Biology subjects

Poddar, S. M.

Publications and source records attributed to Poddar, S. M..

3 recordsLinked to original sources

bioRxiv2023

An amphipathic helix facilitates direct membrane binding of Mycoplasma FtsZ

Cell division in bacteria is initiated by constriction of the Z-ring comprising two essential proteins FtsZ and FtsA. Despite our knowledge about the crucial function of the Z-ring in bacterial division, the precise roles and mechanism of how FtsZ and FtsA drive cell constriction remain elusive. FtsZ/FtsA in wall-less bacteria like mycoplasmas is an ideal model system for obtaining mechanistic insights into Z-ring constriction in the absence of cell wall machinery. In this study, we have analyzed FtsZ and FtsA sequences of 113 mycoplasma species and compared with the corresponding protein sequences in cell-walled bacteria. We report a phylogenetically distinct group of 12 species that possess FtsZs without the canonical FtsA interacting conserved C-terminal peptide (CCTP) motif. Interestingly, these FtsZs contain a putative membrane-binding amphipathic helix as an N-terminal or C-terminal extension to the globular FtsZ domain. As a proof-of-concept, we experimentally show that the proposed C-terminal amphipathic helix in M. genitalium FtsZ binds liposomes in vitro as well as localizes to E. coli membrane in vivo. Additionally, we identify a putative cholesterol recognition motif within the C-terminal amphipathic helix region of M. genitalium FtsZ. Our study catalogues the functional variations of membrane attachment by the FtsZ and FtsA system in cell wall-less mycoplasmas and provides a new perspective to study novel functions of FtsZ/A system in cell division. ImportanceZ-ring and peptidoglycan synthesis machinery both play crucial roles in bacterial cell division. Currently, our knowledge about how FtsZ and FtsA, the two primary components of the Z-ring, function, is limited to cell-walled bacteria where ring constriction is coupled to peptidoglycan synthesis. Cell wall-less bacterial FtsZ/A system is an excellent model to study the mechanism of Z-ring constriction in the absence of cell wall synthesis machinery. Here, we analysed FtsZ protein sequences across mycoplasma species and identified their characteristic sequence features. Our study reveals a novel group of FtsZs from mycoplasma with an inherent membrane binding and probable cholesterol sensing amphipathic motif, which serves as a new paradigm to explore fundamental roles of FtsZ and FtsA in Z-ring constriction during bacterial division.

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↗