Biology subjects

Rath, D.

Publications and source records attributed to Rath, D..

6 recordsLinked to original sources

bioRxiv2026

Using Arabian Sea Coastal Water as a Model to Study Plasmid-mediated AMR Dissemination driven by Anthropogenic Impacts

Background Antimicrobial resistance (AMR) is a growing global public health threat. The water bodies act as both reservoirs and medium for dissemination of AMR. Coastal water driven by anthropogenic effects accumulate antibiotics, heavy metals, microplastics, and disinfectants which act as stressors, increasing the overall AMR. Therefore, it is necessary to understand the AMR entities and its transmission in coastal water bodies. Methods and Results Mumbai, mirroring most cosmopolitan cities of the world, was used as a model to understand how rapid urbanization disseminate AMR in coastal waters of the Arabian Sea and can pose substantial risk to human health. Bacteria isolated from coastal waters were screened and found to be multi-drug resistant. Several isolates carried multiple plasmids of high molecular weight (>10 kb). Plasmid sequencing and analysis using various bioinformatic tools identified plasmid replicons like IncFIA, IncFII, rep7a and repUS43. Analysis showed a high burden of antibiotic resistance genes (ARGs) conferring resistance to antibiotic classes such as monobactams (blaTEM-116), carbapenems (blaNDM-5), vancomycin (vanA), disinfectants and antiseptics (qacG). Additionally, clinically relevant mecA and mecR1 associated with methicillin-resistant Staphylococcus sp., rpsL associated with streptomycin resistant Mycobacterium tuberculosis and the colistin-resistance genes mcr-10.1 and mcr-10.4 were also detected. We also report for the first-time presence of a plasmid-borne mupB gene in coastal waters, that confers high resistance to mupirocin. Conclusions Despite the moderate sample size, the presence of diverse plasmid replicons and clinically important ARGs suggests urban coastal environment as a potent reservoir of AMR, with potential for global transmission through active waterway trade and transport.

bioinformatics↗

bioRxiv2026

CRISPRi-mediated silencing of racR reveals molecular strategies for surviving lethal stress coupled to Rac prophage excision

Cryptic prophages, abundant in bacterial genomes, often carry regulatory modules that rewire host physiology contextually. E. coli K-12 Rac prophage harbors a genetic module consisting of a RacR repressor that tightly represses the toxin genes ydaS/T. Dysregulation of racR and expression of YdaS/T lead to growth arrest, rapid loss of viability, and morphological defects. We investigated the physiological consequences of racR silencing and the host adaptive responses to lethal stress imposed by YdaS/T. We show that racR depletion establishes an inner membrane-centered stress characterized by selective hyperpolarization, increased permeability, and arrest of cytokinesis downstream of FtsZ-ring assembly, triggering copious filamentation. Transcriptomic analysis showed extensive metabolic reprogramming, revealing a coordinated, energy-conserving response marked by respiratory remodelling, repression of proton motive force-intensive pathways, and activation of global stress pathways. We show that recovery is accompanied by remarkable physiological heterogeneity with divergent filament fates, including lysis, persistent arrest, and recovery by loss of Rac. Thus, heterogeneous membrane dysfunction and metabolic states appeared to determine the balance between recovery and cell death. These findings support a model in which racR silencing activates a prophage-host stress circuit interlinking inner membrane energetics, activation of envelope stress, cell division control, and heterogeneous recovery.

molecular biology↗

bioRxiv2023

MSMEG_0311 is a conserved essential polar protein involved in mycobacterium cell wall metabolism

Cell wall synthesis and cell division are two closely linked pathways in a bacterial cell which distinctly influence the growth and survival of a bacterium. This requires an appreciable coordination between the two processes, more so, in case of mycobacteria with an intricate multi-layered cell wall structure. In this study, we investigated a conserved gene cluster and show that knockdown of most of the genes in this cluster leads to growth defects. We further characterised one of the genes, MSMEG_0311. The repression of this gene not only imparts severe growth defect but also changes colony morphology. We demonstrate that the protein preferentially localises to the polar region and show its influence on the polar growth of the bacillus. A combination of permeability and drug susceptibility assay strongly suggests a cell wall associated function of this gene which is also corroborated by transcriptomic analysis of the knockdown. Considering the gene is highly conserved across mycobacterial species and appears to be essential for growth, it may serve as a potential drug target.

molecular biology↗

bioRxiv2022

CRISPRi in Deinococcus radiodurans

The extremely radiation resistant bacterium, Deinococcus radiodurans, is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting. In view of this, the CRISPR-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A Cas9 system in D. radiodurans. An assayable acid phosphatase gene, phoN integrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown of ssb, a gene important for radiation resistance resulted in poor recovery post irradiation. Targeting the Radiation and Desiccation Resistance Motif (RDRM), upstream of the ssb, prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down both phoN and ssb expression simultaneously. The programmable CRISPRi tool developed in this study will facilitate study of essential genes, hypothetical genes, cis-elements involved in radiation response as well as enable metabolic engineering in this organism. Further the tool is amenable for implementing high-throughput approaches for such studies.

molecular biology↗

bioRxiv2019

Determination of Cas9/dCas9 associated toxicity in microbes

The CRISPR-Cas9 system has been used extensively in eukaryotic and prokaryotic systems for various applications. In case of the latter, a couple of previous studies had shown Cas9 protein expression associated toxicity. We studied the same in five microbes, viz Escherichia coli, Salmonella typhimurium, Mycobacterium smegmatis, Xanthomonas campestris and Deinococcus radiodurans. Transformation efficiency of plasmids carrying genes coding for Cas9 or dCas9 was used to gauge toxicity associated with Cas9 protein expression. Results showed differential levels of Cas9 toxicity among the bacteria and lower transformation efficiency for cas9/dcas9 bearing plasmids compared to controls in general. This indicated lethal effect of Cas9/dCas9 expression. While E. coli and S. typhimurium seemed to tolerate Cas9/dCas9 fairly well, in GC rich microbes, M. smegmatis, X. campestris and D. radiodurans, Cas9/dCas9 associated toxicity was acute.

microbiology↗

bioRxiv2018

Type I-E CRISPR-Cas system as immune system in a eukaryote

Defense against viruses and other mobile genetic elements (MGEs) is important in many organisms. The CRISPR-Cas systems found in bacteria and archaea constitute adaptive immune systems that acquire the ability to recognize MGEs by introducing nucleic acid samples, spacers, in the CRISPR locus. The CRISPR is transcribed and processed, and the produced CRISPR RNAs guide Cas proteins to degrade matching nucleic acid sequences. No CRISPR-Cas system is found to occur naturally in eukaryotic cells but here we demonstrate interference by type I-E CRISPR-Cas system from Escherichia coli introduced in Saccharomyces cerevisiae. The designed CRISPR arrays are properly expressed and processed in S. cerevisiae. Targeted plasmids display reduced transformation efficiency, indicative of DNA cleavage. Unlike e.g. Cas9-based systems, which can be used to inactivate MGEs in eukaryotes by introducing specific mutations, type I-E systems processively degrade the target. The type I-E system thus allows for defense without knowledge of MGE gene function. The reconstituted CRISPR-Cas system in S. cerevisiae can also function as a basic research platform for testing the role of various factors in the interference process.

microbiology↗