Biology Direct

Microalgae are key primary producers in marine ecosystems, and their interactions with the surrounding environment rely on the secretion of intracellular metabolites and macromolecules, particularly proteins, supporting essential functions such as nutrient acquisition, environmental sensing and biotic interactions. Most abundant and ecologically relevant seawater algae are secondary endosymbionts, where multiple endosymbiotic events extensively reshaped plastids and intracellular membrane systems, requiring adaptation of protein trafficking mechanisms. This study presents the identification of signal peptides that direct protein secretion in the seawater microalga Nannochloropsis oceanica. Their expression in frame with a fluorescent tag enabled to reconstruct the protein secretion pathway in this organism. Proteins channelled for export are first targeted to the periplastidial compartment, an exclusive structure of secondary endosymbiotic algae, that acts as hub for protein trafficking. Subsequently, vesicle-mediated transport directs proteins through the endoplasmic reticulum into the periplasmic space between the cell membrane and the cell wall, from where they are released upon cell division. These findings reveal an evolutionarily remodeled protein secretion pathway, in which host- and endosymbiont-derived trafficking mechanisms merged into an integrated functional system. Significance StatementThe most abundant and ecologically relevant marine algae are secondary endosymbionts whose evolution required extensive re-adaptation of multiple cellular processes. Among them, protein secretion is essential for the interaction with external environment, and required specific re-shaping to the increased cellular complexity associated with endosymbiosis. This work uncovers protein secretory pathway in the secondary endosymbiont seawater alga Nannochloropsis oceanica showing that is does not follow a direct route, but proteins are first accumulated in the periplastidial compartment, a unique structure derived from its endosymbiotic history, before being directed for secretion. The final pathway integrated components derived from both the host and endosymbiont, highlighting how evolution was able to merge different biological modules to build an integrated and functional system.

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

The phylogenetic position of Nanohaloarchaea has been debated, these nanosized archaea being alternatively proposed as sister group to Haloarchaea, members of the DPANN-Archaea, or sister group to Methanocellales. Screening a set of universal proteins, we identified four insertions located at critical locations in three ribosomal proteins and one RNA polymerase subunit that support the branching of Nanohaloarchaea as sister group to Aenigmarchaea within DPANN cluster II (sensu Dombrowski et al., 2020). Insertion analyses and phylogeny of the monomeric primase specific to DPANN-Archaea confirm the existence of a robust clade grouping Undinarchaea, Naiadarchaea and DPANN cluster II, that we propose to call Nanostetteria. Our insertion analysis also supports including Altiarchaea within DPANN-Archaea and suggest a new clades that has not been recovered in phylogenetic analyses, one grouping DPANN-Archaea with Stygia (Hadarchaea and relative) and an even large one grouping these lineages with Acherontia (Thermococci and relatives). The insertion defining this larger clade, present in the ribosomal protein uS7, is also present at the same position in Thaumarchaea, Korarchaea and a subgroup of Asgardarchaea. Whereas the insertion in Thaumarchaea is certainly due to an independent event, we discuss alternative hypotheses that can explain those present in Korarchaea and Asgardarchaea. Finally, we noticed several cases of MAGs misannotations, indicating that insertion analysis can be useful to identify protein with misleading affiliations. The existence of insertions in otherwise highly conserved universal proteins involved in translation or transcription could partly explain the high rate of protein evolution in some archaeal lineage, especially in DPANN-Archaea.

Most cyanobacterial genomes are rich in the GCGATCGC octamer, also known as Highly Iterated Palindrome 1 (HIP1). Despite its description over three decades ago, the biological function of this highly abundant sequence is only beginning to be elucidated. HIP1 is recognized by two DNA methylases, DmtA and DmtC, and is characterized by its evolutionary conservation and a quasi-periodic distribution within genomes. However, whether the phylogenetic distribution of HIP1 correlates with the presence of functional categories of protein families remains unknown. Here we investigated whether certain protein families share a phylogenetic distribution with this abundant palindromic sequence across cyanobacterial genomes. Our analysis revealed a strong phylogenetic correlation between several proteins of the Type IV secretion system (T4SS) and the abundance of HIP1. This finding aligns with recent discoveries demonstrating that HIP1 enhances DNA transformation in a methylation-dependent manner in two distinct cyanobacterial species. Consequently, we hypothesize that HIP1 function as a conserved adaptation for horizontal gene transfer (HGT) at the phylum level, potentially by serving as a DNA-uptake recognition sequence in cyanobacteria. Significance statementScientists have long been baffled by the HIP1 sequence, a short, highly common, repetitive DNA pattern found across almost all cyanobacterial genomes. Our study used a whole-genome evolutionary approach and found that the presence of this repetitive pattern is tightly linked to the presence of a cells external DNA uptake system. This tight co-evolutionary relationship suggests that HIP1 isnt just random genomic feature, but a conserved evolutionary adaptation used by the entire cyanobacterial phylum to specifically enhance their ability to acquire new genes from one another.

Filamentous phages infecting Gram-positive bacteria remain largely unexplored. Notably, only two filamentous phages, B5 and Philemon infecting Propionibacterium freudenreichii, have been described to date in the phage-rich dairy ecosystem. Although both were genomically characterized, only B5 was confirmed to be an infective filamentous single-stranded DNA phage. The aim of this study was to isolate and characterize new filamentous phages from Swiss-type cheese to investigate their diversity, structural features, host specificity, and potential adaptation to the dairy environment. Thirty raw and pasteurized milk cheeses from France were screened for phages infecting P. freudenreichii strains. Eleven phages were isolated, nine of which displayed a filamentous morphology. Named MINOG1 to MINOG9, these filamentous phages exhibited genomic features typical of this morphotype, including small single-stranded DNA genomes with collinear genes organized into functional modules. Comparison with B5 and Philemon revealed sequence divergence ranging from 0.1% to 7%. These phages also exhibited a diverse host range. To further explore phage-P. freudenreichii interactions, we screened the genomes of the strains used in this study, as well as additional genomes retrieved from the NCBI database, for CRISPR spacers predicted to target these filamentous phages. Numerous strains contained CRISPR spacers showing 79 to 100% identity to genomic regions of these phages. Two P. freudenreichii strains displayed markedly different phage resistance levels despite exact spacer-protospacer matches with phages B5, MINOG1, MINOG2, and MINOG8. Conversely, several strains were resistant to nearly all tested phages despite lacking CRISPR spacers targeting them suggesting the presence of additional defense systems in P. freudenreichii. IMPORTANCEFilamentous phages can play important roles in bacterial ecology by modulating host physiology, population dynamics, and bacterial adaptation to specific environments. However, filamentous phages infecting Gram-positive bacteria remain among the least explored bacterial viruses, and their diversity, ecology, and interactions with their hosts are still poorly understood. This knowledge gap is particularly relevant in dairy ecosystems, where phages are abundant and can influence microbial communities and fermentation processes. In characterizing nine new filamentous phages infecting Propionibacterium freudenreichii from Swiss-type cheeses, this study expands the known diversity of filamentous phages associated with Gram-positive bacteria and provides new insights into phage-host interactions and bacterial defense strategies in dairy-associated bacteria.

Hepatocytes undergo extensive proliferation to facilitate liver repair after injury, yet early adaptive changes prior to proliferation remain unclear. Here, we report that during early acetaminophen (APAP)-induced liver injury, hepatocytes exhibit transient proliferation suppression, most pronounced in mid-zone hepatocytes due to zonal APAP metabolism. Using spatial transcriptomics (ST), immunohistochemistry, and functional studies, we identified a unique mid-zone stress-response program. Central to this adaptation is the Atf4-Chop axis, which actively suppresses proliferation via the cell cycle inhibitor Btg2, prioritizing cytoprotection over cell division. This transient arrest is a critical survival strategy: halting energy-intensive proliferation during peak injury allows mid-zone hepatocytes to redirect resources towards protection, enhancing their survival in early APAP-induced liver injury. Thus, Atf4-Chop-mediated quiescence preserves a hepatocyte reservoir necessary for subsequent regenerative proliferation and effective repair. Our findings reveal a key adaptive trade-off in mid-zone hepatocytes where transient proliferation arrest promotes early survival to enable repair.

Spaceflight imposes unique physiological stresses on mammals, including microgravity and cosmic radiation, which drive complex molecular adaptations. However, the systemic and temporal dynamics of space-induced epigenetic regulation remain poorly understood. We constructed a comprehensive DNA methylome BodyMap across 12 organs or tissues from mice exposed to long-duration spaceflight across three time points using Reduced Representation Bisulfite Sequencing (RRBS). We also performed RNA-seq for five organs and integrated with DNA methylome. We mapped the methylome and transcriptome landscapes and found that spaceflight induces limited but highly tissue-specific differentially methylated CpGs (DMCs). Most spaceflight-induced methylation changes were reverted toward baseline within one to six months of post-flight. Functional enrichment analysis of DMCs highlighted metabolic and mitochondrial dysregulation commonly across organs, while developmental responses in immune, reproductive, and structural tissues were tissue-specific. Transcriptome data revealed that spaceflight suppressed immune and increased inflammatory responses at the multi-organ level, triggering a phenomenon resembling aging. Our study provides a comprehensive DNA methylome BodyMap across 12 organs/tissues in spaceflight mice, elucidating the tissue specificity of epigenetic changes. These insights are essential for developing biomarkers and countermeasures to safeguard astronaut health during extended missions.

Resource competition theory typically assumes static traits and continuous supply of resources. Yet microbial communities often experience feast-famine cycles and rapid trait change. To investigate coexistence under these nonequilibrium conditions, we integrate modern coexistence theory (MCT) with a genome-scale metabolic model that explicitly links resource use (traits) to metabolic fluxes and growth. MCT partitions competitive interactions into niche and fitness differences, to predict when trait-driven departures from neutrality result in coexistence or exclusion. Using dynamic flux balance analysis, we define a function that maps trait-resource matching to niche and fitness differences between species in a two-species two-resource system. This mapping shows that niche and fitness differences are not independently tunable under resource competition: changes in transporter-mediated resource uptake and changes in resource concentration ratios generate constrained trajectories through coexistence space. Specifically, we show that the minimum niche difference required for coexistence increases linearly with the absolute difference in maximal growth rates on limiting resources, showing how limiting similarity between species can emerge from intracellular metabolic constraints. Furthermore, we find that in batch culture simulations, initial conditions (inoculum size, total resource concentration) determine the timescale of the transient growth phase, with niche differences saturating and fitness differences increasing as the timescale grows, thereby governing competition outcomes. Finally, we test these predictions experimentally using E. coli strains with targeted resource transporter knockouts under both equal and skewed resource concentrations. Our results confirm that transporter-mediated trait changes and resource concentration ratio modulation can be harnessed to engineer coexistence. Together, our work demonstrates that trait-resource matching imposes structured constraints on the joint evolution of niche and fitness differences, thereby shaping biodiversity maintenance in microbial communities under nonequilibrium conditions.

Diurnal changes in light availability are a defining feature of life on Earth. Photoautotrophic organisms therefore store reduced carbon during the day to sustain energy metabolism at night. In cyanobacteria, glycogen is the primary carbon storage compound and supports both energy homeostasis and stress responses. Although glycogen-deficient Synechocystis strains have been studied previously, how these mutants cope with the loss of the major daytime carbon sink and can sustain themselves during the night remains unclear. Using single-cell microfluidics, transcriptomics, and metabolomics, we show that {Delta}glgC mutants exhibit pronounced light sensitivity. At sub-lethal light intensities, daytime transcriptional responses are dominated by downregulation of photosynthesis-related genes, likely preventing NADPH overaccumulation in the absence of a carbon sink. During the night, mutants display severe energy limitation, characterized by reduced ATP levels, altered redox balance, and depletion of central carbon intermediates. In contrast, fumarate and malate accumulate, indicating enhanced respiratory flux through succinate dehydrogenase. These metabolic constraints lead to extended lag phases and delayed cell divisions after the onset of light, demonstrating that glycogen-deficient cells fail to efficiently reinitiate growth after dawn. Overall, our results as a snapshot of the initial response to diurnal regimes highlight glycogen as a central integrator of diurnal physiology in Synechocystis, coordinating energy metabolism, redox balance, and cell division, with implications for metabolic robustness and the evolutionary constraints shaping (endo)symbiosis. Short summaryGlycogen deficiency disrupts day-night energy and redox homeostasis in Synechocystis, revealing constraints on growth, division, and symbiotic potential.

Influenza A virus (IAV) causes significant morbidity and mortality worldwide. Understanding how viral RNAs may regulate host genes through microRNA-like mechanisms can clarify pathogenesis and reveal therapeutic targets. In this study, we screened all eight IAV H3N2 RNA segments (PB2, PB1, PA, HA, NP, NA, M, and NS) using an ab initio computational pipeline; five segments (PB2, PB1, PA, HA, and M) met the VMir scoring threshold for further analysis, while NP, NA, and NS were excluded due to low pre-miRNA scores. Mature miRNAs were identified using MatureBayes, and target genes in the human genome were predicted with the miRDB server. From these targets, we selected two genes per qualifying segment (10 genes total) based on their functional relevance to influenza infection and supporting literature; all selected genes are unique to their respective segment. We identified 10 segment-specific target genes (IFNL1, DDX60, SAMHD1, MAVS, IRF4, BIRC2, AGO1, MAP3K1, NOD1, and TNFAIP1) and one common target across all five analyzed segments (CADM2). Gene Ontology and pathway analyses showed enrichment in interferon signaling, RIG-I-like receptor pathways, antiviral restriction, RNA interference, and inflammatory responses. Literature supports roles for these genes in pulmonary and antiviral innate immunity. Our findings provide a basis for experimental validation and may help the research community better understand influenza virus pathogenesis and identify novel therapeutic candidates. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/725090v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@2b14adorg.highwire.dtl.DTLVardef@5a9b2eorg.highwire.dtl.DTLVardef@81ffc1org.highwire.dtl.DTLVardef@be119b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Viruses constitute a significant proportion of Earths genetic diversity, yet most remain uncharacterized beyond their sequences in viral metagenomes. Linking viral genotypes to phenotypes--especially enzyme function to phage infection dynamics--is challenging due to the lack of cultured virus-host systems. DNA polymerase I (PolA), essential for genome replication in [~]25% of dsDNA phages, provides an opportunity to explore these connections. In phage T7, residue 526 is critical for nucleotide incorporation, with previous in vitro evidence indicating impacts on enzyme efficiency and fidelity. Previous analyses identified three substitutions at this position (Tyr/Y, Phe/F, Leu/L) linked with deeply rooted viral PolA clades. Mutation impacts at residue 526 were tested in vitro and in vivo. The Y526F protein exhibited a 50% reduction in specific activity, and when introduced via High Complexity Golden Gate Assembly into T7 demonstrated a 53% decrease in burst size and significantly longer latent period compared to wild type. The Y526L protein exhibited a 97% decrease in activity, and the Y526L phage was incapable of completing its lifecycle. These findings confirm historical biochemical data, provide in vivo context for these mutations in the T7-E. coli system, and offer experimental support for genotype-to-phenotype associations in viral PolA, informing viral metagenomics studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/726624v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@14395e5org.highwire.dtl.DTLVardef@261504org.highwire.dtl.DTLVardef@2dc1e4org.highwire.dtl.DTLVardef@147a7f_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Keown, R. (2026) https://BioRender.com/mhrmup3

Multicellularity has evolved multiple times across the eukaryotic tree of life, including among protist lineages. Because transposable elements (TEs) strongly influence genome architecture and gene regulation, understanding their potential impact on genome structure and their relationship with gene expression may provide insight into the evolution of multicellularity. Here, we generated a new genome assembly for the facultatively multicellular amoeba Acrasis kona and performed comparative analyses of TE composition, TE diversity, and TE-density organization across diverse protist lineages. Comparative analyses included unicellular and multicellular representatives from across the tree of eukaryotes, (Heterolobosea, Filasterea, Cristidiscoidea, and Chlorophyceae), including Naegleria spp., Tetramitus jugosus, Capsaspora owczarzaki, Pigoraptor spp., Fonticula alba, Parvularia atlantis, Volvox carteri, and Chlamydomonas reinhardtii. To examine relationships between TEs and gene regulation, we integrated transcriptomic datasets from A. kona, Capsaspora owczarzaki, and Volvox carteri with genome-wide TE-density analyses of differentially expressed genes. TE abundance and composition varied substantially among lineages, with species that exhibit more complex developmental or cellular organization generally containing higher TE proportions than closely related unicellular taxa. Patterns of TE-density organization near up-regulated, down-regulated, and non-differentially expressed genes also differed among systems, ranging from strong TE depletion in A. kona to weaker or cell-type-specific patterns in Capsaspora and Volvox. Together, these findings suggest that transposable elements are associated with multicellularity across diverse protist lineages, although the specific roles they play appear to be complex, lineage-specific, and not yet fully understood.

The circadian clock is a highly conserved evolutionary advantage which allows organisms to anticipate regular changes in daily environmental conditions. Clocks from fungi to mammals rely on a transcription-translation feedback loop (TTFL) mechanism. Phosphorylation is understood to be a critical regulatory step for maintaining the period of the circadian clock and feedback loop closure. The role of kinases in the Neurospora clock has been examined extensively; however, phosphatases have not been systematically interrogated. By re-examining the Neurospora genome using current informatic tools we identified the 30 genes previously identified as encoding protein phosphatases as well as 13 novel genes, and we assessed the function of the core circadian clock in 39 non-essential phosphatases using a real-time luciferase reporter. We observed both period lengthening and shortening effects, which are not restricted to a single phosphatase family or fold. All but one deletion mutant maintained a rhythmic core clock. In addition, we observed a new temperature compensation defect in the previously studied knockout of phosphatase pph-4, the result of nutritional growth conditions.

Microalgal metabolism relies on their chloroplasts, and involves both nucleus and plastidial-encoded proteins of various evolutionary origins. The plastidial ATP synthase complex is a key player in photosynthesis, and has been extensively studied in plants. However, our knowledge in other photosynthetic eukaryotes remains limited, despite their importance in marine environments. Here, we report the characterisation of a novel homologue of the F-type ATP synthase alpha subunit, hereby named xATPA, widespread in microalgae but absent from other photosynthetic organisms. Comparisons of xATPA sequences and predicted structures revealed a specific feature, the bump domain, and highlighted the absence of an ATP-binding site. We assessed xATPA prevalence in microalgae in the global ocean using environmental data from Tara Oceans, with a particular focus on diatoms, and demonstrate that its expression is associated with polar summer conditions. Using a reverse genetic approach in the model diatom Phaeodactylum tricornutum, we show that xATPAP t has a plastidial localisation, and that xATPA KO mutants exhibit growth deficiencies in a combination of low temperature, low salinity and constant light, consistent with environmental analysis. Surprisingly, both RNAseq and physiological assays suggest that xATPA is not involved in ATP synthase functions. On the other hand, xATPA interacts with other F1 ATP synthase subunits in vitro, which we suggest forms transient unassembled complexes. This study hence represents a comprehensive analysis of a novel protein from the environment to the lab, and reveals a new player in the plastidial physiology of eukaryotic microalgae.

Chromosome spatial organization plays critical roles in transcriptional regulation and DNA protection. In cyanobacteria--photosynthetic bacteria that experience dramatic fluctuations in light intensity--chromosome reorganization could facilitate rapid transcriptional reprogramming and protect DNA from photodamage. However, chromosome organization in these polyploid organisms has remained technically challenging to observe, leaving light-dependent responses unexplored. Here, we show that higher-order chromosome organization in Synechocystis sp. PCC 6803 is associated with light intensity, revealing a previously unrecognized light-dependent adaptation in cyanobacteria. We established fluorescence in situ hybridization (FISH) methods for this model cyanobacterium carrying multi-copy genomes, together with a computational pipeline to assign paired FISH signals to individual genome copies. The slope relating genomic and spatial distance was steeper under standard conditions ({beta} = 0.972 nm/kbp, R{superscript 2} = 0.12) than under high-light conditions ({beta} = 0.450 nm/kbp, R{superscript 2} = 0.02), indicating that local chromosome organization is substantially disrupted by elevated light intensity. The spatial distribution of the multiple genome copies also differed between conditions, independently supporting condition-dependent chromosome reorganization. Hi-C analysis corroborated these findings, revealing reduced chromosomal interactions within the 10-100 kbp range under high-light conditions. Together, these results demonstrate that light intensity is a previously unrecognized determinant of higher-order chromosome organization in a photosynthetic bacterium.

The origin of eukaryotes is a key event in the evolution of cellular life hypothesized to involve a symbiotic integration between a member of the Asgard archaea and the Alphaproteobacteria. Recent work has provided evidence for additional genetic input from other prokaryotes to the eukaryotic proteome yet the extent and sources of these contributions remain debated. Here we aimed to further resolve the prokaryotic origins of eukaryotic genes to inform our understanding of eukaryogenesis. Specifically, we developed a phylogenetic framework to investigate the origins of eukaryotic gene families associated with metabolism and informational processing for comparison. We found that informational processing genes were predominantly derived by archaea whereas eukaryotic metabolism is highly chimeric in its origin. In contrast to previous studies, we report a substantial number of archaeal origins of diverse metabolic enzymes including key metabolic regulators. This highlights an overlooked participation of archaeal metabolism and pinpoints potential metabolic integrations during eukaryogenesis. Apart from the alphaproteobacterial contributions to the eukaryotic metabolism, we found an additional dominant phylogenetic signal of genes potentially derived from Myxococcota, especially for gene families associated with lipid metabolism. By systematically analysing the origins of eukaryotic metabolism, this research offers novel insights into the origin of eukaryotic membranes and refine our current models for the origin of the eukaryotic cell.

Rhodococcus opacus is an oleaginous actinobacterium with considerable potential for lipid-based bioproduction, as well as for utilising a variety of carbon sources as substrates, including renewable, cost-effective resources. Although its capacity for triacylglycerol accumulation is well established, the regulatory logic that governs its fatty acid and mycolic acid biosynthesis is still poorly understood. Here, we investigated the transcriptional control of the type II fatty acid synthase (FASII) pathway in R. opacus PD630, revealing a regulatory architecture that is more complex than previously assumed. Differential gene expression analysis showed that environmental cues, including temperature, pH, carbon-to-nitrogen ratio and the presence of free fatty acids influence the FASII gene cluster expression in a non-uniform manner. This phenomenon suggests the presence of internal transcription start sites and modular regulation within the cluster. We identified three lipid-responsive transcription factors, MabRRO, FadR1RO and FadR2RO, that are all capable of binding the fasII promoter in vitro. DNA binding of FadR1RO and FadR2RO was disrupted by long-chain acyl-CoA molecules, indicating ligand-dependent control. Together, these findings reveal previously unrecognised layers of transcriptional regulation in the R. opacus FASII pathway and highlight both conserved and divergent regulatory features within the Mycobacteriales lineage. Featured Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/678588v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@456a6dorg.highwire.dtl.DTLVardef@14e3d1forg.highwire.dtl.DTLVardef@18ed368org.highwire.dtl.DTLVardef@1d979a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cyanobacteria manage photosynthetic and environmental stresses through transcriptional programs controlled by regulators also affecting carbon flux, growth states, and metabolic output that bioproduction seeks to optimize. This regulatory architecture and its most influential nodes remain incompletely characterized. We hypothesized two influential regulator layers: a conserved core responding to common stresses, and species-specific regulators mediating strain-level niche adaptations. Mapping both layers underpins understanding genome[->]regulatory-network[->]phenotype flow, enabling global transcription machinery engineering for reliable bioproduction. To test our hypothesis, we constructed conserved-core and species-specific gene regulatory networks (GRNs) for three cyanobacteria, Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803 and Picosynechococcus sp. PCC 7002, integrating a manually curated multi-pipeline regulator inventory with 1,098 harmonized transcriptome states for the 1,362-gene tri-homolog core genome. We quantified each regulator influence using local (degree, k-core), global (betweenness, closeness), and community-aware (eigenvector) centrality measures, and an Integrated Centrality score aggregating influence across complementary topological measures. High-influence regulators are predicted to exert broad metabolic effects when manipulated, making them priority candidates for single-target engineering interventions that modulate multiple genes and reprogram complex phenotypes. Across the three cyanobacteria, the two GRN layers proved topologically distinct: the conserved core concentrated influence in stress-related hubs (11 of its top 15 by Integrated Centrality were stress-related), while species-specific networks spread influence across functionally diverse regulators. Stress-coupled enrichment also held per individual centrality measure: regulators ranking top in both the core and species-specific GRNs by the same measure were mostly stress-related (15 of 19 instances), including the multi-stress regulators RpaB, Rre1, and BolA, the heat-shock HrcA, and the nitrogen NtcA. In species-specific GRNs, stress-related regulators remained the leading category alongside circadian, carbon-metabolism, morphology, and housekeeping regulators, including PlmA, Pex, TetR, and SrrB in PCC 7942; KaiC3, Sycrp1, Rre28, and Bhl in PCC 6803; and Zur, Sycrp1, and NarL in PCC 7002. High-influence putative regulators included the iron-stress AraC-family paralogs IutR1-IutR3, OmpR-family paralogs OmpR1-OmpR2, and chromosome- or plasmid-encoded Xre-family, AraC, and HypP. Stress regulation emerges as a recurring high-influence axis across these networks. The conserved core identifies universal regulatory programs, and species-specific layers reveal strain-level innovations for cross-strain transfer to support engineering of robust bioproduction. ImportanceCyanobacteria are studied as platforms for sustainable, carbon-recycling production of fuels and chemicals from sunlight, water, and atmospheric carbon dioxide. Their reliable deployment in industrial settings is limited by environmental stresses that depress photosynthetic efficiency and product yields. The same regulatory proteins that govern stress responses also control how cells partition carbon, switch growth states, and direct metabolic output, making them natural levers for engineering robust production strains. Yet systematic, cross-species maps of these regulators have been missing. We present the first comparative regulatory map spanning three biotechnologically important model cyanobacteria, Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803, and Picosynechococcus sp. PCC 7002, and identify the conserved regulators most influential across all three. The resulting catalog prioritizes candidate targets for experimental validation, and the supporting datasets and analytical framework are released for reuse to support efforts to engineer cyanobacterial strains for reliable industrial bioproduction.

Archaea of the order Sulfolobales exhibit a highly ordered cell cycle progression similar to that of eukaryotes. It has been demonstrated that degradation of the cell division protein CdvB by the proteasome controls the progression of cell division. However, how the proteasome itself is regulated during the cell cycle is not fully understood. Recently, the cyclically expressed ArsR family transcription factor CCTF1 (cell cycle transcription factor 1) was shown to represses expression of PAN (proteasome-activating nucleotidase) in Sulfolobus acidocaldarius. Here, using biochemical approaches, comparative transcriptomics and proteomics, we provide further insights into the CCTF1 function and show that it regulates PAN expression in Saccharolobus islandicus by binding to an AT-rich palindromic sequence within the pan promoter. We also reassessed the role of the cyclically expressed protein kinase aCcrK in regulation of proteasome activity and conclude that CCTF1, rather than aCcrK, is the primary factor controlling the proteasome activity. These findings advance our understanding of the proteosome-mediated cell cycle regulation in archaea.

Despite advances in knowledge and medicine, hepatitis C virus (HCV) infection remains a global challenge. The viral life cycle heavily depends on lipid metabolism; therefore, HCV infection is associated with profound changes in host lipid homeostasis. The transcription factor nuclear factor erythroid 2 related factor-1 (Nrf1) is one of the regulators maintaining this homeostasis. Nrf1 exists in multiple proteoforms that differ in their capacity to serve as cholesterol sensor, activator or inhibitor of gene expression. We have previously identified that the amount of full-length Nrf1 protein in HCV-replicating cells is significantly reduced. Here, we investigate whether HCV affects the formation of the different proteoforms and their functionality using Western blot, qPCR, CLSM and FRET acceptor-photobleaching methods. We report that HCV infection does not alter the onset of Nrf1 proteoforms generated through proteasomal cleavage of the protein. However, the amount of different Nrf1 proteoforms is significantly reduced in HCV-positive cells due to enhanced Nrf1 turnover. Furthermore, the Nrf1 proteoforms with transcriptional activator functions are prevented from translocation into the nucleus. Reduced Nrf1 activity contributes to elevated cholesterol levels and favors lipid droplets formation, which serve as a central platform for viral morphogenesis. Conversely, rescue of Nrf1 activity in HCV-replicating cells is associated with decreased intracellular cholesterol levels, reduced number of lipid droplets and impaired viral release, which is reflected by intracellular accumulation of the core protein and intact viral particles. Taken together, our results characterize the so far not investigated complex interplay between HCV and Nrf1. HCV-mediated inhibition of Nrf1 functionality leads to intracellular cholesterol accumulation, resulting in enhanced lipid droplet formation that supports the HCV life cycle and contributes to HCV-associated pathogenesis. Author SummaryThe lack of a vaccine and limited access to effective drugs (pan-genotypic direct-acting antivirals) for curing hepatitis C virus (HCV) infection means that HCV remains an ongoing and urgent challenge worldwide. In light of this, a deeper understanding of the virus-host interaction is required. In this study, we investigate the interplay between HCV and lipid metabolism, focusing on the uncharacterized role of the cholesterol sensor and transcription factor Nrf1 in this interaction. We observe the inhibition of Nrf1 activity in HCV-replicating cells, which leads to enhanced intracellular cholesterol accumulation and lipid droplet formation, resulting in microenvironment favorable for viral morphogenesis. We reveal the underlying mechanisms and describe their relevance to the viral life cycle and virus-associated pathogenesis.