Biology subjects

Flores, E.

Publications and source records attributed to Flores, E..

At least 19 recordsLinked to original sources

bioRxiv2026

TAp63-regulated oncogenic long non-coding RNA-8 ( TROLL-8 ) regulates human breast cancer progression through CPT1A-mediated fatty acid oxidation

Metabolic reprogramming is a crucial hallmark of cancer, supporting tumor growth and adaptation to cellular stress. Although fatty acid oxidation (FAO) has emerged as an important regulator in cancer, the mechanisms that control the FAO machinery remain poorly understood. Here, we demonstrate that the TAp63-regulated long non-coding RNA TROLL-8 is a key regulator of FAO in breast cancer. Using metabolomics and protein microarray assays followed by immunoprecipitation-mass spectrometry, we mechanistically demonstrate that TROLL-8 binds the FAO enzyme CPT1A and promotes the formation of a complex with ACSL1 and VDAC1, thereby enabling efficient fatty acid processing. Loss of TROLL-8 destabilizes this complex, leading to impaired FAO, decreased metabolic fitness, and suppression of tumorigenic phenotypes, such as anchorage-independent growth. Notably, higher levels of CPT1A and VDAC1 are associated with worse survival in breast cancer patients. Given the emerging role of CPT1A in therapy resistance, these findings suggest that targeting TROLL-8 could be a promising approach to selectively disrupt the hyperactive FAO machinery in breast cancers and other tumor types that rely on FAO for their progression.

Cancer Biology↗

bioRxiv2026

Metabolic commitment and nitrogen control of diazotrophy in the diazoplast-containing diatom Epithemia adnata

Earths nitrogen cycle is central to sustaining ecosystem productivity and global biogeochemical balance. Although biological N2-fixation is well characterized in prokaryotes and plant symbioses, in other eukaryotic lineages it remains poorly understood. Diatoms of the family Rhopalodiacea harbor diazoplasts, endosymbiotic spheroid bodies specialized for N2-fixation. This makes these diatoms genuine N2-fixing eukaryotes that represent a unique model for organelle evolution, parallel but distinct from haptophyte nitroplasts. Here, we report the isolation and stable cultivation of an Epithemia adnata strain, the sequencing of its diazoplast genome and its proteomic profile when growing diazotrophically in the light or darkness, or upon exposure to ammonium. Our analyses reveal that ammonium induced broad down-regulation of diazoplast proteins, particularly those linked to N2-fixation, ATP synthesis, and central carbon metabolism underscoring a general regulatory commitment toward diazotrophic metabolism tightly coupled to host carbon and nitrogen status. The pentose phosphate pathway and ferredoxin-NADP oxidoreductase appear as likely source of reductant to nitrogenase. A striking enrichment of chaperones, peroxiredoxins, bacterioferritin-like proteins, and DpsA might stabilize nitrogenase and buffer against oxidative stress during light-driven diazotrophy. Importantly, we identified a plasmid-encoded GlpF as a putative glycerol transporter, pointing to glycerol-mediated host-symbiont metabolic integration in the extant symbiosis and possibly a crucial innovation during the early evolutionary stages of its establishment. Thus, diazoplast activity is not autonomous but requires integration with host carbon and nitrogen status, establishing glycerol transport, reductant supply, stress mitigation, and nutrient-responsive regulation as pivotal mechanisms of nitrogenase activity and host integration. These findings have broad implications for biogeochemical cycling, organellogenesis, and synthetic biology strategies aimed at engineering N2-fixation in crop plants. SignificanceN2-fixing eukaryotes are increasingly recognized as abundant algae containing bacterial-derived diazotrophic endosymbionts, representing an underappreciated component of global N cycling. Diazoplasts in rhopalodiacean diatoms represent a compelling example of such endosymbionts specialized for N2-fixation. By combining genomic sequencing and proteomic analysis, we demonstrate their metabolic specialization, host integration, and regulatory commitment to diazotrophy. These findings reinforce the emerging view that diazoplasts function as organelle-like entities dedicated to N2-fixation, dependent on host-supplied carbon while contributing fixed N in return. Beyond giving evolutionary insights into organellogenesis, this work establishes a framework for translational applications, such as engineering N2-fixation into agricultural plants. Such advances could reduce reliance on synthetic fertilizers, influence biogeochemical cycles, and promote sustainable food production.

plant biology↗

bioRxiv2026

Quinolinic acid phosphoribosyl transferase moonlights as an apoptosis regulator to empower lung cancer progression

Although nicotinamide adenine dinucleotide (NAD) metabolism is fundamental for cancer cell survival, the role of the de novo NAD biosynthetic pathway, particularly in non-small cell lung cancer (NSCLC), remains largely unknown. Here, we describe a non-canonical role for the rate-limiting enzyme in de novo NAD+ biosynthesis, quinolinate phosphoribosyltransferase (QPRT), in NSCLC progression. We show that QPRT is highly expressed in late-stage tumors and required for NSCLC growth; however, its suppression does not change NAD levels or elicit compensatory NAD biosynthetic activity. Instead, QPRT interacts with caspase-3 and suppresses its activation, protecting NSCLC cells from apoptosis. This reveals a moonlighting function for QPRT in apoptosis regulation independent of its enzymatic activity in tryptophan catabolism. Together, these findings, redefine QPRT as a protein with dual functionality and reveal it as a potential therapeutic target in NSCLC, highlighting the importance of non-canonical roles of metabolic enzymes in cancer biology. SignificanceThis study reveals that QPRT supports NSCLC progression by directly inhibiting caspase-3-mediated apoptosis independent of NAD biosynthesis, redefining its role and highlighting non-enzymatic functions of metabolic enzymes as therapeutic targets.

cancer biology↗

bioRxiv2026

Genetic access to an obligate cyanobacterial endosymbiont within the shoot meristem of fern hosts

Eukaryote-associated microbes are ubiquitous, but their essential roles in the development and ecology of their host is yet to be fully understood, partly because complex associations cannot be reconstituted and, in many instances, the genetic tools to elucidate those roles are not available. Here, we report the conjugative transfer of DNA into Nostoc azollae within two Azolla fern hosts. N. azollae is a filamentous, N2-fixing, heterocyst-forming cyanobacterium which is an obligate endosymbiont of the complex microbial community associated with the floating ferns of the genus Azolla. The cyanobiont provides fixed nitrogen to its host, supporting maximum growth rates without any N-fertilizer and making Azolla symbioses both ecologically and agriculturally important. Triparental mating protocols and fluorescent reporter detection were optimized for the cyanobiont isolated from the fern, allowing further demonstration of heterologous gene expression in N. azollae driven by several promoters, including some of a CRISPR-associated transposon (CAST) system. Azolla was then treated with a cytokinin hormone to render fern shoot apices amenable to in planta conjugation, permitting DNA transfer to, and stable gene expression in two distinct developmental stages of N. azollae within Azolla. These included (i) cells of filaments from the Shoot Apical Nostoc colony, the only cyanobacterial stem-cell population vertically transmitted across fern generations, and (ii) cells from differentiated filaments in early formed Azolla leaf cavities. Our approach represents a technically groundbreaking advance for the genetic engineering of cyanobacterial endosymbioses that may be useful for other symbiotic systems, opening a pathway to investigate these important biological entities.

microbiology↗

bioRxiv2025

Photosynthesis regulation impacts carbon and nitrogen assimilation in the diazotrophic cyanobacterium Anabaena sp. PCC 7120

O_LIDiazotrophic cyanobacteria fix both atmospheric carbon (C) and nitrogen (N) into biomass, but the two assimilation pathways are not compatible. Species like Anabaena sp. PCC 7120 physically separates C and N assimilation in different cell types. Even if separated, they are strongly intertwined, as N assimilation relies on the C skeletons and reducing power from photosynthesis, that in turn depends on N rich molecules as pigments and proteins. C_LIO_LIWhereas the two pathways have been extensively studied individually, here we investigate their interaction by analysing photosynthetic properties upon exposure to changes in light, CO2 and N availability, including the contribution of photosynthetic electron fluxes. C_LIO_LIGrowth depended on the availability of both light and CO2, while the N2 fixation activity mainly on the C supply. Upon diazotrophic conditions, the total photosynthetic electron transport activity increased, with a modified contribution of different electron pathways. A mutant strain affected in the vehiculation of fixed N between cell types showed that the modulation of photosynthesis depended on the metabolic connection between assimilation pathways. C_LIO_LIOverall, data showed that the regulation of photosynthetic electron fluxes is a major component of the synergic metabolic relationship between C and N assimilation pathways upon dynamic environmental conditions. C_LI

plant biology↗

bioRxiv2025

Intercellular communication in the fern endosymbiotic cyanobacterium Nostoc azollae

The water fern Azolla spp. harbors as an endobiont the N2-fixing, filamentous, heterocyst-forming cyanobacterium Nostoc azollae. N. azollae provide the fern with fixed nitrogen permitting its growth in nitrogen-poor environments. In the filaments of heterocyst-forming cyanobacteria, an intercellular exchange of regulators and metabolites occur in which heterocysts provide vegetative cells with fixed nitrogen and vegetative cells provide heterocysts with reduced carbon. Intercellular molecular exchange takes place by diffusion through septal junctions and can be probed by fluorescence recovery after photobleaching (FRAP) analysis with fluorescent markers such as calcein and 5-carboxyfluorescein. The septal junctions traverse the septal peptidoglycan through nanopores that can be visualized in isolated septal peptidoglycan disks by electron microscopy. Here we obtained N. azollae material from Azolla plants, which contains the symbiotic cyanobacterium in a viable state and with different morphologies, including heterocyst-containing filaments. FRAP analysis showed effective transfer of the fluorescent markers between vegetative cells as well as from vegetative cells to heterocysts. Interestingly, communicating and noncommunicating vegetative cells and heterocysts could be distinguished showing conservation in the endobiont of a regulatory mechanism capable of opening and closing septal junctions. Peptidoglycan sacculi were also isolated and showed septal disks with arrays of nanopores that conform to those visualized in other heterocyst-forming cyanobacteria. However, a wider range of septal disk size was observed in N. azollae. In spite of its eroded genome, N. azollae maintains the intercellular communication system that is key for its growth as a multicellular organism. ImportanceThe water fern Azolla constitutes a unique symbiotic system in which cyanobacterial endobionts capable of fixation of atmospheric nitrogen provide the plant with the nitrogen needed for growth. This symbiosis is an important fertilizer for rice crops worldwide, thereby reducing the reliance on fossil fuel-derived nitrogen fertilizers. The symbiotic cyanobacterium, Nostoc azollae, is a heterocyst-forming strain in which a filament of cells is the organismic unit of growth. Here we show that the intercellular molecular exchange function necessary for the multicellular behavior of the organism is conserved in the endobiotic Nostoc azollae.

microbiology↗

bioRxiv2024

Goliath clades and in vivo tracking of clonal dynamics show three phases of UV-induced skin carcinogenesis

While the genetic paradigm of cancer etiology has proven powerful, it remains incomplete as evidenced by the widening spectrum of non-cancer cell-autonomous "hallmarks" of cancer. Studies have demonstrated the commonplace presence of high oncogenic mutational burdens in homeostatically-stable epithelia. Hence, the presence of driver mutations alone does not result in cancer. Here, we reveal additional forces governing the eco-evolutionary dynamics of carcinogenesis. Using a UV-driven mouse model of cutaneous squamous cell carcinoma, we tested our central hypothesis that cancer initiation occurs in three phases: 1) tissue disruption and the emergence of unusually large "goliath" clades (ecological driver), 2) clonal selection within a subset of these goliaths as evidenced by the presence of areas of unusually high local densities of cells (termed "micro-lumps") with higher mutational burdens (evolutionary driver), and 3) emergence of macroscopic lesions. We tracked these tissue level ecological and evolutionary drivers of cancer initiation via in-vivo serial imaging and 3-D reconstruction of fluorescently labeled keratinocyte clades, yielding 25,085 clade measurements from 14 mice over 3 months, and 14,525 clades from 4 of these mice over 6 months. While median and mean clade sizes differed little between UV and non-UV exposure (cc. 65,000 {micro}m3), our ecological survey revealed the emergence of large goliath clades (> cc. 4.2x106 {micro}m3), almost exclusively within UV-exposed skin. Goliath clades emerged by month 2, increased dramatically in number by months 3-4, and plateaued between months 5-6. Goliath clades arose as serpentine structures, intercalating between adjacent keratinocytes. Unexpectedly, targeted DNA sequencing revealed mutations with very low variant allele frequencies within clades, but substantial differences among clades, suggesting that positive selection for these mutations is superfluous to the development of goliaths early in carcinogenesis. scRNAseq analysis of both bulk skin and sorted clades revealed epidermal de-differentiation and immune suppression as early events. By month 7, mutational burdens were significantly larger in goliath clades, particularly for those with micro-lumps. Finally, lesions began to emerge between months 6 and 7, only in UV-exposed skin. To confirm that goliath clades are orders of magnitude more likely to spawn lesions, we randomly selected 21 goliath clades at months 6-7, prior to the time of the emergence of detectable lesions, and followed them over time. Remarkably, 2 of these developed into macroscopic lesions. Our adaptation of the Drake equation estimated the probability of this to be <10-6. Taken together, our results support the presence of three phases of cancer initiation, the earliest of which presage the acquisition of driver mutations and also explains why cancers are relatively rare in relation to the degree of somatic mosaicism present in UV-exposed skin.

cancer biology↗

bioRxiv2024

iModMix: Integrative Module Analysis for Multi-omics Data

SummaryIntegrative Module Analysis for Multi-omics Data (iModMix) is a biology-agnostic framework that enables the discovery of novel associations across any type of quantitative abundance data, including but not limited to transcriptomics, proteomics, and metabolomics. Instead of relying on pathway annotations or prior biological knowledge, iModMix constructs data-driven modules using graphical lasso to estimate sparse networks from omics features. These modules are summarized into eigenfeatures and correlated across datasets for horizontal integration, while preserving the distinct feature sets and interpretability of each omics type. iModMix operates directly on matrices containing expression or abundances for a wide range of features, including but not limited to genes, proteins, and metabolites. Because it does not rely on annotations (e.g., KEGG identifiers), it can seamlessly incorporate both identified and unidentified metabolites, addressing a key limitation of many existing metabolomics tools. iModMix is available as a user-friendly R Shiny application requiring no programming expertise (https://imodmix.moffitt.org), and as an R package for advanced users (https://github.com/biodatalab/iModMix). The tool includes several public and in-house datasets to illustrate its utility in identifying novel multi-omics relationships in diverse biological contexts. Availability and implementationShiny application: https://imodmix.moffitt.org. The R package and source code: https://github.com/biodatalab/iModMix.

bioinformatics↗

bioRxiv2024

Correlating Disordered Activation Domain Ensembles with Gene Expression Levels

Transcription factor proteins bind to specific DNA promoter sequences and initiate gene transcription. In eukaryotes, most transcription factors contain intrinsically disordered activation domains (ADs) that regulate their transcriptional activity. Like other disordered protein regions, ADs do not have a fixed three-dimensional structure and instead exist in an ensemble of conformations. Disordered ensembles contain sequence-encoded structural preferences which are often linked to their function. We hypothesize this link exists between the structural preferences of disordered AD ensembles and their ability to induce gene expression. To test this, we used FRET microscopy to measure the ensemble dimensions of two activation domains, HIF-1 and CITED2, in live cells, and correlate this structural information with transcriptional activity. We find that point mutations that expanded the HIF-1 ensemble increased transcriptional activity, while those that compacted it reduced activity. Conversely, CITED2 showed no correlation between ensemble dimensions and activity. Our results reveal a sequence-dependent relationship between AD ensemble dimensions and their transcriptional activity. WHY IT MATTERSTranscription factors have activation domains (ADs) that bind to coactivator complexes to initiate gene transcription. Despite their key role, a comprehensive understanding of what drives their transcriptional activity has remained elusive. Efforts to understand AD activity have largely focused on their amino acid composition. In recent years, it is increasingly realized that the structural ensembles of disordered proteins contain biases that dictate their structural properties. For ADs, ensemble structures remain poorly explored, especially in relation to their activity. Here we report a mutational study of two ADs, HIF-1 and CITED2, that examines how ensemble dimensions correlate with activity. Our findings suggest that ensemble dimensions may drive activity in some ADs, and that AD ensemble dimensions can be modulated not only through mutations, but also through changes in the cellular environment.

biophysics↗

bioRxiv2024

Using gene complementation to identify a SulP-family bicarbonate transporter in an N2-fixing cyanobacterial endosymbiont of an open ocean diatom

Diatom-Diazotrophic Associations (DDAs) contribute significantly to new and primary production in the worlds oceans, yet the understanding of how production is sustained is poorly resolved. These symbioses involve diatoms and N2-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 CO2 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 CO2 uptake systems inactivated, which is unable to grow in air levels of CO2. 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 CO2 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.

microbiology↗

bioRxiv2023

Evolution of Multicellularity Genes in the Lead Up to the Great Oxidation Event

Cyanobacteria are among the most morphologically diverse prokaryotic phyla on Earth. Their morphotypes range from unicellular to multicellular filaments, yet mechanisms underlying the evolution of filamentous morphologies remain unknown. Here, we implement phylogenomic, Bayesian molecular clock and gene-tree-species-tree reconciliation analyses to estimate when genes encoding cell-cell joining structures first evolved. We also characterise septal structures and measure intercellular communication rates in non-model and early-branching filamentous strains. Our results suggest that genes encoding septal proteins (namely sepJ, sepI, and fraE) and potentially pattern formation (hetR) evolved in the Neoarchaean [~]2.6-2.7 billion years (Ga) ago. Later, at the start of the Great Oxygenation Event [~]2.5 Ga, genes involved in cellular differentiation (namely hetZ, patU3 and hglK) appeared. Our results predict that early-branching lineages like Pseudanabaena were capable of intercellular communication, but further innovations in cellular differentiation were needed to drive ecological expansion on a scale large enough to permanently oxygenate Earths atmosphere.

evolutionary biology↗

bioRxiv2023

Colonization of larval zebrafish (Danio rerio) with adherent-invasive Escherichia coli prevents recovery of the intestinal mucosa from drug-induced colitis

Inflammatory bowel disease (IBD) is a broad term for a range of chronic intestinal disorders, including Crohns disease and ulcerative colitis. The global prevalence of IBD is rising, with over one million patients affected in the US alone. Adherent-invasive E. coli (AIEC) is a pathobiont frequently found in IBD biopsies. AIEC adhere to and invade epithelial cells, and can survive inside phagocytes in vitro. However, how AIEC contribute to IBD in vivo remains unclear. Here, we established a larval zevbrafish (Danio rerio) model to study the interplay between pre-existing intestinal inflammation and AIEC colonization of the gut. We used the pro-inflammatory drug dextran sulfate sodium (DSS) to induce colitis. This was followed by food-borne infection of larvae with AIEC using the protozoan Paramecium caudatum, a natural prey, as a vehicle. We show that AIEC more robustly colonizes the zebrafish gut, and persists for longer, compared to non-pathogenic E. coli. In addition, DSS induced colitis increases both bacterial burden and persistence in the larval gut. We benchmark our model against existing rodent models using two mutants deficient in the known AIEC virulence factors FimH and IbeA, which have virulence defects in both rodent and the larval zebrafish model. Finally, we show that AIEC colonization exacerbates DSS induced colitis and prevents recovery from inflammation. In conclusion, we established a high-throughput, genetically tractable model to study AIEC-host interactions in the context of chronic inflammation. IMPORTANCEAlthough inflammatory bowel diseases are on the rise, a lot remains to be learned about the link between IBD severity and the underlying cause. Although host genetics, microbiome, and environmental factors have all been shown to correlate with the development of IBD, cause and effect are difficult to disentangle in this context. For example, AIEC is a known pathobiont found in IBD patients, but it remains unclear if gut inflammation during IBD facilitates colonization with AIEC, or if AIEC colonization makes the host more susceptible towards pro-inflammatory stimuli. To develop successful therapeutics, it is critical to understand the mechanisms that contribute to AIEC infections in a susceptible host. Here, we show that the larval zebrafish model recapitulates key features of AIEC infections in other animal models, and can be utilized to address these gaps in knowledge.

microbiology↗

bioRxiv2023

An autochthonous model of lung cancer in the Naked Mole-Rat (Heterocephalus glaber)

Studies on cancer resistance in the naked mole-rat (NMR) have generally failed to interrogate possible resistance mechanisms in a physiological context. Here, we provide evidence that the NMR presents as a novel model of tumor initiation. We developed an endogenous lung cancer model in NMRs, driven by an oncogenic Eml4-Alk fusion protein introduced through CRISPR- mediated genome editing. While this is sufficient to drive tumorigenesis in mice, the development of progressive disease in NMRs required the additional loss of key tumor suppressors. Our results show that tumor initiation in NMRs more closely recapitulates that of human tumors. This suggests that the proposed "resistance" of NMRs to cancer development may stem from tumor initiation events that are likely to be comparable to the mechanisms in human cells. One-Sentence SummaryTumor development in the cancer-resistant naked mole-rat more accurately represents the tumor initiation process in humans.

cancer biology↗

bioRxiv2023

Genetic Screening Of Halothane Gene On Selected Philippine Native Pig Herds

Establishment of nucleus herds (NHs) of Native Pigs (NPs) at various R&D stations in the Philippines is currently being undertaken for food security and genetic conservation. Marker-assisted selection (MAS) is being utilized to identify individuals carrying favorable alleles of genes associated with production traits and screen-out genetic defects (GD) for breeding purposes. Porcine Stress Syndrome (PSS) caused by a mutation in Halothane (HAL) gene is a GD frequently found in commercial breeds that when expressed, causes pale, soft, exudative (PSE) meat. PSE is inferior quality meat undesirable in the market causing economic loss to the swine industry. Thus, this study was conducted to screen HAL gene using mutagenically separated-polymerase chain reaction (MS-PCR) in selected NP herds and assessed its repeatability in local breeds. Results showed that out of 577 screened individuals, 543 (94.11%) were normal (NN), 0 (0%) were homozygous mutant (nn) and 34 (5.89%) were heterozygous carriers (Nn). Therefore, the optimized PSS screening protocol using MSPCR is also applicable to local breed as described in the previous study. As such the availability of genetic test for PSS could be useful in improving the breeding selection and elimination of PSS mutant in the nucleus herd of Philippine Native Pig.

genetics↗

bioRxiv2023

Spatio-temporal coherence of circadian clocks and gating of differentiation in Anabaena filaments

Circadian clock arrays in multicellular filaments of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 display remarkable spatio-temporal coherence under nitrogen-replete conditions. To shed light on the interplay between circadian clocks and the formation of developmental patterns, we followed the expression of a clock-controlled gene under nitrogen deprivation, at the level of individual cells. Our experiments showed that differentiation into heterocysts took place preferentially within a limited interval of the circadian clock cycle; that gene expression in different vegetative intervals along a developed filament was discoordinated; and that the circadian clock was active in individual heterocysts. Furthermore, Anabaena mutants lacking the kaiABC genes encoding the circadian clock core components produced heterocysts but failed in diazotrophy. Therefore, genes related to some aspect of nitrogen fixation, rather than early or mid-heterocyst differentiation genes, are likely affected by the absence of the clock. A bioinformatics analysis supports the notion that RpaA may play a role as master regulator of clock outputs in Anabaena, the gating of differentiation by the circadian clock and the involvement of the clock in proper diazotrophic growth. Together, these results suggest that under nitrogen deficient conditions, the functional unit in Anabaena is reduced from a full filament under nitrogen-rich conditions, to the vegetative cell interval between heterocysts.

biophysics↗

bioRxiv2023

Heterologous expression of genes from a heterocystous cyanobacterial endosymbiont highlights organic carbon exchange with its diatom host

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, 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 N2 fixation. This knowledge is key to understand the physiology of the globally significant H. hauckii-R. euintracellularis symbiosis. SIGNIFICANCEDiatom diazotroph associations (DDAs) between diatoms and N2-fixing bacteria (diazotrophs) have a relevant impact on N2 fixation-based production, but the mechanisms underlying their integrated N2 and CO2 fixation remain unstudied. In the association between the diatom Hemiaulus hauckii (host) and the N2-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.

molecular biology↗

bioRxiv2022

YAP condensates are highly organized hubs for YAP/TEAD transcription

YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. The transcription factor TEAD1 actively stabilizes YAP condensates, which also recruit BRD4, a coactivator that is enriched at active enhancers. Using single molecule tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.

cell biology↗

bioRxiv2022

Towards genome-engineering in complex cyanobacterial communities: RNA-guided transposition in Anabaena

In genome engineering, integration of incoming DNA has been dependent on enzymes produced by dividing cells which has been a bottle neck towards increasing DNA-insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here we developed Golden-Gate vectors to test CAST in filamentous cyanobacteria and show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing the CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide(sg)-RNA encoding the leading, but not the reverse complement strand of the target were effective with the protospacer associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci, therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.

synthetic biology↗