Biology subjects

Cohen, E. J.

Publications and source records attributed to Cohen, E. J..

6 recordsLinked to original sources

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↗

bioRxiv2021

Periplasm homeostatic regulation maintains spatial constraints essential for cell envelope processes and cell viability

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics and a synthetic lethal screen we show that lengthening Lpp to the upper limit does not change the spatial constraint, but rather impacts the load-bearing capacity across the outer membrane. Our findings demonstrate E. coli expressing elongated Lpp homeostatically counteracts periplasmic enlargement with a combination of tilting Lpp and reducing Lpp abundance. By genetic screening we identified all of the genes in E. coli that become essential in order to enact this homeostasis, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to enact this homeostasis. We observed increased levels of factors determining cell stiffness, decrease membrane integrity, increase membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling and protein translocation

cell biology↗

bioRxiv2019

Campylobacter jejuni motility integrates specialized cell shape, flagellar filament, and motor, to coordinate action of its opposed flagella in viscous media

Campylobacter jejuni rotates a flagellum at each pole to swim through the viscous mucosa of its hosts gastrointestinal tracts. Despite their importance for host colonization, however, how C. jejuni coordinates rotation of these two opposing flagella is unclear. As well as their polar placement, C. jejunis flagella deviate from the Enterobacteriaceael norm in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand how C. jejunis opposed motors coordinate, and what contribution these factors play in C. jejuni motility, we developed strains with flagella that could be fluorescently labeled, and observed them by high-speed video microscopy. We found that C. jejuni coordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament has evolved to facilitate this wrapped-mode swimming. Unexpectedly, C. jejunis helical body is important for facile unwrapping of the flagellar filament from the cell body during switching of swimming trajectory. Our findings demonstrate how multiple facets of C. jejunis flagella and cell plan have co-evolved for optimal motility in high-viscosity environments.

microbiology↗

bioRxiv2019

A tetRA-based promoter system for the generation of conditional knockouts in Campylobacter jejuni

Campylobacter jejuni is responsible for tens of millions of cases of gastroenteritis each year. Despite its prevalence and impact on human health, the repertoire of genetic tools available for researchers to study C. jejuni remains limited. In order to expand upon the genetic toolkit in this species, we have engineered a system for generating conditional knockouts based on the tetRA tetracycline-resistance cassette. This system exhibits tight repressibility and titratability of target-gene expression and will be useful for future research on this important human pathogen.

microbiology↗