Biology subjects

Kalita, A.

Publications and source records attributed to Kalita, A..

6 recordsLinked to original sources

bioRxiv2023

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

The trace metal ion manganese in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. The addition of manganese activates the riboswitch, thereby restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. Deletion of the riboswitch abolishes Rho-dependent termination and renders bacteria sensitive to manganese due to overexpression of mntP. The high mntP expression is associated with reactive oxygen species (ROS) production, slow growth, and cell filamentation phenotypes. We posit that manganese-dependent transcriptional activation in the absence of Rho-dependent termination leads to the observed toxicity arising from excessive MntP expression, a membrane protein. Thus, we identified a novel regulatory role of Rho in preventing membrane protein toxicity by terminating at the riboswitch element.

biochemistry↗

bioRxiv2023

Deletion of Xist upstream sequences alters TAD interactions and leads to defects in Xist coating and expression

The topological organization of the genome plays an important role in regulating gene expression. However, the connection between the two remains poorly understood. X-chromosome inactivation is a unique model system to explore the interlink between topologically associated domains (TADs) and gene expression. TADs are largely lost upon X-inactivation, and the inactive-X gets bipartitely reorganized into two large mega domains. However, the X-inactivation center (XIC) harbors two TADs - at the locus of long non-coding RNA Xist (Xist-TAD) and Tsix (Tsix-TAD). Xist is the master regulator of X-inactivation, which coat the inactive-X and facilitates heterochromatinization. Here, we deleted Xist upstream sequences ([~]6 kb) near the Xist TADs boundary in extraembryonic endoderm stem cells (XEN), which undergo imprinted X-inactivation. This deletion led to the major rearrangement of TADs and affected the expression of genes located within Xist and Tsix TAD, specially the expression of Xist was upregulated, suggesting TADs are essential for proper transcriptional regulation. On the other hand, Xist-upstream deletion on the inactive-X resulted in dispersal of Xist coating and loss of enrichment of repressive chromatin marks on the inactive-X but no effect on X-linked gene silencing. However, we found that autosomal genes were dysregulated in Xist-upstream deleted cells, probably because of misregulation of genes located in Xist and Tsix-TAD, specially Xist. We conclude that Xist upstream sequences are necessary for proper organization of the TADs at the XIC, maintenance of Xist coating/expression and autosomal gene expression.

genetics↗

bioRxiv2022

An intrinsic alkalization circuit turns on mntP-riboswitch under manganese stress in Escherichia coli

The trace metal manganese in excess affects iron-sulfur cluster and heme-protein biogenesis eliciting cellular toxicity. The manganese efflux protein MntP is crucial to evading manganese toxicity in bacteria. Recently, two Mn-sensing riboswitches upstream of mntP and alx in Escherichia coli have been reported to mediate the upregulation of their expression under manganese shock. As the alx-riboswitch is also responsive to alkaline shock administered externally, it is intriguing whether mntP-riboswitch is also responsive to alkaline stress. Furthermore, how both manganese and alkaline pH simultaneously regulate these two riboswitches under physiological conditions is a puzzle. Using multiple approaches, we show that manganese shock activated glutamine synthetase (GlnA) and glutaminases (GlsA and GlsB) to spike ammonia production in E. coli. The elevated ammonia intrinsically alkalizes the cytoplasm. We establish that this alkalization under manganese stress is crucial for attaining the highest degree of riboswitch activation. Additional studies showed that alkaline pH promotes a 17 to 22-fold tighter interaction between manganese and the mntP-riboswitch element. Our study uncovers a physiological linkage between manganese efflux and pH homeostasis that mediates enhanced manganese tolerance. Significance statementRiboswitch RNAs are cis-acting elements that can adopt alternative conformations in the presence or absence of a specific ligand(s) to modulate transcription termination or translation initiation processes. In the present work, we show that how manganese and alkaline pH both are necessary for maximal mntP-riboswitch activation to mitigate the manganese toxicity. This study bridges the gap between earlier studies that separately emphasize the importance of alkaline pH and manganese in activating the riboswitches belonging to the yybP-ykoY-family. This study also ascribes a physiological relevance as to how manganese can rewire cellular physiology to render cytoplasmic pH alkaline for its homeostasis.

biochemistry↗

bioRxiv2021

Free spermidine evokes superoxide radicals that manifest toxicity

Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Besides, a specific mechanism of SpeG function conferring pathogenic fitness to Staphylococcus aureus USA300 strain is unknown. Here, we provide evidence that although spermidine mitigates oxidative stress by lowering hydroxyl radical and hydrogen peroxide levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the E. coli {Delta}speG strain as well as naturally SpeG-deficient S. aureus RN4220 strain. However, wild-type E. coli and S. aureus USA300 with a horizontally-acquired speG gene tolerate applied exogenous spermidine stress. Furthermore, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Superoxide radicals thus generated, further affects redox balance and iron homeostasis. Since iron acquisition and metabolism in the host tissues is a challenging task for S. aureus, the current findings helped us explain that the evolutionary gain of SpeG function by S. aureus USA300 strain allows it to neutralize exogenous spermidine- and spermine-mediated toxicity towards iron metabolism inside the host tissues.

biochemistry↗

bioRxiv2021

Isonicotinamide extends yeast chronological lifespan through a mechanism that diminishes nucleotides

Isonicotinamide (INAM) is an isomer of the NAD+ precursor nicotinamide (NAM) that stimulates the enzymatic activity of Sir2, an NAD+-dependent histone deacetylase from the budding yeast, Saccharomyces cerevisiae. Supplementing INAM into growth media promotes the replicative lifespan (RLS) of this single cell organism by maintaining intracellular NAD+ homeostasis. INAM also extends yeast chronological lifespan (CLS), but the underlying mechanisms remain largely uncharacterized. To identify interacting genes, a chemical genomics screen of the yeast knockout (YKO) collection was performed for mutants sensitized to growth inhibition by INAM. Significant Gene Ontology (GO) terms included transcription elongation factors, metabolic pathways converging on one-carbon metabolism, and de novo purine biosynthesis, collectively suggesting that INAM may perturb nucleotide metabolism. Indeed, INAM caused dose-dependent depletion of intracellular cytidine, uridine and guanosine, ribonucleosides derived from the breakdown of nucleotide monophosphates by a set of nucleotidases (Phm8, Sdt1, Isn1) or the alkaline phosphatase Pho8. Direct inhibition of recombinant Sdt1 and Phm8 nucleotidase activity by INAM was confirmed in vitro, as was inhibition of alkaline phosphatase activity. Each of these enzymes can also convert nicotinamide mononucleotide (NMN) to nicotinamide riboside (NR), consistent with an accumulation of NMN and NAD+ upon inhibition by INAM. Taken together, the findings suggest a model whereby partial impairment of nucleotide salvage pathways can trigger a hormetic stress response that supports enhanced quiescence during chronological aging.

biochemistry↗

bioRxiv2020

A cell non-autonomous mechanism of yeast chronological aging regulated by caloric restriction and one-carbon metabolism

Caloric restriction (CR) improves healthspan and lifespan of organisms ranging from yeast to mammals. Understanding the mechanisms involved will uncover future interventions for aging associated diseases. In budding yeast, Saccharomyces cerevisiae, CR is commonly defined by reduced glucose in the growth medium, which extends both replicative and chronological lifespan (CLS). We found that conditioned media collected from stationary phase CR cultures extended CLS when supplemented into non-restricted (NR) cultures, suggesting a potential cell non-autonomous mechanism of CR-induced lifespan regulation. Chromatography and untargeted metabolomics of the conditioned media, as well as transcriptional responses associated with the longevity effect, pointed to specific amino acids enriched in the CR conditioned media (CRCM) as functional molecules, with L-serine being a particularly strong candidate. Indeed, supplementing L-serine into NR cultures extended CLS through a mechanism dependent on the one-carbon metabolism pathway, thus implicating this conserved and central metabolic hub in lifespan regulation.

cell biology↗