Hiatus

I was hoping to avoid this by maybe writing some future-posts to appear during the holidays, but time just ran out on me. So I'll be going on holiday-induced hiatus for the next two weeks, while I enjoy some time with my family and turn off the science brain for a bit (and work my way through the most AMAZING CHRISTMAS PRESENT EVER aka the complete works of Shakespeare =D )

I've got plans for when I get back though! Starting from January I'm going to try and keep to the whole twice-a-week-blogging I managed last term, which will include, among other things:

• Book reviews. I've been catching up on all the science books that I never quite got around to reading, and I'm planing on writing proper reviews for them. Expect some Stephen Gould, and Schrodinger's 'What is life' among others. (I might even attempt the Stephen Hawking).
  
• The molecular clock. I've been wanting to write about this for a while. Hopefully I'll manage to get my thoughts and references together manage something half-way decent.
• Plants. The course I've chosen for next term is primarily about plant and chloroplast evolution and diversity. As I'll have to read around this subject I'll probably be dissecting some of the papers in my blog, so there will probably be quite a heavy plantsci slant to the papers coming up.
• More bacteria. As always, I'll be reading papers concerning my project and my major interests, so those will feature as usual.
• Shakespeare quotes. Oh yes.

I've had a great time blogging this term. Hopefully I can keep it up next year!

Lab Rat would like to wish everyone who is having a holiday a Happy-Holiday!

Plastic from bacteria

I'm on holiday at the moment, so today's post is another section from my long essay last year, about the potential uses of biorefineries. It was written for a more scientific-based audience so might be a little harder to decipher than my usual posts.

Bioplastics

Bioplastics are polyesters that accumulate intracellularly in microorganisms in storage granules. They are usually built up from hydroxyl-acyl CoA derivatives through a range of different pathways in different microorganisms. As they are both biodegradable and biocompatible they have found numerous applications within medical and surgical fields, as well as having a greater environmental advantage over petroleum based plastics. The main disadvantages of bioplastics for commercial use are their high production and recovery costs.

The most widely produced bioplastics are poly(3-hydroxybutyrate) and poly(hydroxyalkanoic acid), referred to as PHB and PHA respectively. These both contain different β-oxidation intermediates as monomers, which are enzymatically polymerised through a condensation reaction. The structure of PHA is shown below (the 'n' indicates that the section show below is repeated multiple times):The first bioplastic to be described was PHB, found in Bacillus megaterium in 1926 by Lemoigne. It is stored in polymer form in granules within the cell.In order to decrease the recovery costs of the PHB granules, several attempts have been made to produce the secreted monomers, for polymerization outside the bacterial system. This has been achieved by expressing recombinant genes in E. coli

There are a large number of PHA polymers, ninety-one of which have been fully characterised. They are produced by both Gram negative and Gram positive bacteria via at least five different metabolic pathways. The main enzyme involved in polymer formation is PHA synthase (of the α/β hydrolase family), which polymerizes the monomers by connecting the coenzyme A thioesters of one monomer to the hydroxyl groups at positions 3, 4, 5 or 6 of the acyl moiety of the second monomer. There are four classes of PHA synthase, which are distinguished by their primary structures, substrate specificity and subunit composition. PHA synthases are found on the surface of the PHA storage granules, along with other proteins, and phospholipids.

(The structure of a PHA granule is shown above, image taken from Rehm 2003)

Engineering of recombinant bacteria that are capable of producing bioplastics requires both the transfer of a functional PHA synthase enzyme (there is no evidence as yet to suggest that any post-translational modifications of the enzyme are important for its function), and the engineering of suitable substrates that provide the enzyme with suitable substrates and sufficient concentrations. While the enzyme has been successfully transferred into model organisms such as E. coli, S. cerevisiae and even some transgenic plants, the provision of substrates is a more difficult problem as it involves dealing with large numbers of interlinked metabolic pathways. Metabolic flux analysis, carried out in transgenic E. coli, has substantially increased the carbon flux towards the production of PHB without detriment to the health of the bacteria, however this form of analysis has not yet been carried out on more complex PHA polymers.

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Madison LL, & Huisman GW (1999). Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiology and molecular biology reviews : MMBR, 63 (1), 21-53 PMID: 10066830

Steinbuchel, A., & Valentin, H. (1995). Diversity of bacterial polyhydroxyalkanoic acids FEMS Microbiology Letters, 128 (3), 219-228 DOI: 10.1111/j.1574-6968.1995.tb07528.x

Rehm BH (2003). Polyester synthases: natural catalysts for plastics. The Biochemical journal, 376 (Pt 1), 15-33 PMID: 12954080

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

Lab Rat guide to Fourier Transformations

Protein crystallography is one of those mysterious things that I always feel I should know more about. It starts with protein crystals, fair enough, and then heads rapidly out beyond the Magical Event Horizon leaving me with a picture of set of small fuzzy dots which mysteriously resolve themselves into an electron density map. Somewhere in all this someone will mention the dreaded Fourier transformation, at which point I know all hope is lost and a might as well stop listening.

With the help of a good lecture and an utterly outstanding website though, I'm starting to get the hang of it. X-ray crystallography works by shooting X-rays at a protein crystal. As the crystal is in a regular lattice structure, the X-rays are scattered in a regular way. Each scattered ray can be characterised by the amplitude and the phase, as shown below:Each dot on the resulting image (which usually looks something like the figure shown on the right) corresponds to a scattered X-ray. The position of the dot gives (by way of a set of clever equations) the amplitude of the wavelength. However in order to calculate the position of an atom you need both the amplitude and the phase, and you can't get the phase from just the position of the dot. And here is where the dreaded Fourier transformation comes in, to help give information about the phase.

The Fourier transformation is an equation, which gives reciprocal information about a molecule, for example if the molecule is a single small point, the Fourier transformation (i.e what it looks like after plugging its positional coordinates into the Fourier equation) will be a large fuzzy blob, as shown below (all pictures from now on are taken - with permission - from Kevin Cowtan's Book of Fourier) . Molecule is on the left and its Fourier transformation on the right:
For the more mathematically minded, what the Fourier transformation actually does is take a function and express it as the sum of a set of different sine and cosine waves. Apparently this can be done for any continuous function. By using this transformation you can get not only the amplitude, but also the phase of any given molecule inside an atom. You can also make the equation go backwards as well, turning the red fuzzy blob on the left back into the individual point. As most molecules contain many, many atoms there are various tricks you have to do in order to make this easier for large molecules, but first a quick proof of this concept using my favourite pictures on the Cowtan website.

Here is a picture of a duck:
And here is the Fourier transformation of the duck, with the amplitude represented by the brightness of the colour and the phase represented by the actual colour:
In order to show that the Fourier transformation really does show the phase, this transformation is mixed with the Fourier transformation of a picture of a cat. Rather than mix them both equally, the amplitude (brightness) of the duck transformation is mixed with the phase (actual colour) of the cat to give the following Fourier transformation:Performing the Fourier equation on this blob (to turn it back into an animal again) gives the following result, hopefully not unsurprising if I've managed to explain this alright:
It's a cat! A rather blotchy cat, true, with a fairly trippy background, but nevertheless the Fourier transformation has faithfully reproduced the phases, rather than the amplitudes.

So how does this help when looking at molecules? It turns out that the fuzzy black-and-gray-dots picture that is the end result of X-ray crystallography (shown above and reproduced here on the right) is the Fourier transform of the atomic electron clouds inside the protein crystal. That picture is like the fuzzy coloured blobs that the duck and cat images came out of. In the same way that those turned into animals, this picture can be turned into the approximate shape of the electron clouds surrounding a molecule. For low resolution images this can show the secondary structure of a protein, the positions of alpha helices and beta sheets and a general idea of protein shape. For high resolution images, individual amino-acid residues can be seen, allowing a much more detailed view of the structure to be generated.

It isn't always perfect. Sometimes you do get the equivalent of a blobby cat with a trippy background and have to play around with homologous comparisons and allowed bond-angles to get a meaningful structure. There are plenty of strategies that exist to help you get a better image as well, particuarly for larger molecules which need more help resolving phases. From what I've heard though, once you've actually got the crystal, the rest seems like childs play in comparison. I know people who have spent their whole PhD's, and longer, just trying to isolate and concentrate a single protein crystal...

Mitochondrial Networks

Having taken a brief look at some plastids last week, I thought I should probably balance things out by writing about mitochondria; the energy generating centres of the (eukaryote) cell. Like plastids, mitochondria are thought to originate from endosymbiosed bacteria-like organisms and are often shown as looking something like the picture on the right. As well as creating energy in the form of ATP mitochondria are also involved in the B-oxidation of fatty acids (producing energy from fats), Iron-sulfur cluster generation, oxygen metabolism, cell death control and Calcium ion buffering and signalling.

Mitochondria are surrounded by two membranes, an inner (the Inner Mitochondria Membrane - IMM) and an outer (OMM). The inner membrane is the main barrier to the outside world and contains most of the energy-making apparatus either embedded in it or present in the inter-membrane space. The outer membrane is used to coordinate function with signalling, and plays a part in apoptosis, or organised cell death.

In the cell however, mitochondria are often not found in the neat little packages as shown above, but instead fuse together to form long cell-wide tubular arrangements. Usually, they can be found in both vesicle and tube like states, constantly fusing or breaking up depending on cell circumstances, the type of cell, or the functional state of the mitochondria. The result is shown below; a network of mitochondria within the cell:Two processes are involved in the creation and maintenance of this tubular network. Fusion; the joining of mitochondria, and fission; where they split apart.

Fusion: mitochondrial fusion is controlled by large GTP proteins, which use the hydrolysis of GTP to produce energy to join the two membranes together. OMM localised proteins Mfn1 and 2 carry out the initial tethering and joining of the OMM by forming coiled-coil type interactions of their C-terminals which acts to bring two mitochondria together. Once the outer membranes have fused, OPA1 (a soluble protein found in the inter-membrane space) fuses together to join the inner membranes. The fusion of both the outer and inner membrane is usually highly synchronous, although studies in yeast have shown the two fusions can be decoupled.

Fission: in contrast, fission is controlled not by GTPases, but by a protein related to dynamin called Drp1. Drp1 is usually found in the cytosol, but it can localise to the mitochondria in clumps which lead to active fission sites. The Drp1 forms polymers which wrap spirally around the mitochondrial tubule and lead to it splitting. It is thought to be recruited to specific sites by the mitochondrial-bound protein Fis1, although this still requires further study.

As aberrations in mitochondria dynamics are generally associated with neurodegenerative disorders, the control and organisation of these processes are vital. A variety of cofactors and inhibitors have been found that can regulate the Mfn proteins involved in fusion, and OPA1 is thought to be controlled by alternative proteolysis to create different isoforms. There are a number of different protein kinases that are able to phosphorylate the Drp1 fission protein, which not only allows a large degree of control, but means that this control can be synchronised with different intracellular signaling pathways, giving more integrated cellular control.

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Benard G, & Karbowski M (2009). Mitochondrial fusion and division: Regulation and role in cell viability. Seminars in cell & developmental biology, 20 (3), 365-74 PMID: 19530306