Page 104 - Annual report 2021-22

Annual Report 2021-22 |

Rakesh Sharma

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Rakesh Sharma’s lab is involved in understanding microbial defense systems to ecological conditions.
Metagenomics is one of the niche areas of his lab.

Using this approach his lab works towards identification of new CRISPR-Cas systems. In a recent study
by his lab, metagenomes of hot spring microbial mats were assembled and binned to obtain
metagenome assemble genomes (MAGs) of the bacteria present in the samples. The MAGs were
annotated with RAST and analyzed for the presence of CRISPR-Cas systems. A MAG belonging to
unclassified Saprospiraceae bacterial species was found to have CRISPR-Cas systems and a Cas9.
CRISPR repeats and spacer sequences were identified. Other MAGs are also being screened for the
presence of new CRISPR-Cas systems. CAS9 enzymes with best features as per bioinformatics analyses
will be selected for cloning and characterization in the future.
Metal ions and the diversity in their abundance across ecological niches requires specialized strategies
in microbes to respond, scavenge, store, and export them. One of the projects in Rakesh Sharma’s lab
is to understand the regulation of the putative metal transporter genes in Mycobacterium spp. Ctp E
is a calcium uptake transporter in mycobacteria. The regulation of CtpE was analysed by
semiquantitative RT-PCR. The cultures were grown in calcium-deficient and calcium-sufficient
condition expression of ctpE and echA genes was checked. Both genes were found to be up-regulated
in calcium-deficient conditions indicating inducible expression of ctpE. Disruption of CtpE resulted in
differences in cell aggregation, biofilm formation and sensitivity to polymyxin. These results indicated
that calcium is important for cellular physiology and specifically for maintenance of cell envelope.

The effect of CtpE disruption on the cell envelope was evaluated by purifying various components of
the cell wall. Various polar and apolar components were purified and separated by TLC from wild type
and mutant along with mutant complemented with the ctpE gene from a plasmid. Only difference
observed was the increased amount of diacylglycerol (DAG) in the mutant in comparison to wild type
and this change was rescued in the complemented strain confirming that the increase in DAG is indeed
due to disruption of the ctpE. Calcium could also rescue the phenotype while use of EGTA (a calcium
ion chelator) increased the DAG in mutant as well as in wild type strain. DAG along with CAM kinase
play an important role in regulation in eukaryotes but its similar role is not known in bacteria. Another
possibility of DAG increase can be defect in utilization for DAG in other membrane components
synthesis but they could not observe any difference in these components. These results show the role
of intracellular calcium in DAG homeostasis and the impact of this change would be studied in future.