Biological organisms can be difficult to study since they are constantly changing and evolving. In 1976, this prompted a founder of molecular biology, Jacques Monod, to suggest the existence of a factor that could explain the behaviour of the bacteria he studied. Finally, 45 years later, the factor has been found, and this work potentially explains far more than just the behaviour of a specific type of bacteria. For example, it may explain why studying cancer outside the human body is so difficult.
Studying children’s physiology may seem pointless if you want to know about adults. Many researchers face a similar challenge in studying the growth of bacteria or the mechanisms of cancer cells. The cells are cultured under conditions of growth and division, even though this is not their normal state. Instead, they are most often in a steady state, which differs physiologically from the growth state.
“We examined the carbon metabolism of bacteria and found a molecule whose existence was proposed 45 years ago. The discovery also suggests the mechanism behind frequently observed phenomena in both microbial evolution and cancer: mutations often aggregate in genetic hotspots and may have transient effects. The effects often disappear again if we culture cells under conditions other than those in which the mutations arose. This is important knowledge for ensuring that we study cancer under the right conditions but also when we try to artificially alter bacteria so that they can produce new substances,” explains Morten H. H. Nørholm, Senior Researcher, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby.
Bacteria under pressure
The new study builds on previous studies in which researchers showed how bacteria test new mutations using retromutagenesis. The classical theory of evolution is based on the fact that small random errors occur continually in DNA and are then passed on to future generations. However, the bacteria have developed a special mechanism in which the cell translates some DNA mutations into RNA and proteins. The changes are then conserved in DNA and passed on only if they turn out to be beneficial.
“This mechanism is especially advantageous in ageing bacterial cultures, since energy and food sources may be lacking and the bacteria cannot afford to conserve harmful mutations,” says Morten H. H. Nørholm.
The researchers examined the carbon metabolism in Escherichia coli bacteria to determine what happens in ageing bacterial cultures. They especially focused on the cAMP receptor protein (Crp), a transcription factor that binds to DNA molecules to ensure that the right genes are expressed at the right times. One function of Crp is to activate carbon metabolism, but it regulates hundreds of genes in bacteria.
“Crp is one of the first and most thoroughly studied transcription factors, but even though molecular biology pioneer Jacques Monod tried to understand how it was regulated already in the 1970s, we are still discovering new details. We found that when E. coli starves and ages, new mutations occur in Crp. More specifically, the mutations occur when cytidine accumulates over time. This applies pressure to the bacteria, which creates mutations in Crp to keep the bacteria alive. Cytidine is thus the modulator factor that Monod was seeking,” explains Morten Nørholm.
A new survival strategy
The new study also provides clues to another unsolved mystery in living cells. Cytidine is not a unknown substance but a variant of one of the four bases of DNA: adenine (A), thymine (T), guanine (G) and cytosine (C). But unlike the other three, which living cells can easily make themselves, cytosine can only be produced using a base of RNA – uracil.
“The C’s in DNA are synthesised in a very strange way. Living cells cannot from basic metabolism produce cytidine or cytosine or the phosphorylated versions, CMP and CDP, which are used to build DNA. Instead, the C’s are formed when RNA is degraded. CTP is created from UTP and then embedded in RNA. So RNA is needed to make DNA. Why? Our research indicates that cytidine acts not only as a building block in DNA and RNA but also as an important stress signal in the body,” says Morten Nørholm.
Cytidine thus accumulates in the nutrient-poor medium in which the cells are put under stress and therefore have to adopt a new survival strategy. The researchers made the bacteria age and mutate in the nutrient-poor and cytidine-rich environment and then put them in fresh growth medium.
“When the mutants are recultured with fresh nutrients, the cytidine levels fall and thus the effect of the Crp mutations. This creates even more mutations in Crp. Now mutations that give independence from cytidine suddenly become beneficial. Metabolism is the key to understanding why many mutations are generated in the same gene,” explains Morten Nørholm.
Cancer cells organise themselves differently
According to the researchers, the new experiments are “a cautious narrative that mutations should be studied in exactly the environment in which they originate”. Given this fact, the researchers realised that they needed to adopt a special method: robots that can very accurately track the growth of bacterial cultures on agar plates.
“The robots help us monitor precisely when certain types of interesting mutations occur,” says Morten Nørholm.
For example, Morten Nørholm mentions that cancer cells typically age under conditions in which special mutations occur. The cancer cells would therefore be expected to behave differently, and new mutations could occur if they are transferred to a new and nutrient-rich culture medium. Most biological processes do not take place in growth but in a steady state, in which they have to struggle to obtain nutrients.
“If we do not properly culture cells in the laboratory, we risk studying the wrong mechanisms and finding inappropriate ways to fight cancer. The same applies when we create certain changes in microorganisms so that they can tolerate a certain substance or perform a process. When the bacteria are cultured under other conditions, such as in large production tanks, they might mutate or function differently than in a test tube,” concludes Morten Nørholm.