Stress-induced molecular globs increase bacterial fitness

W.hen two different liquids arise from a previously mixed solution – for example when oil and vinegar separate in a salad dressing – physicists call the process liquid-liquid phase separation (LLPS). Aside from spices, this phase separation also takes place on a microscopic scale inside cells, where it concentrates biomolecules and facilitates their reactions. While there are several examples of LLPS in eukaryotic cells, the process has been studied less intensively in bacteria because of their small size. Consequently, questions remain as to when, how, and why LLPS occurs in prokaryotes.

Thanks to a report in Scientific advances yesterday (October 20th) “LLPS is now. . . firmly confirmed in prokaryotic systems ”, writes Frederic Rousseau, who studies protein aggregation at the KU Leuven in Belgium and was not involved in the research, in an email The scientist. It underscores “how fundamental LLPS is to cellular life and perhaps its origins,” he adds.

See “These organelles have no membranes”

Conventional microscopy methods have discovered protein aggregates that are believed to be formed by LLPS – called condensates – in bacteria, says chemist and biophysicist Julie Biteen of the University of Michigan, who was also not part of the research team but understands the mechanisms by which they are formed and whether they are liquid or solid, “was a real challenge,” she says. This is partly because the condensates are only slightly, if at all, larger than the diffraction limit of microscopy – the minimum distance at which two objects can be seen separated or resolved (about 200 nm for fluorescence microscopes). The new paper is therefore “a really important one. . . Demonstration of how state-of-the-art high-resolution imaging can be used for these problems. “

The biophysicists Fan Bai from Peking University and Mark Leake from the University of York had previously shown that protein condensates, which they call aggresomes, form in them Escherichia coli Cells when the bacteria are stressed by antibiotic treatment or the availability of ATP, a form of molecular energy, is reduced. In the team’s latest study, they examined the dynamics of aggressive education, as well as its function.

Using fluorescently labeled proteins that the team had previously shown to accumulate in aggresomes, the researchers found that when ATP is depleted, aggresomes form gradually over several hours, usually resulting in one at each end of the rod-shaped bacteria leads. Using a special type of fluorescence microscopy called fast super-resolved single-molecule tracking that follows the movements of individual labeled proteins, they also showed that the proteins diffuse freely within the aggresomes, so that they can pass through a move fluid environment.

Further evidence that the aggresomes are liquid came from experiments showing that they could be dissolved by an organic solvent that cannot dissolve solids and that fluorescently labeled proteins diffuse from one half of one aggresome to the other. In addition, computer simulations showed, among other things, that this is not an active process, but that no energy supply is required for the formation of aggresomes – d LLPS.

Although the team doesn’t yet know how the aggresomes form, they suspect it might have something to do with ATP itself. The molecule is a hydrotrope, explains Leake, which means that it has the ability to make hydrophobic molecules, including some proteins, soluble in aqueous environments. Therefore, its depletion could stimulate hydrophobic proteins to accumulate.

Regardless of the mechanism, “the most important result of our work is to show that proteins in aggresomes tend to stick together, but not rigidly together. They still show a certain amount of movement. That’s why we call it a liquid protein droplet, not a solid protein ball, ”wrote Bai in an email The scientist.

So why does it matter whether an aggresome is solid or liquid?

“It tells you how they form and what molecular interactions drive them,” says Stephanie Weber from McGill University, who studied the spatial organization of cells and was not involved in the research. If they were solid, they would be less dynamic and limit the types of interactions or reactions the proteins within them could perform, she explains. In addition, liquid droplets can form and dissolve quickly compared to solid structures, explains Leake, a clever strategy for adapting to the timescale of their rapidly changing environment. “

The team went on to show that these aggresomes were not unique E. colithat is produced in response to reduced ATP in eight other types of gram-negative bacteria, and that the aggresomes promoted cell survival. mutant E. coli which failed to form aggresomes (found by screening a library of mutated strains) but whose viability was otherwise apparently unaffected, had poorer survival when ATP was depleted when compared to controls. Since the mutations only impaired the formation of aggresomes and did not completely eliminate it, the team could see that “if [a mutant cell] had observable aggresomes, they were more likely to survive, ”says Leake.

“It is really exciting to show that not only condensates form in bacteria, but that they have a functional influence on survival and resistance to stress,” says Weber. In addition to elucidating the mechanism that triggers the LLPS, however, it would be important to know exactly what proteins are in the liquid clumps, she says. “That’s the million dollar question.”