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

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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.