Biology subjects

Drobnic, T.

Publications and source records attributed to Drobnic, T..

6 recordsLinked to original sources

bioRxiv2026

Bridging the lipid divide: archaeal ESCRT-III binds phosphoinositol and polarises the cytokinetic membrane

All cells remodel their membranes to divide. The highly conserved ESCRT-III system forms contractile polymers which, through direct interactions with membrane lipids, remodel membranes across the tree of life. In exploring how ESCRT-III divides the chemically and structurally unique archaeal membrane, we reveal that the homologue CdvB1 is required for the establishment of a distinct membrane domain within the division bridge of Sulfolobus acidocaldarius, associated with an accumulation of membrane-spanning inositol phosphate lipids. We show that CdvB1 associates with phosphoinositides in vitro and that this interaction aids cytokinesis in vivo. Together, we suggest that although eukaryotes inherited their membrane lipids from bacteria during eukaryogenesis, key features of the ESCRT-III:membrane interface that allow these polymers to bind, organise, and remodel eukaryotic membranes, may originate in archaea.

cell biology↗

bioRxiv2025

Molecular structure of the ESCRT III-based archaeal CdvAB cell division machinery

Most prokaryotes divide using filaments of the tubulin-like FtsZ protein, while some archaea employ instead ESCRT-III-like proteins and their filaments for cell division and cytokinesis. The alternative archaeal system comprises Cdv proteins and is thought to bear some resemblance to ESCRT-III-based membrane remodelling in other domains of life, including eukaryotes, especially during abscission. Here we present biochemical, crystallographic and cryo-EM studies of the Sulfolobus Cdv machinery. CdvA, an early non-ESCRT component, adopts a PRC-domain/coiled-coil fold and polymerises into long double-stranded helical filaments, mainly via hydrophobic interfaces. Monomeric CdvB adopts the canonical ESCRT-III fold in both a closed and a distinct "semi-open" conformation. Soluble CdvB2 filaments are composed of subunits in the closed state, appearing to transition to the open, active state only when polymerised on membranes. Short N-terminal amphipathic helices in all CdvB paralogues, B, B1 and B2, mediate membrane binding and are required for liposome recruitment in vitro. We provide a molecular overview of archaeal ESCRT-III-based cytokinesis machinery, the definitive demonstration that CdvB proteins are bona fide ESCRT-III homologues and we reveal the molecular basis for membrane engagement. Thus, we illuminate conserved principles of ESCRT-mediated membrane remodelling and extend them to an anciently diverged archaeal lineage. Significance statementMembrane remodelling by ESCRT-III proteins is a fundamental and conserved process across the tree of life. The archaeal ESCRT-III-based cell division system (Cdv) drives cytokinesis in many archaeal groups, yet the molecular architecture of its components remained unknown, making it difficult to decipher the molecular mechanisms employed for cell division and cytokinesis in these organisms. We present structures of the Cdv machinery in Sulfolobus organisms that have been used previously to study the cell division process. We show that CdvA forms unexpected antiparallel helical filaments, while the ESCRT-III homologues retain the canonical fold we show they share with eukaryotic proteins. We demonstrate that N-terminal helices mediate membrane binding and that membrane contact, rather than polymerisation alone, likely triggers activation of Cdv ESCRT-IIIs.

molecular biology↗

bioRxiv2025

Functional genomics of Campylobacter-host interactions in an intestinal tissue model reveals a small lipoprotein essential for flagellar assembly

Campylobacter jejuni is the leading cause of bacterial food-poisoning, with motility being an essential virulence factor. While many aspects of flagella biogenesis have been studied, a complete picture of the components and regulators of these multi-protein machines is still missing. To identify genes crucial for C. jejuni pathogenesis and motility, we applied transposon sequencing in a humanized tissue model. This revealed three largely uncharacterized genes (pflC , pflD , pflE ) as essential for motility. While PflC/D turned out to be components of the flagellar motor disk structures, PflE is a small protein of only 57 aa. PflE strikingly affects motor biogenesis, with complete loss of motor structures upon its deletion. We demonstrate PflE is a lipoprotein and supports outer-membrane localization of the main basal-disk protein FlgP. With motility as a critical Campylobacter virulence factor, our work demonstrates that deletion of a small protein can bring a bacterial pathogen to a halt.

microbiology↗

bioRxiv2024

Structure of the complete extracellular bacterial flagellum reveals mechanism for flagellin incorporation

The bacterial flagellum is essential for motility, adhesion, and colonization in pathogens like Salmonella enterica and Campylobacter jejuni. Its extracellular structure comprises the hook, hook-filament junction, filament, and filament cap. The native structures of the hook-filament junction and the cap remain elusive, leaving the molecular details of cap-mediated filament assembly largely uncharacterized. Here, we report the structure of the complete extracellular flagellum, encompassing the hook, hook-filament junction, filament, and cap. This structure reveals intermediates of filament assembly, providing a molecular blueprint for flagellin folding and insertion at the filament tip. Mutagenesis and functional assays demonstrate the crucial roles of the caps terminal regions in flagellin incorporation, and of the structural integrity of the hook-filament junction. Finally, the structure of the cap and hook-filament junction prior to filament assembly reveals the structural basis for the initiation of filament assembly. Collectively, this study provides comprehensive insights into flagellum assembly and how flagellin incorporation is coupled with its secretion.

microbiology↗

bioRxiv2023

Evolution of a large periplasmic disk in Campylobacterota flagella facilitated efficient motility alongside autoagglutination

Although the bacterial flagella of Escherichia coli and Salmonella enterica are distributed around the cell body, many bacteria instead place their flagella at their poles. This widespread form of flagellar motility is relatively poorly understood, but these polar flagellar motors invariably feature periplasmic disk structures of unknown function. The flagellar motor of Campylobacter jejuni features a 100 nm-wide periplasmic disk associated with scaffolding a wider ring of motor proteins to increase torque, but the size of this disk is excessive for a role solely in scaffolding motor proteins. Here we show that the basal disk in C. jejuni is a flange that braces the motor during disentanglement of the flagellar filament from interactions with the cell body and other filaments, interactions that are otherwise important for host colonization. Our results reveal an entanglement of co-dependencies in the evolution of flagellar motor structure and cell plan in the Campylobacterota (previously epsilonproteobacteria). Note that this manuscript has a sibling manuscript titled Molecular model of a bacterial flagellar motor in situ reveals a "parts-list" of protein adaptations to increase torque that describes a molecular model of the Campylobacter jejuni flagellar motor discussed here.

microbiology↗

bioRxiv2023

Molecular model of a bacterial flagellar motor in situ reveals a "parts-list" of protein adaptations to increase torque

One hurdle to understanding how molecular machines work, and how they evolve, is our inability to see their structures in situ. Here we describe a minicell system that enables in situ cryogenic electron microscopy imaging and single particle analysis to investigate the structure of an iconic molecular machine, the bacterial flagellar motor, which spins a helical propeller for propulsion. We determine the structure of the high-torque Campylobacter jejuni motor in situ, including the subnanometre-resolution structure of the periplasmic scaffold, an adaptation essential to high torque. Our structure enables identification of new proteins, and interpretation with molecular models highlights origins of new components, reveals modifications of the conserved motor core, and explain how these structures both template a wider ring of motor proteins, and buttress the motor during swimming reversals. We also acquire insights into universal principles of flagellar torque generation. This approach is broadly applicable to other membrane-residing bacterial molecular machines complexes.

molecular biology↗