Biology subjects

Govindarajan, S.

Publications and source records attributed to Govindarajan, S..

15 recordsLinked to original sources

bioRxiv2026

A phiKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases

Bacteria employ diverse DNA-targeting systems, including restriction-modification (R-M) and CRISPR-Cas, to cleave invading bacteriophage genomes. In response, phages encode counter-defense strategies that block or mitigate DNA damage. Here, we screened a panel of Pseudomonas aeruginosa phages against native and heterologous DNA-targeting systems and identified the Phikmvvirus phage genus as broadly resistant to multiple CRISPR-Cas and R-M systems. Following CRISPR-Cas12a exposure, most protospacer sequences remained genetically unchanged. However, at an intergenic protospacer, mutations accumulated with high frequency at the Cas12a cleavage site rather than within PAM or seed sequences, resembling repair-associated indels observed after genome editing in eukaryotic cells. Genetic screens to isolate Cas12a- and EcoRI-sensitized phage mutants revealed perturbations to the phage DNA ligase. A Cas12a-sensitive mutant phage was rescued by DNA ligase expression in trans, which was also sufficient to reverse CRISPR targeting of an unrelated phage. Together, our results support a model in which phiKMV-like phages tolerate certain nucleases through ligase-dependent repair of nuclease-induced double-stranded breaks.

microbiology↗

bioRxiv2026

Cell fusion reprograms tumor cells and promotes RUNX1-mediated invasion and dissemination in colorectal cancer

Metastasis remains the primary cause of cancer-related morbidity and mortality, despite significant advances in targeted therapies. Although metastatic dissemination requires tumor cells to escape the primary lesion and colonize distant organs, the mechanisms by which primary tumor cells gain metastatic competence remain poorly understood. Increasing evidence demonstrates that fusion of tumor (i.e., neoplastic) and immune (e.g., macrophages) cells generate a distinct population of tumor-immune hybrid cells with enhanced functional ability to migrate and disseminate into peripheral blood. Herein, our study investigates tumor-macrophage hybrid cells, an underexplored population of disseminated tumor cells, and their inherent heterogeneity and acquisition of molecular mechanisms underlying their dissemination as metastatic effectors in colorectal cancer (CRC). Through hybrid cell phenotyping utilizing integrative single-cell RNA sequencing (scRNA-seq), cyclic immunofluorescence (cyCIF) and functional assays with an in vitro model of CRC hybrid cells, we identify Runt-related transcription factor 1 (Runx1) as a central regulator of hybrid cell motility and invasion. Runx1 depletion in hybrid cells suppressed functional protease expression, chemotactic activity and extracellular matrix (ECM) invasion. Furthermore, pharmacologic inhibition of RUNX1 in an in vivo model reduced hybrid tumor growth and dissemination into peripheral blood, key attributes of metastatic spread of disease. In patients with CRC, RUNX1+ hybrid cells were identified in both primary tumor and peripheral blood, where circulating hybrid cells (CHCs) exhibited enriched migratory and epithelial-to-mesenchymal transition (EMT) phenotypes. Taken together, these findings reveal a mechanistic role for RUNX1 in driving invasive behavior of tumor-immune hybrids and highlight disseminated CHCs as an under-recognized contributor to metastatic spread and a promising noninvasive biomarker for tumor progression.

cancer biology↗

bioRxiv2026

miniMTI: minimal multiplex tissue imaging enhances biomarker expression prediction from histology

Virtual multiplexing from routine histology has advanced rapidly, yet morphology alone provides limited access to molecular state, imposing an intrinsic ceiling on H&E-only inference. Here, we introduce miniMTI, a molecularly anchored virtual staining framework that determines the minimal set of experimentally measured markers required, alongside H&E, to accurately reconstruct large multiplex tissue imaging (MTI) panels while preserving biologically and clinically relevant information. miniMTI learns from paired same-section H&E-MTI data using a unified multimodal generative model that can condition on arbitrary combinations of measured marker channels, coupled with an iterative panel selection strategy to rank informative molecular anchors. Across colorectal and prostate cancer cohorts spanning two MTI platforms and over 40 million cells, miniMTI reduces a 40-marker MTI assay to H&E plus as few as three measured molecular markers, while accurately recovering withheld markers, preserving cellular phenotypes and spatial tissue architecture, and disease-associated molecular programs, including Gleason grade-linked signatures. By integrating histology context with sparse molecular grounding, miniMTI overcomes the limitations of morphology-only virtual staining and provides a scalable, cost-effective approach for expanding MTI-level biomarker coverage with retained biological interpretability and clinical relevance.

bioinformatics↗

bioRxiv2026

Periplasmic proteostasis enables bacterial survival during MreB cytoskeletal disruption

The bacterial actin homolog MreB is essential for various cellular processes, including cell wall biosynthesis, membrane organization, and cell polarity determination. Given its multifaceted roles, MreB is considered a potential target for antibiotic development. However, the bacterial response to MreB inhibition and the factors contributing to bacterial survival under such conditions are not well understood. In this study, RNA sequencing (RNA-seq) was used to identify genes that are differentially expressed in response to MreB inhibition by the A22 antibiotic or to deletion of the mreBCD operon. We identified 6 upregulated genes and 24 downregulated genes under both conditions. To determine whether the upregulated genes contribute to bacterial survival during MreB inhibition, we performed A22 antibiotic susceptibility assay on mutants deleted for each of the 6 upregulated genes. Our findings reveal that cells lacking DegP, a periplasmic serine protease, are highly suceptible to A22 treatment. Complementation analysis showed that wild-type DegP, but not a protease-defective mutant, mitigated the effects of A22. The morphological defects in DegP-deficient cells caused by A22 were reduced by ectopic expression of related periplasmic proteases, such as DegQ and DegS. Furthermore, elevated temperatures could alleviate the effects of A22 in a DegP-dependent manner. Overall, our study provides a comprehensive analysis of the global transcriptome-wide effects of MreB inhibition, offering new insights into bacterial response to cytoskeleton disruption. The findings highlight the critical role of DegP in bacterial survival during MreB inhibition and suggest potential avenues for developing novel combinatorial antibiotic strategies targeting MreB and DegP. HighlightsO_LIMreB disruption by A22 or mutation causes major transcriptome alterations C_LIO_LISix genes are consistently upregulated under both MreB disruption conditions C_LIO_LIDegP, a periplasmic protease, is crucial for bacterial tolerance to A22 C_LIO_LIElevated temperatures alleviate A22 toxicity in a DegP-dependent manner C_LI

microbiology↗

bioRxiv2025

Crowding drives terminal investment in a generalist pest

Apart from genotype and environment, several fitness related traits of individuals are also influenced by the conditions experienced by their parents, i.e., by parental effects. To uncover the underlying physiological or molecular mechanisms, most studies analyze specific drivers in isolation, such as parental age or the degree of crowding experienced by the parents. However, potential interaction effects between different aspects of parental context are poorly understood. We analyzed the combined effect of parental age and population density on the number, fitness and physiology of offspring, in the red flour beetle Tribolium castaneum. Both factors independently reduce fitness, leading us to predict that old parents from high density should produce fewer offspring of the lowest quality. Surprisingly, older parents exposed to crowding produced more eggs than expected. These eggs were larger, had higher survival in an optimal as well as a suboptimal resource, and were provisioned with more lipids. This result is consistent with terminal reproductive investment in response to high density. The interaction between two ecologically relevant contexts -- ageing and crowding -- thus drives terminal investment via nutrient provisioning, perhaps contributing to the success of this generalist pest.

evolutionary biology↗

bioRxiv2025

Language of Stains: Tokenization Enhances Multiplex Immunofluorescence and Histology Image Synthesis

Multiplex tissue imaging (MTI) is a powerful tool in cancer research, allowing spatially resolved, single-cell phenotype analysis. However, MTI platforms face challenges such as high costs, tissue loss, lengthy acquisition times, and complex analysis of large, multichannel images with batch effects. To address these challenges, we propose a novel computational method to model the interactions between dozens of panel markers and Hematoxylin & Eosin (H&E) staining, enabling in-silico generation of marker stains. This approach reduces the reliance on experimentally measured markers, bridging low-cost H&E data with MTIs high-content information. Our approach uses a two-stage frame-work for channel-wise bioimage synthesis: first, vector quantization learns a visual token vocabulary, then a bidirectional transformer infers missing markers through masked language modeling. Comprehensive bench-marking across different MTI platforms and tissue types demonstrates the effectiveness of our method in improving marker prediction while maintaining biological relevance. This advance makes high-dimensional multiplex tissue imaging more accessible and scalable, supporting deeper insights and potential clinical applications in cancer research.

bioinformatics↗

bioRxiv2025

KilR of E. coli Rac prophage is a dual morphogenetic inhibitor of bacterial cell shape

Bacterial cryptic prophages encode genes that reduce the viability of the host, upon induction, but also contribute to host survival during stress conditions. Rac is a cryptic prophage of Escherichia coli and it encodes a toxic protein KilR which causes morphological defects to the host. But the mechanistic basis of its action is not well understood. In this study, we provide evidence that KilR is a dual morphogenetic inhibitor that affects cell division and cytoskeletal organization. We show that KilR expression is highly toxic, as demonstrated previously, and its predicted C-terminal unstructured region plays a crucial role in its function via a length-dependent manner. Low levels of KilR expression lead to cell filamentation and disruption of Z-rings, while high levels result in rod-shaped defects and mislocalization of the MreB cytoskeletal protein. Using fluorescent fusions, we observed that KilR is diffusively localized in the cytoplasm. When MreBCD proteins are overexpressed, KilR co-localizes with them, forming membrane-associated filaments, indicating a physical association. However, overexpressed MreBCD proteins does not alleviate the KilR-associated growth defect, unlike FtsZ. Finally, we present evidence that chromosomal KilR contributes to the co-inhibition of FtsZ and MreB localization in response to oxidative stress. Our data indicate that KilR inhibits MreB-associated cytoskeletal system, in addition to its effect on FtsZ-associated cell division system. We propose that dual inhibition activity of KilR contributes to its high level of toxicity and to its function in SOS-independent DNA damage tolerance during oxidative stress. IMPORTANCEKilR is a Rac cryptic prophage encoded toxic protein which contributes to host survival during oxidative stress conditions. It is known to inhibit cell division by targeting the tubulin homolog, FtsZ. In this study, we show that KilR is a dual morphogenetic inhibitor that affects FtsZ-mediated cell division and MreB-mediated cell elongation. Simultaneous inhibition of cell division and cell elongation are known to be crucial for bacterial survival during stress conditions like oxidative stress. Our study identifies KilR as a dual morphogenetic inhibitor, offering insights into how bacterial-phage coevolution drives the emergence of cryptic prophage elements, with specific genes enhancing bacterial fitness.

microbiology↗

bioRxiv2024

Probing the Molecular Interactions of A22 with Prokaryotic Actin MreB and Eukaryotic Actin: A Computational and Experimental Study

Actin is a major cytoskeletal system that mediates the intricate organization of macromolecules within cells. The bacterial cytoskeletal protein MreB is a prokaryotic actin-like protein governing cell shape and intracellular organization in many rod-shaped bacteria including pathogens. MreB stands as a target for antibiotic development, and compounds like A22 and its analogue, MP265, are identified as potent inhibitors of MreB. The bacterial actin MreB shares structural homology with eukaryotic actin, despite lacking sequence similarity. It is currently not clear whether small molecules that inhibit MreB can act on the eukaryotic actin due to their structural similarity. In this study, we investigate the molecular interactions between A22 and both MreB and eukaryotic actin through molecular dynamics approach. Employing MD simulations and free energy calculations with an all-atom model, we unveil robust A22-MreB interaction and substantial binding affinity with eukaryotic actin. Experimental assays reveal A22s toxicity to eukaryotic cells, including yeast and human glioblastoma cells. Microscopy analysis demonstrates profound effects of A22 on actin organization in human glioblastoma cells. Overall, this integrative computational and experimental study advances our understanding of A22s mode of action and highlights its potential as a versatile tool for probing actin dynamics and as a candidate for therapeutic intervention in pathological conditions like cancer.

biophysics↗

bioRxiv2022

An international scholastic network to generate LexA enhancer-trap lines for Drosophila

Conditional gene regulation in Drosophila through binary expression systems like the LexA-LexAop system provides a superb tool for investigating gene and tissue function. To increase the availability of defined LexA enhancer trap insertions, we present molecular, genetic and tissue expression studies of 301 novel Stan-X LexA enhancer traps derived from mobilization of the index SX4 line. This includes insertions into distinct loci on the X, II and III chromosomes that were not previously associated with enhancer traps or targeted LexA constructs, an insertion into ptc, and eleven insertions into natural transposons. A subset of enhancer traps was expressed in CNS neurons known to produce and secrete insulin, an essential regulator of growth, development and metabolism. Fly lines described here were generated and characterized through studies by students and teachers in an international network of genetics classes at public, independent high schools, and universities serving a diversity of students, including those underrepresented in science. Thus, a unique partnership between secondary schools and university-based programs has produced and characterized novel resources in Drosophila, establishing instructional paradigms devoted to unscripted experimental science.

genetics↗

bioRxiv2021

NiftyTorch: A Deep Learning framework for NeuroImaging

We present NiftyTorch a Deep Learning Framework for NeuroImaging. The motivation behind the development of such a library is that there are scant amount of centralized tool for deploying 3D deep learning for NeuroImaging. In addition, most of the existing tools require expert technical knowledge in Deep Learning or programming, creating a barrier for entry. The goal is to provide a one stop package using which the users can perform classification tasks, Segmentation tasks and Image Transformation tasks. The intended audience are the members of NeuroImaging who would like to explore deep learning but have no background in coding. In this article we explore the capabilities of the framework, the performance of the framework and the future work for the framework.

neuroscience↗

bioRxiv2020

The evolutionary history of topological variations in the CPA/AT superfamily

CPA/AT transporters consist of two structurally and evolutionarily related inverted repeat units, each of them with one core and one scaffold subdomain. During evolution, these families have undergone substantial changes in structure, topology and function. Central to the function of the transporters is the existence of two non-canonical helices that are involved in the transport process. In different families, two different types of these helices have been identified, reentrant and broken. Here, we use an integrated topology annotation method to identify novel topologies in the families. It combines topology prediction, similarity to families with known structure, and the difference in positively charged residues present in inside and outside loops in alternative topological models. We identified families with diverse topologies containing broken or reentrant helix. We classified all families based on 3 distinct evolutionary groups that each share a structurally similar C-terminal repeat unit newly termed as "Fold-types". Using the evolutionary relationship between families we propose topological transitions including, a transition between broken and reentrant helices, complete change of orientation, changes in the number of scaffold helices and even in some rare cases, losses of core helices. The evolutionary history of the repeat units shows gene duplication and repeat shuffling events to result in these extensive topology variations. The novel structure-based classification, together with supporting structural models and other information, is presented in a searchable database, CPAfold (cpafold.bioinfo.se). Our comprehensive study of topology variations within the CPA superfamily provides better insight about their structure and evolution.

bioinformatics↗

bioRxiv2020

Identification of a novel fold type in CPA/AT transporters by ab-initio structure prediction

Members of the CPA/AT transporter superfamily show significant structural variability. All previously known members consist of an inverted duplicated repeat unit that folds into two separate domains, the core and the scaffold domain. Crucial for its transporting function, the central helix in the core domain is a noncanonical transmembrane helix, which can either be in the form of a broken helix or a reentrant helix. Here, we expand the structural knowledge of the CPA/AT family by using contact-prediction-based protein modelling. We show that the N-terminal domains of the Pfam families; PSE (Cons_hypoth698 PF03601), Lysine exporter (PF03956) and LrgB (PF04172) families have a previously unseen reentrant-helix-reentrant fold. The close homology between PSE and the Sodium-citrate symporter (2HCT) suggests that the new fold originates from the truncation of an ancestral reentrant protein, caused by the loss of the C-terminal reentrant helix. To compensate for the lost reentrant helix one external loop moves into the membrane to form the second reentrant helix, highlighting the adaptability of the CPA/AT transporters. This study also demonstrates that the most recent deep-learning-based modelling methods have become a useful tool to gain biologically relevant structural, evolutionary and functional insights about protein families.

bioinformatics↗

bioRxiv2020

Direct observation of genome surveillance by CRISPR-Cas in bacteria

CRISPR-Cas systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remains largely unaddressed. Here, we used fluorescent fusion proteins to study the localization of the Type I-F CRISPR-Cas system native to Pseudomonas aeruginosa. When targeted to an integrated prophage, the crRNA-guided (Csy) complex and a majority of Cas3 molecules in the cell are recruited to a single focus. When lacking a target in the cell, however, the Csy complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. Nucleoid association for the Csy proteins is crRNA-dependent, and inhibited by expression of anti-CRISPR AcrIF2, which blocks PAM binding. The Cas9 nuclease is also nucleoid localized, only when gRNA-bound, which is abolished by PAM mimic, AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in Type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex.

microbiology↗

bioRxiv2020

Tyrosine phosphorylation-dependent localization of TmaR, a novel E. coli polar protein that controls activity of the major sugar regulator by polar sequestration

The poles of E. coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a novel protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization of the general PTS protein Enzyme I (EI) by preventing it from exerting its activity by binding and polar sequestration, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results argue that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release. SIGNIFICANCEIn recent years, we have learnt that bacterial cells have intricate spatial organization despite the lack of membrane-bounded organelles. The endcaps of rod-shaped bacteria, termed poles, are emerging as hubs for sensing and responding, but the underlying mechanisms for positioning macromolecules there are largely unknown. We discovered a novel protein, TmaR, whose polar localization depends on a phospho-tyrosine modification. We show that TmaR controls the activity of EI, the major regulator of sugar metabolism in most bacteria, by polar sequestration and release. Notably, TmaR is essential for survival in conditions that E. coli often encounters in nature. Hence, TmaR is a key regulator that connects tyrosine phosphorylation, spatial regulation, sugar metabolism and survival in bacteria and the first protein reported to recruit proteins to the E. coli cell poles.

microbiology↗

bioRxiv2019

CRISPR-Cas immunity repressed by a biofilm-activating pathway in Pseudomonas aeruginosa

CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB/AlgB), previously characterized in regulating Pseudomonas aeruginosa virulence and biofilm establishment, as a regulator of the biogenesis and activity of the Type I-F CRISPR-Cas system. Downstream of the KinB/AlgB system, activators of biofilm production AlgU (a {sigma}E orthologue) and AlgR, act as repressors of CRISPR-Cas activity during planktonic and surface-associated growth. AmrZ, another biofilm activator, functions as a surface-specific repressor of CRISPR-Cas immunity. Pseudomonas phages and plasmids have taken advantage of this regulatory scheme, and carry hijacked homologs of AmrZ, which are functional CRISPR-Cas repressors. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.

microbiology↗