Researchers headed by teams at the Leibniz Institute for Natural Product Research and Infection Biology at the Max Planck Institute for Evolutionary Anthropology, and at Harvard University, have reconstructed the bacterial genomes of previously unknown bacteria, from the calcified dental plaque of human and Neanderthal remains. Using these genetic blueprints, the scientists built a biotechnology platform to revive the ancient bacteria’s natural products, which they reconstructed in living bacteria in the lab. The results offer up new insights into previously undescribed Pleistocene bacterial metabolites.
“In this study, we have reached a major milestone in revealing the vast genetic and chemical diversity of our microbial past,” said Christina Warinner, PhD, associate professor of Anthropology at Harvard University, group leader at the Max Planck Institute for Evolutionary Anthropology (MPI-EVA), and affiliate group leader at the Leibniz Institute of Natural Product Research and Infection Biology (Leibniz-HKI). Added Pierre Stallforth, PhD, Professor of Bioorganic Chemistry and Paleobiotechnology at Friedrich Schiller University Jena and Head of the Department of Paleobiotechnology at the Leibniz-HKI. “Our aim is to chart a path for the discovery of ancient natural products and to inform their potential future applications.” Warinner and Stallforth are co-senior authors of the researchers’ published paper in Science, which is titled “Natural products from reconstructed bacterial genomes of the Middle and Upper Paleolithic.”
Microbes are nature’s greatest chemists, and the high structural and functional diversity of microbial natural products make such products “… an ideal source of therapeutic drugs, antimicrobials and other functional compounds,” the authors noted. Producing these complicated chemical natural products is not straightforward, however, 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 three billion years, there is an enormous diversity of past natural products with therapeutic potential that have to date remain unknown. “Characterizing the natural products encoded in biosynthetic gene clusters (BGCs) and synthesized by ancient microbial communities would provide both insights into past microbial lifestyles and access to previously hidden chemical, structural and functional diversity,” the team stated. However, they continued, “the direct detection and characterization of metabolites from ancient sources has met only limited success.”
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 struggled with the fact that most ancient DNA cannot be matched to anything known today. “To date, most studies in the field of ancient microbial research have focused on sequence alignment to databases of reference genes or genomes, thus restricting findings to known taxa and their close relatives.”
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—in order 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,” explained Alexander Hübner, PhD, postdoctoral researcher at the MPI-EVA and co-lead author of the study. Three years of testing and optimization later, Hübner says they reached a breakthrough, achieving stretches of reconstructed DNA more than 100,000 base pairs in length and the recovery of a wide range of ancient genes and genomes. “We can now start with billions of unknown ancient DNA fragments and systematically order them into long-lost bacterial genomes of the Ice Age.”
For their reported study the team focused on reconstructing bacterial genomes encased within dental calculus—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 the lifetime, turning living dental plaque into a graveyard of mineralized bacteria. The researchers reconstructed 459 bacterial metagenome-assembled genomes (MAGs) including numerous oral bacterial species, and those of other species whose genomes had not been described before.
Among these was an unknown member of Chlorobium—“a genus not typical of oral microbiota or burial sediments,” the authors noted—whose highly damaged DNA showed the hallmarks of advanced age, and which was found in the dental calculus of seven Paleolithic humans and Neanderthals. “Known members of Chlorobium are photolithoautotrophic obligate anaerobic green sulfur bacteria that typically perform photosynthesis in anoxic water columns” the investigators explained. They speculated that, as Cholrobium species are photoautotrophs, the anaerobic colonization for the dental plaques occurred either during life or shortly after death, but prior to burial, through transient exposure to contaminated freshwater sources.” All seven Chlorobium genomes identified in the ancient samples were found to contain a biosynthetic gene cluster of unknown function.
“The dental calculus of the 19,000-year-old Red Lady of El Mirón, Spain yielded a particularly well-preserved Chlorobium genome,” said Anan Ibrahim, PhD, a postdoctoral researcher at the Leibniz-HKI 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.”
The team then used synthetic molecular biotechnology tools to generate the chemicals encoded by the ancient genes in living bacteria. 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, and which their results indicated “… could be involved in regulating the bacterial photosynthesis … The analysis and comparison of the product spectrum of ancient and modern paleofuran BCG homologs provide key opportunities to explore BGC evolution.” Martin Klapper, PhD, postdoctoral researcher at the Leibniz-HKI and co-lead author of the study, added, “This is the first step towards accessing the hidden chemical diversity of earth’s past microbes, and it adds an exciting new time dimension to natural product discovery.”
The success of the study is the direct outcome of an ambitious collaboration between archaeologists, bioinformaticians, molecular biologists, and chemists to overcome technological and disciplinary barriers and break new scientific ground. “With funding from the Werner Siemens Foundation, we set out to build bridges between the humanities and natural sciences,” said Stallforth. “By working collaboratively, we were able to develop the technologies needed to recreate molecules produced a hundred thousand years ago,” said Christina Warinner. Looking towards the future, the team hopes to use the technique to find new antibiotics.
“Our approach can, in principle, be applied to any ancient metagenomes, thus providing a roadmap for future BGC exploration and metabolite discovery,” the authors wrote. “… by merging metagenomics, genome mining, gene synthesis, and metabolic analysis with the field of aDNA research, we chart a path for the discovery of ancient natural products to gain evolutionary insights on their formation and origin, as well as to inform their potential future applications.”