Breakthroughs in ancient genome reconstruction and biotechnology are now revealing the rich molecular secrets of Paleolithic microorganisms.

In a transdisciplinary study, scientists are rebuilding microbial natural products up to 100,000 years old using dental calculus of humans and Neanderthals. New techniques of reconstructing bacterial genomes encased within the dental calculus, also known as tooth tartar, from Neanderthals and early forms of anatomically modern humans could lead to the discovery of new antibiotics, according to a new study.

The scientists are led by Pierre Stallforth of the Friedrich Schiller University and the Leibniz Institute for Natural Product Research and Infection Biology in Germany, and Christina Warinner of Harvard University and the Max Planck Institute for Evolutionary Anthropology, also in Germany.

Red Lady's skeleton
The Red Lady's skeleton

Among the dental calculus used in the research was that of the Red Lady of El Mirón Cave in Spain. The Red Lady was found in 2010 during excavations by Emeritus Leslie Spier Distinguished Professor at The University of New Mexico Lawrence Straus and David Cuenca, who is now a lecturer at the Universidad de Cantabria in Spain. Manuel Gonzalez Morales, Emeritus Professor at the Universidad de Cantabria, is co-director of the project. 

The woman — possibly one of special status in a group of hunter-gatherers — died around 19,000 years ago and was buried in El Mirón Cave in northern Spain. In 1996, archaeologists started exploring the cave, finding abundant evidence of prehistoric people. In 2010, Straus and then-student David Cuenca found the woman’s remains, including her jaw, after Straus had what he described as “a hunch” to dig in an area behind an engraved block at the back of the cave’s huge vestibule.

The prehistoric woman’s age is estimated to be 35-40 years at death and her bones were coated with ochre, a red iron oxide pigment, prompting Straus to name her the Red Lady of El Mirón. Since that finding, the Red Lady has continued to offer a torrent of information and data to archaeologists and bioanthropologists.

In a new study published in Science, a team of researchers reconstructed bacterial genomes of previously unknown bacteria dating to the Pleistocene. Using their genetic blueprints, they built a biotechnology platform to revive the ancient bacteria’s natural products. The technique might eventually be used to find new antibiotics.

Microbes are nature’s greatest chemists, and among their creations are many of the world’s antibiotics and other therapeutic drugs. Producing these complicated chemical natural products is not straightforward, and to do so bacteria rely on specialized kinds of genes that encode enzymatic machinery capable of making such chemicals. At present, scientific study of microbial natural products is largely limited to living bacteria but given that bacteria have inhabited the earth for more than 3 billion years, there is an enormous diversity of past natural products with therapeutic potential that remain unknown to us – until now.

A billion-piece jigsaw puzzle

When an organism dies, its DNA rapidly degrades and fragments into a multitude of tiny pieces. Scientists can identify some of these DNA fragments by matching them to databases, but for years microbial archaeologists have known that most ancient DNA cannot be matched to anything known today. This problem has long vexed scientists, but recent advances in computing are now making it possible to refit the DNA fragments together – much like the pieces of a jigsaw puzzle – to reconstruct unknown genes and genomes. The only problem is that it does not work very well on highly degraded and extremely short ancient DNA from the Pleistocene.

“We had to completely rethink our approach,” said Alexander Hübner, postdoctoral researcher at the Max Planck Institute and co-lead author of the study. Three years of testing and optimization later, Hübner said they reached a breakthrough, achieving long stretches of reconstructed DNA and the recovery of a wide range of ancient genes and genomes, adding, “We can now start with billions of unknown ancient DNA fragments and systematically order them into long-lost bacterial genomes of the Ice Age.”

Exploring the microbial Paleolithic

The team focused on reconstructing bacterial genomes encased within dental calculus, also known as tooth tartar from 12 Neanderthals dating to ca. 102,000–40,000 years ago, 34 archaeological humans dating to ca. 30,000–150 years ago, and 18 present-day humans. Tooth tartar is the only part of the body that routinely fossilizes during life, turning living dental plaque into a graveyard of mineralized bacteria. The researchers reconstructed numerous oral bacterial species, as well as other more exotic species whose genomes had not been described before.

Among these was an unknown member of Chlorobium, whose highly damaged DNA showed the hallmarks of advanced age, and which was found in the dental calculus of seven Paleolithic humans and Neanderthals. All seven Chlorobium genomes were found to contain a biosynthetic gene cluster of unknown function.

“The dental calculus of the 18,800-year-old Magdalenian Red Lady of El Mirón Cave yielded a particularly well-preserved Chlorobium genome,” said Anan Ibrahim, postdoctoral researcher at the Leibniz Institute and co-lead author of the study. “Having discovered these enigmatic ancient genes, we wanted to take them to the lab to find out what they make.”

Basically, Straus explained, “The Red Lady’s dental cementum was extremely rich — richer than any other individual in the sample of Neanderthals and early Homo sapiens sapiens from the Upper Paleolithic — in this bacterium and it seems to have gone extinct after the time of the Miron Red Lady at the end of the Ice Age.”

The team used the tools of synthetic molecular biotechnology to allow living bacteria to produce the chemicals encoded by the ancient genes. This was the first time this approach had been successfully applied to ancient bacteria, and it resulted in the discovery of a new family of microbial natural products that the researchers named “paleofurans.”

The success of the study is the direct outcome of an ambitious collaboration between archeologists, bioinformaticians, molecular biologists, and chemists to overcome technological and disciplinary barriers and break new scientific ground.

“By working collaboratively, we were able to develop the technologies needed to recreate molecules produced a hundred thousand years ago,” said Warinner, co-senior author of the study and associate professor of Anthropology at Harvard University. Warinner was a Journal of Anthropological Research Distinguished Lecturer at UNM in 2022 to whom Straus had suggested in 2016 the possible genetic study of the Red Lady calculus. Looking towards the future, the team hopes to use the technique to find new antibiotics.

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