Biology subjects

SRINIVASAN, R.

Publications and source records attributed to SRINIVASAN, R..

3 recordsLinked to original sources

bioRxiv2024

Mutational analysis of the F plasmid partitioning protein ParA reveals novel residues required for oligomerisation and plasmid maintenance

Mobile genetic elements such as plasmids play a crucial role in the emergence of antimicrobial resistance. Hence, plasmid maintenance proteins like ParA of the Walker A type cytoskeletal ATPases/ ParA superfamily are potential targets for novel antibiotics. Plasmid partitioning by ParA relies upon ATP-dependent dimerisation and formation of chemophoretic gradients of ParA-ATP on bacterial nucleoids. Though polymerisation of ParA has been reported in many instances, the need for polymerisation in plasmid maintenance remains unclear. In this study, we provide novel insights into the polymerisation of ParA and the effect of polymerisation on plasmid maintenance. We first characterise two mutations, Q351H and W362E, in ParA from F plasmid (ParAF) that form cytoplasmic filaments independent of the ParBSF partitioning complex. Both mutants fail to partition plasmids, do not bind non-specific DNA and act as super-repressors to suppress transcription from the ParA promoter. Further, we show that the polymerisation of ParAF requires the conformational switch to the ParA-ATP* state. We identify two mutations, R320A in the C-terminal helix-14 and E375A helix-16 of ParAF, that abolish filament assembly and affect plasmid partitioning. Our results thus suggest a role for higher-order structures or polymerisation of ParA in plasmid maintenance.

microbiology↗

bioRxiv2022

Structures of FtsZ from a cell-wall less bacterium Spiroplasma provide a mechanism for kinetic polarity

FtsZ, the tubulin homolog essential for bacterial cell division, assembles as Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we characterized the FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). SmFtsZ was found to be a slower GTPase and has higher critical concentration (CC) for polymerization compared to Escherichia coli FtsZ (EcFtsZ). In FtsZs, a conformational switch from R (close)- to T (open)- state favors polymerization. In FtsZs, a conformational switch from R (close)- to T (open)- state favors polymerization. We identified a residue, Phe224, located at the cleft between N-terminal domain (NTD) and C-terminal domain (CTD) of SmFtsZ, which is crucial for R- to T-state transition. The mutation F224M in SmFtsZ cleft resulted in higher GTPase activity and lower CC, whereas the corresponding M225F in EcFtsZ resulted in cell division defects in E. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization. Our structural analysis of interdomain interactions suggests that R- to T-state transition likely follows addition of a GTP-bound monomer to the filament through interaction of the preformed NTD. Hence, the addition of monomers to the NTD-exposed end of filament is slower in comparison to the C-terminal domain end, thus supporting the phenomenon of kinetic polarity in a single protofilament assembly.

biochemistry↗

bioRxiv2022

A salt bridge mediated resistance mechanism to FtsZ inhibitor PC190723 revealed by a single step cell-based screen

Bacterial cell division proteins, especially the tubulin homolog FtsZ, have emerged as strong targets for developing new antibiotics. Here, we have utilized the fission yeast heterologous expression system to develop a cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ. The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. Based on sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt-bridge interactions between the central H7 helix and beta-strands S9 and S10 mediate resistance to PC190723 in FtsZ. The single-step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay serves as a potent tool to rapidly identify novel compounds targeting polymeric bacterial cytoskeletal proteins like FtsZ to understand how they alter polymerization dynamics and address resistance determinants in targets.

cell biology↗