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Dr. Ranjan Sen
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Current Research Interests

Laboratory of transcription is engaged in understanding the mechanism of transcription termination and antitermination in prokaryotes. A wide range of techniques from biophysics (spectroscopy, thermodynamics, fast kinetics, etc.), biochemistry (protein purification, chemical and enzymatic foot-printing of protein and nucleic acids, cross-linking, etc.), molecular biology (recombinant DNA techniques, site-directed mutagenesis), bacterial genetics and genomics are used in the laboratory to solve these intellectually challenging problems.

Projects:

  1. Mechanism of transcription termination by transcription termination factor Rho.
  2. Mechanism of Rho-NusG interaction in vivo and in vitro.
  3. In vivo nature of Rho-dependent terminators.
  4. Physiological roles of Rho-dependent terminations.
  5. Super-resolution microscopy of the transcription machinery.
  6. Fast-kinetics approach to study the transcription termination processes.
  7. Isolation of mycobacteriophage derived inhibitors of the Mycobacterium sp.
  8. Design of antimicrobial peptides from bacteriophage proteins.

Collaborators::

  • Prof Markus Wahl, Freie Universität Berlin, Germany (Cryo-EM).
  • Udayaditya Sen, SINP, Kolkata (Crystallography).
  • Prof. Agnieszka Szalewska-Pałas, Uniwersytet Gdański, Poland.

    Research Highlights

  • The Rho-dependent transcription termination is involved in broad-spectrum antibiotic susceptibility in Escherichia coli (Front. Microbiology, 2020).

    One of the major ways of acquiring multidrug resistance in bacteria is via drug influx and efflux pathways. Here, we show that E. coli with compromised Rho-dependent transcription termination function has enhanced broad-spectrum antibiotic susceptibility, which arises from the inefficient TolC-efflux process and increased permeability of the membrane. The Rho mutants have altered morphology, distinct cell surface, and increased levels of lipopolysaccharide in their outer membrane, which might have rendered the TolC efflux pumps inefficient. These alterations are due to the upregulations of poly-N-acetyl-glucosamine and lipopolysaccharide synthesis operons because of inefficient Rho functions. The Rho mutants are capable of growing on various dipeptides and carbohydrate sources, unlike their WT counterpart. Dipeptides uptake arises from the upregulations of the di-peptide permease operon in these mutants. The metabolomics of the Rho mutants revealed the presence of a high level of novel metabolites. Accumulation of these metabolites in these Rho mutants might titrate out the TolC-efflux pumps, which could further reduce their efficiency. We conclude that the transcription termination factor, Rho, regulates the broad-spectrum antibiotic susceptibility of E. coli through multipartite pathways in a TolC-dependent manner. The involvement of Rho-dependent termination in multiple pathways and its association with antibiotic susceptibility should make Rho-inhibitors useful in the anti-bacterial treatment regimen.


    A mycobacteriophage genomics approach to identify novel mycobacteriophage proteins having Mycobactericidal properties (Microbiology 2019)

    The Mycobacteriophages specific to mycobacteria are the sources of varieties of effector proteins capable of eliciting bactericidal responses. We describe a genomics approach combining with bioinformatics to identify mycobacteriophage proteins that are toxic to mycobacteria upon expression. A genomic library made from the collections of phage genomes is screened for the clones capable of killing the M. smegmatis strain mc2155. We identified four unique clones; clones 45 and 12N (from the mycobacteriophage D29), clones 66, and 85 (from the mycobacteriophage Che12). The gene products from the clones 66 and 45 were identified as Gp49 of Che12 phage and Gp34 of D29 phage, respectively. The gene products of the other two clones, 85 and 12N, utilized novel ORFs coding for synthetic proteins. These four clones (clone 45, 66, 85, and 12N) upon expression caused growth defects in M. smegmatis and M. Bovis. Clones having Gp49 and Gp34 also induced growth defects in E. coli indicating that they target conserved host-machineries. Their expressions induced various morphological changes indicating that they affected DNA replication and cell division steps. We predicted Gp34 to be a Xis protein required in phage DNA excision from the bacterial chromosome. Gp49 is predicted to have an HTH motif having DNA-bending/twisting properties. We suggest that this methodology is useful to identify new phage proteins having desired properties without laboriously characterizing the individual phages. It is universal and could be applied to other bacteria-phage systems. We speculate that the existence of a virtually “unlimited” number of phages and their unique gene products could offer a cheaper and less hazardous alternative to explore new antimicrobial molecules.


    Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions (Nature Communications, 2019).

    In bacteria, transcription-coupled repair of DNA lesions initiates after the Mfd protein removes RNA polymerases (RNAPs) stalled at the lesions. The bacterial RNA helicase, Rho, is a transcription termination protein that dislodges the elongation complexes. Here, we show that Rho dislodges the stalled RNAPs at DNA lesions. Strains defective in both Rho and Mfd are susceptible to DNA-damaging agents and are inefficient in repairing or propagating UV damaged DNA. In vitro transcription assays show that Rho dissociates the stalled elongation complexes at the DNA lesions. We conclude that Rho-dependent termination recycles stalled RNAPs, which might facilitate DNA repair and other DNA-dependent processes essential for bacterial cell survival. We surmise that Rho might compete with, or augment, the Mfd function.

    Projects in progress:

    1. Nature of the Rho-dependent terminators in vivo.
    2. Understanding the physiological consequences of Rho-dependent termination.
    3. Understanding the role of the omega subunit of RNAP in Rho-dependent termination
    4. In vivo localization of Rho and NusG by super-resolution microscopy.
    5. Identification Rho-RNAP functional interaction domains.
    6. Isolation and characterization of anti-mycobacterial proteins from mycobacteriophages.
    7. Design of antiterminator peptides from Psu protein.
    8. Computational approaches to understanding the conformational changes of Rho and NusG during the termination process.

    Extramural Funding:

    1. DST-Indo-Polish Grant (2020-2022).
    2. Indo-German ICMR grant (2020-2023).
    3. DST-SERB Grant (2020-2023).
    4. DBT-Tata Innovation Fellowship (2019-2022).
    5. DBT grant (2019-2022).
    6. The grant from DBT COE on "Microbial Physiology" (2014-2020).

    Awards/Recognition:

    1. 2002-2007: GRIP research grant award from NIH, USA.
    2. 2003-2008: Wellcome Trust, UK, Senior Research Fellowship.
    3. 2007: DBT Bioscience carrier development award.
    4. 2007: Elected member of GRC.
    5. 2008: DST Swarnajayanti Research Fellowship.
    6. 2011: Elected fellow of NASI, Allahabad.
    7. 2015: Member DST- SERB, task force.
    8. 2018: Elected Fellow INSA, New Delhi.
    9. 2018: Elected fellow IASc, Bangalore.
    10. 2018: Fellow of Telangana Academy of Sciences.
    11. 2019: DBT TATA Innovation Fellowship.

    Editorial Board Member:

  • Journal of Applied Genetics (microbial Genetics section).

    Reviewer of Journals/grants/Thesis:

    1. Nature Communications, TIBS, Journal of Molecular Biology, Molecular Microbiology, Journal of Bacteriology, Science Reports, Microbiology, PLOS one, Indian Journal of Biophysics and Biochemistry, Journal of Bioscience etc.
    2. The reviewer of grants for different granting agencies like DBT, DST, DRDO, Indo-French programs, Marsden Fund, New Zealand. etc.
    3. The reviewer of the thesis of the Ph.D. students from renowned institutions like SINP, Kolkata; Bose Institute, Kolkata; IISc., Bangalore; IMTech, Chandigarh, HCU, JNU, etc.

    Patients:

    1. ‘NOVEL SYNTHETIC PEPTIDES’; Indian Patent Application No. 201841048582 filed on December 20, 2019.

  • Contact Information
    Email: rsen<at>cdfd.org.in
    Phone: +91-40-27216103
    Fax: +91-40-27216006
    Last updated on : Wednesday, 11th November, 2020.

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