Faculty of Applied Sciences

20 February 2020

Monitoring the development of a tumour using the memory of bacteria

Researchers at Delft University of Technology have discovered how certain proteins keep a bacterium's memory up to date. This is essential for bacteria, since their memory protects them from attacks by bacteriophages, their natural enemies. It is also interesting for us humans, since the system can be converted into a 'DNA recorder': a kind of biological logbook that keeps track of what happens in a cell. Such a DNA recorder could, for instance, be used to monitor how a tumour develops over time. The first DNA recorders have already been built, but they are inefficient and do not work in human cells. The new fundamental knowledge of bacterial memory formation gained by the Delft researchers can be used to develop the next generation of DNA recorders. The findings have been published in Nature . Despite the fact that researchers all over the world are using CRISPR-Cas9 for gene editing, there are still many open questions about the precise functioning of CRISPR systems and the proteins involved. This is because CRISRP-Cas9 is not a man-made molecular tool, but a biological defence mechanism that protects bacteria against their arch-enemies: bacteriophages, the viruses of bacteria. Race against time When bacteriophages encounter a bacterium in nature, they cling to it and inject their DNA into the cell. Then, a race against time begins. If a bacterium does not recognize and destroy the hostile DNA in time, the bacteriophage hijacks the cell and uses it to make copies of itself, finally causing the bacterium to explode. The newly released bacteriophages then start looking for their next victim. In order to recognise hostile DNA before it’s too late, a bacterium relies on its memory. Certain Cas proteins are tasked with storing pieces of hostile DNA in the bacterial genome. "This is done in a place called the 'CRISPR array'," says group leader Chirlmin Joo of Delft University of Technology. The CRISPR array is a kind of logbook that describes which enemy attacked the bacterium, in the form of fragments of hostile DNA separated by a recurring string of nucleotides. If a bacterium’s memory contains a fragment of the DNA of a bacteriophage, it has the ability to quickly recognise and render harmless any hostile DNA in the event the same bacteriophage mounts another attack. Bypass "The problem for bacteria is that bacteriophages constantly evolve, thus trying to bypass the bacterial immune system," says Joo. "Therefore, it is essential for bacteria to keep their memory up to date." Two cooperating proteins are involved in this process: Cas1 and Cas2. These proteins trim pieces of hostile DNA, which makes it possible for the genetic information to be stored in the memory of the bacterium - the CRISPR array. The Delft researchers have now discovered how Cas 1 and 2 proteins do this. It appears that more is required than just the two Cas proteins. Among other things, the researchers have discovered that an enzyme that also play an important role in other cellular processes is involved. This enzyme is called ‘DNA-polymerase III’ and it is known for its role in DNA replication, the copying of DNA. "DNA polymerase has a sort of backspace function for when it makes an error while copying DNA," Joo explains. "We've discovered that it also uses this function to trim fragments of hostile DNA in such a way that they can be incorporated into the CRISPR array." In addition to discovering that DNA polymerase plays a key role in memory formation in bacteria, the researchers also discovered what properties the fragments of hostile DNA need to have in order to allow the Cas proteins to do their work. They also found out how the proteins actually incorporate these DNA fragments into the bacterial memory. Biological logbook This new fundamental knowledge about memory formation in bacteria is important for the further development of a new technique called 'DNA recording'. "In recent years, several research groups have shown that it is possible to build a kind of biological logbook based on these systems," says research leader Sungchul Kim. The idea is that, with such a system, information about cellular processes can automatically be stored in the DNA of that cell. Since Cas1 and Cas2 store information in chronological order, one could determine exactly how, for instance, a tumour has developed by removing some cells from the tumour and consulting their biological logbook. Doctors could then use this valuable diagnostic data to make a tailor-made treatment plan. At the moment, DNA recording is still in its infancy. "Simple systems have already been developed, but they are not efficient and cannot, for example, follow multiple processes at the same time," says Kim. Moreover, they do not yet function in human cells. "But knowing how Cas1 and Cas2 work now gives us the information we need to understand where things went wrong with the first DNA recorders," Kim continues. "This allows us to design more efficient recorders that also work in human cells." Watch a scientific animation explaining the findings here. Selective loading and processing of prespacers for precise CRISPR adaptation, Sungchul Kim, Luuk Loeff, Sabina Colombo, Slobodan Jergic, Stan J. J. Brouns & Chirlmin Joo, Nature DOI: 10.1038/s41586-020-2018-1. Dr. Chirlmin Joo +31 15 27 83220 C.Joo@tudelft.nl Joo Lab Read more: CRISPR-Cas9

19 February 2020

Researchers discover new mechanism for the coexistence of species

Researchers from the AMOLF institute in Amsterdam and Harvard show how the ability of organisms to move around plays a role in stabilizing ecosystems. In their paper published today in Nature, they describe how the competition between 'movers' and 'growers' leads to a balance in which both types of bacteria can continue to exist alongside each other. We are all too familiar with the threats to our planet’s ecosystems: global warming, forest fires, nitrogen deposition, biodiversity decline, and even mass extinctions. But what actually makes ecosystems stable or fragile? What prevents one species from outcompeting all others, and hence drive them to extinction? These questions have captivated biologists since Darwin. We have learned that food-webs and cooperation between species are key pieces to this puzzle, because they help explain how species then depend on one another to survive. Now a group of biophysicists from the Netherlands and the US have advanced a startling finding: the active movement of organisms can also drive ecosystem diversity and stability – through a remarkably simple mechanism that does not require food-webs or cooperation. Slowed-down movie of motile E. coli cells. Credits: AMOLF. "Movement is fundamental to essentially all organisms – even plants move by seed dispersal," says Sander Tans at the AMOLF institute in Amsterdam. "Bacteria are well known to move actively. Our experiments show how this movement can keep different bacterial species, typically called strains, together in a larger population. There is a rich literature on the possible roles of movement in such coexistence of species, but direct experiments that can exclude other explanations were lacking. The coexistence paradox – what prevents extinctions in a competitive world? To find bacterial species that might form a minimal stable ecosystem in the lab, the PhD student Sebastian Gude took two species (also called strains) from the gut of the same animal. If both survived there, perhaps they would also do so in his experiments. To follow their competition, he colored one blue and the other red. However, all his first attempts failed in the beginning. One of the two strains always lost the competition when grown together, with these bacteria always producing fewer offspring than the other. This blue ‘loser’ strain would thus become outnumbered by the red ‘winner’ strain, and was ultimately driven to extinction. Cartoon of experiment showing coexistence between bacteria (red and blue) that cyclically colonize nutrient patches (green). Credits: AMOLF. Gude’s luck took a dramatic up-turn, however, when he changed the design of the experiment. He took the sugary liquid that the bacteria normally grew in, and turned it into a gel, reminiscent of a jelly desert. When the blue ‘loser’ bacteria became outnumbered by the reds when growing on this gel, they started to produce more offspring than the reds, and were thus winning. But in turn the reds also became more competitive when rare. In this way both strains thus escaped extinction, and hence coexisted together. These results underscore the fundamental paradox of the coexistence debate: what causes losers that are close to extinction to suddenly start winning? Bacterial strains A and B were show to both outcompete the other when rare, and hence coexist. Credits: AMOLF. Getting territorial To resolve this conundrum, Gude followed the competition by making movies. "The results were quite striking," says Tom Shimizu. “We saw the populations migrating into the gel like a wave, where they multiplied using the sugars they encountered. Initially, red dominated the expansion and blue was barely seen. But then the red advance suddenly stopped - just when the blues emerged and were seen to overtake the reds’ front, where they formed just a thin layer. After that, the wave was blue only. The blue bacteria could thus proliferate alone in the deeper regions of the gel, freed from competition from the reds that could not reach that far. This also explained the coexistence: whenever blue were rare, they could build up their population in the deeper regions of the gel.” Fluorescence microscopy of bacterial populations across the gel after the competition. Credits: AMOLF. Should I spread or should I grow? But how did the blue bacteria organize themselves and efficiently confine the red? Did they signal to each other or secrete toxins, as is known for some bacteria? In trying to address these questions the team discovered a rather different mechanism. The blue bacteria were indeed worse at proliferating, as they lost in a direct competition. But they compensated by migrating faster. By reaching the farther regions first, they could finish the local sugars. Hence, they gave the red no chance there, and could thus block their advance, a bit like in a scorched earth strategy. Accelerated movie of competing bacterial strains. Credits: AMOLF. Tans: “Some bacteria apparently are good at proliferating, and others at migrating. But they cannot excel at both. This makes sense because both activities cost a lot of energy. Such specialization is often observed, though effects on coexistence are often difficult to prove. Here we could manipulate the capacity to migrate and proliferate by genetic engineering, and show it alone is enough for coexistence. Other mechanisms like exchanging toxins or dependencies like in food-webs are thus not required per se.” Disturbance ecology Out in the real world, bacteria of course cannot count on encountering bowls of jello left out of the fridge. Luckily for them they don’t need to, as pristine pastures of fresh nutrients do arise more often than one might expect. Much like the vegetation that colonize cleared soil after forest fires, many bacteria grow on patches of resources: from windfallen fruits to decomposing animals, or – for those microbes that inhabit your gut – the lunch you just had. Shimizu: "We demonstrated this migration-proliferation mechanism in motile bacteria. But what we found is for instance evocative of models of plant ecology, where fast-growing plants compete with plants that invest more in spreading their seeds." He added that there is a lot to be learned about more complex scenarios. "There is a lot of interest in explaining the diversity of bacteria in the human gut, which we now know, thanks to modern DNA analysis, is intimately related to our health. Our findings suggest that looking at motility genes and their spatial distribution within the gut might help explain some of that diversity." More information Bacterial coexistence driven by motility and spatial competition Sebastian Gude, Erçağ Pinçe, Katja M. Taute, Anne-Bart Seinen, Thomas S. Shimizu, and Sander J. Tans, Nature 2020 DOI : 10.1038/s41586-020-2033-2 www.nature.com/articles/s41586-020-2033-2 Prof. S. J. Tans +31 20 754 7255 tans@amolf.nl sandertanslab.nl AMOLF institute Amsterdam Delft University of Technology Prof. T.S. Shimizu shimizu@amolf.nl AMOLF institute Amsterdam Free University

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