Page 55 - Biennial Report 2018-20 Jun 2021

additional responder strains from India is required for possible association with drug resistance
and/or geographical speciation.

A Methylome map of T. interdigitale strain was constructed. The Oxford nanopore sequencing
enables detection of methylation sites directly in DNA from reads without extra laboratory
techniques. More than 100X coverage was achieved with nanopore sequencing that helped train
a hidden Markov model to distinguish 5-mC from unmethylated cytosine. Using this model, more
than 20,000 methylation sites were identified in the whole genome of T. interdigitale clinical
isolate (lab annotation BK4). About 15% of the methylations could be mapped onto regulatory
regions of the genetic regions. Identification of methylations via nanopore sequencing is a new
and fast-evolving method and now another 'Deepsignal' method is available for identifying
methylated bases in the genomic DNA. These potential methylations will be validated before
further investigations of these epigenetic signals in the fungal genome.

ELUCIDATING THE MOLECULAR BASIS FOR HIGHLY SPECIFIC RECOGNITION OF
16s rRNA NUCLEOTIDES BY COGNATE METHYLTRANSFERASES OF
Mycobacterium tuberculosis

The overall objective of this study by Bhupesh Taneja is to investigate the specific modes of
recognition by RNA methyltransferases of M. tuberculosis through structural biology methods.
The specific objectives include structure determination of mycobacterial 16S rRNA
methyltransferases and investigation of the role of ribosomal proteins on methyltransferase
activity. As antibiotic resistance is a looming problem, the effect of nucleotide methylations on
antibiotic response is particularly important.

Structure-function studies on the following mycobacterial 16S ribosomal methyltransferases was
reported. Diffraction data of Rv3919c (homolog of RsmG methyltransferase) was obtained at low
resolution (~3.7 Angstroem). Efforts to solve the structure by molecular replacement or
experimental phasing by SAD/SIRAS methods were initiated. Deletion of the RsmG- homolog of
M. smegmatis was carried out in M. smegmatis mc2155 using pPR27 as a suicidal vector. RsmG-
deleted M. smegmatis show increased resistance to ribosome-targeting antibiotics, specifically
to streptomycin. Deletion of the RsmE-homolog of M. smegmatis was carried out in M.
smegmatis mc2155. RsmE-deleted M. smegmatis were found to have reduced growth in
response to pH or temperature stress and showed resistance to several ribosome-targeting
antibiotics, which was restored in the RsmE-complemented cells. There was no change in
response to antibiotics having non-ribosomal targets. In order to understand how mycobacterial
proteins cope with extreme stress conditions in the host, the effects of pH on the structure and
stability of Rv2966c were studied under varying pH conditions (pH 2.0-pH 11.0). The protein
maintains its native structure in neutral to alkaline pH range but adopts a structure with
increased beta-sheet content at extremely low pH. A comparison of thermal stability of RsmD at
pH 7.5 and pH 2.0 shows a structure highly resistant to low pH.

Structure determination of mycobacterial 16S rRNA methyltransferases was carried out. The
crystal structure of Rv3919c (RsmG-like methyltransferase) was determined up to a resolution
of 2.02Å. The overall structure consists of a SAM-methyltransferase fold in the protein with a
short sub-domain consisting of two helices at the N-terminus as the likely substrate binding

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