Carbon carbon everywhere...

A while ago I wrote about the Great Oxidation Event, the point way back in the history of the earth where a lot of little blobby organisms suddenly discovered the trick of using sunlight as energy, and in the process producing a reaction that liberated oxygen (photosynthesis). This (as I discussed in the previous post) had a major impact on all the rest of the life on earth, but it also rather majorly effected the photosynthesising organisms themselves, not so much because of the increase in oxygen, but because of the semi-simultaneous decrease in carbon dioxide.

When photosynthetic organisms first developed, there was a lot more carbon dioxide in the environment for them to use (with the world so new and all...) and therefore they weren't particularly bothered about getting hold of it. However when the carbon dioxide levels dropped (along with a potential rise in temperature) they were suddenly in very real danger of suffocating. These were marine organisms, and there just isn't that much carbon dioxide in seawater. There's plenty of carbon floating around, certainly, but it's all in bicarbonate form (HCO3- rather than CO2) and Rubisco, the main enzyme involved in photosynthesis, doesn't know how to use bicarbonate, it relies exclusively on carbon dioxide.

Our lecturer called it the 'Ancient Mariner paradox'; the ocean is full of carbon, but the photosynthesis machinery just couldn't use it:

"Water, water, everywhere,

And all the boards did shrink;

Water, water, everywhere,

Nor any drop to drink."

This left the little suffocating blobs with three options. They could stay remain tiny (as the picoplankton did) to minimise diffusion differences and therefore still survive dispite the low carbon dioxide levels. Or they could try to change the way Rubisco worked, but Rubisco has a rather compromised active site as it is, having to both distinguish between carbon dioxide and oxygen and trying to keep carbon dioxide processing levels high. Rubisco is often criticised as being an 'inefficient' enzyme, and compared to other enzymes it is, but with carbon dioxide levels at the level they are in the sea it's only ever working at half of its maximum speed. Carbon dioxide is the clear limiting factor.

So instead, these photosynthetic organisms started to develop ways to get carbon dioxide into the cell and concentrating it around the Rubisco. The main factor in this was the enzyme carbonic anhydrase, which converts bicarbonates back into carbon. However doing that inside the cell just leads to the carbon dioxide diffusing right back out again and therefore today almost all photosynthetic bacteria (and chloroplasts inside plants) contain a special internal compartment, a protein coat surrounding the Rubisco, and all the carefully hoarded carbon dioxide:

Figure above shows TEM of bacteria with carboxysomes pointed out by arrows. The scale bar on the bottom right is 100nm

Photosynthesising bacteria (apart from the picoplankton) contain a compartment called a carboxysome, which consists of a protein coat which contains carbonic anhydrase enzyme and Rubisco, allowing carbon dioxide to be produced right where it's most needed. The addition of a number of bicarbonate transporters on the outside of the cell allows bicarbonate to be brought into the cell, and the whole assembly is known as a Carbon Concentrating Mechanism, or CCM.

When these photosynthesising proto-bacteria were then picked up by free-moving proto-algae to become chloroplasts, they kept their CCMs. The CCM of eukaryotic chloroplasts is called a pyraniod, and can be seen in the picture below (from Dartmouth College) as the dark black blob in the upper left hand corner. The white things that it's surrounded by are starch grains. The big fuzzy blob below it is the cell nucleus, and the little grey membrane-filled circles are the mitochondria. The long black threads are either thylakoid membranes (inside the chloroplast) or endoplasmic reticulum:

The first algae would have been marine as well, and would have needed the CCMs in their chloroplasts in order to produce energy. Sea water tends to be alkaline, which means that the biocarbonate: carbon dioxide ration is insanely large. Gasses don't diffuse very well in water either, carbon dioxide takes about about 10 000 times longer to get anywhere in liquid compared to air.

In fact the best thing to do to get as much free carbon dioxide as possible is to leave the water altogether, and head out onto the land. But that is different story.

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Tanaka S, Kerfeld CA, Sawaya MR, Cai F, Heinhorst S, Cannon GC, & Yeates TO (2008). Atomic-level models of the bacterial carboxysome shell. Science (New York, N.Y.), 319 (5866), 1083-6 PMID: 18292340

Half plant, half predator, all weird.

I was planning on brushing up my knowledge of chloroplasts today, as next week I'm starting a plantsci course for my options lectures, but I got sidetracked by Captain Skellet alerting me to Hatena. I've heard of several organisms containing proto-plasmids; symbiotic chloroplasts which haven't completely been endosymbiosed, but Hatena was a new one so I went to look it up. And I'm very glad I did, because it's pretty amazing.

Hatena, taken from the reference. Green blob is the symbiont. Scale bar is 10um

Quick background: chloroplasts are little membrane enclosed vesicles in plants which carry out photosynthesis. Current theory for how they developed is that they were once free-living bacterial type organisms (cyanobacteria) which were engulfed by a larger cell and over time lost their own identity and became little photosynthesising factories inside the larger cell. (I've got another post on it here for anyone particularly interested in the subject.)

Hatena arenicola doesn't have a chloroplast, but it does have a symbiotic relationship with another organism; nephroselmis. The nephroselmis is always found in the same place in the Hatena, and carries out photosynthesis to provide energy for both of them. Unlike regular chloroplasts, nephroselmis has it's own proper nucleus and even it's own mitochondria although most of the internal cellular organisation and any kind of motile apparatus (such as flagella) has been lost.

The weirdest thing about these two organisms though, is their replication cycles. When Hatena replicates, the nephroselmis doesn't, and as a result only one of the offspring gets the photosynthesising symbiont. The other organisms remains colourless and develops a complex feeding apparatus at the apex of the cell, presumably as it can no longer rely on the symbiont for food. This wierd 'half plant, half predator' lifecycle is shown below. (Picture taken from the reference, scale bar 10um):


That's just weird. Seriously odd. The Hatena is able to move seemingly freely between being a predator consuming other cells for food, and being a plant-like organism, once it settles down with it's symbiotic partner. The grey non-symbiont organisms can be induced to take up free-moving nephroselmis and (in the words of the paper) "tentitavely" maintain a symbiotic relationship with them.

The paper suggests that Hatena cycles between these two modes of living, depending on circumstance. Thus the 'predator' grey cell shown above will continue eating fellow cells until it consumes a nephroselmis, at which point it degrades its complex feeding apparatus, accepts energy from the symbiont until it's ready to divide. One of the daughter cells will then go through the whole cycle again while the other remains as a non-predating plant. The authors freely admit that there is little evidence for much of these stages, but it seems a reasonable way to explain what is going on.

As this is clearly a very early stage in symbiotic capture it has important implications for the endosymbiotic theory of chloroplast evolution. Along with various other 'intermediate' symbionts (such as Karenia mikimotoi and Lepidodinium viride) the Hatena helps to show how chloroplasts might have first formed in the cellular ancestor of plants. Hatena and its symbiont have already acquired an intimate structural association, only the coordination of their cell cycles would be required to turn the nephroselmis into an internally replicating plastid.

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OKAMOTO, N., & INOUYE, I. (2006). Hatena arenicola gen. et sp. nov., a Katablepharid Undergoing Probable Plastid Acquisition Protist, 157 (4), 401-419 DOI: 10.1016/j.protis.2006.05.011

Endosymbiosis - a big tangled mess of algae

Next term I'm taking a short course on plastid evolution (e.g chloroplasts), as it was the choice that came closest to my beloved bacteria. While I hold no great love for the inner workings of multicellular creatures, I'm forced to admit that there is something quite special about eukaryotic cells. They're full of little compartments, closed off organelles and selfishly horded genomes, including bacterial genomes within their mitochondria and chloroplasts. Also Psi Wavefunction writes about plastid-containing things a lot, and has convinced me that while they'll never be bacteria, they are pretty amazing in their own right.

The theory of endosymbiosis is that mitochondria and plastids (pigmented organelles such as chloroplasts) were once free-living bacteria-type organisms, which were engulfed by larger cells. Rather than being subsequently digested, these organisms managed to survive inside the larger cells, providing energy for them in return for a safe place to stay:

Diagram 'borrowed' from last years lecture notes

However this state of affairs immediately creates a problem. Not one of space, or resources, but of nuclei. Two nuclei are now present in the same organism, creating problems of control. The plastid cannot simply divide, replicate and produce energy whenever it wants, and the same is true of the surrounding cell, the two must work together, which means putting at least some of their genes under the same system of central control.

What usually happens is that over time the internal plasid's genes migrate to the nucleus of the surrounding organism (although apparently some protists will hold fairly epic genomic battles about who ends up with the majority of the genome). Genomic analysis shows the plastid or mitochondrian genes sitting happily in the nucleus, although the organelle will retain some of its genes in it's own little chromosome, probably for the same reason that the USA remains a federal government.

Doing further genetic analysis however, particularly on the chromalveolates (a large group of protists which include, among others, the red-tide producing dinoflagellates and the photosynthesising marine diatoms) shows that the story is not quite so clear cut. The chromalveolates are believed to all originate from an ancestor containing an engulfed red-algae plastid for photosynthesis. Analysis of the genome of the diatome P. tricornutums does indeed show red algal genes, however it also shows genes from green algae. In fact, large-scale phylogenetic analysis of algae and diatoms revealed over 1700 green algae genes in the P. tricornutums nucleus, outnumbering the red algae genes.

How did they get there?

One theory presented is that this diatom has in fact had two endosymbiotic events in its past; a green algae that later somehow disappeared or was lost, and then the red algae. However as there are many different branches of the chromoveolates containing varying amounts of red/green algae material this seems to make a rather large assumption about the ease of endosymbiosis...it sounds like algae are being absorbed and lost surprisingly easily, and quickly. The discovery that some chromoveolates without any plastids also have plastid genes in their nucleus doesn't help matters. How are the genes getting in, and is multiple rounds of endosymbiosis, followed by subsequent plastid loss really a realistic answer?

There are other explanations, although as yet no real answers. Green algae are not the most well-sampled of organisms, and the genomic record is distinctly patchy. This makes it a lot harder to determine where genes truly come from. Also there is the matter of horizontal gene transfer. Before being engulfed by the chromalveolates, the algae would have been able to share genes in much the same way bacteria do. The green algae genes might have got into the red algae before they were engulfed.

Horizontal gene transfer can also occur in some ciliates, which may explain the presence of plastid genes in their nucleus, despite the fact that they don't have any plastids. Ciliates often form symbiotic relationships with algae. This means that they will often come into contact with lysed algae, and may have been able to pick up genetic material from them. There also may be a certain 'background' of algae-like genes which in reality have nothing to do with algae. Some plastid-like genes have been found in amoeba, which really don't have any reason to have them, so it might just be an artifact.

Even so, the evolution and origin of plastids is clearly a wonderfully convoluted and undetermined area. Away from the weirdness of algae-containing protists there are still many questions to be answered. In the plant model-organism arabidopsis there is still a fascinating interplay between the genomes of the chloroplast and the nucleus. The chloroplast uses several nuclear genes that it never even supplied in the first place, it seems to have hijacked some of the nuclear genes for it's own purposes. The arabidopsis nucleus has returned the favour, with less than half of the genes supplied by the chloroplast being used for chloroplast-related purposes.

I can't wait to start studying it. (Especially because it means the endlessly boring set of 'techniques' lectures will finally be over.)

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Elias M, & Archibald JM (2009). Sizing up the genomic footprint of endosymbiosis. BioEssays : news and reviews in molecular, cellular and developmental biology, 31 (12), 1273-1279 PMID: 19921698

Protists and their plastids

A quick skim through this blog reveals fairly quickly that I have a slight fixation on bacteria. I like to research them, read about them, and then blog about them, most specifically about their cell walls. However life contains more than just bacteria, and occasionally, strange though it might seem, people write papers about such non-bacterial things, and they end up on my desk with a small post-it attached reminding me that I have a presentation for my supervision group coming up.

So for the sake of my supervision, and to prevent myself becoming too scientifically blinkered, I took a quick foray this weekend into the murky world of protists, the strange and wonderful organisms that occupy the taxonomic equivalent of the 'misc.' draw in a filing cabinet. The creatures that are neither plant, nor animal, nor demonstrably bacteria. Many of them are single celled, some of them photosynthesise, and they all seem to occupy little evolved niches of their own, producing proteins with no noticeable homologues in any other branch of life.

The paper has the rather terrifying title of : "Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium". And I am not ashamed to admit that I had to go double-check the meaning of several of those words.

Dinoflagellates are little organisms that live in water, and mostly look a little like the picture on the right. Many of them are marine organisms, making up a large amount of the photosynthesising biomass in the ocean, and occasionally blooming to form 'red tides', leading to whole sweeps of water turning bright red (possibly occasionally on biblical command). The photosynthetic ones contain chloroplasts, which are wrapped up in three membranes, rather than the usual two. These, like all chloroplasts, contain their own genetic material (known as plastid genes), although unlike plant plastids, they don't seem to contain very many, and those that they do posess are found on little minicircles.

What the paper is interested in is whether there are any other genes in the chloroplast which aren't in minicircle form. There are, afterall, only 12 genes encoded on the minicircles, which is a small amount for a plastid. In order to explore this, it uses a characteristic property of the dinflagellate species it's working with. All organisms, when making proteins, make them from an mRNA copy of the genetic code. This mRNA copy tends to have a long string of adenosine residues added to the end, in order to prevent the mRNA getting degraded. This happens in our dinoflagellate species as well, but it doesn't happen to the plastid genes.

However instead of getting multiple adenosine repeats the plastid genes get multiple uracil repeats. It's just a different base, but it allows the mRNA made in the nucleus, and the mRNA made by the chloroplast to be separated. You can probe for adenosine enriched and adenosine depleted mRNA as shown on the gel below (A and B show different species). The psbA mRNA is clearly strongly present A+ (adenosine enriched) and therefore codes for a nuclear encoded protein. Conversely, the 23S RNA is A- (adenosine depleted) and is coded for in the chloroplast, from a plastid gene.

(Image taken from reference below)

The paper selected 300 random poly-uridine mRNAs (A-) and sequenced them to see if they corresponded to genes found in minicircles, or whether they might be plastid genes held in some different architecture. All the A- mRNA corresponded to the 12 genes discovered in the minicircle. They carried out rarefaction analysis to see if their sample size was large enough, apparently it was, in fact 300 clones was way in excess of the amount needed to find a further, non-minicircled-gene.

This suggests that minicircles are the only architecture for plastid genes and, importantly, that there really are only 12 genes contained in the chloroplast of the dinoflagellate Lingulodinium. This is a very small number of genes, all the rest have somehow migrated to the nucleus, leaving these 12 behind. And it's still very much an open question about why these have been left behind. The paper, in its discussion section puts forward the possibility of size. The genes that have been left behind all code for some of the longer proteins usually found in chloroplasts, although the paper does have the good grace to admit that that's not the most convincing of arguments.

It's worlds away from my little bacteria. But still just as fascinating.
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Wang, Y. (2006). Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium Nucleic Acids Research, 34 (2), 613-619 DOI: 10.1093/nar/gkj438