Biology subjects

Misra, H. S.

Publications and source records attributed to Misra, H. S..

11 recordsLinked to original sources

bioRxiv2026

A comparative analysis of urinary microbiome identifies putative probiotics

Urinary tract infections (UTIs) are the most prevalent bacterial infections globally, and their management increasingly challenged by antimicrobial resistance (AMR). Probiotics offer a promising approach to mitigate AMR by competitively excluding uropathogens and enhancing host immunity by producing immune modulators. Despite being potential, key gaps persist between the discovery of uroprotective probiotic strains and optimization of formulations for urinary tract delivery. Here, we analyzed the urinary microbiome of UTI patients and healthy individuals to identify potential probiotic candidates for the prevention and management of UTIs. Publicly available 16S rRNA amplicon sequencing data of the urinary tract were processed using a standardized pipeline for sequence quality assessment, taxonomic assignment, and microbial function prediction. Comparative analysis showed a significant shift in microbial composition between UTI patients and healthy controls. The dominated phyla identified included Acidobacteriota, Actinobacteriota, Bacteroidota, Campylobacterota, Cyanobacteria, Firmicutes, Fusobacteriota, Patescibacteria, Proteobacteria, and Synergistota. Overall differential abundance analysis revealed Escherichia coli as the predominant UTI-associated species, while Lactobacillus crispatus was enriched in healthy samples. Additionally, predictive functional analysis indicated that metabolic pathways associated with beneficial microbes were enriched in the healthy group. Overall, the study highlights the association of distinct urinary microbiome signatures with infection status, which supports L. crispatus as the most promising probiotic for UTI prevention and control.

bioinformatics↗

bioRxiv2023

Natural transformation specific DprA coordinate DNA double strand break repair pathways in heavily irradiated D. radiodurans

Deinococcus radiodurans exhibits remarkable survival under extreme conditions, including ionizing radiation, desiccation, and various DNA-damaging agents. It employs unique repair mechanisms, such as single-strand annealing (SSA) and extended synthesis-dependent strand annealing (ESDSA), to efficiently restore damaged DNA fragments. In this study, we investigate the regulatory role of the NT-specific protein DprA in DNA repair pathways following acute gamma radiation exposure. Our findings demonstrate that the absence of DprA leads to rapid repair of gamma radiation-induced DNA double-strand breaks (DSBs), with diminished involvement of the ESDSA pathway. Furthermore, our data suggest that the SSA pathway becomes the primary mechanism for DNA DSB repair in the absence of DprA. Overall, our results highlight the regulatory function of DprA in modulating the choice between SSA and ESDSA pathways for DNA repair in the radiation-resistant bacterium D. radioduransx.

cell biology↗

bioRxiv2022

FtsK, a DNA motor protein, coordinates the genome segregation and early cell division processes in Deinococcus radiodurans

FtsK/SpoIIIE protein family are DNA translocases known as the fastest DNA motor proteins that use ATP for their movement on DNA. Most of the studies in single chromosome-containing bacteria have established the role of FtsK in chromosome dimer resolution (CDR) connecting the bacterial chromosome segregation process with cell division. But, only limited reports are available on the interdependent regulation of genome segregation and cell division in multipartite genome harbouring (MGH) bacteria. In this study, for the first time, we report the characterization of FtsK from the radioresistant MGH bacterium Deinococcus radiodurans R1 (drFtsK). drFtsK shows the activity characteristics of a typical FtsK/SpoIIIE/Tra family. It stimulates the site-specific recombination catalyzed by Escherichia coli tyrosine recombinases. drFtsK interacts with various cell division and genome segregation proteins of D. radiodurans. Microscopic examination of different domain deletion mutants of this protein reveals alterations in cellular membrane architecture and nucleoid morphology. In-vivo localization studies of drFtsK-RFP show that it forms multiple foci on nucleoid as well as on the membrane with maximum density on the septum. drFtsK coordinates its movement with nucleoid separation. The alignment of its foci shifts from old to new septum indicating its cellular dynamics with the FtsZ ring during the cell division process. Nearly, similar positional dynamicity of FtsK was observed in cells recovering from gamma radiation exposure. These results suggest that FtsK forms a part of chromosome segregation, cell envelope, and cell division machinery in D. radiodurans. ImportanceDeinococcus radiodurans show extraordinary resistance to {gamma}-radiation. It is polyploid and harbours a multipartite genome comprised of two chromosomes and two plasmids, packaged in a doughnut-shaped toroidal nucleoid. Very little is known about how the tightly packed genome is accurately segregated and the next divisional plane is determined. FtsK, a multifunctional protein, helps in pumping the septum-trapped DNA in several bacteria. Here, we characterized FtsK of D. radiodurans R1 (drFtsK) for the first time and showed it to be an active protein. The absence of drFtsK causes many defects in morphology at both cellular and nucleoid levels. The compact packaging of the deinococcal genome and cell membrane formation is hindered in ftsK mutants. In-vivo drFtsK is dynamic, forms foci on both nucleoid and septum, and coordinates with FtsZ for the next cell division. Thus, drFtsK role in maintaining the normal genome phenotype and cell division in Deinococcus radiodurans is suggested.

molecular biology↗

bioRxiv2022

FtsZ phosphorylation brings about growth arrest upon DNA damage in Deinococcus radiodurans

The polymerization/depolymerization dynamics of FtsZ plays the pivotal role in cell division in majority bacteria. Deinococcus radiodurans, a radiation resistant bacterium, shows an arrest of growth in response to DNA damage, despite no change in the level of FtsZ. This bacterium does not deploy LexA/RecA type of DNA damage response and cell cycle regulation, and its genome does not encode homologs of E. colis SulA, which attenuate FtsZ functions in response to DNA damage in different bacteria. A radiation responsive Ser/Thr quinoprotein kinase (RqkA) characterized for its role in radiation resistance in this bacterium, could phosphorylate several cognate proteins including FtsZ (drFtsZ) at Serine 235 (S235) and Serine 335 (S335) residues. Here, we report the detailed characterization of S235 and S335 phosphorylation effect in the regulation of drFtsZ functions, and demonstrated that the phospho-mimetic replacements of these residues in drFtsZ had grossly affected its functions that could result in cell cycle arrest in response to DNA damage in D. radiodurans. Interestingly, the phospho-ablative replacements were found to be nearly similar to drFtsZ while phospho-mimetic mutant showed the loss of signatures characteristics to wild type enzyme including the arrest in its dynamics under normal conditions. The post-bleaching recovery kinetics for drFtsZ and phospho-mimetic mutant was nearly similar at 2h post irradiation recovery but found to be different under normal conditions. These results highlighted the role of S/T phosphorylation in the regulation of drFtsZ functions and cell cycle arrest in response to DNA damage and is first time demonstrated in this prokaryote.

molecular biology↗

bioRxiv2022

DivIVA phosphorylation at threonine affects its dynamics and cell cycle in Deinococcus radiodurans.

RqkA, a {gamma} radiation responsive Ser/Thr quinoprotein kinase, is characterized for its role in radioresistance in Deinoocccus radiodurans. DivIVA is a cell division protein involved in determination of cell pole and division site in bacteria. RqkA phosphorylated cognate DivIVA (drDivIVA) at Threonine 19 (T19) residue located in its pole recognition motif. The phospho-mimetic replacement of T19 (T19E) functioned differently than phospho-ablative (T19A) and drDivIVA proteins. T19E-RFP expressing in wild type background showed arrest in dynamics of drDivIVA, and loss of interaction with genome segregation protein. divIVA is shown to be an essential gene in this bacterium. The allelic replacement of divIVA with T19E-RFP was not tolerated unless drDivIVA was expressed episomally while there was no effect of this replacement with T19A-RFP and drDivIVA-RFP. These results suggested that the T19 phosphorylation in drDivIVA by RqkA has affected in vivo functions of DivIVA that would render cell cycle arrest in this bacterium.

microbiology↗

bioRxiv2022

RNase H1 exists as phase-separated assemblies in association with elongating RNA Polymerase II during transcription

R-loops are three stranded nucleic acid structures consisting of a RNA/DNA hybrid and a single stranded displaced DNA. RNase H1 is an endonuclease which specifically degrades the RNA moiety in RNA-DNA hybrids. Here, we report that RNase H1 interacts with transcription elongation machinery during active transcription in a liquid-liquid phase separation (LLPS) dependent manner. We show that RNase H1 interacts with nascent RNA, and transcription elongation machinery in Hela cells, using in-situ nascent RNA labelling and Proximity ligation assay. Further, RNase H1 was found to exhibit properties of liquid-like condensates both in vitro and in vivo. Interestingly, RNase H1 interaction with elongating RNA Polymerase II can be disrupted by chemicals that perturb LLPS. Importantly, we show that LLPS is important for regulation of R-loop levels in the cell. Based on our results, we propose that RNase H1 exists as phase-separated assemblies in association with elongating RNA Polymerase II during active transcription.

cell biology↗

bioRxiv2022

Genome-wide analysis of bromodomain gene family in Arabidopsis and Rice

The bromodomain containing proteins (Brd-proteins) belongs to family of epigenetic mark readers, integral to epigenetic regulation. The Brd-members contain a conserved bromodomain (BRD/BRD-fold: interacts with acetylated-lysine in histones), and several additional domains, making them structurally/functionally diverse. Like animals, plants also contain multiple Brd-homologs, however the extent of their diversity and impact of molecular events (genomic duplications, alternative splicing, AS) therein, is relatively less explored. The present genome-wide analysis of Brd-families of Arabidopsis thaliana and Oryza sativa showed extensive diversity in structure of genes/proteins, regulatory elements, expression pattern, domains/motifs, and the bromodomain (length/sequence, location) among Brd-members. Orthology analysis identified 13 ortholog groups, three paralog groups and four singleton members. While more than 40% Brd-genes were affected by genomic duplication events in both plants, AS-events affected 60% A. thaliana and 40% O. sativa genes. These molecular events affected various regions (promoters, untranslated regions, exons) of different Brd-members with potential impact on expression/structure-function characteristics. RNA-Seq data analysis of Brd-members, and RT-qPCR of duplicate Brd-genes showed differences in tissue-specificity and response to salinity. Phylogenetic analysis based on conserved BRD-region placed the A. thaliana and O. sativa homologs into clusters/sub-clusters, consistent with ortholog/paralog groups. The bromodomain-region displayed conserved signatures in key BRD-fold elements (-helices, loops), and variations (1-20 sites) including indels among the Brd-duplicates. Homology modeling and superposition identified structural variations in BRD-folds of divergent and duplicate Brd-members, which might affect their interaction with chromatin and associated functions. The study also showed contribution of various duplication events in Brd-family expansion among diverse plants, including monocots and dicots.

plant biology↗

bioRxiv2020

Topoisomerase IB interacts with genome segregation proteins and is involved in multipartite genome maintenance in Deinococcus radiodurans

Deinococcus radiodurans, an extremophile, resistant to many abiotic stresses including ionizing radiation, has 2 type I topoisomerases (drTopo IA and drTopo IB) and one type II topoisomerase (DNA gyrase). The role of drTopo IB in guanine quadruplex DNA (G4 DNA) metabolism was shown in vitro. Here we report that D. radiodurans cells lacking drTopo IB ({Delta}topoIB) show sensitivity to G4 DNA binding drug (NMM) under normal growth conditions. The activity of G4 motif containing promoters like mutL and recQ was reduced in the presence of NMM in mutant cells. In mutant cells, the percentage of anucleated cells was more while the ploidy numbers of genome elements were less as compared to wild type. Protein-protein interaction studies showed that drTopo IB interacts with genome segregation and DNA replication initiation (DnaA) proteins. The typical patterns of cellular localization of GFP-PprA were affected in the mutant cells. Microscopic examination of D. radiodurans cells expressing drTopo IB-RFP showed its localization on nucleoid forming a streak parallel to the old division septum and perpendicular to newly formed septum. These results together suggest the role of drTopo IB in genome maintenance in this bacterium.

microbiology↗

bioRxiv2020

DivIVA is essential in Deinococcus radiodurans and its C terminal domain regulates new septum orientation during cell division

FtsZ assembly at mid cell position in rod shaped bacteria is regulated by gradient of MinCDE complex across the poles. In round shaped bacteria, which lack predefined poles and the next plane of cell division is perpendicular to previous plane, the determination of site for FtsZ assembly is intriguing. Deinococcus radiodurans a coccus shaped bacterium, is characterized for its extraordinary resistance to DNA damage. Here we report that DivIVA a putative component of Min system in this bacterium (drDivIVA) interacts with cognate cell division and genome segregation proteins. The deletion of full length drDivIVA was found to be indispensable while its C-terminal deletion ({Delta}divIVAC) was dispensable but produced distinguishable phenotypes like slow growth, altered plane for new septum formation and angular septum. Both wild type and mutant showed FtsZ foci formation and their gamma radiation responses were nearly identical. But unlike in wild type, the FtsZ localization in mutant cells was found to be away from orthogonal axis with respect to plane of previous septum. Notably, DivIVA-RFP localizes to membrane during cell division and then perpendicular to previous plane of cell division. In trans expression of drDivIVA in {Delta}divIVAC background could restore the wild type pattern of septum formation perpendicular to previous septum. These results suggested that DivIVA is an essential protein in D. radiodurans and the C-terminal domain that contributes to its interaction with MinC determines the plane of new septum formation, possibly by controlling MinC oscillation through orthogonal axis in the cells.

microbiology↗

bioRxiv2020

Overlapping oriC and centromere-like functions in secondary genome replicons determine their maintenance independent of chromosome I in Deinococcus radiodurans

The Deinococcus radiodurans multipartite genome system (MGS) consists of chromosome I (ChrI) and secondary genome elements; Chr II and megaplasmid (MP). The sequences upstream to parAB operons in Chr II (cisII) and MP (cisMP) helped an E. coli plasmid maintenance in D. radiodurans and showed sequence specific interactions with DnaA and ParBs. The cells devoid of cisII ({Delta}cisII) or cisMP ({Delta}cisMP) showed reduced {gamma} radiation resistance and copy number of Chr II and MP. Fluorescent Reporter-Operator System (FROS) developed for ChrI, ChrII and MP in {Delta}cisII or {Delta}cisMP mutants showed no change in wild type pattern of Chr I localization. However, the relative copy numbers of Chr II and MP had reduced while anucleate cells had increased in mutants. These results suggested that cisII and cisMP elements contain both ori and centromere-like functions, and like other MGS bacteria, the Chr I and secondary genome are maintained independently in D. radiodurans.

cell biology↗

bioRxiv2020

PprA interacts with replication proteins and affects their physicochemical properties required for replication initiation in Deinococcus radiodurans

The deletion mutant of pprA, a gene encoding pleiotropic functions in radioresistant bacterium Deinococcus radiodurans, showed an increased genomic content and ploidy in chromosome I and chromosome II. We identified oriC in chromosome I (oriCI) and demonstrated the sequence specific interaction of deinococcal DnaA (drDnaA) with oriCI. drDnaA and drDnaB showed ATPase activity while drDnaB catalyzed 5'[->]3' dsDNA helicase activity. These proteins showed both homotypic and heterotypic interactions. The roles of C-terminal domain of drDnaA in oriCI binding and its stimulation of ATPase activity were demonstrated. Notably, PprA showed ~2 times higher affinity to drDnaA as compared to drDnaB and attenuated both homotypic and heterotypic interactions of these proteins. Interestingly, the ATPase activity of drDnaA but not drDnaB was inhibited in presence of PprA. These results suggested that PprA influences the physicochemical properties of drDnaA and drDnaB that are required for initiation of DNA replication at oriCI site in this bacterium.

molecular biology↗