Bacteria produce a variety of exciting chemicals called natural products, including numerous antibiotics and other therapeutic agents. For science, they are one of the most important sources of new medicines – but so far researchers have only been looking for them in bacteria that are alive today. But since bacteria have colonized the earth for more than three billion years, there is an enormous variety of natural products with therapeutic potential in bacteria that are now extinct.
Researchers led by the chemist Pierre Stallforth from the Leibniz Institute for Natural Product Research and Infection Biology in Jena and the archaeogeneticist Christina Warinner from the Max Planck Institute for Evolutionary Anthropology in Leipzig and Harvard University have joined forces to tap into this source . “For the first time, we have succeeded in reproducing substances that were produced by bacteria a hundred thousand years ago – the paleofurans,” says Stallforth. Bacterial DNA served as the basis: This contains the blueprints for enzymes, which in turn can assemble chemical compounds. “With this study, we have reached an important milestone in uncovering the enormous genetic and chemical diversity of our microbial past,” adds Warinner.
Jigsaw puzzles made up of billions of pieces
When an organism dies, its DNA is rapidly degraded and broken down into a multitude of tiny fragments. Scientists can identify some of these DNA fragments by comparing them to databases of modern-day organisms. But much of the DNA belongs to unknown microorganisms that may now be extinct.
However, recent advances in computer science make it possible to reassemble the DNA fragments, much like the pieces of a jigsaw puzzle, to reconstruct even unknown genes and genomes. A major challenge with the heavily degraded, extremely short DNA fragments from the Stone Age: “We had to completely rethink our approach,” explains Alexander Hübner, postdoc at the Max Planck Institute for Evolutionary Anthropology. Three years of testing and optimization later, according to Hübner, one of the first authors of the study, they have made a breakthrough: they have succeeded in reconstructing DNA sections with a length of more than 100,000 base pairs and in restoring a large number of old genes and genomes. “We can now begin to systematically classify billions of unknown ancient DNA fragments into long-lost Stone Age bacterial genomes.”
Exploring the Microbial Stone Age
To get to the DNA of Stone Age microorganisms, the team used tartar from Neanderthals who lived around 100,000 to 40,000 years ago and from humans who lived between 30,000 and 150 years ago. Tartar is the only part of the body that petrifies over time, turning living plaque into a graveyard of mineralized bacteria. Using the latest bioinformatic methods, the researchers reconstructed the genomes of numerous bacterial species. “The major bioinformatics challenge was to correct errors in the degraded DNA and to rule out contamination, for example by younger DNA,” says Anan Ibrahim, a postdoc at the Leibniz Institute for Natural Product Research and Infection Biology and also the first author of the study.
In addition to many bacteria that still colonize the human oral flora today, she found an unknown member of the genus Chlorobium. Its severely damaged DNA showed all the hallmarks of advanced age and was found in the tartar of seven Stone Age humans and Neanderthals. All seven chlorobium genomes contained a biosynthetic gene cluster – the blueprint for enzymes – with an unknown function. A particularly well-preserved chlorobium genome was reconstructed from the tartar of the approximately 19,000-year-old “Red Lady of El Mirón”, Spain. The skeleton found in a Spanish cave in 2010 is the oldest evidence of a Magdalenian burial on the Iberian Peninsula.
“Once we discovered these mysterious ancient genes, we wanted to find out what they do,” says Ibrahim. Using state-of-the-art biotechnological methods, the researchers succeeded in inserting the genes into living bacteria, which actually formed functional enzymes from them. They are the first to successfully apply this approach to bacterial DNA that is tens of thousands of years old. The reactivated enzymes, in turn, produce a new family of microbial natural substances, which the researchers have dubbed “paleofurans”. “This is the first step in unlocking the hidden chemical diversity of the microbes of Earth’s history,” says Martin Klapper, postdoc at the Leibniz Institute for Natural Product Research and Infection Biology and another first author of the study.
Novel collaboration to create a new research area
This achievement is the direct result of a unique collaboration between researchers in archaeology, bioinformatics, molecular biology and chemistry, who wanted to break down technological and disciplinary barriers and break new scientific ground. “With the funding from the Werner Siemens Foundation, we want to build a bridge between the humanities and the natural sciences. It enabled us to set up the new research area of palaeobiotechnology,” says Pierre Stallforth. And Christina Warinner adds: “In this way, we were able to develop technologies to create new molecules that were produced a hundred thousand years ago.” The team hopes to be able to use this approach to search for new antibiotics in the future.