Social Evolution in Bacteria - SGM series

This is the fourth post in my latest SGM series.

The social behaviour of bacteria is something that I get very excited about. From the wolf-pack hunting strategies of Myxococcus xanthus to the terminal differentiation of cyanobacteria, it's something that I never get tired of writing about. As well as providing interesting quirks of bacterial behaviour, living within a colony also gives new scope for exploring the evolution of bacteria; not just as single entities but as a fully functioning social group.

One of the differences of living within a social colony as opposed to alone means that altruistic-type behaviour has to be adopted. Bacteria living within a biofilm need to excrete the sticky goo that holds the biofilm together, which is problematic because synthesising and secreting goo takes up a lot of energy. So within this colony, there will be 'cheaters' - those bacteria that live in the surrounding goo produced by others, while making none themselves.



A bacterial biofilm, showing individual bacteria in green. Image taken from the FEI website, shown there courtesy of Paul Gunning, Smith & Nephew



As with all colonies, cheating might benefit the individual but has no benefit for the colony as a whole. Too many cheaters and there won't be any biofilm. And recently an even more subtle form of cheating has been shown within the biofilms of the bacteria Pseudomonas aeruginosa, with bacteria that don't just refuse to make vital sticky chemicals, but also abstain from the entire process of forming a biofilm.

Bacteria use a complex communication system called quorum sensing in order to determine how many other bacteria they are surrounded by. Once enough bacteria are present, all signalling their existence, the biofilm will start to form. However some bacteria isolated from the biofilm were shown not to be taking part in any quorum sensing at all. Quorum sensing appears to be quite a burden for a growing cell - cells with the quorum sensing genes knocked out tend to grow a lot faster that the socially conscious cells that allow biofilms to form.

The paper that goes through this (reference one) highlights it as a form of social cheating, with bacteria avoiding quorum sensing to benefit themselves while mooching off the quorum sensing behaviour of others. I'm not entirely certain that this is the case though. It may just be an good example of job allocation within the bacterial society. Clearly not all bacteria are required to be continually quorum sensing, so why should they all have to? Would it not be more sensible to have some exempt from that task, so that they can concentrate on growing, dividing, and spreading the colony? This may be more a case of tax-breaks than of benefit-cheats.

Social evolution doesn't just take place within species, but also between them, and like every other organism bacteria are in a constant state of coevolution with both their 'prey' and their predators. Most predator-prey interactions take long periods of time to study, but the beauty of bacteria is that you can go through several generations in the course of one week's growth. Studies of the bacteria Pseudomonas fluorescens and its bacteriophage parasite showed that both the bacteria and the bacteriophage evolved far quicker when interacting together than they did when competing against a non-changing opponent.

Bacteriophage surrounding a bacteria. Image from wikimedia commons

'Evolve' here means that the bacteria and the bacteriophage showed a greater change in their genetic makeup, and a greater genetic divergence from bacteria not pitted against the phages. Unsurprisingly, the genes that changed the most were those involved in host-phage interaction. This study (reference 2) is also a great example of the usefulness of whole genome sequencing. Whole populations of bacteria and phage were allowed to evolve both together and separately and then just sent away for sequencing with the results analysed at the end.

You really can't be an anti-evolutionist while studying bacteria. They just do it so damn quickly and often you can see it happening.

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Sandoz, K., Mitzimberg, S., & Schuster, M. (2007). From the Cover: Social cheating in Pseudomonas aeruginosa quorum sensing Proceedings of the National Academy of Sciences, 104 (40), 15876-15881 DOI: 10.1073/pnas.0705653104

Paterson S, Vogwill T, Buckling A, Benmayor R, Spiers AJ, Thomson NR, Quail M, Smith F, Walker D, Libberton B, Fenton A, Hall N, & Brockhurst MA (2010). Antagonistic coevolution accelerates molecular evolution. Nature, 464 (7286), 275-8 PMID: 20182425

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Nanofibre paint that kills MRSA

MRSA, the antibiotic resistant form of Staphylococcus aureus is a major problem in hospitals. The antibiotic resistance makes it hard to erradicate, not just from patients, but in the surounding environment, on surfaces, on medical equipment, on the walls of the hospital. In order to minimise the numbers of dangerous bacteria found in hospital surroundings, quite a lot of research has gone into creating antibacterial coverings or coatings that would reduce the number of bacteria p. Currently however, many of these coating substaces work by either using biocides (such as silver) or releasing antibiotics and antimicrobials, which doesn't work on bacteria that have gained resistance.

Scanning electron microscope picture of clusters of Staph aureus

However medical scientists aren't the only ones trying to kill bacteria, virus's known as bacteriophages are also interested in breaking open bacterial cells and they do it using a cocktail of different enzymes to break open the cell wall. Many of these enzymes feature a two-domain structure with bacteria-specific cell wall targeting and catalytic domains. The enzymes Lst was found to be particularly good at breaking apart Staph aureus cell walls with one end of the enzyme (C terminus) recognising and binding to the bacterial cell wall while the other end (the N terminus) breaks the protein bridges between the sugar componants of the peptidoglycan layer, which is a major componant of the cell wall.

Schematic of the cell wall

Work from the Rensselaer’s Center for Biotechnology has been looking at incorporating these enzymes into nanofibres to create stable bactericidal paint films. The molecular-level curvature of carbon nanotubes stabilizes a wide range of enzymes and the lab was able to successfully create Lst-containing nanocomposite films which achieved >99.9% killing of MRSA upon contact within 2 h. They also explored incorporating these into a latex paint, which retained the bactericidal properties of the nanofibres. This paint could theoretically be spread over hospital surfaces to reduce the numbers of Staph aureus within the hospital environment. Incorporating this enzyme into the nanofibres (rather than directly mixing with the latex) gives added stability and helps the enzyme stay within the coating for longer. Films which were stored dry at room temperature showed >99% bactericidal activity against S. aureus after 30 days.

It remains to be seen how effective this technique will be outside of a laboratory setting but at the moment it looks like a highly promising step to help reduce the incidence of a dangerous pathogen. The speed and likelihood of resistance also remains to be seen, but it's heartening that bacteriophages have been using these enzymes against the bacteria for far longer than we've been using antibiotics. This is unlikely to be any kind of magical anti-MRSA cure, but it could certainly be very useful in helping to reduce the incidence of the disease.

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Pangule RC, Brooks SJ, Dinu CZ, Bale SS, Salmon SL, Zhu G, Metzger DW, Kane RS, & Dordick JS (2010). Antistaphylococcal nanocomposite films based on enzyme-nanotube conjugates. ACS nano, 4 (7), 3993-4000 PMID: 20604574
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Jumping DNA

Here's a bit of random information while I wait for my agar to melt...

Bacteria have a remarkable genome plasticity. They are able to mop up spare DNA in the environment, take pieces from circling bacteriophages (viruses that infect bacteria for the uninitiated) and exchange parts of their genome with bacteria from another species. The species boundary can be very wide as well, the bacterial equivalent of a mouse nicking bits of DNA from an elephant and incorporating it into it's genome.

In order to jump from one genome to the other (bacteriophage and other viruses) the DNA must be flanked by so called 'transposable elements' usually shortened to 'transposons' because molecular biologists are lazy when it comes to saying unnecessary words. (also, I suspect because 'transposons' sounds more scifi and scientists have a distressing tendency to be geeky like that). These transposons code for enzymes that cut the DNA out and paste it elsewhere, essentially allowing it to jump around between various genomes, being expressed and replicated in different bacteria.

ooop, there goes the sodding microwave. We have a new one after the old one stopped working (about three weeks after I entered the lab. PURE COINCIDENCE) and this one hits a pitch which is just slightly higher than the comfort level.

Incidentally, does anyone know how to do those fancy 'cut' things in blogs? Like when there's a blue underlined 'read more' label which whisks you away to the rest of the blog post. I'd really like to do that but I don't know how...

And why have you used 84 plates?

The thing about scientific protocols is that they are meant to be exact and precise. Every step must be explained concisely and there should be a reason for all the methodology.

For example, for every protocol you use, you should be able to answer random questions about why you did what you did at each step. Why was the bacteria incubated for four hours? Why was the temperature kept at 50 degrees? Why was the product stored on ice?

The answers to these questions should be sensible and scientific. Temperatures, amounts and incubation's are used to optimise reactions. Every step should be planned to get the best possible result in the most efficient way.

At the moment, we're growing phages on agar plates. At one stage of the protocol, we use exactly 84 plates to grow them. Two of the plates are controls and four are for dilutions but the fact remains that every time we use exactly 84 plates, no matter what we're doing.

The answer (as can probably be guessed) is not scientific in the least. Science is not a cold and clinical organised space, no matter how much scientists want it to be. It is a wild and crazy world full of human error and things going random and even more human error. The problems are not just scientific, they are also spacial and temporal; incubators are only so big, parts of the lab are only open during certain hours.

And the big jars we use for incubating will only physically fit 42 plates. We've tried to stuff more in but the lids won't shut. And while we have three of them in total the incubator is quite small and only fits two at a time.

42 x 2 = 84

Which wouldn't seem so bad if it weren't for the dilutions and the controls, because once you've put them in there's only room for 39 plates of actual phage sample per jar. 42 is at least a nice round number, but a protocol always looks a bit odd if it starts with the phrase '39 plates were taken...' It begs the question, 'why 39?' and the answer is, scientifically, faintly embarrassing.

Science would be a lot more precise if the world stopped getting in the way.

But far less fun =D

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 :)

What to do with DNA

Now we've actually managed to extract our DNA (like this) we get to cut it all up into tiny pieces. It might seem a bit pointless, but this is how to determine that we actually have the right bits of DNA in our extracts. Cutting DNA with little enzymes called restriction enzymes produces different restriction patters depending on what DNA you have.

Restriction enzymes are naturally produced by most bacteria, and what they do is cut pieces of DNA at very specific points. EcoRI, for example, cuts DNA after the G in the DNA sequence GAATTC. As each viral genome has a different DNA sequence, each one will produce a different restriction map, producing a characteristic number of bands on a gel:

(this picture is not from my research, it is from here)

Each band is a blob of a certain size of DNA lit up with ethidium bromide (which is a dye, nothing very exotic). Different restriction enzymes, and different genomes, will produce different band patterns on the gel.

So, what do the bacteria need to produce DNA cutting enzymes for? The answer (naturally) is bacteriophages! One way the bacteria can protect themselves against viral invasion is to have lots of these enzymes around. As soon as the viruses inject their DNA into the bacteria cell, the restriction enzymes chop it all up.

But bacteria also contain DNA, and unlike people (and other eukaryotes), they don't keep it all tucked up in a nuclear membrane. So how do they stop the restriction enzymes from cutting up their DNA? One of the most common ways is to methylate the DNA, essentially sticking a methyl group (a carbon atom attached to three hydrogen atoms) onto some of the bases. in the example shown above, therefore, the restriction enzyme is looking for the sequence GAATTC. It sees this in invading DNA and slices it up, but in the bacterias own DNA it sees GA(methylated)A(methylated)TTC, which it doesn't recognise. And therefore, does not cut.

Restriction enzymes were first discovered my Daniel Nathans, Werner Arber, and Hamilton Smith. They won the Nobel Prize for it in 1978. (see here)

On the specificity of phages

One of the main objections that I have heard to the use of bacteriophages as therapeutic agents is that phages tend to be very very specific. Each phage usually only attacks one type of bacteria, e.g a bacteriophage for a certain type of e. coli will usually only attack that specific e. coli type and no other bacteria. As there are a large number of different varieties of e. coli (and indeed many other pathogenic bacteria), this specificity could be a problem for targeting infections.

(as an aside, phages are often so specific that they are used to 'type' bacteria. If you find a bacteria in, say, sewage, one way to find out what it is is to attack it with different phages and see which one digests it. This can give a very specific pinpoint of what bacteria you have).

The usual response to the problem of phage specificity is to suggest that a cocktail of phages can be used, one for each potential type of attacking bacteria. In some ways, this can be even more useful for controlling infection; a broad-spectrum antibiotic will knock out any bacteria it comes across while a cocktail of phages will target specifically the unwanted ones. Going back to the example of e.coli: there are lots of e. coli living happily in your gut. If a pathogenic strain gets in, the phage therapy that you are given could be designed to target just the pathogens, rather than the bacteria that are already there (and are necessary for correct digestion).

I was quite surprised therefore to come across this paper while randomly searching PubMed (as you do). Maddeningly, there seems to be no way to get the the actual paper, but what it says is that a phage has been found (and named KVP40 for those interested) which has quite a wide host range. Not only does it attack a variety of Vibrio phages (both pathogenic and non-pathogenic) it also is able to attack a Photobacterium as well (Photobacterium leiognathi). A further paper states in the introduction that the receptor that the Vibrio uses to bind to the surface of its bacterial hosts is the OmpK outer membrane protein. I am not entirely certain what this protein does, but I have found that it is involved in vibrio bacterium immunoprotection and is also present in the photobacterium species. As it elicits a large immune response, it is also being considered as a vaccination, if not in people then at least in the yellow croaker (which is a fish).

And this is where phage therapy could come in useful. I don't know how dangerous the vibrio phage is in yellow croakers (this paper seems to make quite a thing of it, but that may be linked to funding purposes). With enough commercial interest (if it hasn't already happened), it's only a matter of time before someone starts looking for an antibiotic for Vibrio harveyi, the vibio species that attacks the poor fish. Essentially this means that a large amount of time and effort will be spent looking for something that will attack the vibrio species, find it on the basis of the OmpK protein, and then destroy it.

Completely ignoring, of course, the fact that such a thing already exists, in the form of the KVP40 vibriophage. There are millions of them floating around in the sea. They've been isolated as well, in pure phage form.

More people need to be working on this stuff...

Contamination!

I came in this morning, all bright and ready for phage propagation (harvesting phages from plates is actually quite fun) and what did we find? All the plates from yesterday were bad. To make a phage plaque you plate out a smooth 'lawn' of bacteria onto an agar plate and then pour phage over it. Where one phage lands it kills and multiplies until it forms a little clear spot or 'plaque' on the plate. (you can't see the phage plaques very well in the picture, but you can see the bacterial lawn)

This morning, all our plates had little fuzzy marks on them. Which is fine, could be phages except...so did the control plate. The control plate had no phage on it at all, it was meant to have just the bacteria but itstead it too had little fuzzy marks.

*sigh*

Without good controls you can't trust your results. At first we thought it was just badly grown host until my supervisor noticed that the tryptone we'd been working with (tryptone broth is one medium that you can grow the bacteria in) was cloudy. Not good. If a substance that is usually clear starts looking cloudy then it means it's got bacteria in it.

Our plates were all contaminated. The fuzzy marks were phage, bad host and/or another bacterial contaminant. Basically we have to do all of yesterdays work all over again today and we need a lot more media (agar for plates, tryptone for bacteria, etc).

Guess who gets to make up all the new media?

I do love working in labs. But it can sometimes be the more frustrating thing on earth. And media pouring is just mindnumbingly boring and faintly awkward. Which is why, of course, the lab rat gets to do it.

Phages

So...as this is my first post, I might as well start with what I'm working on. Bacteriophages.

Bacteriophages (usually shortened to phages) are viruses that attack bacteria. They tend to look like little lunar landing modules; essentially they 'land' on the bacteria, inject in their DNA and replicate it using the bacterial DNA replication proteins. They propagate inside the bacterial cell and then, when finished, burst out of it. This kills the bacteria and leads eventually to nice little plaques forming on an agar plate.

The most interesting thing about phages is that they attack bacteria exclusively. This is because bacteria are prokaryotic, and have a very different internal structure too eukaryote cells (like yours, or mine, or that of a sheep etc). Prokaryotes have no nucleus, and no proper chromosomes, usually just circular loops of DNA. They also use different types of replication proteins and have very different cell surface proteins (although some bacteria can mimic host cell proteins, mostly for infection purposes).

This means that the best thing about phages is that they can potentially be used to treat bacterial infections. Once the phages reach the site of infection they can multiply, kill off the bacteria, and then quietly depart (I am still not exactly sure what actually happens to phages once they've killed all the bacteria, but what I do know is that they are not dangerous in any way). This is a wonderful idea, saves the use of antibiotics, but also carries a slight social stigma with it, after all technically it involves giving people viruses. Even though these viruses don't actually do people any harm at all.

Then I read this:
phagetherapy

For those unwilling to trawl through the link, the basic information is that not only is phage theory a good idea in practise, it is also an idea that works. While Western Europe has been fiddling around with antibiotics and coping with MRSA and other resistant bugs, there are labs in Eastern Europe that are already researching and testing phage therapy. They have very small labs and the conditions and funding are both fairly appaling but the work is being done. This, I think, was my favourite sentance:

"Each soldier in the Georgian army carried a spray-on phage cocktail which they used to disinfect their wounds "

The stuff is actually out there, it works and it's relatively safe (more on that in a bit), why isn't it being researched and tried more often?

Safety:
There are very few side affects associated with phage therapy. As far as I know there's really only one although it is potentially fairly hazerdous. Phages work by killing the bacteria (as mentioned before) which unfortunately leaves a large amount of bacterial debris. In some cases, this is picked up on by the immune system which assumes it's under massive bacterial invasion and over-reacts. This produces what's known as a 'cytokine storm' where lots of immune chemicals (e.g cytokines) are released causing a result similar to anaphalaxis (which is what happens with asthma and allergic reactions).

This is not, however, a novel side effect. It already happens with some antibiotics. And phage therapy has so many more benefits. Bacterial resistance happens less often, and as the phages mainly use the virulence factor surface proteins (the proteins which produce the things that make you feel ill) of bacteria as markers for landing and invasion, resistant strains will be less dangerous. Phages can also evolve with the bacteria, changing to adapt to resistant mutants.

That's why I decided to work on phage therapy.