The findings come from a long-standing project between Colin Kleanthous at the University of Oxford and Christoph Baumann at the University of York. Together with lead author Patrice Rassam and key collaborators Mark Sansom at Oxford and Jacob Piehler at the University of Osnabrück.
Gram-negative bacteria are a major cause of disease, in part because they have a robust outer membrane that protects against the immune system and certain antibiotics. They can live in a broad range of environments, which for E.coli includes river water as well as humans and animals.
The bacteria have intricate regulatory mechanisms for ensuring they have the right complement of outer membrane proteins – known as OMPs – for a particular habitat. But little is known about how OMPs are replaced in the outer membrane when large scale remodelling of these proteins has to occur on adapting to changes in growth conditions.
The new research describes how bacteria are able to change the proteins in their outer membrane and how this is intimately linked to the process of protein insertion in the membrane.
It was an initial focus on colicins – toxins produced by some strains of E.coli – which led Kleanthous and Baumann to their current finding.
The researchers were tracking how colicins make their way into bacteria via specific OMP receptors and had developed fluorescent versions of colicins to do this. Using single-molecule fluorescence microscopy, they noticed that the colicin-bound receptors behaved in an unusual way in the membrane.
'We spent many years trying to figure out what might be causing the receptors to behave in this way, as if something was boxing them in,' said Professor Kleanthous. They felt that an understanding of this would help unravel a basic property of the outer membrane, a key component of pathogenesis in Gram-negative bacteria.
After discounting many potential mechanisms, they discovered that the receptors bunch together in the outer membrane into structures they call 'OMP islands'. It was this that was causing the 'boxing in' they had noticed originally. They estimate that there are hundreds of OMPs in these islands and up to 40 OMP islands in a typical cell.
The scientists could replicate the apparently complex behaviour of OMPs in Gram-negative bacteria using purified proteins in an artificial membrane system in which they found that OMPs have a natural tendency to self-associate.
The next step was to find out where the machinery that inserts OMPs within the outer membrane of Gram-negative bacteria is located.
'We were not surprised to find the OMP insertion machinery in the islands,' explains Dr Baumann. 'But it was completely unexpected to discover this machinery shuts down as the outer membrane matures.' Although the reason for this is unclear, it is an important part of the new mechanism as it means 'old' and 'new' OMPs do not intermix.
When the group explored this further, they found that OMP biogenesis occurs as a gradient that is highest in the central regions of the cell and diminishes towards the ends of the cell. The cumulative effect is that old OMPs are pushed to the ends of growing cells as new OMPs take their place. This results in what the researchers call 'binary OMP partitioning': after two cell divisions, cells appear that do not have any of the original OMPs. In simple terms, a bacterium like E. coli can change its outer membrane protein coat in just two generations.
The research demonstrates that there is a surprising amount of spatial and temporal organisation of the outer membrane within a Gram-negative bacterium and this lies at the heart of how OMPs are replenished in the membrane.
Professor Kleanthous and Dr Baumann believe that their ongoing collaborative work, which began when the former was at the University of York, will have a major impact on our understanding of the bacterial outer membrane.
'It offers up many new avenues of research and also suggests new potential targets for antibiotic development, for example the disruption of OMP islands,' Professor Kleanthous said.