Biology subjects

Mitra, N.

Publications and source records attributed to Mitra, N..

8 recordsLinked to original sources

bioRxiv2024

Mutational analysis of the F plasmid partitioning protein ParA reveals novel residues required for oligomerisation and plasmid maintenance

Mobile genetic elements such as plasmids play a crucial role in the emergence of antimicrobial resistance. Hence, plasmid maintenance proteins like ParA of the Walker A type cytoskeletal ATPases/ ParA superfamily are potential targets for novel antibiotics. Plasmid partitioning by ParA relies upon ATP-dependent dimerisation and formation of chemophoretic gradients of ParA-ATP on bacterial nucleoids. Though polymerisation of ParA has been reported in many instances, the need for polymerisation in plasmid maintenance remains unclear. In this study, we provide novel insights into the polymerisation of ParA and the effect of polymerisation on plasmid maintenance. We first characterise two mutations, Q351H and W362E, in ParA from F plasmid (ParAF) that form cytoplasmic filaments independent of the ParBSF partitioning complex. Both mutants fail to partition plasmids, do not bind non-specific DNA and act as super-repressors to suppress transcription from the ParA promoter. Further, we show that the polymerisation of ParAF requires the conformational switch to the ParA-ATP* state. We identify two mutations, R320A in the C-terminal helix-14 and E375A helix-16 of ParAF, that abolish filament assembly and affect plasmid partitioning. Our results thus suggest a role for higher-order structures or polymerisation of ParA in plasmid maintenance.

microbiology↗

bioRxiv2022

A cobB like protein in Oryza sativa indica regulates the mitochondrial machinery under stress conditions

Sirtuins are ubiquitous in nature and are known to play an important role as metabolic regulators. In plants, these NAD+ dependent deacetylases are not well characterized. In this study, we are reporting a new member of sirtuin in Oryza sativa indica. It shares approx. 89% sequence identity with bacterial sirtuin, a class III sirtuin member. This protein is mostly present in mitochondria with trace amounts in the nucleus. It can physically interact with histones H3 and H4 and can specifically deacetylate histone H3 at K9 and K18 positions. In mitochondria, acetyl coA synthetase (AcS) and isocitrate dehydrogenase (IDH2) are the targets for its deacetylation. This removal of acetyl group is the mode of regulation under certain stress conditions. Thus, this is the first mitochondrial cobB targeting important plant machinery under changing growth environment. The OscobB deacetylase activity is not majorly affected by its products, NAM & ADP ribose but are sensitive to certain metal ions like Fe2+ and Mg2+. In contrast to some class III members, it does not have any ADP ribosyl transferase activity. In response to abiotic stress conditions like dehydration and low temperature, this enzyme can also mobilize to the nucleus to regulate the plant metabolism. HighlightsO_LIA new class III member of the sirtuin family found in Oryza sativa indica. C_LIO_LIUnder normal conditions, this protein is localized mostly in mitochondria, with trace amounts in the nucleus. C_LIO_LIThis enzyme is capable of using both NAD+ and NADP+ as a substrate for catalysis. C_LIO_LIIt specifically deacetylates the nuclear histone H3 at K9 and K18 positions. C_LIO_LIMitochondrial proteins acetyl coA synthetase (AcS) and isocitrate dehydrogenase (IDH2) are the regulatory targets of OscobB. C_LIO_LIUnder certain stress conditions in plants like dehydration, pathogenesis and low temperature, there is localisation of OscobB from mitochondria to nucleus. C_LI

plant biology↗

bioRxiv2022

A salt bridge mediated resistance mechanism to FtsZ inhibitor PC190723 revealed by a single step cell-based screen

Bacterial cell division proteins, especially the tubulin homolog FtsZ, have emerged as strong targets for developing new antibiotics. Here, we have utilized the fission yeast heterologous expression system to develop a cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ. The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. Based on sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt-bridge interactions between the central H7 helix and beta-strands S9 and S10 mediate resistance to PC190723 in FtsZ. The single-step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay serves as a potent tool to rapidly identify novel compounds targeting polymeric bacterial cytoskeletal proteins like FtsZ to understand how they alter polymerization dynamics and address resistance determinants in targets.

cell biology↗

bioRxiv2021

Biochemical analysis of deacetylase activity of rice sirtuin OsSRT1, a class IV member in plants

The role of plant sirtuins is slowly unwinding. There are only reports of H3K9Ac deacetylation by OsSRT1. This belongs to class IV sirtuin family with a longer C-terminus. Here C-terminus is required for ligand binding and catalysis. OsSRT1 can deacetylate the lys residues at the N terminal tail of both H3 and H4. It can also target the non-histone target, OsPARP1 playing a role in DNA damage repair pathway. Changes in the extent of different histone deacetylation by OsSRT1 is also related with different abiotic stress conditions. NAM and ADP-ribose has negative effect on OsSRT1 deacetylation. HighlightsO_LIOsSRT1 is capable of deacetylating various lysine residues of histone H3 and H4 as well as OsPARP1. C_LIO_LIThe extra long C-terminus of OsSRT1 is required for its substrate binding and thus, its catalysis. C_LIO_LIOn plants exposure to H2O2 and Arsenic toxicity, there is a relationship between increased expression of OsSRT1 and increased deacetylation of H3 and H4. C_LI

plant biology↗

bioRxiv2021

Understanding the mechanism of monoADP ribosylation in OsSRT1 and its linkage to the DNA repair system under stress conditions.

The role of sirtuins in plants are slowly unraveling. There are only reports of H3K9Ac deacetylation by OsSRT1. Here our studies shade light on its dual enzyme capability with preference for mono ADP ribosylation over deacetylation. OsSRT1 can specifically transfer the single ADP ribose group on its substrates in an enzymatic manner. This mono ADPr effect is not well known in plants, more so for deacetylases. The products of this reaction (NAM and ADP ribose) have immense negative effect on this enzyme suggesting a tighter regulation. Resveratrol, a natural plant polyphenol proves to be a strong activator of this enzyme at 150 M concentration. Under different abiotic stress conditions, we could link this ADP ribosylase activity to the DNA repair pathway by activating the enzyme PARP1. Metal stress in plants also influences these enzyme activities. HighlightsO_LIOsSRT1 can transfer a single moiety of ADP-ribose on itself as well as other nuclear proteins like histones H3 and H2A. C_LIO_LINAM, ADP-ribose and certain metal ions negatively regulate this ADP-ribose transfer. C_LIO_LIADPr of OsPARP1 and OsPARP2 links OsSRT1 to DNA damage repair pathways. C_LIO_LIOsSRT1 positively regulates the activity of OsPARP1 by ADP ribosylating it. C_LIO_LIOn plants exposure to H2O2 (oxidative stress) and Arsenic toxicity, there is a link between the increased activity of the players of DNA damage repair system and overexpression of OsSRT1. C_LI

biochemistry↗

bioRxiv2021

Filament dynamics driven by ATP hydrolysis modulates membrane binding of the bacterial actin MreB

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. While the eukaryotic actin utilizes ATP hydrolysis to drive filament treadmilling, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we report mechanistic insights into the interplay between nucleotide-binding, ATP hydrolysis and membrane-binding of Spiroplasma citri MreB5 (ScMreB5). Antiparallel double protofilament assembly of ScMreB5WT with ATP, ADP or AMPPNP and an ATPase deficient mutant ScMreB5E134A demonstrate that the filaments assemble independent of ATP hydrolysis. However, capture of the filament dynamics revealed that efficient filament formation, bundling through lateral interactions and filament disassembly are affected in ScMreB5E134A. Hence, the catalytic glutamate (Glu134 in ScMreB5) plays a dual role - it functions as a switch by sensing the ATP-bound state for filament assembly, and by assisting hydrolysis for triggering disassembly. Glu134 mutation also exhibits an allosteric effect on membrane binding, as observed from the reduced liposome binding compared to that of the wild type. Thus, ATP hydrolysis can modulate filament length and bundling, and consequently the orientation of MreB filaments on the cell membrane depending on the curvature. Binding of ScMreB5 with liposomes is mediated by surface charge-based interactions, demonstrating paralog and organism specific features for MreB function. We conclude that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis has been repurposed for modulating curvature-dependent organization of filaments on the membrane.

biochemistry↗

bioRxiv2020

A rapid, low cost, and highly sensitive SARS-CoV-2 diagnostic based on whole genome sequencing

Early detection of infection with SARS-CoV-2 is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset. Here, we introduce a low-cost, high-throughput method for diagnosing and studying SARS-CoV-2 infection. Dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), this method amplifies the entirety of the SARS-CoV-2 genome. This contrasts with typical RT-PCR-based diagnostic tests, which amplify only a few loci. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing and de novo genome assembly. Using this method, we can reliably (>95% accuracy) detect SARS-CoV-2 at a concentration of 84 genome equivalents per milliliter (GE/mL). Almost all diagnostic methods currently authorized for use by the United States Food and Drug Administration with the Coronavirus Disease 2019 (COVID-19) Emergency Use Authorization require larger concentrations of the virus to achieve this degree of accuracy. In addition, we can reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy. The genotypic data contained in these genome assemblies enable the more effective analysis of disease spread than is possible with an ordinary binary diagnostic. These data can also help identify vaccine and drug targets. Finally, we show that the diagnoses obtained using POLAR of both positive and negative clinical nasopharyngeal swab samples 100% match the diagnoses obtained in a clinical diagnostic lab using the Center for Disease Controls 2019-Novel Coronavirus test. Using POLAR, a single person can manually process 192 samples over an 8- hour experiment at the cost of [~]$36 per patient (as of December 7th, 2022), enabling a 24-hour turnaround with sequencing and data analysis time. We anticipate that further testing and refinement will allow greater sensitivity in this approach.

genomics↗

bioRxiv2019

Cohesin depleted cells pass through mitosis and reconstitute a functional nuclear architecture

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is debatable. Using live-cell and super-resolved 3D microscopy, we demonstrate that cohesin depleted cells pass through an endomitosis and rebuild a single multilobulated nucleus (MLN) with chromosome territories (CTs) pervaded by interchromatin channels. CTs contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. Splicing speckles are located nearby within the lining channel system. These clusters form microscopically defined, active and inactive compartments, which correspond to A/B compartments, detected with ensemble Hi-C. Functionality of MLN despite continuous absence of cohesin was demonstrated by their ability to pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.

cell biology↗