Biology subjects

Das, A. K.

Publications and source records attributed to Das, A. K..

At least 19 recordsLinked to original sources

bioRxiv2026

RAP2.6 negatively regulates Arabidopsis submergence tolerance through GSNOR1-dependent redox and hormonal signaling

Group VII ETHYLENE RESPONSE FACTORs (ERFVIIs) are central regulators of plant responses to oxygen deprivation. However, the contributions of many other APETALA2 (AP2)/ERF transcription factors to hypoxia and flooding responses remain poorly understood. Here, we identify the group X ERF transcription factor RAP2.6 as a negative regulator of submergence tolerance in Arabidopsis thaliana. RAP2.6 expression declined under hypoxia, and plants overexpressing RAP2.6 exhibited reduced survival following dark submergence, whereas the rap2.6-1 loss-of-function mutant showed enhanced tolerance to submergence, hypoxia, and ethanol treatment. Enhanced tolerance of rap2.6-1 was associated with reduced membrane injury, maintenance of photosynthetic pigments and soluble sugars, and extensive metabolic remodeling. RAP2.6 overexpression altered nitric oxide (NO), S-nitrosothiol (SNO), and ethylene accumulation during submergence and was associated with greater oxidative damage and enhanced abscisic acid (ABA)-responsive gene expression. Genetic analyses using S-NITROSOGLUTATHIONE (GSNO) REDUCTASE 1 (GSNOR1) gain- and loss-of-function backgrounds demonstrated that GSNOR1-dependent GSNO homeostasis strongly modified RAP2.6-dependent submergence responses. Increased GSNOR1 activity suppressed the submergence-sensitive phenotype of RAP2.6-overexpressing plants, reduced nitro-oxidative stress, and restored carbohydrate reserves, whereas loss of GSNOR1 activity abolished the enhanced tolerance of rap2.6-1. GSNOR1 also modified RAP2.6-dependent expression of genes associated with oxygen sensing and ABA signaling, while its protective effect was not explained solely by changes in ethylene accumulation. Together, these findings identify RAP2.6 as a regulatory component of the low-oxygen response and reveal a functional interaction between RAP2.6 and GSNOR1-dependent redox signaling that coordinates plant hormonal, metabolic, and oxidative responses during submergence.

plant biology↗

bioRxiv2026

Integrative genetic and structural analyses reveal nitric oxide-driven regulation of ERFVII stability in Arabidopsis under low O2

Nitric oxide (NO) is a key signaling molecule that regulates diverse physiological responses, including adaptation to hypoxia in plants. Although stabilization of group-VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factors under low O2 is known to be facilitated by the N-dragon pathway, whether NO directly mediates N-terminal cysteine (Cys) oxidation to regulate ERFVII stability remains unresolved. Here, we combined genetic, computational, and structural approaches to investigate NOs role in proteasomal degradation of ERFVII during flooding stress. Arabidopsis mutants with elevated S-Nitrosoglutathione (GSNO) levels compromised ERFVII activation during dark submergence but increased expression of genes encoding N-degron pathway enzymes. Although direct detection of in vivo NO-mediated S-nitrosylated proteins remains technically challenging, the GPS-SNO 1.0 tool predicted the conserved N-terminal second Cys residue as a high-confidence S-nitrosylation site. Structural modeling using the Schrodinger Suite 2024-4 further revealed that conversion of the Cys thiol (Cys-SH) to S-nitrosothiol (Cys-SNO) induced notable conformational changes in ERFVII TFs. Molecular docking further demonstrated that Cys-SNO-modified ERFVII peptides exhibited stronger binding affinities and altered interaction networks with N-degron pathway enzymes, supporting a role for NO-mediated structural remodeling in ERFVII degradation. Elevated GSNO also disrupted energy balance efficiency for overcoming O2 deficiency, altered the expression of sugar starvation-responsive genes, and impaired ATP binding capacity of Cys-SNO ERFVII proteins, as evidenced by docking and molecular dynamics simulations. Collectively, these results support a model in which NO-mediated modification of the conserved N-terminal Cys promotes ERFVII degradation, thereby linking NO signaling to hypoxia-responsive transcriptional regulation and metabolic adaptation during flooding stress.

plant biology↗

bioRxiv2026

AP2/ERF transcription factor RAP2.6 regulates early flowering in Arabidopsis thaliana by altering S-nitrosothiol levels and cytokinin responses

RAP2.6, an AP2/ERF transcription factor (TF), regulates plant stress responses; however, its role in floral transition remains unexplored. Here, we evaluated RAP2.6s role in flowering and the associated transcriptional changes in Arabidopsis thaliana under long-day conditions. RAP2.6-overexpressing line showed early flowering with fewer rosette leaves, whereas rap2.6-1 mutant flowered later, had more rosette leaves, and higher expression of the floral repressor FLOWERING LOCUS C (FLC). Early flowering in the overexpressing line was accompanied by transcriptional activation of the floral integrators GIGANTEA (GI), FLOWERING LOCUS T (FT), and COSTANS (CO), potentially through RAP2.6 interaction with GCC/DRE cis-regulatory elements. RAP2.6-mediated floral transition depended on nitric oxide (NO), with flowering time largely varying based on NO bioactivity. RAP2.6 was found to be a downstream regulator of Arabidopsis S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1) in controlling S-nitrosothiol (SNO) levels, flowering time, and silique formation. The NITRIC OXIDE-ASSOCIATED 1 (NOA1)-dependent reduction in NO levels abolished early flowering in 35S::RAP2.6 plants without affecting silique formation. Furthermore, enhanced cytokinin sensitivity and upregulation of cytokinin biosynthetic genes suggest cytokinin involvement in RAP2.6-mediated flowering. Together, these findings highlight the crucial role of RAP2.6 in regulating flowering time by integrating redox and hormonal signaling to coordinate reproductive development in A. thaliana.

plant biology↗

bioRxiv2025

Shaping the active site from a distance: Unravelling the regulatory role of the conserved N-terminal β-flap of thiol peroxidases through structure-function characterization of the Staphylococcal ortholog

Bacterial thiol peroxidases (Tpxs) are members of atypical two-cysteine peroxiredoxin, which detoxify hydroperoxides during host-pathogen interactions. Despite extensive research on bacterial Tpxs, the precise structure-based functional characterization of Staphylococcal Tpx (SaTpx) remains elusive. Herein, we cloned and purified SaTpx along with its plausible physiological redox partners to reconstitute its electron relay system. We solved the high-resolution crystal structure of SaTpx, revealing the essential active-site catalytic triad (Cys 60, Thr 57, Arg 128). The crystal structure of SaTpx reveals the presence of the highly conserved N-terminal {beta}-flap, absent in its mammalian orthologs (Peroxiredoxin 5). To investigate the plausible role of this highly conserved {beta}-flap, we cloned and characterized a deletion mutant, N{Delta}15-SaTpx, lacking this region. N{Delta}15-SaTpx showed reduced ability to mitigate organic hydroperoxides in vitro; partially impaired to prevent ROS-mediated ds-DNA nicking and failed to protect host E. coli cells from exogenous ROS stress. The ultra-high-resolution crystal structures of N{Delta}15-SaTpx and N{Delta}15-SaTpx with substrate mimic revealed a distorted catalytic triad (with flipped Arg 128), explaining its partially impaired catalytic activity. This study, for the first time, elucidates the critical regulatory role of the N-terminal {beta}-flap in stabilizing the catalytic triad of bacterial Tpx, providing insights for future inhibitor designing. HighlightsO_LIUltra high-resolution crystal structures of SaTpx and N{Delta}15-SaTpx C_LIO_LIUltra-high resolution crystal structure of hydroperoxide substrate mimic bound N{Delta}15-SaTpx C_LIO_LI{beta}-flap deletion mediated distortion of catalytic triad in N{Delta}15-SaTpx C_LIO_LIThe novel regulatory role of the conserved N-terminal {beta}-flap of Thiol peroxidases. C_LI

biophysics↗

bioRxiv2025

Beyond species means - the intraspecific contribution to global wood density variation

Wood density is central for estimating vegetation carbon storage and a plant functional trait of great ecological and evolutionary importance. However, the global extent of wood density variation is unclear, especially at the intraspecific level. We assembled the most comprehensive wood density collection to date (GWDD v.2), including 109,626 records from 16,829 plant species across woody life forms and biomes. Using the GWDD v.2, we explored the sources of variation in wood density within individuals, within species, and across environmental gradients. Intraspecific variation accounted for up to 15% of overall wood density variation (sd = 0.068 g cm-3). Sapwood densities varied 50% less than heartwood densities, and branchwood densities varied 30% less than trunkwood densities. Individuals in extreme environments (dry, hot, acidic soils) had higher wood density than conspecifics elsewhere (+0.02 g cm-3, [~]4% of the mean). Intraspecific environmental effects strongly tracked interspecific patterns (r = 0.83) but were only 20-30% as large and varied considerably among taxa. Individual plant wood density was difficult to predict (RMSE > 0.08 g cm-3; single-measurement R2 = 0.59). We recommend (i) systematic within-species sampling for local applications, and (ii) expanded taxonomic coverage combined with integrative models for robust estimates across ecological scales.

plant biology↗

bioRxiv2025

A global map of wood density

Wood density influences how quickly woody plants grow, how long they live and how much carbon they store, yet its global variation remains poorly mapped. Here we combined 109,626 wood density measurements from 16,829 species with 300,949 vegetation plots to produce a km-scale map of community-weighted wood density for every woody biome. Our model led to a prediction accuracy 32-51 % higher than previous global products, and a 1.8-3.7-fold wider wood density range (0.28-1.00 g cm-3; global mean: 0.57 g cm-3) than previously assumed. Spatial cross-validation showed low bias ({+/-}2.5 % of the mean), and uncertainties decreased from 20% in poorly sampled drylands and boreal regions to 5% in data-rich temperate forests. Mean annual temperature was the best predictor of community-weighted mean wood density, increasing by 0.01 g cm-3 for every 1{degrees}C change. We deliver a low-bias, high-resolution wood density layer for Earth system models, together with spatially explicit error maps. This study represents a major step forward for carbon accounting and trait-based forecasts of vegetation change.

plant biology↗

bioRxiv2025

Hylocereus polyrhizus peel extract restricts DMBA- Croton oil induced skin carcinogenesis: An integrated in vivo and in silico approach

Hylocereus polyrhizus, locally known as red dragon fruit, is valued for its nutritional benefits including high levels of antioxidants and is gaining popularity as both a food and a medicinal plant. The present study addressed the in vivo and in silico chemopreventive potential of Hylocereus polyrhizus peel extract (HPPE) in DMBA-croton oil induced skin carcinogenic model mice. The mRNA expression level of pro-inflammatory cytokines and inflammatory mediators in tumor mass were estimated by real time-RT-qPCR. In addition, molecular docking and molecular dynamic simulation analyses were conducted on the reported compounds. In the in vivo chemopreventive activity assessment, the peel extract at 500 mg/kg was found effective in reducing total tumor number, yield, burden, incidence, and weight. Total proteins and endogenous antioxidants (GSH, SOD, CAT) levels in liver and skin tissues from mice were significantly (P<0.05) elevated. In addition, the HPPE at 500 mg/kg dose significantly reduced (P<0.05) the gene expression of pro-inflammatory cytokines such as TNF-, IL-1{beta}, IL-6, IL-18, and inflammatory mediators like TGF-{beta}1, COX-2, and NF{kappa}B. In the molecular docking studies, reported compounds including Quercimeritrin, Rutin, and Kaempferol 3-O-{beta}-D-glucopyranoside were identified as the top-performing compounds, with a docking score of - 7.4, -7.1 and -7.0 kcal/mol against TGF-{beta}1 protein. This indicates stronger binding interactions compared to vincristine (-5.3 kcal/mol). In drug-likeness assessment, all compounds demonstrated the most favourable ADMET and pharmacokinetic profile. Furthermore, MDS data showed greater dynamic stability for Kaempferol 3-O-{beta}-D-glucopyranoside and Rutin, while vincristine exhibited higher fluctuations. The results suggest that HPPE may serve as a potential inhibitor of skin carcinogenesis through upregulating endogenous antioxidants as well as suppressing different proinflammatory and inflammatory cytokines. Quercimeritrin, Rutin, and Kaempferol 3-O-{beta}-D-glucopyranoside might be the probable leads responsible for this chemopreventive activity. Highlights{checkmark} Hylocereus polyrhizus peel extract (HPPE) demonstrated notable chemopreventive potential against skin cancer. {checkmark}HPPE significantly increased endogenous antioxidants (GSH, SOD, CAT) in liver and skin tissues, suggesting enhanced cellular defense mechanisms. {checkmark}Gene expression of pro-inflammatory cytokines (TNF-, IL-1{beta}, IL-6, IL-18) and inflammatory mediators (TGF-{beta}1, COX-2, NF{kappa}B) were notably suppressed by HPPE. {checkmark}Quercimeritrin (CID: 5282160) demonstrated strong binding to TGF-{beta}1 with a docking score of -7.4 kcal/mol, outperforming vincristine (-5.3 kcal/mol). {checkmark}Kaempferol 3-O-beta-D-glucopyranoside (CID: 5282102) and rutin (CID: 5280805) showed the best ADMET, pharmacokinetic profiles, and molecular stability, supporting their potential as lead compounds.

cancer biology↗

bioRxiv2025

Bringing the Ends together: Cryo-EM structures of mycobacterial Ku in complex with DNA define its role in NHEJ synapsis

Non-homologous end joining (NHEJ) is the sole pathway for repairing double-strand breaks in Mycobacterium tuberculosis during dormancy, relying on mycobacterial Ku (mKu) and ligase D, with mKu as the rate-limiting factor. Despite its essential role, the lack of structural information on prokaryotic Ku has hindered understanding of the molecular mechanisms underlying bacterial two-component NHEJ machinery. Here, we present the first cryo-EM structures of mKu in DNA-bound and higher-order super-complex forms, revealing a Ku-mediated DNA synapsis mechanism unique to prokaryotes. Integrating cryo-EM with hydrogen-deuterium exchange mass spectrometry, we define key mKu-mKu dimerization, DNA-binding, and synapsis interactions essential for efficient NHEJ, bridging structure with function. Structure-guided in silico mutagenesis, coupled with electrophoretic mobility shift assays, identifies residues essential for DNA binding and synaptic assembly, which are crucial for NHEJ. Forster resonance energy transfer confirms DNA-dependent mKu oligomerization in solution, while live-cell imaging captures its spatiotemporal dynamics during DSB repair. These findings provide fundamental insights into the architecture and function of prokaryotic NHEJ, positioning mKu as a potential therapeutic target against tuberculosis and offering a framework for understanding DNA repair across bacterial species.

biochemistry↗

bioRxiv2025

A Proposed Statistical Approach for Conducting Longitudinal Assessment of Circulating Tumor DNA

As circulating tumor DNA (ctDNA) may reflect cancer progression, understanding its temporal evolution can inform clinical decision-making in precision oncology. Temporal changes in ctDNA often exhibit complex patterns, varying significantly between and within patients. Factors such as patient characteristics and treatment regimens can further impact these changes. Traditional statistical methods may fall short in adequately characterizing patient-level ctDNA evolution over time, highlighting the need for advanced approaches. In this study we provide a framework to guide the identification of optimal models for the analysis of genetic biomarkers in liquid biopsy settings. Specifically, we focus on hierarchical mixed-effects models as they provide both cohort and patient-level insights. To illustrate the versatility of these models, we conduct an exploratory analysis of a real-word data consisting of patients with advanced colorectal cancer. In our analysis, we discovered that a hierarchical linear spline mixed-effects model was most optimal. Based on this finding, we used the model to generate cohort and patient-level response patterns, where patient-level results compare ctDNA trajectories using different combinations of patient demographics, medical history, treatments regimen, and outcomes. Finally, we discuss how results could potentially assist in constructing a patient monitoring system to help inform patient care.

genomics↗

bioRxiv2025

Seek-a-Seq: Optical detection of target nucleic acids with attomolar sensitivity

We introduce Seek-a-Seq, an enzyme-free, PCR amplification-free, optical method to detect microbial nucleic acids based on fluorescent readout of hybridization between target sequences and complementary probes immobilized on the surface of customized sample chambers. We demonstrate the ability to detect ultralow concentration (~ 1 attomolar) of short target DNA and synthetic SARS-CoV-2 RNA with ease and specificity. Seek-a-Seq can enormously reduce costs, accelerate diagnostic workflows, and outperform existing enzyme-dependent methods.

biophysics↗

bioRxiv2024

A ML Framework for Genetic Sequence Identification using 2D Electrical Conductance Probability Distributions from Mixed Data Sets

Genetic sequence identification from electrical characterization of single molecules has emerged as a promising alternative to traditional approaches. Since electrical data on single molecules is extremely noisy due to the limitations of even state-of-the-art approaches, achieving high detection rates is challenging, particularly when the task involves being able to distinguish a sequence from its single base-pair mismatches. To address this issue, we propose an architecture based on combining a convolutional neural network with an ensemble learning method, XGBoost. In addition, four different input feature representations are considered, 1D conductance probability distributions and 2D conductance versus distance probability distributions which can be viewed as images, with or without averaging over the experimental parameters. The with averaging case corresponds to feature matrices derived from mixed datasets. We find that 2D probability distributions are helpful with respect to classifier accuracy, but averaged conductance probability distributions are much more impactful and significantly enhance prediction accuracy. Our quantitative analysis of multiple sequences shows an impressive performance increase of approximately 10% for all sequences. While the basis of our analysis is conductance data of DNA strands for COVID-19 Alpha, Beta, and Delta variants and their single base-pair mismatches, our method is generally applicable to other single-molecule identification based on their conductance.

bioinformatics↗

bioRxiv2024

Subtractive proteomics unravel the potency of D-Alanine-D-Alanine Ligase as the drug target for Burkholderia pseudomallei

Melioidosis, also known as Whitmores disease, is caused by the deadly pathogen Burkholderia pseudomallei and remains a significant global health concern, particularly in South Asia. The disease is contracted through exposure to contaminated soil, water, air, and food. Infected individuals often present with abscesses in internal organs such as the lungs, spleen, and liver, and in soft tissues, with severe cases leading to septic shock and acute pneumonia. The rising incidence and mortality rates, coupled with B. pseudomalleis ability to form biofilms and develop resistance to antibiotics like cephalosporins, make treatment increasingly challenging. This highlights the urgent need for novel therapeutic approaches. D-Alanine-D-Alanine ligase (Ddl), a crucial enzyme involved in the final stage of bacterial cell wall synthesis, which protects the pathogen from the hostile cellular environment of the host. While many bacteria have two isoforms of this enzyme, B. pseudomallei possesses only the DdlB isoform, presenting a significant vulnerability. Our study represents the first successful attempt to target DdlB through a combination of molecular docking and molecular dynamics simulations. These investigations provide strong evidence that Conivaptan acts as an effective inhibitor of DdlB, offering a novel therapeutic approach for combating melioidosis.

bioinformatics↗

bioRxiv2024

Designing a novel Scaffold-Based Multi-Epitope Vaccine to Combat Melioidosis Caused by Burkholderia pseudomallei: An In-silico and Immunoinformatics approach

Burkholderia pseudomallei, the gram-negative bacteria causing melioidosis, is becoming a serious threat to healthcare settings. In recent years, B. pseudomallei has been identified as an emerging and significant etiological agent responsible for localized pyogenic infections primarily observed in India and South Asia. At present, no vaccine against melioidosis is available in the treatment system. This study has undertaken an in-silico reverse vaccinology approach to design a novel multi-epitope vaccine for treating B. pseudomallei-mediated infections. B-cell and T-cell epitopes have been predicted and stitched to develop a multi-epitope vaccine. The predicted vaccine is found to be non-toxic, non-allergic, and immunogenic in nature. Immune simulation results indicate that the designed vaccine can generate an immune response resembling a real-life scenario. The 610 amino-acid long vaccine construct has been codon-optimized and could be cloned in the E. coli K12 system. These findings from this immunoinformatics study offer a foundation for developing a tailored, safe, and potent vaccine targeting B. pseudomallei.

bioinformatics↗

bioRxiv2024

Cdc25A phosphatase is activated and mediates neuronal cell death by PUMA via pRB/E2F1 pathway in a model of Parkinson's disease

Parkinsons disease (PD) is a predominant movement disorder caused mainly due to selective loss of the dopaminergic neurons in the substantia nigra pars compacta of the mid brain. There is currently no cure for PD barring treatments to manage symptoms. The reasons might be due to lack of precise understanding of molecular mechanisms leading to neurodegeneration. Aberrant cell cycle activation has been implicated in neuronal death pathways of various neurodegenerative diseases including PD. This study investigates the role of cell cycle regulator Cell division cycle 25A (Cdc25A) in a PD-relevant neuron death model induced by 6-OHDA treatment. We find Cdc25A is rapidly elevated, activated and is playing a key role in neuron death by regulating Rb phosphorylation and E2F1 activity. Knockdown of Cdc25A via shRNA downregulates the levels of pro-apoptotic PUMA, an E2F1 target and cleaved Caspase-3 levels, suggesting Cdc25A may regulate neuronal apoptosis through these effectors. Our work sheds light on the intricate signaling networks involved in neurodegeneration and highlights Cdc25A as a potential therapeutic target for mitigating aberrant cell cycle re-entry underlying PD pathogenesis. These novel insights into molecular mechanisms provide a foundation for future development of neuroprotective strategies to slow or prevent progression of this debilitating disease.

neuroscience↗

bioRxiv2024

Designing a Novel 3D Scaffold for Multiepitope Vaccine Development: Engineering Ag85a Protein for Enhanced Stability and Antigenicity

Designing multi epitope vaccine (MEV) by reverse vaccinology has become immensely important in the area of vaccine research due to the emergence of new pathogens as well as rise of multi drug resistant old evils like tuberculosis. Administering a vaccine may have the best possibility to save mankind from these unforeseeable events. The strategy of designing a MEV in-silico lies in a few basic steps, including procuring the amino acid sequence of the B cell and T cell epitopes from literature search, bioinformatics approach to construct a potent immunogen capable of eliciting both humoral and cell mediated response. But the challenge lies in the construction of a stable protein with a compact tertiary structure. Merely joining the epitopes one after another, may not be sufficient to achieve this. In this study, a methodology has been detailed to tackle this great challenge using a simple approach of protein engineering. A scaffold based MEV has been designed for the very first time against Mtb by converting a vaccine candidate protein, Ag85A into a scaffold by truncating its non-immunogenic regions so the gaps could be filled by the highly immunogenic epitopes. The stability of the MEV was estimated by molecular dynamics simulation.

biochemistry↗

bioRxiv2023

Apolipoprotein-E transforms intracellular Amyloid-β oligomers to a more toxic state

It is poorly understood why ApoE variants are major genetic risk factors in Alzheimers disease (AD), which is associated with the aggregation of amyloid beta (A{beta}). Here we directly image specific changes in small A{beta} oligomers in rat brain cells that correlate with the cellular ApoE content. An inhibitor of A{beta}-ApoE interaction suppresses this change and concomitantly reduces A{beta} toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and the stoichiometry of the oligomers. hiPSC-derived neural stem cells from Alzheimers patients also show similar changes. Interaction with ApoE therefore changes the oligomeric state, membrane affinity, and toxicity of A{beta} oligomers, and can be directly read out in live cells. Our findings suggest a rapid and quantitative assay for AD drug discovery. One-sentence summaryApoE causes specific toxicogenic modifications of A{beta} oligomers, and these changes can be directly imaged in live cells.

biochemistry↗

bioRxiv2023

Putative staphylococcal enterotoxin possesses two common structural motifs for MHC-II binding

Staphylococcus aureus has become a significant cause of health risks in humankind. Staphylococcal superantigens (SAgs) or enterotoxins are the key virulent factors that can exhibit acute diseases to severe life-threatening conditions. Recent literature reports S. aureus has steadily gained new enterotoxin genes over the past few decades. In spite of current knowledge of the established SAgs, several questions on these putative enterotoxins are still remaining unanswered. Keeping that in mind, this study sheds light on a putative enterotoxin SEl26 to characterize its structural and functional properties. In-silico analyses indicate its close relation with the conventional SAgs, especially the zinc-binding SAgs. Additionally, important residues have been predicted that are vital for T-Cell receptor (TcR) and major histocompatibility complex class II (MHC-II) interaction and compared them with established SAgs. Besides, our biochemical analyses exhibited the binding of this putative enterotoxin with MHC-II, followed by regulating pro-inflammatory and anti-inflammatory cytokines.

biochemistry↗

bioRxiv2023

The alternative sigma factor RpoD4 pulses at division and links the circadian clock and the cell cycle in Synechococcus elongatus

The cyanobacterial circadian clock is an extensive regulator of gene expression, generating a 24-hour rhythm in the majority of genes in Synechococcus elongatus PCC 7942. This raises the question of how the cyanobacterial clock regulates, and receives input from, the diverse cellular processes it controls. Alternative sigma factors, which bind to the core RNA polymerase enzyme and direct the synthesis of specific sets of transcripts, are one mechanism for the clock to set the time of its outputs. In this work, we use single-cell time-lapse microscopy to reveal the transcriptional dynamics of RpoD4, an alternative sigma factor that was previously reported as either arrhythmic or circadian. We find instead that RpoD4 pulses at cell division, dynamics missed by previous bulk averaging. The circadian clock modulates the amplitude of RpoD4 expression pulses, as well as the timing of pulses through its control of division timing. In turn, a rpoD4 mutation causes a reduction in clock period. Further, a rpoD4 mutant results in smaller cell size, and increasing expression of RpoD4 results in larger cell sizes in a dose-response manner, allowing tunable control of cell size. Thus, our single-cell analysis has revealed pulsing gene expression dynamics in cyanobacteria, linking the clock, RpoD4 and the cell cycle.

systems biology↗