Pushing the envelope: atomic force microscopy imaging of the bacterial outer membrane during growth and division
Lead Research Organisation:
University of Oxford
Department Name: Biochemistry
Abstract
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Technical Summary
The outer membrane (OM) is a crucial barrier that protects Gram-negative bacteria against environmental insult and restricts entry of drugs, thereby contributing to antibiotic resistance. Recent fluorescence microscopy data suggest that the OM is assembled according to previously unsuspected organisational principles, including protein clustering at length scales of ~100 nm. Due to technical limitations of fluorescence microscopy, however, it has remained hard to probe the nature and mechanisms of such outer membrane organisation, and particularly how it is modulated to facilitate growth and division in living cells.
As we have recently reported (Benn et al., PNAS USA 2021), atomic force microscopy (AFM) has revealed another layer of OM organisation, showing living Gram-negative bacteria (E. coli) at a spatial resolution of a few nm, covered with a densely packed network of trimeric outer membrane proteins (OMPs) interspersed by OMP-depleted domains that are enriched in lipopolysaccharides (LPS).
With the research proposed here, we will develop methodology to perform such high-resolution AFM experiments on cells that are growing and dividing. We will next use this to map OM organisation and its dynamics over the entire cell cycle, thereby determining how the solidity/fluidity of the OM is modulated and how the OM biosynthesis machinery is spatially and temporarily organised to facilitate growth and division.
As we have recently reported (Benn et al., PNAS USA 2021), atomic force microscopy (AFM) has revealed another layer of OM organisation, showing living Gram-negative bacteria (E. coli) at a spatial resolution of a few nm, covered with a densely packed network of trimeric outer membrane proteins (OMPs) interspersed by OMP-depleted domains that are enriched in lipopolysaccharides (LPS).
With the research proposed here, we will develop methodology to perform such high-resolution AFM experiments on cells that are growing and dividing. We will next use this to map OM organisation and its dynamics over the entire cell cycle, thereby determining how the solidity/fluidity of the OM is modulated and how the OM biosynthesis machinery is spatially and temporarily organised to facilitate growth and division.
