Archaea, Eukaryotes and the evolution of DNA replication complexes

The relationship between bacteria, archaea and eukaryotes is an interesting one, and made slightly harder to approach as people tend to lump archaea and bacteria into the one grouping of 'prokaryotes' which is not much more than a scientific word for "blobs I don't care about". Delving deeper into the biochemistry of all three superkingdoms shows that while the metabolic pathways used by archaea are more similar to those in bacteria, their core DNA processes (such as replication and protein synthesis) are more similar to the processes in eukaryotes. (I talk more about the distinction between the three superkingdoms here)

There was an interesting paper in PLoS ONE lately that was looking at the evolution of DNA replication complexes in archaea, and seeing as this blog has been rather heavily bacteria-biased (i.e I haven't talked about archaea for a while) I decided to take a look at it. They were focussing on three main complexes that help in DNA replication and are found in both archaea and eukaryotes: proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacteria do use corresponding proteins, but they are far more distantly related.

Schematic of the structure and subunits of the three complexes.

The MCM complex is thought to act as a helicase; unwinding the two DNA strands to allow them to split in two to be replicated. The PCNA and RCF are known as the clamp and clamp loader and help to attach the RNA primer for replication to the Polymerase, which uses the primer to start replicating the DNA.

All of these three complexes consist of separate subunits, which are almost identical. PCNA, for example, is a trimer (in the diagram above each subunit is a separate colour). In eukaryotes these the subunits are identical, but in archaea variations are found between them. This general pattern, that subunit composition was far more variable within the archaea, was found in all three of the complexes. This method of gene duplication followed by gene modification to create two different proteins is an important one for evolution, and in the case of DNA replication it seems to have been exploited far more in archaea than in eukaryotes.

Changing some subunits also allows these complexes to carry out different tasks. It's been suggested that for some archaea there may be a functional difference between PCNA with all subunits the same (homotrimers) and PCAN with differing subunits (a heterotrimer). This allows multiple functions to be generated through simple DNA duplications - although all the functions are likely to relate to DNA replication in some way.

This brings forth the interesting point of view that the truly 'ancestral' forms of these genes and proteins may be more like the proteins seen in the eukaryotes rather than the archaea! Archaea (and bacteria) can tolerate a lot more genetic change than eukaryotes can, and have a far shorter generation time, allowing them to change and evolve more quickly than the larger, less genetically mutable eukaryotes. On the other hand the lack of change and high level of conservation in eukaryotes means that the complexes remain very similar to those of the ancestral eukaryote from which they evolved. They may even be closer to the forms found in the last common ancestor between eukaryotes and archaea, before the eukaryotes gained a nucleus and became unable to share genes with the surrounding organisms.

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Chia N, Cann I, & Olsen GJ (2010). Evolution of DNA replication protein complexes in eukaryotes and Archaea. PloS one, 5 (6) PMID: 20532250

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Niches of Sunlight

In every environment there will be competition for resources, and there are generally two ways organisms deal with this; generalise or specialise. To generalise, you try to cope with as many different conditions as possible, so that if you get out-competed in one area you can try to cope with the conditions elsewhere. To specialise, you get damn good at using the conditions in your little niche, in the hope that you'll be better than anyone else who comes along, and be able to out-compete them.

There are many resources that need fighting over, and in the sea one of the major ones for photosynthetic organisms is sunlight. Other nutrients such as nitrogen, phospherous and trace metals such as iron and copper can exist in different forms at different levels in the ocean (as shown below), but once you start getting below a certain depth, sunlight quickly becomes a finite and rapidly diminishing resource:

Diagram showing availability of nutrients at different depths.

Taken from the reference below.

Different organisms can cope with the lack of sunlight in different ways. Some (especially the larger algae species) have generalised, they contain a whole range of different light capturing pigments which can absorb a range of light wavelengths, including those in the darker depths. But little photosynthesising bacteria like Procholorococcus (which I mentioned in this post) which have the smallest genomes of all photosynthesising organisms, don't have that option. Instead they have to specialise, so that different strains in a species are adapted to different levels of light.

Work done by Rocap (paper reference below) looked at two different strains of Prochlorococcus: MED4 and MIT9313 (which I will just call MED and MIT). The MED strain was found only in the surface waters, while MIT was found much lower down; a phenomenon known as 'vertical niche partitioning'. Despite their genomes differing by only 3%, and despite being technically the same species (although 'species' is an uncertain word in the world of bacteria) they have optimised themselves to completely different levels of not just light but also nutrients, trace metals and virus specificities.

MED (the one near the surface) has a slightly smaller genome than MIT, yet contains twice as many genes dedicated to high-light-inducible proteins, many of which seem to have arisen by gene duplication. It also has genes specialised for the nitrogen sources found near the surface of the water, and organic phosphates (which again are found predominantly on the surface).

MIT on the other hand has fewer genes for ultraviolet damage repair, but more light harvesting genes, for example it possesses two copies of the hight-harvesting chlorophyll binding antenna protein. This helps it to gather as much light as possible, despite being further below the surface. it's also adapted for its specific nitrogen source and increased ability to use orthophosphate, rather than organic phosphates.

Both genomes have lost the ability for photoacclimatisation, that is the ability to change to suit different light conditions. By taking up vertical niche positions, they have forfeited the ability to change their response, meaning that a strict horizontal partition between them must be maintained at all times. Any Prochlorocuccus found at lower levels will be of the MIT variety, while those at higher levels will be MED. It's even thought that there might be further strict niche partitioning; with different ecotypes of MED adapted to use different iron sources, or different temperatures.

For the photosynthetic organisms that inhabit it, seawater is more than just a blue shifting salty mass. It's a whole range of niches and environments, partitioned in three dimensions depending on the surrounding conditions and nutrients.

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Rocap G, Larimer FW, Lamerdin J, Malfatti S, Chain P, Ahlgren NA, Arellano A, Coleman M, Hauser L, Hess WR, Johnson ZI, Land M, Lindell D, Post AF, Regala W, Shah M, Shaw SL, Steglich C, Sullivan MB, Ting CS, Tolonen A, Webb EA, Zinser ER, & Chisholm SW (2003). Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature, 424 (6952), 1042-7 PMID: 12917642

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Wonderful Life - book review

This is my first attempt at a proper scientific book review, so any feedback would be much obliged. Thanks!

The Burgess Shale is a collection of fossils of soft-bodied invertebrates that was formed soon after the Cambrian Explosion, an apparently rapid appearance of a many groups of complex animals. Initially discovered in 1909 by Charles Walcott, the fossils were later examined by three researchers in Cambridge; Charles Whittington and two graduate students, Derek Briggs and Simon Conway Morris. What began as an exercise to catalogue a group of ancient arthropods turned into the discovery of several new phyla of organisms, and ultimately changed the way evolution and selection were viewed. “Wonderful Life” by Stephen Jay Gould is a documentation of this process, exploring and explaining this new view of evolutionary change through Deep Time.

Although Gould was not personally involved in the cataloguing of the Burgess Shale organisms, he was in close communication with the people who were and his clear enthusiasm for the subject shows through. In the preface Gould sets out three main aims; to chronicle the intellectual drama that *was* the Burgess Shale examination, to explore the implications that this change in the perceived workings of evolution brought about, and finally to briefly look at *why* the discovery of the Burgess Shale seems to have passed so unnoticed by the general public, and even non-paleontological scientists.

The book is divided into five sections. The first sets up the central theme that the book is out to destroy; the iconographic idea of the March of Progress, that evolution is a kind of ‘onwards and upwards’ affair, with each generation leading to greater complexity. This was the idea that the discovery of new creatures in the Burgess Shale, creatures that belonged to no known phylum (and could therefore not be simpler and less developed forms of current animals) began to destroy. The second section covers background information considered necessary for an understanding of the Burgess Shale, a quick course on the Paleontological timeline and arthropod anatomy. Being the rather badly specialized microbiologist that I am, I read through these dutifully and promptly forgot them, so can safely say that it’s perfectly possible to enjoy the book without a great depth of scientific understanding.

The third section was for me the most exciting, as it was the actual description of the Burgess Shale creatures, written in order of their discovery (by Wittington, Briggs and Conway Morris). As they slowly discovered more new creatures, they began to realize that these were animals that had never been seen before, that had been wiped out by some extinction event. Furthermore, there was no particular evolutionary *reason* for certain animals to have been saved, the ‘March of Progress’ was beginning to look more like a lottery of chance. The last part of this section discusses the implications of this point of view, that humanity is not a strived-for evolutionary point of perfection, but simply a small twig on a tree of life which has had several branches snapped off altogether at different points in time.

The fourth section leaves the Burgess Shale (in a rather anticlimactic and in my opinion a slightly disappointing shift) to discuss Walcott, the man who found the fossils in the first place. Being a busy man, who in later life was caught up with various family tragedies, Walcott never properly had time to examine his fossils, and in the few papers he did write about them, he ‘shoehorned’ every fossil into modern phyla. Although I couldn't find the discussion of the life of an Edwardian scientist anywhere near as exciting as the beautiful fossil-creatures of the Shale, this section allowed a detailed examination of the shift in the way evolution was viewed as a theory, and why the original iconography of the March of Progress was so seductive and successful. In the fifth section Gould takes a brief but fascinating look at how things might have changed had life had a chance to play out a second time, if different branches of the tree of life had been cut off at different stages in history.

Overall I really enjoyed the book; I was especially pleased as this is (embarrassingly) the first book by Stephen Jay Gould I’ve ever read. The writing style is easily accessible, even to people with a very sketchy view of Deep History and the importance of arthropods, and is, a little surprisingly, highly immersive. The creatures of the Burgess Shale are so beautiful and wonderful that they stand up perfectly well on their own, and Gould lets them do so, his writing concentrating on exploring the philosophies surrounding their discovery rather than over-elaborate descriptions. The many accompanying pictures, most of which are the original drawings made by Wittington taken straight from the fossil samples, help to provide a wonderful visual image of the amazing and sometimes quite frankly weird creatures that populated the Burgess Shale.

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For those interested in such things, I now have a twitter. Like the blog, it will be strictly sciency, rather than personal and will be updated far more often.

Bacterial evolution: negative to positive?

The most commonly used distinction in bacterial populations is that of Gram negative and Gram positive bacteria. They are named after the method used to distinguish them: the Gram stain (developed by Hans Christian Gram). Gram positive bacteria have larger peptidoglycan cell walls, and therefore retain the crystal violet stain, whereas Gram negative bacteria have two membranes with a thin peptidoglycan wall between them, do not retain the crystal violet stain, and pick up the safranin counter-stain. The practical upshot of this is that you end up squinting at small blotched shapes under the microscope, trying to work out whether they look more pink or purple:

Gram +ve on the left, Gram -ve on the right.

As well as hinting that Mr Gram was one of those people who knows what shade 'fuchsia' is, the Gram stain is also one of the most important ways of telling what kind of bacteria you're dealing with. Despite being seemingly arbitrary, the composition of the cell wall plays a major role in determining behaviour. Gram negative bacteria (small cell wall, two cell membranes, see the picture below) tend to be motile, opportunistic, and able to colonise a wider range of environments. Gram positives on the other hand (big cell wall) are not so motile, but tend to have a huge range of excretory proteins to make up for this; almost all known antibiotics come from Gram positive bacteria.

Again, Gram +ve on the left, -ve on the right. Image from soil microbiology webpage

One thing that I've never really considered before is which one of them evolved from which. I haven't done much taxonomy, and the only time I really covered bacteria (unrelated to lab work) was in my Pathology course, which didn't seem too concerned about where different types of bacteria had come from, only what they were currently up to. The few times I did vaguely think about this though, I would have gone for the positive to negative direction. After all, surely you start with one cell membrane, and move on to two.

I recently came across a paper that came to the complete opposite conclusion, and therefore was too interesting not to read. The thing about bacterial taxonomy is that a lot of the major changes to morphology took place in Deep Time, and bacteria leave precious few fossils. Bacteria (and archaea...) had somewhere in the region of over one billion years to evolve before eukaryotic-things even started to be considered. That's a lot of time to try and sort out. To put that into context, one billion years ago from now things were just about starting to think about going multicellular. No dinosaurs, no plants even; the most complex form of life was something resembling a sofa cushion.

So how to sort out what was going on in that billion years or so? There are four main ways of going about it:

• Paleontological evidence. Bacteria don't form a huge number of fossils, but they can occasionally leave some physical evidence of their presence. For example, bacteria that eat iron will leave behind little fossilised iron cases; those that eat rocks can leave microscopic drilling holes. These provide temporal evidence for changes in structure and metabolism.
• Transition analysis. This is used to polarise major changes by turning them into a simple before-or-after question, and uses comparative, developmental, and selective arguments for determining answers. For example: did legs or wings develop first? Or, in bacterial cases: Which came first, Gram negative or Gram positive?
• Congruence testing. This searches for similarities across whole evolutionary trees, enabling loss or gain of evolutionary abilities (wings, feathers, second membranes etc) to be identified and polarised. As this is a comparison of many species, it allows potential mistakes from the arguments made in transition analysis to be found.
• Sequence trees. Sequence trees are ... problematic, but at the same time indispensably useful. They are formed by taking DNA sequences from a range of organisms and then using algorithms to tell the 'relatedness' between sequences and using these 'relatedness' levels to make evolutionary trees. They tend to be biased towards your sample distribution, undirectional, unable to properly account for generation times, and go somewhat screwy when you try to introduce horizontal gene transfer. Nevertheless they were instrumental data in showing that archaea and bacteria are two very distinct super-kingdoms (and I will freely admit that most of my distrust for them occurs because I can't get the damn things to work whenever I try them)

So...using these techniques can we get a clearer idea what was happening with bacterial membranes during those 1 billion-odd years before the arrival of eukaryotes? On the face of it; positive-to-negative seems to make more sense: start with one membrane, gain a second, possibly by gene duplication.

However like many evolutionary stories, that one falls apart a little when closer examined. Because Gram positive bacteria are not simply 'one cell membrane' they also have a massive cell wall surrounding them. Developing a second cell membrane on top of that seems absurd. And then why would the cell wall shrink? And how would anything get through this suddenly developed cell membrane. Transport proteins for the outer membrane tend to form a protein structure called a beta-sheet, while those for the inner membrane form an alpha-helix. That's a whole new system of protein folding that has to evolve pretty quickly, because otherwise the bacteria will starve, nothing can get through its outer membrane (which is balancing precariously on top of the huge cell wall...)

In view of this, the schematic seen on the right starts to make a little more sense (figure taken from the reference below). 'Murein' means 'peptidoglycan cell wall' and the cytoplasm denotes the inside of the cell. In this scenario, the double-membraned proto-bacteria (which has spend the last half-a-billion years or so evolving a well adjusted double membrane system) suddenly looses the outer membrane. A very simple genetic change would lead to a massively overgrown cell wall, which would rip the outer membrane away. The cell looses all it's outer membrane porins, and signal systems, but in return gains a highly protective cell wall, which potentially allows it to survive in different niches. How these aspects are lost genetically is another matter, and the paper rather hand-waves away by saying that unused genes tend to get lost eventually. Which is true in bacteria, they have such a small genome they don't want it getting filled up with unnecessary genes, but I have a feeling genes tend to leave something behind. Even so, the question of where the now-unnecessary genes go is possibly one of the weaker parts of this arguments (to my untrained student eyes at least.).

One thing that would really support this hypothesis would be to show that Gram positive bacteria formed a 'mono-clade' i.e came from a single universal common ancestor. Unfortunately this data is proving hard to pin down, not helped by the bacterial trick of swapping DNA around with all and sundry. Another confounding factor is the sheer space of time. Trying to determine whether a range of different modern bacteria all came from the same blob several million years ago is a daunting task. You can sort of get RNA sequence trees that support the mono-cladal Gram positives, but only if you close one eye and squint, which is not generally accepted scientific practise.

I don't think I'll ever end up going into taxonomy, even of bacteria. but it does produce fascinating ways to look at the world; how it changed, how it evolved, and how it finally turned into the way it is now. Orwell wrote, fairly famously, "He who controls the past commands the future", and when you're trying to figure out how bacterial resistance works, and preferably how to stop them getting it, that phrase takes on a whole new meaning beyond the political.

(It's not a perfect quote for this post. "Understands the past" would work better. But I'm not quite pretentious enough to go trawling through the quote archives to find something better. Any suggestions would be appreciated :p )

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Cavalier-Smith T (2006). Rooting the tree of life by transition analyses. Biology direct, 1 PMID: 16834776

On the classification of blobs

The first forays into microscopy revealed a whole world of blobs, tiny microscopic organisms that were invisible to the naked eye. These went through a range of different names, from the 'animalcule' denomination given by Anton van Leeuwenheok (which he used to describe everything small he saw under the microscope, including his own sperm) through 'monera' (a more specific name for certain types of blobs, namely those that weren't eukaryotic) to 'prokaryotes', a name that still stands.

Prokeryote means, literally, 'no nucleus', and it's use allows the world of living organisms to be split up into two groups: Prokaryotes and Eukaryotes. Eukaryotes are things with a nucleus, a membrane covered partition to hold DNA, as well as many separate organelles existing within their cells, such as mitochondria, endoplasmic reticulum, the Golgi apparatus...

Prokaryotes are...uh...everything else.

Which means that the label 'prokaryote' was always waiting to fall apart. After all, they may just be blobs but there are a lot of them, and some are very different blobs. Around the 1970's people started noticing that there were a group of the prokaryotes that behaved differently, mainly through studies done by Carl Woese and George E. Fox who created classification tables based on the genetic sequences of ribosomal RNA (the part of the genome most likely to be conserved, this is often used for classification, especially of things in Deep Time). This showed that there was a distinct group of prokaryotes with a mostly separate evolutionary history (more on the mostly later) to the rest of the prokaryotes. They were originally named 'archaebacteria', and together with 'eubacteria' (true-bacteria) were put in the prokaryotes group. They were blobs without a nuclei, and that was where they belonged.

However, things started to get a bit more complicated the more people looked at archaebacteria. They weren't just a group of slightly odd bacteria, they were something else. Something different. Although their metabolic pathways are similar to bacteria, their methods of turning DNA into proteins more resembles eukaryotic processes. Their flagella (tentacle like structures used for movement) have a markedly different structure from bacterial flagella. Like bacteria, they reproduce asexually and (also like bacteria) they can share their DNA around, in fact they can also share there DNA with bacteria, which makes taxonomists tear their hair out. It's very difficult to classify something when it keeps giving its DNA away, and collecting bits from other sources.

It is proposed in the SGM journal (Society for General Microbiology-journal not available on line) that the term 'prokaryote' should be scrapped altogether. As well as being an incorrect label for a large group of organisms it also produces an incorrect evolutionary perspective. The use of the eukaryote/prokaryote terms suggests a very human based linear "One upon a time there were blobs with no nuclei and then they got nuclei and then they were better" sort of story. A more correct view is that of all three superkingdoms; bacteria, archaea and eukaryotes splitting away from each other. Eukaryotes safely packaging their DNA away, allowing a more complex system to build up, yet forfeiting the ability to share bits of DNA. The archaea and bacteria on the other hand, continued to share their genetic material, just became more selective about it as they diverged (hense the 'mostly' seperate history).

Or maybe not. It might be that the archaea/eubacteria formed a very selective group of blobs, which then split further when some developed a nucleus, while the others continued to share their DNA with the bacteria, picking up different metabolic secrets. It's hard to work out; especially given that similarities between the DNA of archaea and bacteria does not necessarily show their relatedness; it might be a gene that has remained conserved in both of them for millions of years, or it might just be one that was exchanged last week.

There a several arguments against removing the 'prokaryote' as a naming system but most of them boil down to the very multicellular-centric argument of: "but they're all just blobs!" The three superkingdoms of archaea, bacteria and eukaryote are a far more accurate, and scientifically and taxonomically correct way of looking at things than the prokaryote/eukaryote model.

My only complaint is that I spent ages in secondary school trying to learn how to spell 'prokaryote'... removing the name means I could have spent that time doing something far more useful...like building paper planes and reading 'Redwall'.

Taxonomists vs. Phages

Taxonomy is one of those wonderful subjects that at first seems very simple (and very boring). The word comes from Greek - taxis meaning 'order' and nomos meaning 'law', or 'science' and is at it's most basic the science of classification. Most people do varying amounts of taxonomy at school; dividing living things into plants, animals and fungi, dividing the vertebrates into birds, mammals, amphibians, fish and reptiles etc.

Putting the dubious accuracy value of these classification labels aside (particularly the bit about reptiles) taxonomy is, at it's most basic level, a simple system for ordering things and putting them in little boxes. Glorified stamp collecting, as it were. But there are still plenty of arguments and various feuds about the exact relationships of things, most often at the species level, and even whether the whole 'five kingdom' model is any use (the five kingdoms are animals, plants, fungi, prokaryotes - roughly bacteria, and protoctista - which are basically the equivalent of the filing draw marked misc.). In fact, the more you delve into the science of taxonomy, the more complications and problems you start to encounter. Even something that would seem fairly simple, such as what defines a 'species' is a matter of hot debate.

And when you get down to the level of single-celled organisms the whole system goes a bit haywire. The distinction between 'animals', 'plants' and 'fungi' completely breaks down, there are single-celled things that are motile and animal-like but photosynthesise, things that only photosynthesise at the right time of day, or are perfectly sessile and plant-like except they don't photosynthesise. Most protozoa (single celled thingys, more information under the link) are now broken up into a whole new set of labels, very few of which seem to relate to the larger multi-cellular organisms.

When it gets to things like bacteria and bacteriophages, of course, taxonomists just break down and cry. Because bacteria, unlike most other things, don't even maintain their genetic integrity. Bacteria can share bits of their genome quite happily, even with bacteria that are seemingly very unrelated, making the whole 'species' concept break down a bit anyway. Phages merrily incorporate various bits of bacteria into themselves, splice bits out, even splice themselves in to bacterial genomes and sit there for a while. It's a complete headache to try and organise the things.

Various attempts have been made, of course, since the first discovery of phages in 1915 by the wonderfully python-esquely named Frederick Twort. (to give all and full credit they were also discovered completely independently in 1917 by Felix d'Herelle). The first system was based on morphology, what the phage looked like, and was greatly helped by the electron microscope. Although most phages adopt the 'lunar landing module' look, there is plenty of variation within that. Length of tail fibres, size of capsid, symmetry of the capsule, alright, not very much variation, but still something for taxonomists to hang onto.

Size and shape are never good indications of relatedness, a dolphin is more related to a hyena than to a shark, however similar the two might look. Nucleic acid research during the 1960's shook up the whole discipline of taxonomy by providing lots of new exciting DNA information. Phages could now be classified by the amount and type of DNA, which, added to the morphological data, provided a system (albeit a slightly wobbly one).

One of the most currently most widely popular methods to classify things is to examine the genetic DNA that codes for the ribosomes. Ribosomes are complexes of RNA and protein that are used to turn the genetic code into proteins. They are very highly conserved and are therefore very useful in determining evolutionary relatedness and taxonomy.

The problem is, of course, that bacteriophages don't have any ribosomes. They use the bacterial ones; they harness the bacterial internal machinery for replicating DNA and making proteins and therefore don't need to carry any of their own. In view of this, one of the most recent attempts to organise phage taxonomy has focused on looking at their proteins. The relatedness of the proteins has been used to create clever sounding things like distance matrices and the highly impressively named 'phage proteome tree'. Of course there are several problems, possibly the main one being that phages, especially the ones that sit inside bacteria, have a distressing tendency to pick up bits of DNA that aren't theirs. Which translates into proteins that aren't theirs and makes the whole procedure just that little bit more awkward.

There's been some work comparing the genomes of just the structural components, in the hope that there won't be too much genetic exchange going on with the genes that are actually needed to build the phage. The people doing it seem fairly confident, and have managed to isolate about five separate genera. There's a paper from them here, hopefully should be accessible (in form if not in content).

The whole thing is really all a bit up in the air, with some fairly amusing piss-ups between the different schools of thought. Horizontal gene transfer can be a bitch sometimes :)