The results of adaptive evolution can be seen throughout the natural world, from zebra stripes to camouflage in moth wings, but the events that lead to adaptations are often impossible to track. A study published on November 13 in Nature* presents a breakthrough. Harvard scientists marked yeast with barcodes and tracked their progeny in an evolution experiment that lasted 1000 generations. While the course that evolution took in this experiment generally agreed with existing theoretical models, certain events in the evolutionary process turned out to be more important than previously thought.
Although all organisms evolve, when it comes to studying
evolutionary processes in the lab, single-celled microorganisms are easiest to work
with. The authors of this study used yeast, a single-celled model organism that
reproduces by budding. In laboratory yeast evolution experiments, the yeast jostle
for survival in a test tube. As they grow and reproduce, they accumulate mutations,
some of which affect how well they grow. A yeast ancestor and its progeny form
a lineage. Over time, some yeast lineages take over while others die out.
DNA Barcoding
To keep track of the evolution going on in test tubes, researchers
must be able to keep track of the yeast. This can be challenging from a technical
standpoint, given that microbial evolution experiments can involve millions of
cells and thousands of generations. In this study, the authors overcame these
difficulties by improving upon an existing technique. They used DNA barcoding,
in which unique identifying sequences are inserted into an organism’s DNA. The authors
designed an updated version of this technique, adding a new barcode sequence
into each yeast every 100 generations. After hundreds of generations, each
yeast in the experiment contained a series of barcodes, arranged one after the other,
in a single location in its DNA.
The Travelling Wave
This reneweable barcoding technique is powerful. Through it, the yeast lineages could be divided more finely into sublineages. When some yeast got mutations that made them more successful, the increasing numbers of yeast in those sublineages could be tracked by their corresponding series of barcodes. Upon analyzing the data, the authors generally saw what evolutionary theory predicts for such an experiment: some yeast gained beneficial mutations and formed successful sublineages while other yeast lineages died out; however, the average overall fitness of the yeast in the experiment increased with every generation. This phenomenon is known as “the travelling wave” – the idea that fitness (represented by a bell-like curve) travels along an axis in the direction of increasing fitness.
Leapfrogging yeast disrupt the travelling wave.
While their results largely support this model, the authors
did notice one surprising thing. Organisms are predicted to gain fitness gradually
– the positive effects of beneficial mutations stack onto those of existing
beneficial mutations. Sometimes, however, an event called “leapfrogging” can
occur. In leapfrogging, a lineage will get a mutation that suddenly makes it
much fitter, causing its fitness to “leapfrog” over that of other, previously
fitter, lineages. These events weren’t thought to be very important, but in
this experiment they happened regularly. A mutation would appear in a lineage,
creating a sublineage, and within a few generations, yeast from that sublineage
would make up a significant proportion of the test tube’s population. Contrary
to what is predicted by the usual travelling wave model, in this experiment, small
numbers of mutations had big impacts.
In the future, the renewable barcoding technique can be
applied to answer a variety of questions. For example, to test the effects of
mutations that are predicted to affect an organism’s ability to adapt. Overall,
this experiment shows that there is more to learn about evolutionary processes,
and that new technology can help.
Link to paper: “High-resolution lineage tracking reveals
travelling wave of adaptation in laboratory yeast” https://www.nature.com/articles/s41586-019-1749-3#Sec9
*In the spirit of declaring conflicts of interest, my partner is the lead author.
One barrier to distributing life-saving vaccines in developing countries is cold storage. Vaccines are typically made up of delicate components that can go bad over time, making it difficult to transport vaccines to remote locations where refrigeration is not available. In a recently released paper, a group of scientists from Northwestern University and Cornell describe one way to circumvent this problem. They demonstrate a method where the components of a vaccine can be mixed in a tube, freeze-dried, then reconstituted with water and be ready to inject after an hour. This paper has not yet been peer-reviewed, which means that it still needs to be approved by experts in the authors’ field of study. Despite this, I think it is worth looking at. The problem the paper tries to solve and the techniques the authors use are largely not new (although the way they are being used is). They stand alone as interesting methods, regardless of whether this paper gets published in a journal.
Vaccines come in many types, and the type the paper focuses
on is conjugate vaccines. Conjugate vaccines are produced by fusing together
two different molecules: one is an antigen found on the pathogen against which
the vaccine protects, and the other is a carrier molecule. The carrier molecule
acts as a sort of helper; it is used to elicit long-lived immunity while the weak
antigen “teaches” the immune system what it should recognize and fight. Conjugate
vaccines are used when an antigen can’t by itself produce an immune response
that provides long-term protection. For this reason, the antigen conjugated to
the carrier molecule is sometimes called the “weak antigen”.
In this paper, the weak antigen the researchers focus on comes
from the pathogen Francisella tularensis subsp. tularensis (type A) strain Schu S4.
(Quite a mouthful.) F. tularensis causes tularemia and is considered a potential
bioterrorism agent because of how quickly and easily it can be spread, and the
high mortality rate associated with it. Also included in this study were antigens
from pathogenic E. coli. These were used to demonstrate the modularity
of this method.
Back to the problem at hand. Most vaccines need to be kept
in the cold to remain effective. They also require specialized equipment to
make, which usually means they can’t be made at the location where they are
administered. A method that can be used to overcome these barriers is cell-free
protein synthesis (CFPS). Whereas proteins (and other biomolecules) are usually
made inside living cells, CFPS takes cells out of the equation. In this method,
only the components of the cell used to make proteins, sugars, and other
biomolecules are required. When it came to making a conjugate vaccine, the authors
of this paper found that they could make part of the vaccine inside cells and
then use CFPS to make the final product. A key point is that CFPS can be done
even after freeze-drying and reconstituting the mixture. Therefore, combining
freeze-drying and CFPS can give vaccines a longer shelf-life.
The authors of this paper call their vaccine production
platform iVAX (in
vitro bioconjugate vaccine expression). The process goes as follows:
The weak antigen is made inside living cells. Because this antigen needs to be linked to the carrier molecule, a protein that will do the job of linking them is produced too.
The cells are then broken open, leaving only the synthetic machinery.
DNA that codes for the carrier molecule is added.
The mixture is freeze-dried and is ready to be stored or shipped.
After reconstitution with water, the carrier molecule gets synthesized and the linking protein combines the weak antigen and carrier molecule to make the final vaccine. (This is the actual iVAX part.)
Although this vaccine elicited an immune response in mice,
more work needs to be done before this method can be applied to commercial vaccines.
This method also suffers from some drawbacks, such as the need for freeze-drying
– a process that not all molecules survive. However, it makes for an exciting
potential advance in vaccine production and distribution.
Bacteria have been studied for so long you’d think the most important parts of their biology would be well understood. That’s mostly true, but there is still more to learn. For example, the making of bacterial proteins, which make up the machinery and building blocks of bacterial cells, involves steps that are still not well understood.
Proteins are made in a process called translation. During translation, a sequence of nucleotides, which make up an mRNA molecule, is translated into a sequence of amino acids, which make up the protein molecule. Translation is carried out by the ribosome, which clamps down on the mRNA, brings in amino acids one by one, connects them, and releases the newly-made protein chain. Translation has 3 main stages: the initiation stage, where the components involved in translation are assembled, the elongation stage, where the amino acids are linked together, and the termination stage, in which the protein chain is released from the translation machinery.
A study published about one week ago shows a view of translation not seen before. The researchers were able to capture several snapshots of the initiation stage as it was happening. In the future, this information could be used by scientists to design antibiotics that stop the bacterial translation reaction from progressing further.
Dancers frozen in place
Capturing the rearrangement of molecules in real-time was possible because of a technique called time-resolved cryo-electron microscopy (TR cryo-EM). Electron microscopy allows scientists to see entities as tiny as proteins, but to do this they bombard the samples with destructive high energy electron beams. In order for biological samples to survive the EM process, they are quickly frozen in extremely cold liquid ethane. TR cryo-EM takes advantage of this rapid freezing, which happens within milliseconds, to trap biochemical reactions as they are happening. However, it still takes a little bit of time to prepare the sample before freezing, and so TR cryo-EM was once only used to study the slower biochemical reactions.
Because the reactions these authors were monitoring happen very quickly, they added another layer to the TR cryo-EM process. The components of the chemical reaction were first mixed together in a special chamber and sprayed onto a surface that was then plunged into liquid ethane. By varying the amount of time the reaction components spent together, the authors were able to capture snapshots of different time points during a series of biochemical reactions.
The visuals they produced show how the different components of the translation machinery arrange and rearrange themselves during the translation initiation stage. This improved model of translation can be used to look for new antibiotics using computational and experimental means. Ultimately, TR cryo-EM should benefit not only fundamental science but also translational research.
Link to paper: www.nature.com/articles/s41586-019-1249-5
In the “list of unsolved problems in biology”, one of the biggest unknowns is the origin of life. We don’t yet know how living things arose from non-living matter, but there are other origin questions that have partly been answered. One of these is the problem of how the more “complex” life forms arose.
“Complexity” is a loaded term in biology, but it is sometimes useful to refer to some organisms as more complex than others because they just seem to be made of more types of stuff. This is the case with eukaryotes. It is generally accepted that at one point in ancient history, one organism engulfed another and the engulfee continued to survive and thrive inside the host cell. This was a key step in the genesis of modern eukaryotes. Eukaryotes belong to a group called the Eukarya, a name given to one of the main groups of living organisms (the other two groups are Archaea and Bacteria) and includes pretty much all living things that can be seen without a microscope.
Reconstructing the timeline
While the identity of the organism that was engulfed is
more-or-less known, the nature of the host organism is still controversial. Bacteria,
Archaea, and Eukarya make up the three domains of life, which are used to sort all
living organisms. Members of the Bacteria and Archaea are single-celled
organisms that are different in how they are structured. The
eukaryotes are even more obviously different. Some members of this group are
multicellular while others are single-celled. On top of that, unlike archaea
and bacteria, eukaryotes enclose their DNA in a compartment called the nucleus.
Eukaryotes also have other compartments that are pretty critical to
their survival, and at least some of these are thought to have come from
organisms that were free-living.
There are two major structures inside eukaryotic cells that are thought to once have been bacteria: mitochondria and chloroplasts. Mitochondria are found in all eukaryotic cells and act as energy factories. Plants (a subgroup within the Eukarya) have chloroplasts in addition to mitochondria. Chloroplasts are the sites of photosynthesis, which is the process through which plants make food for themselves using water, CO2, and sunlight. Mitochondria likely came from a member of the alphaproteobacteria while the chloroplast was probably a cyanobacterium 1,2. And the organism that engulfed them? We now know what it was. Probably.
Two competing ideas
Three-domain versus Two-domain tree of life.
In one hypothesis, the host organism which engulfed the mitochondria looked a bit like both an archaeon and an eukaryote, and was an ancestor of both 3. There is also a competing hypothesis: it states that the organism that engulfed the bacteria was an archaeon 4–7. This was dubbed the “Eocyte hypothesis”. The name comes from the Crenarchaeota (the Eocytes), which were proposed to be the archaea from which modern eukaryotes evolved. Around a decade ago (when this author was an undergraduate and researching this topic for an essay) the Eocyte hypothesis was very controversial 8–10. Some thought that Eocytes were simply too different from Eukaryotes to be likely eukaryote progenitors. All of this changed a few years ago following a tantalizing new discovery.
The Asgard superphylum
Archaea have a habit of living in the most inhospitable of environments:
hot springs, ultra-acidic soils, oxygen-free marsh muds, and
brine are just some of
the places where they thrive – though some also live in totally mundane places.
It therefore wasn’t surprising when a group of scientists surveying microbes in
deep sea vents identified a new group of archaea. Researchers from the
Universities of Uppsala, Vienna, and Bergen took samples of sediments from the
deep sea floor near a field of hydrothermal vents known as Loki’s Castle and
sequenced DNA in the samples. This approach, known as metagenomics, makes it
possible to identify organisms without growing them in the laboratory, and has been game-changing as lots of organisms don’t grow under standard laboratory conditions. Out
of this analysis the researchers pulled a group of archaea and called them the Lokiarchaeota 11. The Lokiarcheota were named
after Loki’s Castle, which in turn was named after the notoriously deceitful Norse
god Loki and refers to the
fact that the location is difficult to find 12.
Asgard archaea form a group with the eukaryotes in the tree of life. Credit: Eva Fernández-Cáceres. Reproduced according to permissions as stated in the corresponding EurekAlert press release.
By comparing DNA sequences, geneticists can figure out which groups of organisms are more closely related to each other and which are less closely related. The Lokiarcheota were particularly fascinating because (at least according to some analyses) they appear to be more closely related to eukaryotes than to other archaea. Since then, more siblings of the Lokiarcheota have been discovered 13. In the spirit of thematic consistency, they’ve been named Thorarchaeota, Odinarchaeota, and Heimdallarchaeota, and together with Loki, make up the Asgard superphylum. All of these were found to be closely related to the Eukarya. Apart from their sequences resembling those of eukaryotes, the Agards also code for cell components that were thought to be eukaryote-unique. These include proteins that give cells shape, help cells divide, and transport molecules across the cell 13–15. Because of these findings, the discovery of the Asgard archaea appeared to provide support for the Eoctye (or two-domain) tree.
Not enough data
DNA comparisons as well as similarities in cell structures do suggest that eukaryotes came from the Archaea, but this assertion has not gone unchallenged. In fact, in one of the first talks I saw about the Asgards, it was challenged during the question period – not everyone was satisfied with this revamped tree of life. While there is increasing evidence for archaea being the original host to a bacterial endosymbiont, those who disagree cite a few reasons. First of all, not all methods of analysis produce the same trees 16–18. For example, a study that published an updated tree of life in 2016 found support for the Eocyte tree using their method of analysis, but got a three-domain tree using a more classical method of analysis 19. Other caveats exist too. For example, eukaryotes and archaea share more types of protein structural folds with bacteria and viruses than they do with each other 20. Structural folds change more slowly over time than DNA sequences, and can serve as another measure of relatedness 21. That archaea and eukaryotes don’t share many types of protein structures between them might imply they are not that closely related, but other interpretations also exist.
What’s new?
The identity of the eukaryotic ancestor remains shrouded in
mystery. As one study put it “…further analyses…will be strengthened with the availability of genomes for a
greater diversity of organisms” 19. Fortunately, new archaea are
constantly being sequenced 22,23. Furthermore, as scientists
become better at growing archaea in the lab, it might become possible to
perform experiments on the Asgards and to see if they exhibit eukaryote-like behavior, and not just eukaryote-like genes.
The question of how eukaryotes arose is not just an esoteric conundrum. It could help shed light on what kind of environments favoured the emergence of eukaryote-like organisms. As more species are discovered, as more rare microorganisms are pulled out of deep sea vents and other inhospitable nooks, scientists might get the information they need to put this debate to rest once and for all.
Citations and more reading 1. Roger, A. J., Muñoz-Gómez, S. A. & Kamikawa, R. The Origin and Diversification of Mitochondria. Curr. Biol. CB27, R1177–R1192 (2017). 2. Jensen, P. E. & Leister, D. Chloroplast evolution, structure and functions. F1000Prime Rep.6, (2014). 3. Woese, C. R. & Fox, G. E. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc. Natl. Acad. Sci. U. S. A.74, 5088–5090 (1977). 4. Lake, J. A. Origin of the eukaryotic nucleus determined by rate-invariant analysis of rRNA sequences. Nature331, 184 (1988). 5. Cox, C. J., Foster, P. G., Hirt, R. P., Harris, S. R. & Embley, T. M. The archaebacterial origin of eukaryotes. Proc. Natl. Acad. Sci. U. S. A.105, 20356–20361 (2008). 6. Archibald, J. M. The eocyte hypothesis and the origin of eukaryotic cells. Proc. Natl. Acad. Sci. U. S. A.105, 20049–20050 (2008). 7. Williams, T. A., Foster, P. G., Cox, C. J. & Embley, T. M. An archaeal origin of eukaryotes supports only two primary domains of life. Nature504, 231–236 (2013). 8. Cavalier-Smith, T. Deep phylogeny, ancestral groups and the four ages of life. Philos. Trans. R. Soc. B Biol. Sci.365, 111–132 (2010). 9. Poole, A. M. & Neumann, N. Reconciling an archaeal origin of eukaryotes with engulfment: a biologically plausible update of the Eocyte hypothesis. Res. Microbiol.162, 71–76 (2011). 10. Lake, J. A. Eukaryotic origins. Philos. Trans. R. Soc. B Biol. Sci.370, (2015). 11. Spang, A. et al. Complex archaea that bridge the gap between prokaryotes and eukaryotes. Nature521, 173–179 (2015). 12. Scientists Break Record By Finding Northernmost Hydrothermal Vent Field. ScienceDaily Available at: https://www.sciencedaily.com/releases/2008/07/080724153941.htm. (Accessed: 25th March 2019) 13. Zaremba-Niedzwiedzka, K. et al. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature541, 353–358 (2017). 14. Akıl, C. & Robinson, R. C. Genomes of Asgard archaea encode profilins that regulate actin. Nature562, 439–443 (2018). 15. Hennell James, R. et al. Functional reconstruction of a eukaryotic-like E1/E2/(RING) E3 ubiquitylation cascade from an uncultured archaeon. Nat. Commun.8, 1120 (2017). 16. Da Cunha, V., Gaia, M., Gadelle, D., Nasir, A. & Forterre, P. Lokiarchaea are close relatives of Euryarchaeota, not bridging the gap between prokaryotes and eukaryotes. PLoS Genet.13, (2017). 17. Spang, A. et al. Asgard archaea are the closest prokaryotic relatives of eukaryotes. PLOS Genet.14, e1007080 (2018). 18. Da Cunha, V., Gaia, M., Nasir, A. & Forterre, P. Asgard archaea do not close the debate about the universal tree of life topology. PLoS Genet.14, (2018). 19. Hug, L. A. et al. A new view of the tree of life. Nat. Microbiol.1, 16048 (2016). 20. Nasir, A., Kim, K. M., Da Cunha, V. & Caetano-Anollés, G. Arguments Reinforcing the Three-Domain View of Diversified Cellular Life. Archaea2016, (2016). 21. Illergård, K., Ardell, D. H. & Elofsson, A. Structure is three to ten times more conserved than sequence–a study of structural response in protein cores. Proteins77, 499–508 (2009). 22. Zhang, C., Phillips, A. P. R., Wipfler, R. L., Olsen, G. J. & Whitaker, R. J. The essential genome of the crenarchaeal model Sulfolobus islandicus. Nat. Commun.9, 4908 (2018). 23. Seitz, K. W. et al. New Asgard archaea capable of anaerobic hydrocarbon cycling. bioRxiv 527697 (2019). doi:10.1101/527697