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The heat is on – unknown biochemistry in extreme situations

“Most of what is easily accessible has in principle already been discovered,” said Dr. Ivan Berg from the University of Freiburg, explaining why he is investigating the metabolic pathways in extremophilic microorganisms. The researcher and his team are interested in the biochemistry of organisms living in hot volcanic springs and the Dead Sea. Examples of this are two metabolic pathways which the researchers from Freiburg discovered in organisms belonging to the Crenarchaeota. The bacteria use the pathways to assimilate atmospheric carbon dioxide without the risk of poisoning themselves with the breakdown products arising at high temperatures. What can industry learn from extremophilic bacteria? What are the advantages and disadvantages for laboratory applications? And what can we learn from them about evolution on Earth?

The crenarchaeal organism Sulfolobus solfataricus seen under the electron microscope. © www.wikipedia.org

Plants take up CO2 and convert it into sugar as part of the Calvin cycle during photosynthesis. However, many microorganisms cannot make use of this reaction pathway, in particular those that live in inhospitable corners of the Earth like volcanic springs in temperatures of up to 120°C. The reason for this is that some of the intermediary products of the Calvin cycle break down into toxic products at high temperatures that can kill cells. The unicellular organisms within Archaea that are several billion years old and have adjusted to life under extreme conditions have evolved other biochemical solutions to solve the problems that occur in Earth’s “hot spots”. “I am looking for new metabolic pathways,” said Dr. Ivan Berg, head of a group of researchers in the Department of Microbiology at the Institute of Biology II at the University of Freiburg. “I am also interested in the evolution of these metabolic pathways and their ecological importance for the respective organisms.”

Two new metabolic pathways

Berg’s team of researchers are mainly focused on basic research, but they are also interested in the implication of their findings for industry. Lipid-degrading enzymes isolated from thermophilic bacteria have long been used in detergents because of their ability to remove oil at high temperatures. However, enzymes that are effective at low temperatures are increasingly gaining in importance as they have the potential to achieve the desired washing results at lower temperatures and hence consume less energy. There are many examples of the industrial application of such enzymes. However, such issues are not of the highest priority for Berg who is specifically interested in discovering new things and obtaining a global picture. During his postdoctoral period in the laboratory of the microbiologist Prof. Dr. Georg Fuchs from Freiburg, Berg discovered two new metabolic pathways in the Crenarchaeota, one of the two subgroups of Archaea, which use these pathways for the assimilation of carbon dioxide into more complex compounds such as sugar and proteins.

The 3-hydroxypropionate/4-hydroxybutyrate cycle is for example used by Metallosphaera sedula bacteria for the fixation of carbon dioxide. © Dr. Ivan Berg
The most interesting steps in the fixation of carbon dioxide in these previously unknown metabolic pathways are those in which two CO2 molecules are attached to an acetate molecule. Acetate is a derivative of acetic acid (the term also includes the anion in solution) and consists of a backbone of two carbons. Acetate is found in all organisms and is converted into a molecule consisting of four carbon atoms in two different reactions, and this molecule is then transformed into the precursors of amino acids and sugars. In one of these cycles (Fuchs, Berg and their team refer to this cycle as the 3-hydroxypropionate/4-hydroxybutyrate cycle), the key reactions of CO2 fixation are carried out in the presence of oxygen (aerobic). This requires a huge amount of energy as many enzymes and intermediary products need to be protected against the oxygen. On the other hand, the reactions can be performed by crenarchaeal microorganisms like Metallosphaera sedula that live in aerobic habitats. These bacteria thrive well in volcanic environments and are able to tolerate temperatures of around 75°C and acidic environments with a pH of 2.

A smart way of stealing ideas from relatives

The dicarboxylate/4-hydroxybutyrate cycle is for example used by Ignicoccus hospitalis for the fixation of carbon dioxide. © Dr. Ivan Berg
The other pathway (known as dicarboxylate/4-hydroxybutyrate cycle) is anaerobic; it requires less energy but is highly sensitive to oxygen, which is why it is found in Crenarchaeota bacteria (e.g. Ignicoccus hospitalis) that live in oxygen-free environments. “These two different ways of CO2 fixation are mediated by two different enzymes known as carboxylases,” said Berg highlighting that the two cycles are otherwise completely identical. It can therefore be assumed that the ecology of an oxygen environment affected the evolution of the CO2 fixation steps. Nowadays, it is assumed that the original archaea lived in an anaerobic, i.e. oxygen-free environment around 3.5 billion years ago. As the atmosphere gradually filled with oxygen, which is vital for humans, some bacteria had to adjust to new environmental conditions in order to survive. “We assume that these adaptations happened by way of lateral gene transfer,” said Berg. Microorganisms have the ability to take up free-floating DNA from the environment. It seems that some Crenarchaeota bacteria accidentally took up the right genes and integrated them into their genome. The carboxylase enzyme, which mediates the key steps of the aerobic CO2 fixation cycle, is a relatively common protein that is found in bacteria other than those of the Archaea domain. “I have to admit that my assumption that the bacteria found a smart way to steal some genes from others is pure speculation. On the other hand, this the best idea I can come up with in order explain how some Crenarchaeota bacteria evolved the aerobic CO2 fixation cycle.”

A double-edged sword for laboratory application

One thing is clear: the two newly discovered metabolic cycles are optimised for high temperatures. They generate no toxic breakdown products that could damage the bacterial cells. However, the enzymes’ resistance to high temperatures is a double-edged sword when used in the laboratory. “In order to investigate the proteins and metabolic products at the point at which they exert their optimal effect, we need to apply very high temperatures of around 80°C. And this requires expensive equipment and specific materials that can tolerate such high temperatures,” said Berg. On the other hand, the model organism E. coli can be used to produce highly pure crenarchaeal enzymes. “The only thing we have to do is to heat the E. coli cell extract to 80°C, which causes all proteins that do not tolerate such high temperatures to denature,” Berg explained going on to add, “many researchers do not like working with extremophilic bacteria because of the associated disadvantages in terms of expensive equipment etc.,” Berg said. “However, everything that used to be easily accessible has now been discovered. This is why I am now doing research involving extremophilic bacteria,” Berg said, stating that he feels it is now necessary for researchers to increasingly focus on the exotic if they want to make new discoveries. “If I lose some money, I will look for it where I have lost it, and not just in places where there is light, although it is good to have light,” Berg concluded.

It is often only after the basic research work has been completed that new ideas for industrial or environmental applications arise. This is what Berg’s discovery of the aerobic CO2 fixation cycle in Metallosphaera sedula clearly shows. In the meantime, genome analyses have shown that the Thaumarchaeota bacteria, which account for around 20 percent of the microbial biomass in the oceans, also possess this particular metabolic pathway. It can therefore be assumed that this metabolic pathway is also involved in the growth of the global biomass and in the carbon cycle in the biosphere. Therefore, Berg’s findings are far from just exotic knowledge. In addition to focusing on other basic research projects, Berg and his team of researchers are now planning to look at the biochemistry of the Thaumarchaeota bacteria in greater detail.

Further information:
Dr. Ivan Berg
Department of Microbiology
Institute of Biology II
University of Freiburg
Schänzlestr. 1
79104 Freiburg
Tel.: +49 (0)761/ 203 2777
E-mail: ivan.berg(at)biologie.uni-freiburg.de

Website address: https://www.biooekonomie-bw.de/en/articles/news/the-heat-is-on-unknown-biochemistry-in-extreme-situations