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Pushing the envelope: atomic force microscopy imaging of the bacterial outer membrane during growth and division

ReferenceBB/X00760X/1
Principal Investigator / Supervisor Professor Bart Hoogenboom
Co-Investigators /
Co-Supervisors
Institution University College London
DepartmentLondon Centre for Nanotechnology
Funding typeResearch
Value (£) 481,479
StatusCurrent
TypeResearch Grant
Start date 01/04/2023
End date 31/03/2026
Duration36 months

Abstract

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.

Summary

Gram-negative bacteria are among the microbes with the highest potential to develop resistance against commonly used antibiotics, such that previously harmless infections can become severe and potentially life-threatening diseases. This is in part because these bacteria (including A. baumannii, P. aeruginosa, and enterobacteriaceae such as Salmonella and E. coli) are protected by an outer membrane that limits entry of antibiotics into the cell. Based on light - and particularly fluorescence - microscopy, it has been suggested that this outer membrane organises its building blocks in rather sophisticated ways, including the formation of protein islands that may also play a role in how efficiently bacteria clear antibiotics. Quite generally, light microscopy has been a most powerful tool to discover and understand biological phenomena that define the life, growth, division, and death of cells. Yet its resolution on living cells is mostly insufficient to resolve cells at molecular length scales. Atomic force microscopy (AFM) is an alternative technique that can probe single molecules by gently tracing their contours with a sharp probe. We have recently used AFM to resolve the outer membrane of living bacteria at molecular-scale resolution, thereby revealing how the membrane segregates into different protein-enriched and lipid-enriched domains. Here, noting that bacteria can divide every ~20 minutes under favourable conditions, we propose to develop methods that will enable us to carry out such AFM experiments on growing and dividing cells, with the aim to better understand how the outer membrane facilitates/adapts to bacterial growth and division, including the synthesis and insertion of new membrane components.
Committee Research Committee D (Molecules, cells and industrial biotechnology)
Research TopicsX – not assigned to a current Research Topic
Research PriorityX – Research Priority information not available
Research Initiative X - not in an Initiative
Funding SchemeX – not Funded via a specific Funding Scheme
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