Understanding the molecular and structural basis of SepH function during cell division in Mycobacterium smegmatis

Diss, Rebecca Mary (2025) Understanding the molecular and structural basis of SepH function during cell division in Mycobacterium smegmatis. Doctoral thesis, University of East Anglia.

PDF Restricted to Repository staff only until 31 October 2027. Request a copy

Abstract

Bacterial cell division is driven by FtsZ polymers that assemble into a contractile ringlike structure (Z-ring), which is required for assembly of the cell division machinery and coordination of septum formation. While this process is reasonably well characterised in several model organisms, it is much less understood in Actinobacteria, which includes filamentous growing antibiotic-producers Streptomyces and unicellular pathogens such as Mycobacterium tuberculosis. SepH has recently been identified as a positive actinobacterial-specific regulator of FtsZ assembly and bundling. However, the molecular mechanism by which SepH promotes FtsZ self-association and efficient cell division was unclear.

Here, I use a combination of in vitro and in vivo assays to characterise the molecular and structural basis for the FtsZ-SepH interaction in Mycobacterium smegmatis. Using HDX-MS, I mapped the interaction interface between SepH and FtsZ and found that SepH binds to the globular N-terminal domain of FtsZ near to the GTP binding pocket. This analysis identified key amino acid residues involved in the interaction, and I generated alanine point mutants to assess their importance in binding and stimulation of FtsZ bundling in vivo and in vitro. Through a combination of structural modelling and in vitro and in vivo assays, I show that the bundling functionality of FtsZ mediated by SepH is dependent on SepH tetramerisation, specifically via the N-terminal 50 amino acids. This work identifies SepH as a potential functional homolog of ZapA, where tetramers are able to cross-link FtsZ filaments which is required for proper Z-ring assembly during cell division.

Additionally, I used Co-immunoprecipitation to uncover the SepH interactome in M. smegmatis. This uncovered 27 proteins with broad functions from DNA binding to cell envelope remodelling, suggesting that SepH may play a broader regulatory role during the cell division cycle.

Together, this work establishes the biochemical mechanism by which SepH promotes FtsZ filament bundling and Z-ring assembly, advancing our understanding of cell division in actinobacteria. These insights provide a framework for investigating SepH function in pathogenic M. tuberculosis and highlight its potential as a target for antitubercular drug development.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	28 Jan 2026 11:37
Last Modified:	28 Jan 2026 11:37
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101764
DOI:

Actions (login required)

View Item