Biology subjects

Reddy, M.

Publications and source records attributed to Reddy, M..

9 recordsLinked to original sources

bioRxiv2026

In vitro Cleavage Requirements and Specificities of Mycobacterial RNase E

Regulation of RNA pools allows for adaptation to changing environments and stress, which is especially important in pathogenic bacteria such as Mycobacterium tuberculosis. RNA degradation is a significant contributor to RNA abundance, and Ribonuclease (RNase) E has a rate-limiting role in degradation of a majority of mycobacterial transcripts. However, many open questions remain about the RNA substrate requirements and specificities for efficient cleavage by mycobacterial RNase E. Here, using both Mycolicibacterium smegmatis and M. tuberculosis RNase E, we demonstrate that this enzyme is only active on substrates with a minimum length of approximately 27 nt. Furthermore, we show that mycobacterial RNase E prefers substrates with 5 monophosphates rather than 5 triphosphates, and that the positions of cleavage events within substrates are dictated by both sequence and distance from the RNA ends. Our results also suggest that RNase E may be affected by product inhibition. Finally, we show that M. smegmatis RNase E behaves similarly to M. tuberculosis RNase E, validating the use of this model organism for RNA degradation studies.

molecular biology↗

bioRxiv2025

Mycobacterium tuberculosis MutT4 is an RNA pyrophosphohydrolase that forms biomolecular condensates and sensitizes mRNAs to degradation

Bacterial adaptation to stress involves changes in transcription and mRNA degradation rates. In Escherichia coli, the Nudix hydrolase RppH initiates mRNA degradation by removing pyrophosphate from mRNA 5-ends, converting 5-triphosphates to 5-monophosphates. We aimed to identify the RppH homolog in the globally important pathogen Mycobacterium tuberculosis (Mtb). We deleted each non-essential Nudix gene from Mtb to determine their impacts on mRNA phosphorylation states. Deletion of mutT4 (Rv3908) increased the relative abundance of 5-triphosphates on myriad mRNAs across the transcriptome. Purified MutT4 converted mRNA 5-triphosphates into monophosphates, and stimulated degradation by RNase E and RNase J. MutT4 has intrinsically disordered regions (IDRs), a common domain for biomolecular condensate formation. Microscopy showed that MutT4 forms condensates that dissociate upon addition of rifampicin, and that the N-terminal IDR is sufficient for condensate formation. These MutT4 condensates localize with RNase E and RNase J. Deletion of mutT4 in Mtb leads to a higher outer membrane permeability and resistance to oxidative stress. We conclude that MutT4 is the RppH homolog of Mtb, assembling in condensates that may act as degradation hubs. Our data indicate that MutT4 is unlikely to participate in DNA repair or nucleotide pool cleansing, and as such would more accurately be named RppH.

microbiology↗

bioRxiv2024

NEMO: Improved and accurate models for identification of 6mA using Nanopore sequencing

DNA methylation plays a key role in epigenetic regulation across lifeforms. Nanopore sequencing enables direct detection of base modifications. While multiple tools are currently available for studying 5-methylcytosine (5mC), there is a paucity of models that can detect 6-methyladenine (6mA) from raw nanopore data. Leveraging the motif-driven nature of bacterial methylation systems, we generated 6mA identification models that vastly surpass the accuracy of the current best model. Our work enables the study of 6mA at a single-base resolution in new as well as existing nanopore datasets.

genomics↗

bioRxiv2023

Glycan strand cleavage by a lytic transglycosylase, MltD contributes to the expansion of peptidoglycan in Escherichia coli

Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently cross-linked mesh-like polymer made up of several glycan strands cross-bridged to each other by short peptide chains. Because PG forms a continuous mesh around the bacterial cytoplasmic membrane, opening the mesh is critical to generate space for the incorporation of new material during its expansion. In Escherichia coli, the space-making activity is known to be achieved by cleavage of cross-links between the glycan strands by a set of redundant PG endopeptidases whose absence leads to rapid lysis and cell death. Here, we demonstrate a hitherto unknown role of glycan strand cleavage in making space for cell wall expansion in E. coli. We find that overexpression of a membrane-bound lytic transglycosylase, MltD that cuts the glycan polymers of the PG sacculus rescues the cell lysis caused by the absence of essential cross-link specific endopeptidases, MepS, MepM and MepH. Further detailed genetic and biochemical analysis revealed that MltD works in conjunction with cross-link specific endopeptidases to expand the PG sacculus. Interestingly, we find that cellular MltD levels are stringently controlled by two independent regulatory pathways. MltD undergoes regulated proteolysis by NlpI-Prc, a periplasmic adaptor-protease complex that specifically degrades two of the elongation-specific endopeptidases, MepS and MepH. In addition, MltD levels are post-transcriptionally controlled by RpoS, a stationary-phase specific sigma factor. Overall, our results show that coordinated cleavage of the glycan strands and the peptide cross-bridges facilitates the opening of the PG mesh for successful expansion of the cell wall during growth of a bacterium. AUTHOR SUMMARYMost bacteria are protected by a cell wall made up of peptidoglycan (PG), a mesh-like large polymer. PG consists of several linear glycan strands interlinked through short peptide chains to form a continuous meshwork around the bacterial cytoplasmic membrane. Because PG tightly encases the cytoplasmic membrane, the growth of a bacterial cell is coupled to the expansion of PG requiring the activity of hydrolytic enzymes that cleave PG cross-links to make space for incorporation of new PG material. ln E. coli, a set of redundant cross-link specific endopeptidases are known to be crucial for expansion of PG. In this study, we show that cleavage of the glycan polymers by MltD, a glycan cleaving enzyme compensates the absence of cross-link cleavage and contributes to the expansion of PG. Overall, our work shows a previously unknown role of glycan hydrolases in cell wall expansion identifying these as potential targets for development of cell wall-specific antimicrobial agents.

microbiology↗

bioRxiv2022

A novel LC-MS/MS method development and validation for the determination of tezepelumab in rat plasma and its application in rat pharmacokinetic studies

By developing a quick, accurate, reproducible, and straightforward LC-MS/MS system and employing trastuzumab as an internal standard (IS), tezepelumab (TZP)s quantification was achieved. This study explores the most recent advancement in bioanalytical LC-MS/MS technologies utilizing a 150x4.6 mm, 3.5{micro} Waters Symmetry C18 column that was set for isocratic mode at ambient temperature. Methanol (MeoH): 0.1% Formic acid (FA) in 40:60 v/v at 1.0 ml/min was utilized as the mobile phase. The injection volume and runtime were 10 {micro}l and 5 minutes, respectively. TZPs retention time (RT) was 1.944 Minutes, and a chromatographic duration total of 5.0 min. With a correlation value (r2) of 0.99971, the technique was validated for Tezepelumab throughout a linear range of 6.00-120.00 ng/mL. Findings for precision, accuracy, recovery, matrix effect, and stability all occurred within acceptable limits. Below visual abstract is the concise summary of the article. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/520442v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@15b24c1org.highwire.dtl.DTLVardef@ca2ddcorg.highwire.dtl.DTLVardef@189026borg.highwire.dtl.DTLVardef@1fdc1a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Three Isoforms of the Essential Translation Initiation Factor IF2 Differentially Modulate Homologous Recombination in Escherichia coli

Homologous recombination (HR) is critically important for chromosomal replication as well as DNA damage repair in all life forms. In Escherichia coli, the process of HR is comprised of (i) two parallel pre-synaptic pathways that are mediated, respectively, by proteins RecB/C/D and RecF/O/R/Q; (ii) a synaptic step mediated by RecA that leads to generation of Holliday junctions (HJs); and (iii) post-synaptic steps mediated sequentially by HJ-acting proteins RuvA/B/C followed by proteins PriA/B/C of replication restart. Combined loss of RuvA/B/C and a DNA helicase UvrD is synthetically lethal, which is attributed to toxicity caused by accumulated HJs since viability in these double mutant strains is restored by removal of the pre-synaptic or synaptic proteins RecF/O/R/Q or RecA, respectively. Here we show that, as in {Delta}uvrD strains, ruv mutations confer synthetic lethality in cells deficient for transcription termination factor Rho, and that loss of RecFORQ pre-synaptic pathway proteins or of RecA suppresses this lethality. Furthermore, loss of IF2-1 (which is one of three isoforms [IF2-1, IF2-2, and IF2-3] of the essential translation initiation factor IF2 that are synthesized from three in-frame initiation codons in infB) also suppressed uvrD-ruv and rho-ruv lethalities, whereas deficiency of IF2-2 and IF2-3 exacerbated the synthetic defects. Our results suggest that Rho deficiency is associated with an increased frequency of HR that is mediated by the RecFORQ pathway along with RecA. They also lend support to earlier reports that IF2 isoforms participate in DNA transactions, and we propose that they do so by modulation of HR functions. ImportanceThe process of homologous recombination (HR) is important for maintenance of genome integrity in all cells. In Escherichia coli, the RecA protein is a critical participant in HR, which acts at a step common to and downstream of two HR pathways mediated by the RecBCD and RecFOR proteins, respectively. In this study, an isoform (IF2-1) of the translation initiation factor IF2 has been identified as a novel facilitator of RecAs function in vivo during HR.

microbiology↗

bioRxiv2021

Cleavage of Braun lipoprotein Lpp from the bacterial peptidoglycan by a paralog of L,D-transpeptidases, LdtF

Gram-negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM protecting cells from turgor and environmental stress conditions. In several bacteria including E. coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun lipoprotein) that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery Lpp has been studied extensively and although L,D-transpeptidases, the enzymes that catalyse the formation of PG-Lpp linkages have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly-identified paralog of L,D-transpeptidases in E. coli is a murein hydrolytic enzyme that catalyses cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF is earlier presumed to be an L,D-transpeptidase; however, our results show that it is indeed an L,D-endopeptidase that hydrolyses the products generated by the L,D-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG-Lpp cross-links suggesting a role for LdtF in regulation of PG-OM linkages to maintain the structural integrity of the bacterial cell envelope. Significance statementBacterial cell walls contain a unique protective exoskeleton, peptidoglycan, which is a target of several clinically important antimicrobials. In Gram-negative bacteria, peptidoglycan is covered by an additional lipid layer, outer membrane that serves as permeability barrier against entry of toxic molecules. In some bacteria, an extremely abundant lipoprotein, Lpp staples outer membrane to peptidoglycan to maintain the structural integrity of the cell envelope. In this study, we identify a previously unknown peptidoglycan hydrolytic enzyme that cleaves Lpp from the peptidoglycan sacculus and show how the outer membrane-peptidoglycan linkages are modulated in Escherichia coli. Overall, this study helps in understanding the fundamental bacterial cell wall biology and in identification of alternate drug targets for development of new antimicrobials.

microbiology↗

bioRxiv2021

LeafGo: Leaf to Genome, a quick workflow to produce high-quality De novo genomes with Third Generation Sequencing technology

Recent years have witnessed a rapid development of sequencing technologies. Fundamental differences and limitations among various platforms impact the time, the cost and the accuracy for sequencing whole genomes. Here we designed a complete de novo plant genome generation workflow that starts from plant tissue samples and produces high-quality draft genomes with relatively modest laboratory and bioinformatic resources within seven days. To optimize our workflow we selected different species of plants which were used to extract high molecular weight DNA, to make PacBio and ONT libraries for sequencing with the Sequel I, Sequel II and GridION platforms. We assembled high-quality draft genomes of two different Eucalyptus species E. rudis, and E. camaldulensis to chromosome level without using additional scaffolding technologies. For the rapid production of de novo genome assembly of plant species we showed that our DNA extraction protocol followed by PacBio high fidelity sequencing, and assembly with new generation assemblers such as hifiasm produce excellent results. Our findings will be a valuable benchmark for groups planning wet- and dry-lab plant genomics research and for high throughput plant genomics initiatives.

genomics↗

bioRxiv2019

Structural basis for the differential regulatory roles of the PDZ domain in C-terminal processing proteases

Carboxyl (C)-terminal processing proteases (CTPs) participate in protective and regulatory proteolysis in bacteria. The PDZ domain is central to the activity of CTPs but plays inherently different regulatory roles. For example, the PDZ domain inhibits the activity of the signaling protease CtpB by blocking the active site but is required for the activation of Prc (or Tsp), a tail-specific protease that degrades the ssrA-tagged proteins. Here, by structural and functional analysis we show that in the unliganded resting state of Prc, the PDZ domain is docked inside the bowl-shaped scaffold without contacting the active site, which is kept in a default misaligned conformation. In Prc, a hydrophobic substrate sensor distinct from CtpB engages substrate binding to the PDZ domain and triggers a structural remodeling to align the active site residues. Therefore, this work reveals the structural basis for understanding the contrasting roles of the PDZ domain in the regulation of CTPs.

biophysics↗