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bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.02.610904
preprint
1
Article
β-barrel proteins dictate the effect of core oligosaccharide composition on outer membrane mechanics
Fitzmaurice Dylan
Amador Anthony
Starr Tahj
Hocky Glen M.
Rojas Enrique R. http://orcid.org/0000-0003-3388-2794

03 9 2024
2024.09.02.610904https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.02.610904
nihpp-2024.09.02.610904.pdf
Abstract

The outer membrane is the defining structure of Gram-negative bacteria. We previously demonstrated that it is critical for the mechanical integrity of the cell envelope and therefore to the robustness of the bacterial cell as a whole. Here, to determine the key molecules and moieties within the outer membrane that underlie its contribution to cell envelope mechanics, we measured cell-envelope stiffness across several sets of mutants with altered outer-membrane sugar content, protein content, and electric charge. To decouple outer membrane stiffness from total cell envelope stiffness, we developed a novel microfluidics-based “osmotic force extension” assay. In tandem, we developed a simple method to increase throughput of microfluidics experiments by performing them on color-coded pools of mutants. Using Escherichia coli as a model Gram-negative bacterium, we found that truncating the core oligosaccharide, deleting the β-barrel protein OmpA, or deleting lipoprotein outer membrane-cell wall linkers all had the same modest, convergent effect on total cell-envelope stiffness but had large, varying effects on the ability of the cell wall to transfer tension to the outer membrane during large hyperosmotic shocks. Surprisingly, altering lipid A charge had little effect on the mechanical properties of the envelope. Importantly, the presence or absence of OmpA determined whether truncating the core oligosaccharide decreased or increased envelope stiffness (respectively), revealing sign epistasis between these components. Based on these data we propose a specific structural model in which the chemical interactions between lipopolysaccharides, β-barrel proteins, and phospholipids coordinately determine cell envelope stiffness, and the ability of the outer membrane to functionally share mechanical loads with the cell wall.

Statement of Significance

The outer membrane is the defining cellular structure of Gram-negative bacteria, a group that contains many important pathogens like Escherichia coli , Vibrio cholerae , and Pseudomonas aeruginosa . One role of the outer membrane is to block the uptake of small molecules like antibiotics. However, it is becoming increasingly clear that it also functions as a structural exoskeleton that is critical for the cell’s ability to cope with internal and external mechanical forces. Here, we carefully dissect the molecular basis for the load-bearing capacity of the outer membrane by screening a set of mutants with a new cell biophysics assay.
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