challenging because of its broad spectrum autofluorescence (400-700 nm) and relatively small size (6-8 m). We developed a super resolution imaging, Expansion Microscopy (ExM) workflow - a hydrogel-based technique for mechanical enlargement of cells, that reveals previously inaccessible subcellular features in B. minutum. This ExM workflow presents a set of thermic and enzymatic conditions which enables 4-fold expansion of B. minutum, optical clearing of autofluorescence as well as the removal of its thick cellulose rich cell wall. We implemented a recently described platinum (II)-based tri-functional linker 1, to retain in situ hybridised oligonucleotides targeted to 18S rRNA within ExM hydrogel and exploited its azide reactive group for post-ExM fluorophore labelling (DBCO modification). We observed actin patches (a cytoskeletal feature) and calmodulin (a calcium binding signalling protein) that are not previously visualised in B. minutum. This approach overcomes some of the long-standing problems in visualisation of B. minutum using commonly available reagents and commercially available low-cost ExM compatible chemistries. The broader uptake of this tool for the visualisation of diverse species of photosymbionts will pave the way for fundamental discoveries underpinning cellular reorganisation in formation and breakdown of symbiosis.

Microalgae are promising biological platforms for sustainable energy production because they can convert photosynthetic activity into electricity in biophotovoltaic (BPV) systems. Identifying strains that grow well at elevated temperatures and under reduced irradiance is an important step toward BPV applications in seasonally variable environments. Microalgae were isolated from Seondong Lake (Seoul, Republic of Korea) during three sampling periods and screened for growth at different temperatures. Among 45 isolates, KHUC-11 showed the highest weighted growth index based on growth rates at 25, 30, and 35 under dark conditions and was selected for further characterization. Phylogenetic reconstruction of the 45S rRNA gene cluster, internal transcribed spacer 2 (ITS2) secondary-structure comparison, and morphological characterization supported the description of KHUC-11 as Desmodesmus ecotolerans sp. nov. D. ecotolerans produced more biomass than Chlamydomonas reinhardtii CC-125 under both moderate- and low-light conditions. Under low light, its final dry biomass concentrations were 1.81 and 1.58 times those of CC-125 under mixotrophic and photoautotrophic conditions, respectively. Despite its smaller cell size, D. ecotolerans showed higher photosynthetic performance than CC-125, and screen-printed electrode (SPE)-based electrochemical assays demonstrated enhanced anodic current generation and CV-derived estimated power output. Taken together, these findings identify D. ecotolerans as a novel climate-resilient Desmodesmus species with bioelectrochemical properties that support further evaluation for BPV applications.

Background: Long non protein coding (lnc)RNAs are ubiquitous transcripts known to be major regulators of cellular processes in eukaryotes. In diatoms, specific lncRNA expression is tightly correlated to nutrient fluctuations, which suggests function, but their biological relevance remains to be established. In order to fill this knowledge gap, we sought to design a protocol to generate diatom lncRNA knockout mutants by CRISPR-Cas9 enabling subsequent functional studies to shed light into their mechanistic roles. Since lncRNAs do not contain open reading frames that can be targeted by point mutations for the generation of functional knockouts, we designed a multiplexed CRISPR-Cas9 plasmid harboring two independent gRNA for the specific ablation of lncRNA loci in P. tricornutum. As proof of concept, we used our plasmid-based system to target three phosphate depletion responsive lncRNAs. Results: Using the golden gate based uLoop system, we assembled a single plasmid containing all the necessary components to generate CRISPR-Cas9 large genomic ablations of lncRNAs in P. tricornutum using bacterial conjugation. Three intergenic lncRNAs (Linc91, Linc77 and Linc24) responsive to phosphate fluctuations were selected and independently targeted. Around 32% of the exconjugants presented a monoallelic ablation of the targeted loci and 11% presented a biallelic ablation on the first PCR-screening round. Mutated lines, harboring biallelic lncRNAs ablations for each lncRNA targeted, were successfully generated for each of the targeted lncRNAs. Additionally, a first line of phenotypic analysis revealed significant growth rate differences for one of the mutants ({Delta}Lnc24) under both full and phosphate depleted conditions, suggesting its involvement in cell division regulation under nutrient fluctuations. Conclusions: This work provides a simple and efficient method for the generation of lncRNA knock-out mutants in P. tricornutum. Furthermore, this approach can be easily adapted to other microalgae amenable to transformation, enabling the enhancement of our understanding of these important cellular regulatory transcripts in the marine environment.

Sucrose metabolism is an important feature of the physiology of the green lineage of photosynthetic organisms and has therefore been the subject of considerable research on plants, algae, and cyanobacteria. Canonical sucrose biosynthesis pathways often involve the condensation of NDP-glucose and fructose-6-phosphate through the action of sucrose-phosphate synthase, then the dephosphorylation of sucrose 6-phosphate into sucrose via sucrose-phosphate phosphatase (SPP). However, many cyanobacterial genomes encode multiple homologs of SPP proteins (SPP-like), including variants that appear to lack key residues reported to be important for sucrose 6-phosphate binding. Herein, we examine these SPP-like proteins, focusing on the biochemical and physiological characterization of the SPP-like protein encoded by the cyanobacterial model, Synechococcus elongatus PCC 7942. Bioinformatic analysis suggests that the SPP-like family of proteins is highly conserved across cyanobacterial species and forms distinct phylogenetic clades that are more widely distributed than the canonical SPP proteins themselves. Biochemical analysis of the purified S. elongatus PCC 7942 SPP-like protein reveals that it not only retains the capacity to dephosphorylate sucrose 6-phosphate, but it also may have physiologically relevant phosphatase activity on 3-phosphoglycerate (3-PGA). We provide evidence that the SPP-like family of proteins represents a well-conserved group of phosphatases across cyanobacteria and suggest some enzymes in this family may have evolved a broader substrate specificity relative to the well-characterized SPP family. Our results have broader implications for cyanobacterial regulation of sucrose biosynthesis and other key steps of central carbon metabolism.

Green algae in Ulva, Monostroma and similar genera are notoriously difficult to identify morphologically, due to high phenotypic plasticity and the presence of cryptic species - which has historically resulted in a challenging taxonomy, poorly resolved species boundaries, and potentially numerous invalid species descriptions. DNA-based specimen identification, especially combined with analysis of historical herbarium voucher DNA, has huge potential in resolving many unresolved taxonomical problems in these algae. One such example is Monostroma balticum, which has been described as foliose, monostromatic green seaweed known only free-floating, and reported to be endemic to the Baltic Sea. Previous studies have highlighted major uncertainty in the status of this taxon. We conducted an extensive sampling campaign of monostromatic green algae across the Baltic Sea to molecularly, morphologically and ecologically characterise this enigmatic taxon. Additionally, we investigated original exciccate material of M. balticum by using a museomic approach. We show that Monostroma balticum represents a monostromatic, foliose morphotype of the cosmopolitan and ubiquitous species Ulva intestinalis. This foliose growth form is seen in low salinity conditions, and, so far, reported only from the inner basin of the Baltic Sea. Our work shows that using morphology- and DNA-based museomic approaches for contemporary and historical specimens is a powerful combination to resolve long-standing taxonomical uncertainties and to produce new biodiversity information, even in relatively well-studied regions.

Inshore coastal waters are almost invariably supersaturated with respect to methane and are thereby sources of methane to the atmosphere. We investigated floating algal mats and surface waters of four contrasting inshore habitats and quantified methane concentrations, sea-to-air emissions, and microbial community composition of surface waters over a seasonal cycle to determine the potential for in-situ microbial methane production in shallow oxygen-saturated surface waters with floating algal biomass. 16S rDNA sequencing indicated that Archaea belonging to the genera Methanocorpusculum, Methanosarcina, Candidatus Methanomethylophilus, and some genera from order Methanobacteriales occurred in the floating algal mats. qPCR of the genes encoding the methyl coenzyme M reductase mcrA revealed the highest expression levels during the warmest sampling periods supporting active methane production directly in surface water. Co-occurrence of the Archaea sequences and sequences belonging to the cyanobacterium strain Nodularia PCC 9350 suggests a structural relationship. Our study underscores the significant, yet underexplored impact of methane production on the surface in aggregates of floating algal material. While Nodularia and methanogens can exist independently in surface waters, their co-occurrence in algal mats reveals where the layered mat structure creates distinct microenvironments that facilitate direct metabolic exchange and provide physical stability for both groups, thereby potentially enhancing methane production in these shallow coastal systems.

Light quality acclimation is one of the key drivers of cyanobacterial physiology, ecology, and productivity. Although chromatic acclimation type 3 (CA3) is the canonical example of light quality-driven phycobilisome remodeling, full-PAR physiological characterization of CA3 strains has so far been limited. Here, we characterize the spectral acclimation strategies of the cyanobacterium Nostoc sp. CCAP 1453/38 across the full photosynthetically active radiation (PAR) range. Genomic analysis confirmed Nostoc as a CA3 strain capable of dynamically adjusting phycoerythrin (PE) and phycocyanin (PC) content in its phycobilisome (PBS) rods. During cultivation under narrow-band LEDs, PE was upregulated under violet, blue and green light (435-555 nm), optimizing light harvesting primarily in the blue-green part of the PAR spectrum, while PC was upregulated under red light (633-687 nm). Beyond canonical CA3 pigment switching, Nostoc responded to wavelengths poorly absorbed by PBS in two qualitatively different ways. Under growth-constraining blue light (465 nm), the strain upregulated total PBS and photosystem II (PSII) levels and biased phycobilisome coupling toward PSII (an increased PBS-PSII/PBS-PSI ratio). However, this metabolically costly response could not overcome the underlying excitonic imbalance caused by preferential PSI excitation, resulting in a low cell division rate. Under near far-red light (687 nm), PBS-PSII coupling itself was enhanced, yet total PBS content was reduced rather than increased. Specific growth rates remained as high as under red light, suggesting that this PBS-PSII reorganization avoided the metabolic burden of antenna upregulation. These results indicate that the same underlying challenge of PSII under-excitation can trigger qualitatively different acclimation responses, only some of which are energetically affordable. Spectral acclimation thus depends not only on matching pigment composition to incident wavelengths, but also on the metabolic cost of the response. Compared with parallel datasets on CA1 and non-CA strains obtained under identical conditions, our findings extend CA3 characterization beyond the canonical green/red framework, highlight bottlenecks and advantages of light quality acclimation in Nostoc, and provide a physiological basis for optimizing light regimes in controlled cyanobacterial cultivations.

Temperature is a critical factor in microalgal production, yet its impact on microalgae and their microbiome remains poorly understood. Here, we investigated the responses of Chlorella sorokiniana and its microbial community to temperature increases in four wastewater-based cultures over two months. Two Test cultures were subjected to a thermal gradient from 20 {degrees}C to 34 {degrees}C, and two were Controls at 20 {degrees}C. Eukaryotic and bacterial communities were analyzed using 18S and 16S rRNA gene sequencing, and the microalga genome was reconstructed using PacBio HiFi sequencing and functionally annotated. Control cultures remained stable, whereas Test cultures showed a decline in C. sorokiniana relative abundance and biomass from approximately 24 {degrees}C. In contrast, photosynthetic efficiency remained stable until temperatures exceeded 33 {degrees}C. Above 22-24 {degrees}C, the bacterial community, dominated by Fontibacter, Flavobacterium, and Luteolibacter, was replaced by taxa whose relative abundance increased with rising temperature and microalgal decline, pointing to bacteria better adapted to the stress conditions. Candidate predator-prey dynamics with the protist Cercozoa also intensified as temperature increased. Microalgal genome annotation identified 28 candidate genes corresponding to seven of eight literature-informed high-temperature/heat-stress targets, primarily associated with protein homeostasis, Reactive oxygen species protection, membrane lipid remodeling, and protein degradation. Collectively, these genes likely support the microalga's response and adaptation to environmental stress. Overall, culture destabilization resulted from interacting thermal, microbial, and grazing-related processes rather than temperature alone. Early microbiome changes may therefore provide useful warning signals of culture instability and help improve the management of large-scale microalgal production systems.

Temperature plays a central role in marine phytoplankton biogeographical dynamics, physiology and gene expression. Nonetheless, the transcriptional regulatory mechanisms controlling temperature acclimation in marine phytoplankton are yet to be characterized. Ostreococcus tauri was chosen as a model species for green marine phytoplankton due to its cellular and genomic simplicity, as well as its evolutionary position within the green lineage. In this study, epigenomic and transcriptomic data were integrated to characterize changes induced by temperature in the trimethylation of histone 3 at lysines 27 and 4 (H3K27me3 and H3K4me3) epigenetic marks established by the Polycomb (PcG) and Trithorax group (TrxG) complexes, respectively. H3K27me3 was found to be a repressive mark responding to temperature, showing predominantly significant increased levels at high temperatures. While H3K4me3 was associated with active transcription, presenting less evident variations in cultures acclimated to different temperatures. H3K27me3 was found only marginally associated with transposable elements, being mostly involved in the repression of specific biological processes, such as gene expression control by transcription factors, meiosis, motors proteins and cytoskeletal structures. No significant conservation was found between the H3K27me3 gene targets in the model plant Arabidopsis thaliana and Ostreococcus tauri. Nonetheless, transcriptions factors belonging to the MADS-box, WRKY and AP2 families were consistently repressed by H3K27me3 in both species, unveiling that, although the specific downstream targets of this epigenetic mark have diversified during evolution, its role in modulating higher order regulatory nodes remains evolutionary conserved.

Microeukaryotes are abundant, diverse, and morphologically and functionally complex. They represent one of the largest groups of primary producers and are involved in major ecosystem functions such as nutrient transformations and maintenance of food webs. Although ~74,000 species of microeukaryotes are estimated to exist, very few are available in culture and only ~3000 are represented by reference genomes and transcriptomes. This limited representation significantly hinders the study of microeukaryotes in their natural environments. Single-cell transcriptomics with sequencing of full-length transcripts has potential to bypass this limitation. In this study, we employ Smart-seq3xpress for taxonomic identification and functional characterization of non-model mixotrophic freshwater microeukaryotes in a culture-free and reference-free manner, while also enabling inferences to be made for their prokaryotic associations. Computational analyses revealed 22 distinct microeukaryote taxa upon sequencing of 1520 randomly sampled cells, with assemblies of 12 good quality de novo partial transcriptomes, followed by functional annotation using sequence and structure homology. Gene expression analysis of the transcriptome for the most abundant microeukaryote in the sample, Rhodomonas, revealed a transcriptomic dip a few hours after experimental shading, followed by a gradual recovery in the next few days. Compared to samples in illuminated conditions, there was widespread downregulation of photosynthesis-, lysosome-, and carbon metabolism-related pathways in the shade, but the prevailing associations with prokaryotic lineages was unaffected by the light conditions. The different microeukaryotes, all present in the same environmental sample, featured distinct prokaryotic associates that were consistent across illumination conditions. This reference-free and culture-free study of 22 different microeukaryotic taxa sets the stage for high-throughput single cell transcriptomics to study microeukaryotic diversity in complex natural ecosystems, for their population-level taxonomic identity, sub-population-level transcriptomic profiles and metabolic states, and individual-level prokaryotic associations.

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.

The filamentous cyanobacterium Nostoc punctiforme is a key model organism used to study several aspects of cyanobacterial biology, including development, nitrogen-fixing symbioses with plants, and secondary metabolites, among others. While N. punctiforme is amenable to genetic manipulation, traditional approaches for the generation of mutant strains using homologous recombination are slow, requiring prolonged outgrowth under antibiotic selection to ensure isogenic mutant populations. CRISPR-based genome editing using Cpf1 (Cas12a) was recently shown to be an effective means of rapid generation of isogenic mutants in several cyanobacteria. In this study, Cpf1-based genome editing tools were developed for N. punctiforme. A total of 19 unmarked, in-frame deletion mutants were successfully constructed using Cpf1-targeted cleavage along with homology directed repair (HDR). The length of the homology arms (HAs) on the homologous repair template (HRT) used for HDR was found to be a critical factor for successful deletion of target genes, with some requiring up to 4 kb HAs to acquire mutant exconjugants. A strategy for allelic replacement was also developed by introducing an exogenous target site in place of the deleted genes, which could subsequently be targeted for cleavage and repaired with an HRT containing altered alleles of the genes of interest. Additionally, a single-step cloning strategy was devised, allowing for rapid assembly of editing plasmids, and improved conjugation protocols for genetic transfer from E. coli to N. punctiforme were implemented. Collectively, these tools and protocols should enhance the pace and ease of conducting genetic studies in this important model cyanobacterium.