Biology subjects

Roper, D.

Publications and source records attributed to Roper, D..

4 recordsLinked to original sources

bioRxiv2026

Development of a high throughput continuous fluorescence anisotropy-based assay to analyse peptidoglycan-digesting enzymes

Bacterial cell wall processing enzymes have a central role in the control of the construction, remodelling and breakdown of the peptidoglycan wall that protects bacteria from osmotic rupture. Many of these enzymes have biologically essential functions and exert mechanistically diverse activities on a complex polymer substrate. Despite this biological importance they are relatively understudied and there are significant technical challenges in measuring the activity of individual enzymes against the complex chemical structure of the bacterial cell wall peptidoglycan. To generate a suitable substrate, bacterial cells are grown with a fluorescent D-alanine analogue, allowing the isolation of natively fluorescently labelled PG. This material is then used to measure interactions with PG-targeting enzymes and regulatory proteins. The resulting assay enables analysis of multiple enzyme-substrate combinations within minutes while retaining molecular specificity. This approach provides a robust, cost-effective alternative to conventional PG activity assays and could accelerate the identification and characterisation of novel antimicrobial compounds targeting bacterial cell wall metabolism. Our results indicate this assay has the potential to be employed in high-throughput inhibitor discovery, making it effective for a broad-spectrum assay for bacterial peptidoglycan research applications.

biochemistry↗

bioRxiv2024

Characterization of a GpsB-associated regulator of PBP1a reveals the organization of the cell wall remodeling complex of Streptococcus pneumoniae

Class A PBPs (aPBPs) play a key role in the biosynthesis and remodeling of peptidoglycan, the main component of the bacterial cell wall. The human bacterial pathogen Streptococcus pneumoniae produces three aPBPs, which are regulated to maintain the bacteriums ovoid shape. Although their exact functions remain unclear, evidence suggests that PBP1a and PBP2a activities are closely coordinated. In this study, we elucidated the function of an unknown function protein named GarP (GpsB-associated regulator of PBP1a), in the regulation of PBP1a activity. We showed that GarP localizes to the division septum and its absence leads to morphological defects. We further identified a GpsB-binding motif in GarP as well as in PBP2a, the PG deacetylase PgdA and the muramidase MpgA. Our analysis of genetic and protein interactions, combined with cell imaging, supports a model of a molecular complex that coordinates PG remodeling during S. pneumoniae cell division.

microbiology↗

bioRxiv2020

Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

The Gram-negative outer membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here we reveal DolP is a lipoprotein functionally conserved among Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds to anionic phospholipids through an extensive membrane:protein interface providing evidence of subcellular localization of these phospholipids within the outer membrane. This interaction is essential for DolP function and is required for sub-cellular localization of the protein to the cell division site. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

biochemistry↗

bioRxiv2020

Structure-based modelling and dynamics of MurM, a Streptococcus pneumoniae penicillin resistance determinant that functions at the cytoplasmic membrane interface

MurM is an aminoacyl-tRNA dependant ligase that aminoacylates the Lipid II peptidoglycan precursor, in the human pathogen Streptococcus pneumoniae. MurM is required for the generation of branched peptidoglycan precursors enabling indirect cross-links in the peptidoglycan and is found to be essential for penicillin resistance. In this study we have solved the X-ray crystal structure of Staphylococcus aureus FemX, an isofunctional homologue of MurM, and used this as a template to generate a homology model of MurM. Using this model, we perform molecular docking and molecular dynamics to examine the interaction of the protein with the phospholipid bilayer and the membrane embedded Lipid II substrate of MurM. Our model suggests that MurM is associated with the major membrane phospholipid cardiolipin, and we confirm this with experimental evidence that the activity of MurM is enhanced by this phospholipid and inhibited by its direct precursor phosphatidylglycerol. This suggests that the spatial association of pneumococcal membrane phospholipids and their impact on MurM activity may be a critical to the final architecture of the peptidoglycan and the expression of clinically relevant penicillin resistance in this pathogen.

biochemistry↗