Biology subjects

Singh, N.

Publications and source records attributed to Singh, N..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Serine recombinases are conserved genetic markers of antiphage defense systems

Renewed interest in phage therapy has highlighted a need to understand how bacteria subvert phage infection through antiphage defense systems. Traditionally, strategies to identify antiphage defense systems lack throughput or have limitations for bacterial species where antiphage defense systems are understudied. Herein, we developed a bioinformatic pipeline that uses a small serine recombinase to identify known and unknown antiphage defense systems. Using this approach to query reference genomes and metagenomes, we show that small serine recombinase genes are genetically linked to antiphage defense systems and serve as bait for finding these systems across diverse bacterial phyla. Using co-transcription predictions and statistical analysis of protein domain abundances, we experimentally validated our bioinformatic approach by discovering that KAP P-loop NTPases are fused to putative antiphage domains and reinforce prokaryotic Schlafen proteins as a new class of antiphage defense. Our work shows that small serine recombinases are a reliable genetic marker for the discovery of antiphage defenses across diverse bacterial phyla.

microbiology↗

bioRxiv2025

The Liver is an Inflammatory Mediator of Pulmonary Arterial Hypertension

The livers contribution to pulmonary arterial hypertension (PAH) pathogenesis remains unclear. We hypothesized that the liver promotes inflammatory injury to the pulmonary endothelium. PAH patients without liver disease with pulmonary artery endothelial cell (PAEC) biopsies were included. Liver serologies and imaging were analyzed by unsupervised classification and regression tree (CART) to identify subclinical liver dysfunction clusters. Two machine-learning models predicted cluster assignment and informed differential expression. PAEC transcriptomes were compared to liver and lung data from monocrotaline and Sugen-Hypoxia rats. Liver fibrosis was assessed in rat and human PAH livers. Among 25 PAH patients (76% female, median age 61 [30 - 84] years), CART identified clusters distinguished by Model for End-Stage Liver Disease Sodium (MELD-Na) [≥]12, predicting higher pulmonary vascular resistance ({beta}=0.5 Wood units per point increase in MELD-Na, 95% CI 0.2-0.8, p=0.005) after adjustment for right atrial pressure. Subjects with MELD-Na [≥]12 had decreased 6-minute walk distance (353 [120 - 576] m vs. 411[300 - 600] m, p=0.03), with upregulation of apelin, beta-catenin, and immune signaling. Rat lung ECs demonstrated survival and hepatic growth-factor signaling, while rat livers showed immune activation. Rat (20.8 vs 16.6 % area stained, p=0.09) and human PAH livers revealed fibrosis despite absent right ventricular failure, supporting a pathogenic lung-liver axis in PAH.

cell biology↗

bioRxiv2025

Liver angiocrine myeloid-derived growth factor protects against endothelial dysfunction in pulmonary arterial hypertension

Myeloid-derived growth factor (MYDGF) is a hepatic angiokine with protective effects in systemic vascular beds, but its role in pulmonary arterial hypertension (PAH) is unknown. We hypothesized that hepatic MYDGF deficiency contributes to pulmonary endothelial activation in PAH and that recombinant MYDGF could rescue endothelial injury. In the Sugen-hypoxia (SuHx) rat model, hepatic MYDGF expression was decreased, while pulmonary vascular cell adhesion molecule-1 (VCAM-1) expression was increased. Human hepatic sinusoidal endothelial cells exposed to pro-inflammatory macrophage conditioned media downregulated MYDGF, and recombinant MYDGF restored pulmonary artery endothelial cell resistance to inflammatory activation via MAP4K4-NF{kappa}B signaling. In the Brown University PHiNE PAH cohort (n=41 PAH, n=27 controls), plasma proteomics demonstrated increased MYDGF in PAH patients compared with controls, but MYDGF levels declined with worsening liver stiffness and correlated with higher pulmonary vascular resistance. In the independent Servetus PAH cohort (n=117), higher plasma MYDGF was associated with mortality and right ventricular dilation. Together, these findings demonstrate hepatic MYDGF deficiency in experimental PAH, tissue specificity of endothelial MYDGF to the liver, and MYDGFs potential to mitigate pulmonary endothelial inflammation. However, human data suggest a paradoxical association of elevated circulating MYDGF with adverse outcomes, underscoring the complex biology of angiogenic growth factors in PAH. MYDGF may represent a novel hepatic angiokine linking systemic inflammation, liver dysfunction, and pulmonary vascular disease.

molecular biology↗

bioRxiv2025

Multidrug resistant E. coli isolate along with ESBL production and Antimicrobial Susceptibility pattern from patients of Urinary Tract Infection from tertiary care hospital, Delhi

BackgroundThird generation Cephalosporins resistances among Escherichia coli due to production of ESBL, mainly the bla-CTX-M, bla-TEM and bla-SHV genes, poses serious challenges in the clinical utility of these drugs in healthcare settings. This study was undertaken to detect prevalence of ESBL producing MDR E. coli isolates by various phenotypic and molecular method from in or out patients with urinary tract infection (UTI). MethodsA total of 56 non-repetitive isolates of E. coli from in or out patients of UTI were subjected to antimicrobial susceptibility testing. Screening for the production of extended-spectrum {beta}-lactamases (ESBL) was determined in third generation Cephalosporins resistant isolates by minimum inhibitory concentration (MIC) E-strip, followed by phenotypic confirmation by DDST, CDST and ESBL E-strip method. Further confirmation was done by molecular method, PCR was carried out for detection of bla-CTX-M, bla-TEM and bla-SHV. ResultsAll strains (56/56) were observed as ESBL producers by MIC E-strip method, 94.64% (53/56) isolates were observed as ESBL producer by DDST and 67.86% (38/56) were observed by CDST. The overall ESBL production among all MDR E. coli isolates was 89.29% (50/56) which includes any one of the three genes. The prevalence of bla-CTX-M (80.36%, 45/56) was highest, followed by bla-TEM (50%, 28/56) and bla-SHV (10.71%, 6/56). ConclusionIn our study we found bla-CTX-M gene is predominantly circulating among all three ESBL genes in the clinical isolates of MDR E. coli that were producers of ESBL.

microbiology↗

bioRxiv2025

MYC-Driven Activation of USP39 Enhances SRSF1 Stability and Promotes PDAC Progression

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy primarily driven by oncogenic KRAS signaling. The splicing factor SRSF1 plays a key oncogenic role in PDAC, where its tightly regulated expression constrains KRAS-driven signaling under normal conditions, while its upregulation promotes tumorigenesis. SRSF1 expression is regulated in part by proteostasis. However, the precise mechanisms remain unclear. Here, we identify USP39 as a deubiquitinase that interacts with SRSF1 in an RNA-independent manner and stabilizes it by reducing ubiquitination. USP39 expression is elevated in PDAC tumors and precancerous lesions, and correlates with poor patient survival. USP39 knockdown suppresses PDAC cell proliferation and migration, effects that are partially rescued by SRSF1 overexpression. Mechanistically, we show that MYC directly activates USP39 transcription via E-box motifs within its exon 1b promoter, linking MYC-driven transcriptional regulation to post- translational stabilization of SRSF1. Together, these findings define a MYC-USP39-SRSF1 regulatory axis that integrates transcriptional and post-translational mechanisms in PDAC and highlight USP39 as a potential therapeutic target.

cancer biology↗

bioRxiv2025

Valorization of Paneer waste Whey through Fermentation with Pediococcus pentosaceus NCDC 273 Insights from Intracellular Metabolomics by GC-MS

The present study employed an untargeted GC-MS-based metabolomics approach to investigate the intracellular metabolic landscape of Pediococcus pentosaceus NCDC 273 during the fermentation of paneer whey. The goal was to understand the metabolic dynamics that enable this bacterium to efficiently utilize a dairy by-product and produce value-added compounds. Intracellular metabolites were profiled at the early (6 h) and late (18 h) exponential growth phases, revealing a distinct temporal metabolic reprogramming. Multivariate analysis and correlation networks confirmed significant metabolic shifts, where the early phase was characterized by active uptake and utilization of diverse sugars and fatty acids to support rapid proliferation. In contrast, the late phase demonstrated a metabolic reorientation towards energy production, redox balance, and stress adaptation. This was evidenced by the significant accumulation of key metabolites such as lactic acid, nicotinamide, and trehalose, which are crucial for maintaining growth and tolerance in an acidifying environment. Overall, the findings demonstrate that P. pentosaceus NCDC 273 possesses the metabolic flexibility to effectively channel whey-derived sugars into central metabolism, while concurrently deploying adaptive strategies for survival. This research represents the first intracellular metabolomic characterization of this strain during whey fermentation, providing novel mechanistic insights that reinforce its potential for the valorization of paneer whey into functional bioproducts.

microbiology↗

bioRxiv2025

Replisome progression regulates R-loop mediated transcriptional repression

Maintaining cellular proliferation necessitates the synchronized activity of diverse molecular machineries operating in parallel on the genome. Wide-spread transcription and R-loop formation can interfere with genome duplication, causing transcription-replication conflicts (TRCs). Here, we use live-cell imaging for simultaneous monitoring of replication fork progression and transcription dynamics of an R-loop prone gene. While robust replisome progression through R-loops is observed in wild-type cells, it is impaired in RnaseH, Mph1 and Sen1 mutants. Intriguingly, we find that R-loop formation inhibits gene transcription, but this inhibition is reversed by replisome passage, demonstrating the dynamic crosstalk between replication, transcription and R-loops in a single cell cycle. Unexpectedly, we find that R-loops also have beneficial roles in reducing the density of RNAPII molecules on the gene, thereby preventing fork stalling upon high RNAPII occupancy. These findings illuminate that regulating R-loops and RNAPII density together is critical for preventing harmful TRCs.

cell biology↗

bioRxiv2025

B cells specific for polyomavirus-derived oncoprotein are predictive of Merkel cell carcinoma progression

Merkel cell carcinomas typically arise from clonal integration of the Merkel cell polyomavirus. Immunogenic viral oncoproteins then lead to tumorigenesis. Oncoprotein-specific T cells are essential for anti-MCC immunity, but it is unclear whether B cells promote tumor control. Here, we analyzed the frequency and phenotype of viral oncoprotein-specific and total B cells in 47 blood samples and 19 unmatched tumors from MCC patients-- of which 8 out 19 progressed. The phenotype of blood B cells did not correlate with MCC patient outcomes. In contrast, all 11 patients with robust oncoprotein-specific antibody-secreting and/or germinal center B cells in tumors experienced long-term MCC control. In vitro, B cells engineered to be specific for viral oncoproteins increased the sensitivity of oncoprotein-specific CD4+ T cells by over 50-fold. Together, our findings suggest that cancer-specific B cells promote anti-tumor immunity via increased T cell responses and that cancer-specific B cell augmentation could be therapeutically relevant. Statement of SignificanceThe link between cancer-specific B cells in anti-tumor immunity and clinical outcomes remains poorly defined. Here, we show that tumor-associated B cells specific for a viral oncoprotein expressed in MCC patient tumors predict disease control with remarkable accuracy, establishing their potential as active participants in tumor immunity.

immunology↗

bioRxiv2025

Comparative Assessment of Chlorella vulgaris Cultivation in Synthetic, Oligotrophic and Industrial Wastewater for Sustainable Biomass Production

This study evaluates the physiological and biochemical responses of Chlorella vulgaris cultivated in synthetic BG11 medium, oligotrophic water, and industrial wastewater under controlled laboratory conditions with optimized pH and nitrogen-to-phosphorus (N:P) ratios. The highest cell density, growth rate, and pigment accumulation were observed in BG11 at neutral pH and higher N:P ratios. Moderately adjusted N:P ratios in industrial wastewater produced comparable biomass yields and improved photosynthetic efficiency, demonstrating its potential as a cost-effective medium for integrated wastewater treatment and algal biomass production. In contrast, oligotrophic water severely limited growth due to nutrient deficiency. FTIR analyses indicated that C. vulgaris maintains essential biochemical components under nutrient stress through adaptive metabolism. This comparative study provides new insights for designing scalable, sustainable microalgal cultivation systems that link biomass production with wastewater management within a circular bioeconomy framework.

systems biology↗

bioRxiv2025

OocystMeter, a machine-learning algorithm to count and measure Plasmodium oocysts, reveals clustering patterns in the Anopheles midgut

We present OocystMeter, a machine learning-based software developed to automate the segmentation of malaria oocysts from images of mosquito midguts stained with mercurochrome. Existing bioimage analysis tools, including machine learning-based ones, often struggle with the unique staining patterns, complex midgut backgrounds, and variable morphology of oocysts, making the determination of oocyst size and numbers cumbersome. To overcome these challenges, we curated a high-quality dataset comprised of 11,178 Plasmodium falciparum oocysts in Anopheles gambiae midguts annotated by expert parasitologists. Using this dataset, we fine-tuned a Mask R-CNN object detection model to achieve segmentation accuracy comparable to human parasitologists (Spearmans correlation of 0.998 for oocyst counts and 0.978 for size measurements). Applying this tool in conjunction with spatial analysis, we uncovered a non-random, clustered spatial distribution of oocysts independent of the midguts anatomical regions or geometric axes, particularly in infections with fewer than 75 oocysts/midgut. Our workflow significantly accelerates malaria oocyst intensity and size analysis, reduces human bias, and provides spatial coordinates for advanced parasitology studies. OocystMeter is freely available at https://github.com/duopeng/OocystMeter, and as a web tool at http://Oocystmeter.org/, offering a valuable resource for researchers investigating the oocyst stage of malaria development.

bioinformatics↗

bioRxiv2025

The intracellular vacuolar pathogen Coxiella burnetii promotes exocytosis in a type IVB secretion system dependent manner

Coxiella burnetii is an obligate intracellular bacterial pathogen that causes a zoonotic disease known as Q fever. Upon internalization into host cells, Coxiella extensively remodels host vesicle trafficking and replicates within acidic, lysosome-derived, spacious vacuoles by secreting a suite of effectors through the type IVB secretion system (T4BSS), and hijacking the host cell machinery. However, how fundamental lysosomal functions, such as exocytosis, are subverted during Coxiella infection has not been well understood. In this study, we aimed to investigate the regulation and role of exocytosis in the context of Coxiella infection. Biochemical and fluorescence-based imaging approaches indicated that C. burnetii infection promotes release of extracellular vesicles (EVs). Increase in extracellular levels of endolysosomal proteins (LAMP1 and cathepsin D) and surface LAMP1 expression were identified in both phagocytic and non-phagocytic cells, during later stages of infection. Interestingly, infection-induced exocytosis was dependent on the activity of the bacterial T4BSS. Modulating the activity of TRPML1, a host calcium channel that induces exocytosis by regulating the release of calcium from lysosomes into the cytosol, using synthetic agonist/antagonist, led to a decrease in intracellular C. burnetii replication, suggesting a complex, tightly regulated role of TRPML1 during infection. Together, this study demonstrates that infection by the lysosome-adapted pathogen Coxiella burnetii activates exocytosis in a temporal, host and bacterial factors-dependent manner.

cell biology↗

bioRxiv2025

MPK3 mediated phosphorylation of WRKY48 down regulates CIPK6 expression during PstDC3000 challenge in Arabidopsis

WRKY transcription factor (TF) family are one of the key regulators of plant immune responses, functioning as both activators and suppressors depending on the pathogen encountered. Among these, WRKY48, a class IIC TF, functions as a negative regulator of pattern-triggered immunity (PTI) in Arabidopsis thaliana. Our study explicates the regulatory mechanisms underlying the function of WRKY48, revealing its role in repressing the expression of CIPK6, a negative regulator of immune response in Arabidopsis, during Pseudomonas syringae DC3000 (PstDC3000) infection. This suppression is mediated through MPK3-dependent phosphorylation of WRKY48. In vitro phosphorylation assays demonstrated that MPK3 primarily phosphorylates WRKY48 at serine residues S35 and S40. The phosphorylated WRKY48 predominantly localizes to the nucleus, enhancing its DNA-binding affinity and trans-repressive activity at specific W-box motifs in the CIPK6 promoter. Whereas invivo dual luciferase shows that unphosphorylated WRKY48 (S26S35S40 / A26A35A40 ) unable to repress luciferase activity of CIPK6 promoter. The bioinformatics analysis identified two WRKY-binding sites (P1 and P2) on the CIPK6 promoter and electrophoretic mobility shift assays confirms the preferential binding of WRKY48 to the P1 site. Unlike other WRKY TFs (WRKY28 and WRKY8) in the same subgroup, WRKY48 exhibited unique specificity, highlighting the distinct regulatory roles within the WRKY family. Functional studies demonstrated that phosphorylation of WRKY48 enhances its ability to suppress CIPK6 both transcriptionally and translationally, leading to increased reactive oxygen species (ROS) levels and decreased susceptibility to PstDC3000. The reduced expressions of PTI marker genes and ROS-associated genes in wrky48 mutants were associated with enhanced resistance in wrky48 and cipk6 mutants and emphasizes the critical role of WRKY48 in fine-tuning immune responses. Our findings identify a novel MPK3-WRKY48-CIPK6 signaling module that connects calcium-dependent pathways to MAPK signalling to modify plant immunity. HighlightsO_LIWRKY48, a class IIC transcription factor, functions as a negative regulator of pattern-triggered immunity (PTI) in Arabidopsis thaliana during Pseudomonas syringae DC3000 (PstDC3000) infection. C_LIO_LIWRKY48 suppresses the expression of CIPK6, a negative regulator of immune signaling, in response to bacterial infection. C_LIO_LIMAP kinase MPK3 phosphorylates WRKY48 at serine residues S35 and S40, enhancing its nuclear localization and transcriptional repressive activity C_LIO_LIPhosphorylated WRKY48 shows increased binding affinity to W-box motifs in the CIPK6 promoter, especially at the P1 binding site, as confirmed by electrophoretic mobility shift assays (EMSA). C_LIO_LIUnlike WRKY28 and WRKY8, WRKY48 exhibits unique binding specificity, suggesting functional diversification within WRKY subgroup IIC. C_LIO_LISuppression of CIPK6 by phosphorylated WRKY48 leads to: Elevated ROS accumulation, Reduced PTI marker gene expression, Increased susceptibility to PstDC3000 C_LI

molecular biology↗

bioRxiv2025

Aeromonas: Genomic Insights into an Environmental Pathogen and Reservoir of Antimicrobial Resistance

Aeromonads are an ecologically versatile group of bacteria that cause infection in aquatic animals and are recognised as an emerging human pathogen. Despite this, our understanding of Aeromonas diversity, especially the relationship between clinical and environmental strains, remains limited. Here, we present a complete view of the Aeromonas genus, comprising 1,853 genomes, and a detailed comparison of clinical and environmental strains from South Asia, including 996 newly sequenced genomes from Bangladesh and India. Phylogenetic analyses revealed that Aeromonas is a highly diverse genus, with no distinct clade separating clinical and environmental isolates. We identified 28 Aeromonas species and 905 novel sequence types, comprising 72.5% of the genomes. Notably, we show a high incidence of AMR genes across all isolates, including against front and last-line antibiotics. Finally, we highlight frequent misidentification of Aeromonas as Vibrio cholerae, key to cholera-endemic regions where both genera co-exist and are associated with diarrhoeal disease. Our study underscores Aeromonas as an important environmental AMR reservoir and emerging multi-species pathogen capable of spilling over into human populations.

genomics↗

bioRxiv2025

CBL1/9-CIPK6 complex negatively regulates Respiratory burst oxidase homolog D in Arabidopsis thaliana

Plant innate immune response must be a well-balanced process with positive and negative regulations for the plants to survive. Calcium signalling is essential for pathogen-associated molecular pattern (PAMP)-driven Respiratory burst oxidase homolog D (RbohD)-mediated reactive oxygen species (ROS) burst. We show Calcium-sensors Calcineurin B like protein 1 (CBL1) and CBL9 and their interacting protein kinase CIPK6 negatively regulate RbohD activity and immune response in Arabidopsis thaliana. Arabidopsis mutant cbl1cbl9, like cipk6, exhibited enhanced resistance and ROS production when infected with the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). CBL1 and CBL9 enhanced kinase activity of CIPK6. Plasma membrane localization of CBL1 and CBL9 and CIPK6 kinase activity were associated with the ROS production and immune response. CBL1/9-CIPK6 module interacts with RbohD at the plasma membrane and phosphorylates its N-terminal cytoplasmic domain at a non-conserved (Ser33) and a conserve (Ser39) Serine residue. While Ser39 phosphorylation increased RbohD activity, Ser33 phosphorylation drastically reduced it and superseded the effect of Ser39 phosphorylation. Replacement of Ser33 with alanine or aspartic acid made RbohD a super-active or low-active enzyme, respectively. Our study reports a direct mechanism of negative regulation of ROS production and plant immune response by a Calcium-signalling module in Arabidopsis. Overall, this study provides a novel insight into how calcium signaling integrates with immune regulation to prevent excessive ROS accumulation, ensuring a balanced plant immune response. One Sentence SummaryInactivation of RbohD by CBL1/CBL9-CIPK6 complex negatively regulates the plant immunity against Pst DC3000 infection in Arabidopsis.

plant biology↗

bioRxiv2025

Adaptive Example Selection: Prototype-Based Explainability for Interpretable Mitosis Detection

Understanding the decision-making process of black-box neural network classifiers is crucial for their adoption in medical applications, including histopathology and cancer diagnostics. An approach of increasing interest is to clarify how the decisions of neural networks compare to, and perform parallel to, those of highly-trained and knowledgeable clinicians and other medical professionals within their prototypical classes of interest. Motivated by this, we introduce Adaptive Example Selection (AES), a prototype-based explainable AI (XAI) framework that facilitates the interpretability of deep learning models for mitosis detection. AES works by selecting and presenting a small set of real-world mitotic images most informative to a given classification, allowing pathologists to visually assess and understand the neural networks decision by comparing test cases with similar previously annotated examples. AES achieves this by expanding the neural networks confidence/belief function and fitting it to a radial basis function (RBF) approximator, an approach we term Decision Boundary-based Analysis (DBA). This method makes the decision boundary more transparent, offering robust visual insights into the models decisions, and thereby equipping clinicians with the information needed to effectively utilize AI-driven diagnostics. Additionally, AES includes customizable user controls, allowing clinicians to tailor decision thresholds and select prototype examples to better align with their specific diagnostic needs. This flexibility empowers users to engage with the AI model more directly and meaningfully, increasing its practical relevance in clinical settings.

pathology↗

bioRxiv2025

A Generalized Platform for Artificial Intelligence-powered Autonomous Protein Engineering

Proteins are the molecular machines of life with numerous applications in energy, health, and sustainability. However, engineering proteins with desired functions for practical applications remains slow, expensive, and specialist-dependent1-3. Here we report a generally applicable platform for autonomous protein engineering that integrates machine learning and large language models with biofoundry automation to eliminate the need for human intervention, judgement, and domain expertise. Requiring only an input protein sequence and a quantifiable way to measure fitness, this autonomous platform can be applied to engineer virtually any protein. As a proof of concept, we engineered Arabidopsis thaliana halide methyltransferase (AtHMT)4 for a 90-fold improvement in substrate preference and 16-fold improvement in ethyltransferase activity, along with developing a Yersinia mollaretii phytase (YmPhytase)5,6 variant with 26-fold improvement in activity at neutral pH. This was accomplished in four rounds over four weeks, while requiring construction and characterization of fewer than a total of 500 variants for each enzyme. This platform for autonomous experimentation paves the way for rapid advancements across diverse industries, from medicine and biotechnology to renewable energy and sustainable chemistry.

synthetic biology↗

bioRxiv2024

Cryo-EM structure of the conjugation H-pilus reveals the cyclic nature of the TrhA pilin

Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor-recipient interactions, a process known as mating pair formation (MPF). IncH are low-copy-number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from Salmonella enterica serovar Typhi, encodes the conjugative H-pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H-pilus forms long filamentous structures that mediate MPF, and describe its cryo electron-microscopic (cryo-EM) structure at 2.2 [A] resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stochiometric ratio of 1:1. While there were previous reports that the T-pilus from Agrobacterium tumefaciens was composed of cyclic subunits, three recent cryo-EM structures of the T-pilus found no such cyclization. Here, we report that the H-pilin is cyclic, with a covalent bond connecting the peptide backbone between the N- and C-termini. Both the cryo-EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxylate residues. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination. SignificanceA major medical challenge is the spread of bacteria which are resistant to antibiotics. The resistance genes are spread via mobilized DNA, mainly via a process named conjugation. During conjugation, a resistant bacterium (donor), transfers the resistance DNA to another bacterium (recipient) in a contact-dependent manner. The initial donor-recipient interaction is mediated by a hollow filament expressed by the donor, named the conjugation pilus, that binds the recipient. This pilus is built via polymerization of a small protein subunit, pilin. Here, we report the atomic structure of the H-pilus, whose pilin subunit has an unusual cyclic structure where the N- and C-termini of the protein are covalently linked by a peptide bond.

microbiology↗

bioRxiv2024

ELONGATED HYPOCOTYL 5 regulates light-mediated squalene biosynthesis and development in Arabidopsis thaliana

Terpenoids are diverse groups of metabolite families that are crucial for plant development and required in cosmetics and pharmacological industries. Various development processes and environmental factors, including light, have been shown to affect terpenoid biosynthesis. However, regulatory factors involved in such regulation have not been explored much. Squalene synthases (SQSs), key enzymes in the terpenoid pathway, are pivotal for sterol and triterpene biosynthesis across various organisms. In this study, we report that AtSQS1 expression and squalene content are higher in the dark, and light through ELONGATED HYPOCOTYL 5 (HY5) negatively regulates the AtSQS1 gene and squalene biosynthesis in Arabidopsis thaliana. Our study shows that the AtSQS1 gene is unaffected in the hy5-215 mutant during light and dark conditions, but it is down-regulated in WT and HY5OX lines. The histochemical GUS assays and GFP expression patterns indicate a negative regulation of squalene biosynthesis by AtHY5. Yeast one-hybrid assays, EMSA, and ChIP experiments have confirmed the physical binding of AtHY5 on light-responsive elements of the AtSQS1 promoter. We have validated our results by developing AtSQS1 promoter: reporter transgenic lines in WT, hy5-215 mutants, and HY5OX backgrounds and conducted expression, metabolite quantification, and histochemical GUS assays. The metabolites quantification of squalene and phytosterols through GC-MS further confirms that AtHY5 negatively regulates the squalene biosynthesis in a light-dependent manner in Arabidopsis.

plant biology↗