Hot World, from Resurrected Proteins

Categories: Extreme Life Interview

A billion years ago, the ancestors of today’s bacteria thrived in an environment similar to a Yellowstone hot spring, suggesting Earth may have been a much warmer place closer to the time when life originated.

So say two University of Florida scientists who have used the newly developed techniques of ‘paleobiochemistry’ to reconstruct ancient bacterial proteins based on similarities in the genetic sequences of modern proteins. The resurrected proteins proved most stable and functional at temperatures between 130 and 150 degrees Fahrenheit, implying that ancient bacteria lived in a hot springs-like soup warmer than most life can tolerate today.

Protein stick-figure model. The small ball and stick figure represents bonds and protein molecules in a flexible chain that can twist, rotate, expand and collapse during rapid folding events.

The research, which appeared Sept. 18 in the journal Nature, is expected to enliven a longstanding debate about the temperatures of Earth when life consisted only of microbes, long before the appearance of animals about a half billion years ago. The findings also may help narrow the search for life on other planets, said Eric Gaucher, a National Research Council fellow and post-doctoral associate at the University of Florida, who is the lead author of the paper.

"If you’re going to search for life on other planets, you can’t just randomly put a probe down and look for life," Gaucher said. "You want to land in a spot that you think is the most probable for hosting life. So having some idea of the temperature zone where you should put the probe will be helpful."

Extreme Life image
Extreme Life Briefing

  • Hottest: 235 F (113 C) Pyrolobus fumarii (Volcano Island, Italy)
  • Coldest: 5 F (-15 C) Cryptoendoliths (Antarctica)
  • Highest Radiation: (5 MRad, or 5000x what kills humans) Deinococcus radiodurans
  • Deepest: 3.2 km underground
  • Acid: pH 0.0 (most life is at least factor of 100,000 less acidic) pH 5-8
  • Basic: pH ~13(most life is at least factor of 1000 less basic) pH 5-8
  • Longest in space: 6 years Bacillus subtilis (NASA satellite)
  • High Pressure (1200 times atmospheric)
  • Saltiest: 30% salt, or 9 times human blood saltiness. Haloarcula
  • Smallest: <0.1 micron or 500 fit across a human hair width (picoplankton)
    Credit: USGS
  • The Earth is believed to have formed about 4.5 billion years. For 700 million years, asteroids and other celestial bodies smacked and smashed the new planet in an era known as "the heavy bombardment." There is much debate about when life first appeared, but some scientists believe the first evidence – consisting of chemical signatures of microbes found in ancient rocks – dates back to the end of the bombardment around 3.8 billion years ago, Gaucher said.

    The climate on Earth from this period until the "Cambrian explosion" – the appearance of many forms of higher animals 570 million years ago – is thought to have varied widely through time. Evidence of glaciers at the equator suggests a "snowball Earth" much colder than today, while other evidence implies the planet also went through comparatively warm periods, Gaucher said.

    Geologists have dominated research on the topic, probing minerals and rocks in an effort to pen a timeline of Earth’s changing climate. Gaucher and Steven Benner, a UF distinguished professor of chemistry, tried a different approach: recreating the ingredients of ancient life, and then testing their ability to persist and thrive in various temperatures.

    Lynn Rothschild, a research scientist at NASA Ames Research Center and expert in astrobiology, said the approach was creative. "It is one of those clever pieces of work that makes me say, ‘I wish I’d thought of that,’" she said. "While this approach is not unique nor definitive, it is indeed clever and provides a much-needed alternative approach to evolutionary studies."

    The process sounds reminiscent of Jurassic Park, but there’s a difference. Scientists in the popular Michael Crichton novel-turned-movie-series resurrect dinosaurs, which date back only about 60 million years. Gaucher and Benner sought to go much further back (Precambrian) – at least 1 billion years.

    The scientists used a technique called paleogenetics, first proposed in 1963 by famed scientists Linus Pauling and Emile Zuckerkandl. Technology at that time was not up to these authors’ dreams, but thanks to vast increases in the speed of information processing starting in the 1980s and other advancements in the laboratory, the concept became a reality late last decade.

    The method is analogous to historical linguistics, which reconstructs ancient languages by finding similarities in their descendant languages. Instead of words or sounds, scientists match up similarities in the amino acids of various existing proteins to reconstruct the amino-acid sequences of ancient proteins. They then recreate, or "resurrect," these proteins in the laboratory.

    Terrestrial options for early climate. Early earth, snowball, cauldron or temperate?Credit: NASA

    Gaucher started with 55 different modern bacteria, extracting a protein called elongation factor tu, which is shared with other bacteria and most other modern organisms. He chose this bacteria, in part, because its prevalence suggests it appeared in a single or common ancestor and also because it is very stable, or doesn’t appear to have changed much over the eons.

    The next step was to reconstruct the ancient protein. "We’re out at the end of the timeline, and we’re trying to go back," Gaucher noted.

    He sequenced each protein and, using computer analysis, teased out the commonalities among these amino-acid sequences. The result was a digital representation of the ancient protein. The next step was to resurrect it in the physical world. Gaucher used E. coli, a modern bacterium, to make the protein.

    He and Benner then tested what happened to the protein at various temperatures. Between 130 and 150 degrees, it performed best at its task — which involves translating the information in its DNA through RNA into the completed protein. At hotter temperatures, the ancient protein fell apart.

    Benner cautions the findings do not imply that the entire Earth was 130 to 150 degrees a billion years ago or longer, but rather that the bacterium whose genes survived to be relayed into descendant organisms thrived at that temperature. Why it proved so successful is a mystery, he said.

    "For some reason, bacteria living at 130 to 150 degrees have made some innovation which allows them to leave their descendants all over the planet, not the other guys that we presume were living in other environments," he said. "And that’s an astonishment to me."

    Astrobiology Magazine had the opportunity to talk with Eric Gaucher, the lead author of the Nature paper and a NASA Astrobiology Institute / National Research Council Postdoctoral Fellow.

    Astrobiology Magazine (AM): What is paleobiochemistry, and why is it an intriguing field particularly now?

    Hottest living organisms: 235 F (113 C) Pyrolobus fumarii (Volcano Island, Italy)

    Eric Gaucher (EG): The quest to characterize ancient life has historically been played by geologists and chemists. This consists of identifying structures found in rocks or elucidating mechanisms of chemical reactions.

    The genomic revolution has now provided an opportunity for biologists to join in the fun. This is possible because genes can be considered a type of dynamic fossil, in that they can adapt to changing environments while still possessing some of their ancestral properties. The trick is being able to extract this ancient information. This defines the field of paleobiochemistry

    AM: Would the temperature range of 130 to 150 Fahrenheit for these ancient organisms to thrive, would that be comparable genetically to what is found as the modern equivalent of the extreme heat-loving microbe, Pyrolobus fumarii (Volcano Island, Italy) nearer to 235 F?

    EG: There are four categories when considering optimal growth temperature of organisms: Psychrophiles (< 68 degrees F, 20 degrees C), Mesophiles (68-104 F, 20-40 C), Thermophiles (104-176 F, 40-80 C), and Hyperthermophiles (176-239 F, 80-115 C).

    Pyrolobus is considered a hyperthermophile, as are Thermotoga maritime and Aquifex. These last two species branch at the base of the bacterial domain of the Universal Tree, and thus have lead in the idea that the last common ancestor of modern life was a hyperthermophile.

    For the most part, hyperthermophiles live in oceanic thermal vents. You may know that these are highly specialized environments with small niches. The temperature zones are quite narrow. An organism adapted to thrive at 176 F (80 C) may die if it moves a few feet away to a spot that’s 140 F (60 C). Vent microbes are unique in that their translational biomolecules have adapted to these massive fluctuations. This is revealed by the optimal temperature stabilities of these biomolecules. Our assay of the elongation factor (EF) protein from Thermotoga was determined to be optimally active between 122-185 F (50-85 C).

    Deep sea ‘black smoker’ vents give rise to exotic biochemistry. Often featuring great depths, boiling water temperatures and high methane concentration. Often looked to for alternatives to water-oxygen life.
    Credit: U.Wash.

    On the other hand, the optimal temperature stabilities of EFs from the mesophilic E. coli and thermophilic Thermus were much narrower compared to the EF protein from the hyperthermophilic. This is seen in the Nature article. For both E. coli and Thermus, the EF proteins were significantly diminished in their capacities to function at temperatures outside their respective organismal optimal growth temperatures, ca. 140 F (40 C) and 158 F (70 C) respectively.

    Since these organisms live in such temperature stable environments, there are relatively little selective pressures for them to thrive across a board range of temperatures, as seen with vent hyperthermophiles.

    So, to answer your question, given the narrow optimal stability temperatures of my ancestral proteins, the ancestral proteins are not comparable to the modern Pyrolobus. However, the ancestral proteins probably are comparable to extant (modern) organisms living in thermal springs, such as Thermus in Yellowstone Park.

    AM: Do these mechanism of survival at high temperature involve some means of avoiding heat-induced protein denaturation–basically the protein has to be protected against coagulation or cooking?

    EG: Protein stability involves an interplay between the obvious necessity of stability for functional activity and the ability to recycle the protein when it is no longer needed. Denaturing proteins requires energy. The stronger the forces holding a protein together, the more energy an organism must spend to break the forces.

    Given that most proteins optimally function at the same temperature at which their host organism optimally grows, natural selection has resulted in a balance between protein stability and denaturation.

    Longest survivors in unprotected vacuum and cold of space: 6 years Bacillus subtilis (NASA satellite).
    Credit: NASA

    So, yes, organisms must some how avoid heat-induced protein denaturation. This is achieved many ways. The most common is to increase the forces holding a protein together, such a hydrophobic interactions, van der Waals forces, and various bonds. Some hyperthermophilic species also employ chaperone proteins that travel around the cell and help re-fold any proteins that have denatured.

    AM: Can you step through the brief method of sequencing? You get the amino acid composition, for instance, but how do you know the peptide ordering and such to synthesize it recombinantly?

    EG: The analogy here is that of the historical linguist. By analyzing the relationships and commonalities of modern languages, along with a knowledge of the alphabet, these historians are able to reconstruct ancient languages. This is precisely my approach.

    I am a historian of Nature. I built a phylogenetic tree using EF proteins. The leaves of the tree represent modern EF sequences while the interior nodes represent extinct sequence (in other words, the sequences once present in ancient organisms). Recent advances in evolutionary statistically models and computational power enable us to estimate the subsequent arrangement of amino acids (Nature’s alphabet) for the EF protein at the interior nodes of the phylogenetic tree. In particular, I am interested in the oldest point, or deepest node, of the bacterial domain of the Universal Tree. This represents the last common ancestor of modern bacteria, which may have lived ca. 3.5 billion years ago.

    Once the amino acid sequence is reconstructed in silico, we resurrect the ancestral gene using gene synthesis techniques, use a modern organism to express the gene, and then purify the protein product and perform the thermostability assay.

    AM: The leap from local hot conditions to global climate, how is that implied from paleochemistry today?

    EG: This work does not resolve any issues about the global climate. Presumably, there were many distinct local climates present 3.5 billion years ago. Life may have even existed in multiple distinct locations. For some reason, however, the organisms that lived in niches at 131-149 F (55-65 C) were able to survive and seed the diversity of life.

    This may be related to bottlenecks caused by the Snowball Earth.

    The paper’s other authors are J. Michael Thompson and Michelle Burgan, both former students at UF. The research was funded by $100,000 grant from the NASA Astrobiology Institute.