Variability in bacterial flagella re-growth patterns after breakage

Guillaume Paradis,Marc Erhardt,Thibaud T Renault,Fabienne F V Chevance,Simon Rainville,Kelly T Hughes,Willisa Liou

doi:10.1038/s41598-017-01302-5

Guillaume Paradis, Marc Erhardt + Show 5 more

Open Access

https://doi.org/10.1038/s41598-017-01302-5

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Abstract

Many bacteria swim through liquids or crawl on surfaces by rotating long appendages called flagella. Flagellar filaments are assembled from thousands of subunits that are exported through a narrow secretion channel and polymerize beneath a capping scaffold at the tip of the growing filament. The assembly of a flagellum uses a significant proportion of the biosynthetic capacities of the cell with each filament constituting ~1% of the total cell protein. Here, we addressed a significant question whether a flagellar filament can form a new cap and resume growth after breakage. Re-growth of broken filaments was visualized using sequential 3-color fluorescent labeling of filaments after mechanical shearing. Differential electron microscopy revealed the formation of new cap structures on broken filaments that re-grew. Flagellar filaments are therefore able to re-grow if broken by mechanical shearing forces, which are expected to occur frequently in nature. In contrast, no re-growth was observed on filaments that had been broken using ultrashort laser pulses, a technique allowing for very local damage to individual filaments. We thus conclude that assembly of a new cap at the tip of a broken filament depends on how the filament was broken.

Highlights

The flagellum of enteric bacteria consists of three main structural parts: (i) a basal body complex that spans the periplasmic space between the inner and outer membranes and is embedded in the cell wall; (ii) an external, flexible linking structure and (iii) a rigid, helical filament made of several thousand flagellin subunits[1, 2]
In order to determine if flagellar filaments can re-grow, Rosu and Hughes analyzed the dynamics of Class 3 gene expression after flagellar shearing in Salmonella enterica[12]
We demonstrate using 3-color differential fluorescent labeling of flagellar filaments that flagellar filaments are able to re-grow after breakage by mechanical shearing forces

Summary

Introduction

The flagellum of enteric bacteria consists of three main structural parts: (i) a basal body complex that spans the periplasmic space between the inner and outer membranes and is embedded in the cell wall; (ii) an external, flexible linking structure (the hook) and (iii) a rigid, helical filament made of several thousand flagellin subunits[1, 2]. Gene products expressed from Class 2 promoters include the components of the HBB complex, as well as regulatory proteins, e.g. the flagellar-specific, alternative σ28 factor and its cognate anti-σ factor FlgM. Class 3 gene products are needed for completion of the flagellum (e.g. the filament subunits, the filament cap, the motor-force generators) and the chemosensory system. By secreting FlgM protein after the secretion specificity switch, the cell ensures that genes needed after HBB completion are only expressed after a functional HBB structure has been assembled, onto which σ28-dependent gene products such as the filament subunits can polymerize. FliD is likely expressed from its Class 3 promoter in the case of shearing events, which allows the formation of a new cap on the tip of the broken filament and re-growth of a sheared filament. A caveat of their experiment was the inability to distinguish re-growth of sheared filaments from continued growth of nascent, short filaments that were not broken

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