An international team of researchers has captured the sharpest images ever of living bacteria, revealing the complex architecture of the protective layer that surrounds them and offering insight into bacteria’s resistance to antibiotics.
The study, recently published in Proceedings of the National Academy of Sciences of the USA, reveals that bacteria with protective outer layers – called Gram-negative bacteria – may have a range of stronger and weaker spots on their surface.
The team from University College London (UCL), in collaboration with scientists at National Physical Laboratory, UK, King’s College London, University of Oxford, UK, and Princeton University, US, found that the bacteria’s outer membrane contains dense networks of protein building blocks, but also patches that don’t appear to contain proteins. Instead, these patches are enriched in molecules with sugary chains (glycolipids) that keep the outer membrane tight.
It’s an important finding because the tough outer membrane of Gram-negative bacteria prevents certain drugs and antibiotics from penetrating the cell.
“The outer membrane is a formidable barrier against antibiotics and is an important factor in making infectious bacteria resistant to medical treatment,” says corresponding author Professor Bart Hoogenboom from the London Centre for Nanotechnology at UCL and UCL Physics and Astronomy.
“However, it remains relatively unclear how this barrier is put together, which is why we chose to study it in such detail.
“By studying live bacteria from the molecular to cellular scale, we can see how membrane proteins form a network that spans the entire surface of the bacteria, leaving small gaps for patches that contain no protein.
“This suggests that the barrier may not be equally hard to breach or stretch all over the bacterium, but may have stronger and weaker spots that can also be targeted by antibiotics.”
To better understand the architecture of this membrane, the scientists ran a tiny needle over living Escherichia coli (E. coli) bacteria to ‘feel’ their overall shape. With the tip of the needle only a few nanometres wide, it was possible to visualise molecular structures at the bacterial surface.
The resulting images show that the bacterial outer membrane is crammed with microscopic holes formed by proteins that allow the entry of nutrients while preventing the entry of toxins. Although the outer membrane was known to contain many proteins, this crowded and immobile nature was unexpected.
The images also surprisingly revealed many patches that didn’t appear to contain proteins. These patches contained a glycolipid normally found on the surface of Gram-negative bacteria.
“The observed patchiness came as a surprise, as the common view was that the bacterial surface would be more homogeneous,” says Hoogenboom. “Now we have discovered the patchy nature of the E. coli outer membrane, we can and should ask what it means.
“We do not have a full answer to this question yet, but we have several leads, related to another observation we have made in this study – that large areas of the membrane are crammed with proteins. That seems a problem, because bacteria need to insert new membrane material to allow them to grow without creating fissures in the membrane.
“We can speculate, for example, that the protein-free patches are spots where the bacteria can more easily insert new membrane material, including new proteins. This could help us to better understand how bacteria can grow without breaking. Obviously, that is speculation that we would like to validate or invalidate by further experiments.”
In addition, a different type of pimple-like patch formed when parts of the membrane were flipped inside out due to mutations. In this case, the appearance of these defects correlated with enhanced sensitivity to bacitracin, an antibiotic usually only effective against Gram-positive – but not against Gram-negative – bacteria.
“The textbook picture of the bacterial outer membrane shows proteins distributed over the membrane in a disordered manner, well-mixed with other building blocks of the membrane,” says Georgina Benn, who did the microscopy on the bacteria.
“Our images demonstrate that that is not the case, but that lipid patches are segregated from protein-rich networks just like oil separating from water, in some cases forming chinks in the armour of the bacteria.
“This new way of looking at the outer membrane means that we can now start exploring if and how such order matters for membrane function, integrity and resistance to antibiotics.”
Hoogenboom predicts that further research into this textured membrane could ultimately lead to the creation of more effective antibiotics.
“Our observations raise the question of whether we can adapt or develop antibiotics to specifically aim for patches or boundaries between protein-enriched and protein-poor regions,” he says. “The first step in that direction will be to examine if some existing antibiotics show a preference for different areas on the membrane. That represents, of course, another set of experiments that we’d love to do.”