Climbing the Tree of Life

MartinIn this week’s blog, Professor Martin Embley reflects on the  the journey that led to him, his collaborators and his laboratory to fundamentally change our views on evolution and the Tree of Life.

The Early Years

My early career was a bit of a random walk while I tried to figure out what I really wanted to do. After my PhD in Newcastle on bacterial diseases of trout and salmon, I got a job teaching industrial microbiology at North East London Polytechnic in 1984. It was an odd but interesting place, a number of staff appeared to have strong religious beliefs of various sorts and wanted to talk about them, and one colleague thought he could change traffic lights from red to green so he never had to slow down. I was keen to keep doing some research and I was interested in evolution, but like a lot of newly independent researchers I struggled to get any funding. My big break came when I got a “cultural exchange” grant from Newham Council to go to Poland to learn some molecular biology and I met Professor Erko Stackebrandt who was passing through. Erko had worked with Carl Woese in pioneering the use of ribosomal RNA sequences to investigate evolutionary relationships among prokaryotes. I persuaded him to let me visit his lab in Germany to learn the new techniques and in 1991 these skills got me a job at the Natural History Museum in London.

The Museum wanted to set up a lab using molecular sequences to investigate evolutionary relationships. The film Jurassic park was just about to appear and there was tremendous excitement about the potential of ancient DNA. The Museum gave me free rein regarding my own research as long as it had evolution at its core. So I decidedTree of life to work on the early evolution of eukaryotic cells. At the time two ideas were central to views of early eukaryotic evolution. One was that the “three domains tree of life” was an accurate description of the relationships between eukaryotes and prokaryotes (you can see the tree here). The other was that some eukaryotes, including obligate intracellular microsporidian pathogens, had never had mitochondria because they split from other eukaryotes before the mitochondrial endosymbiosis. I’ve been trying to test these two ideas for the past 25 years and while it’s often been difficult and frustrating, it has also been a lot of fun.

A Team Effort

Like most PI’s I’ve relied on attracting talented young scientists to do the work that we have published. Robert Hirt walked into my lab off the street and asked me if he could do a project involving eukaryotic evolution and ecology. He already had a first author paper in Cell and now he wanted to do something different. We didn’t do much ecologMitochondriay together but Robert and I co-supervised PhD student Bryony Williams who showed that microsporidians actually contained a tiny, hitherto overlooked mitochondrion, now often called a mitosome.

Unlike our own mitochondria, the microsporidian mitosome doesn’t make ATP, because it has lost all of the pathways used by classical mitochondria to make energy. Alina Goldberg in our lab – now at Newcastle – and Sabine Molik in the lab of Roland Lill in Germany spent Mitochondria 2the next seven years showing that the mitosome functions in the biosynthesis of essential cytosolic and nuclear Iron/Sulphur (Fe/S) proteins. The discovery of a tiny mitochondrion in microsporidia (Figures 1 and 2) was an important piece of evidence that led to current ideas that the mitochondrial endosymbiosis occurred at the origin of eukaryotes. Thus, it is now thought that all eukaryotes contain an organelle related to mitochondria, and its most conserved function is in Fe/S protein biogenesis, not ATP production.

Figure 1Figure 2Competing Hypotheses

In the three domains tree of life, eukaryotes are a separate domain that is most closely related to the domain Archaea and the host for the mitochondrial endosymbiont is already a eukaryote. Although this hypothesis appears in most textbooks, there have actually been a number of alternative hypotheses published over the years (Figure 3 shows one of them), but these have largely been ignored. Cymon Cox spent three years analysing molecular sequence data to identify which of the competing published hypotheses was best supported and reached the surprising conclusion that it was not the three domains tree but an alternative hypothesis called the eocyte tree (you can read a discussion about the differences between the two trees including a picture of the eocyte tree here). In the eocyte tree, eukaryotes originate from within the Archaea, suggesting that eukaryotes are not a primary domain of life like Archaea and Bacteria, but are instead a product of genetic and cellular contributions from both prokaryote domains. Very excited by these results, we sent Cymon’s paper to Nature where it was reviewed and quickly rejected. We appealed, it was revised, it was reviewed again, and it was rejected again, all in all pretty dispiriting, but a common experience for most scientists. However, after hearing me talk about Cymon’s work at a meeting, we were invited to submit Cymon’s paper to PNAS where it was finally published in 2008.

Figure 3A Mixed Response

Although Cymon’s paper has been highly cited it is true to say that the initial response from the community was very mixed. We received emails suggesting that we had manipulated our results to get the answer we wanted and one of the reviewers told us that it was impossible to infer such ancient events using molecular sequences. In responding we agreed that it was difficult to be confident about anything that happened billions of years ago based upon small amounts of data and even the best methods of analysis, but that people in the field seemed happy to use the same data and worse methods to support the three domains tree. Cymon eventually moved on, scarred but not defeated, and Tom Williams took over the project when he came to Newcastle on a Marie Curie Fellowship in late 2010. Over the next few years, more data was published as new Archaea were discovered and new methods of analysis were developed, and every analysis that Tom, or others, did on these data produced a version of the eocyte tree, so that it is now the best supported hypothesis – at least in our opinion.

More evidence emerges

Hypotheses are only useful when they make predictions that can be tested by further research, and evolutionary hypotheses are no different. The eocyte tree predicts that new species that share more features in common with eukaryotes will be discovered among the Archaea, and this prediction now appears to have been spectacularly fulfilled by recent discoveries from Thijs Ettema’s lab in Sweden. The new paper describes the discovery, so far only from metagenome data, of an archaeal lineage called Lokiarchaeota that contains many genes for proteins that were previously thought to be eukaryotic specific, including homologues of proteins used in the eukaryotic cytoskeleton, in membrane remodeling and in phagocytosis.  This is incredibly exciting and the challenge is now to isolate Lokiarchaeota and other new lineages into culture so that their biology and physiology can be studied in the laboratory.

An Interesting Journey

Scientific work is often written up as if it were a linear progression towards improved understanding, a type of “Whig history” which does not accurately reflect how science is really done. In reality, science is a collaborative endeavour with lots of dead ends, confusion and false trails, and we could easily be walking down some of those still. Nevertheless, the currently prevailing paradigm for eukaryotic evolution is now very different to the popular views held in the 1990s when I started my research career. All eukaryotes are now thought to contain a mitochondrial homologue that generally functions in Fe/S protein biogenesis, and the host for the mitochondrial endosymbiont is thought to have originated from within the Archaea. Eukaryotes are thus viewed as the product of an interaction between (at least) those two prokaryotic partners and are not a primary domain of life but one derived from prokaryotic antecedents. The complex features that we take to define eukaryotic cells including our own, such as the nucleus, large genomes and diversity of RNAs, are thus secondary features that have evolved since those primordial interactions. I’m not sure what my religious former colleagues would have made of the work I’ve done since leaving NELP, but it’s been an enjoyable and interesting journey for me.


The Tree of Life:

Bryony Williams’ paper:

Alina Goldberg and Saline Molik paper:

Mitochondria and Fe/S proteins:

The eocyte tree:

Cymon Cox paper:

Tom WIlliams paper:

Thijs Ettema lab paper:

A novel copper storage protein discovered by ICaMB scientists

In a recent issue of Nature the discovery of a new family of proteins that store copper ions inside bacterial cells was reported. This work was carried out by Prof. Chris Dennison’s lab (ICaMB) in collaboration with Dr. Kevin Waldron (ICaMB), Dr. Arnaud Baslé (ICaMB), Dr. Neil Paterson (Diamond Light Source) and Prof. Colin Murrell (UEA). Here, Kevin tells us about this discovery and what its implications could be for the biotechnological exploitation of methane.

Methane, the main component of natural gas, is produced by both geological and anthropogenic processes on Earth, and can be released into the atmosphere. The biosphere’s primary mechanism for limiting the amount of methane (a powerful ‘greenhouse gas’) that escapes into the atmosphere is through its consumption by methane-oxidising bacteria, or methanotrophs (literally ‘methane-eating’), who utilise methane as both a carbon and energy source.

Transmission electron micrograph of a M. trichosporium OB3b cell showing the intracytoplasmic membranes that house the copper-dependent enzyme pMMO.

Transmission electron micrograph of a M. trichosporium OB3b cell showing the intracytoplasmic membranes that house the copper-dependent enzyme pMMO.

The key enzyme that methanotrophs use to oxidise methane to produce methanol is called methane monooxygenase (MMO). Almost all methanotrophs possess a membrane-bound form of MMO that requires copper (Cu) for activity, called pMMO (some strains also possess an iron-requiring alternative soluble enzyme, called sMMO). Therefore, these bacteria have an unusually high demand for copper, a metal that is essential for most organisms but is used rather sparingly, mainly because it can also be extremely toxic.

Previous studies of methanotrophs have revealed that they utilise unusual mechanisms for acquiring the copper they need for pMMO. For example, they produce and secrete a small, modified peptide called methanobactin that is involved in high affinity copper uptake, analogous to the more familiar siderophore iron uptake systems.

The model methanotroph, Methylosinus trichosporium OB3b, was analysed in the hope of uncovering novel copper proteins. Soluble extracts were resolved by two-dimensional liquid chromatography, with the copper content of resulting fractions monitored. The most abundant copper pool was found to contain a small, cysteine-rich protein that had not been studied previously and Chris Dennison (PI) and I (Co-I) obtained a BBSRC grant to investigate this and related proteins.

The presence of 13 cysteine residues in a mature protein (after cleavage of its signal peptide) with only 122 amino acids was immediately significant, as its thiol sidechain is often found in copper-binding sites. The recombinant protein was extensively studied using a suite of biochemical and biophysical methods to determine its in vitro properties This novel tetrameric protein is capable of binding up to 52 Cu(I) ions with high affinity, consistent with a potential role in the storage of this essential metal and was therefore named Csp1 (copper storage protein 1). A mutant strain of M. trichosporium OB3b that lacks this Csp1 protein (as well as a closely-related protein, Csp2) shows a phenotype consistent with a copper storage role of these proteins in vivo.

The crystal structure shows that the apo-monomer adopts a well-characterised four-helix bundle motif with all 13 cysteine sidechains pointing into the core of the bundle. The protein structure is essentially unchanged in the presence of copper, except that the core of each monomer has filled with 13 Cu(I) ions, bound by the cysteine sidechains. This is an unprecedented structure for metal storage.

The structure of the Cu(I)-Csp1 tetramer, with the 13 Cu(I) ions bound within the core of each four-helix bundle shown as orange spheres.

The structure of the Cu(I)-Csp1 tetramer, with the 13 Cu(I) ions bound within the core of each four-helix bundle shown as orange spheres.

The implications of this discovery could be of wide impact. There is a great deal of interest in using methanotrophs and MMOs in industrial biotechnology. Available methane is on the increase due to ‘fracking’, but importantly methane can also be produced from sustainable sources such as through the degradation of biomatter (biogas). Methanotrophs and the MMOs could in future be exploited using synthetic biology approaches for gas-to-liquid conversion, for the production of liquid fuels as well as bulk and fine chemicals from renewable methane.

Any such biotechnological applications require an understanding of how the production of pMMO is regulated and how this complex enzyme is assembled, including the cellular delivery and insertion of the essential copper cofactor. The work published in Nature shows that Csps are an important piece in that assembly jigsaw.

Furthermore, bioinformatics has shown that cytosolic members of the Csp family are also encoded in other bacterial genomes. Most bacteria are thought not to require copper in their cytoplasm, so the presence of cytosolic Csp homologues in their genomes could re-write our basic understanding of their use and homeostasis of copper.


Nature article:

News & Views:

BBSRC website:

ACS Chemical & Engineering news:

Press release:

Another Cell-ebration

Heath MurrayKevin WaldronLast year we brought you details of the inaugural CBCB Symposium. In July the second CBCB symposium was held, and today we hear from the organisers, Kevin Waldron and Heath Murray, about this latest successful event.

The idea for an annual Centre for Bacterial Cell Biology (CBCB) symposium was originally conceived in 2013, and aimed to showcase both the high quality and the immense breadth of research activity that goes on in this unique Centre. It would also be an excellent opportunity to bring together the CBCB research community based in both the Medical School and in the Baddiley-Clark building to discuss their work and build future collaborations.

It wasn’t long after our 2014 Symposium was over when the organising team (Bernie Shaw, Heath Murray, Kevin Waldron and Jeff Errington) began planning for this year’s second event. Obviously we were delighted with the success of that first meeting, but of course it also applied a little pressure on us as organisers; this year’s event had to achieve a similar level of success. Fortunately, the feedback we had received from the 2014 event included a number of constructive suggestions from the CBCB community about how we might be able to improve the Symposium, and we tried to incorporate as many of these ideas as possible.

One of our postdoctoral researcher speakers, Yoshi Kawai, addresses the Symposium audience on the subject of L-forms

One of our postdoctoral researcher speakers, Yoshi Kawai, addresses the Symposium audience on the subject of L-forms

One suggestion was to include a number of more junior speakers in the Symposium Programme as well as PIs, and we are grateful to those postdocs who volunteered to present their work to the CBCB audience. Alexander Egan told us about his research in the Vollmer lab on the proteins that coordinate biosynthesis of the cell envelope during growth and division. Yoshi Kawai of the Errington lab explained  how L-forms, bacteria that lack their cell wall, can be produced in the lab and how they propagate in a manner independent from the known bacterial cell division machinery, as well as speculating on their implications for early life forms on Earth. Marcin Dembek of the Salgado lab contrasted the mechanisms that govern sporulation in Clostridium difficile, a pathogen that primarily causes infections via spores, and the model organism Bacillus subtilis. Finally Didier Ndeh described his research in the Gilbert lab on how gut bacteria degrade the most structurally complex dietary polysaccharide known, rhamnogalacturonan II. PI speakers covered further topics relating to antibiotic discovery and their mechanisms of action and synthetic biology.

In addition to our CBCB researchers, we also again invited two high-profile external speakers. The day started with Mark Leake (University of York) who told us about his research using state-of-the-art microscopy for in vivo imaging of single molecules within the bacterial cell. And the Symposium was concluded by John Helmann (Cornell University) on the subject of transcriptional stress responses in one of CBCB researchers’ favourite model organisms, Bacillus subtilis.

Poster prize winner Lauren Drage

Poster prize winner Lauren Drage

Another of the suggestions that we incorporated into the Symposium schedule this year was a poster session, which was accompanied by light refreshments (of course!) immediately after the day’s talks. We had a great turnout, with more than 20 posters on display, and the session generated a lot of lively scientific discussion. Again the Symposium organisers are very grateful to all those members of CBCB who participated in the poster session. We awarded three poster prizes, with congratulations to winner Lauren Drage for her excellent poster describing her research in the Aldridge lab looking for biomarkers for diagnosis of urinary tract infections, and to our two runners-up, Martin Sim (Wipat lab) and Clare Wilson (Errington lab); and of course thanks to our poster judges, Lucy Eland and Yulia Yuzenkova.

Finally, we all got to enjoy an informal barbecue dinner and drinks, where the science discussions could continue into the evening.

Jeff Errington and John Helmann in post-symposium discussions

Jeff Errington and John Helmann in post-symposium discussions

Planning has already begun for next year’s Symposium, which will be held on the 8th July 2016, and will feature two more external keynote speakers, Christine Jacobs-Wagner (Microbial Sciences Institute, Yale University) and Prof. Tracy Palmer (Molecular Microbiology, University of Dundee). We welcome your feedback too, so if you attended this year’s Symposium and you have any suggestions about how we might improve next year, please let us know.