Bacteria using bacteria

Editor's Selection IconThere are lots of things I enjoy about studying bacteria. I love their biochemistry and the secret inner workings of their metabolic pathways. I love that everything they do the manage within the confines of a single cell, and I love that you can go in there with a wrench and hit some genes until they make what you want.

But what I'm really enjoying exploring at the moment is more ecological bacteriology; how bacteria interact with their environment. How they respond to changes to stresses and, most importantly, to other bacteria. In my last post I covered how natural throat bacteria can help destroy dangerous pathogens such as MRSA Staph aureus so today I'm going to look at almost the opposite; how some bacteria can give each other a helping hand in order to infect humans.

Campylobacter jejuni is a bacteria that I feel a special affinity for because I've worked with it, back in my first ever summer project. Unfortunately it's not a very nice bacteria and can lead to bad stomach illnesses with some rare but quite threatening complications. It's found in chicken meat and cheese as it is perfectly capible of surviving happily in animals without causing them any diseases.

One of the problems with working with Campylobacter jejuni (henseforth shortened to Campy which is what we called it in the lab) is that it's very fussy about the amount of oxygen it's in. Campy is microaerophilic, which means it needs oxygen, but only small amounts, give it too much and all the cels die on you. This problem was solved in the lab by using tightly sealed containers and special packs of ... stuff ... which were put inside the containers to create the right conditions. But this does raise an important question; if the bacteria is so difficult to culture on a plate in the lab then what the hell is it doing surviving on the surface of chicken meat!

A recent study (reference below) found what you've probably guessed if you were reading this post closely, the Campy were being aided by the surrounding bacteria. The picture below shows both Campy and a bacteria called Pseudomonas putida in close interaction, with long fibrelike structures connecting them. Noone seems to be really sure what the fibre-like structures are, they may be being used for chemical communication, or they may just be keeping the bacteria in close physical contact.

The campy is the more slender and slighly spiral shaped bacteria in the centre, the others are Putida. Image from the reference.

Both bacteria were identified as being in close contact, as well as being seen together under the microscope. Further experiments were done to show that the Putida was required for Campy survival - different Campy strains were grown in both the presence and absence of the supporting Putida to see how long they could survive in completely aerobic conditions. The results are kind of hilarious, without the help of the Putida bacteria the Campylobacter just die, really quickly (image from the reference):

Figure A (top) shows the Campy with Putida grown as well, Figure (B) shows the Campy grown alone. You don't really have to be particularly good at science to interpret that one! Interestingly it was found that the interaction between different strains of both Campy and Putida was fairly specific as well, as you can see in the graph above, only three of the Campy strains have survived past 50 hours with the help of this particular Putida. Three of the Campy's still die, although they surive longer than with no help at all.

As Putida are areobic, the most likely explanation for how they are helping is that they create a microaerophilic microenvironment within their immediate surroundings. This is the kind of environment that it is thought Campy will naturally migrate to. This might be less of a helping relationship and more of a seriously exploitative one, with the Campylobacter swarming as quickly as possible towards the environment created by the Putida and then wrapping them all up in a sticky mesh to stop them moving away.

This special relationship is not applicable for all Campys, in other environments such as in humans and in chicken poo the Campy exist fine on their own, but in the highly aerobic environment of the meat surface they rely on other bacteria to survive. The implications for treatment of bacteria are intreguing (especially for antibiotic resistant strains of Campy) but it is another reminder that despite laboratory conditions bacteria do not just exist in isolation. They inhabit a whole tiny world, with challenges of it's own, surrounded by other bacteria that change their envirnment both for better and for worse.

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Hilbert F, Scherwitzel M, Paulsen P, & Szostak MP (2010). Survival of Campylobacter jejuni under conditions of atmospheric oxygen tension with the support of Pseudomonas spp. Applied and environmental microbiology, 76 (17), 5911-7 PMID: 20639377
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Throat bacteria that destroy invaders

I did a post about a week ago, talking about the relationship between the bodies natural (commensal) bacteria and the immune system. I was quite excited therefore to find a paper (reference below) which found a specific protease enzyme that is used by commensal throat bacteria to prevent harmful biofilm formation by Staphylococcus aureus, the bacteria responsible for MRSA.

The helpful bacteria in question is Staphylococcus epidermidis which lives naturally in the throat and nasal cavity of humans. When culturing these bacteria along with the Staph aureus it was found that some epidermidis cultures were capable of destroying biofilm formation, by using the protease Esp. The diagram below shows the effects of extracted Esp on colonies of Staphylococcus aureus (image from the reference):

Figures g and j show Gram stains of the colonies, the blue dye has just stained where bacteria are present. The remaining figures show scanning electron micrographs of the colonies taken at two different levels of magnification. For those interested, the scale bar for g,h,j and k is 10um and for i and l is 1um.

To double check that this protein was having an effect within the bacteria knockout mutants were made which removed the gene from Esp from the epidermidis. These bacteria were incapable of destroying Staph aureus growth. Adding a plasmid containing the Esp gene back into the bacteria restored their ability to fight off the Staph aureus which seems fairly conclusive. Furthermore this affect also works with VRSA and MRSA; Staph aureus which are resistant to antibiotics.

Below is a diagram of the effect of actual epidermidis bacteria on Staph aureus colonies (image from the reference).
These are nasal swabs taken from volunteers who had Staph aureus infections and were given the commensal epidermidis strains to try and clear them. It can be seen that the number of staph aureus is decreasing, although some bacteria are still present after five days of treatment. That might not necessarily be a bad thing as it allows the immune system to kick in with a response, and make antibodies ready for the next potential attack.

There are several exciting things that come out of this. Firstly the use of purified Esp as a defence against MRSA biofilms has the potential to be of major importance, although there may be clinical reasons why it's not such a good idea to spray proteases all over the inside of someones nose! From a less medically-useful perspective it's a wonderful example of bacterial-colony interaction. The kind of struggle for survival that happens inside your nose is occurring for bacteria everywhere; in soil, in the water, in the air, and even in humans.

From the Staph epidermidis point of view your nasal cavity is just a great place to live (warm, safe, lots of nutrients) and it's not going to give up that kind of living environment without a fight!

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Iwase, T., Uehara, Y., Shinji, H., Tajima, A., Seo, H., Takada, K., Agata, T., & Mizunoe, Y. (2010). Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization Nature, 465 (7296), 346-349 DOI: 10.1038/nature09074

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The Gibson Assembly Song

There are no words to describe how awesome this is. You just have to watch it:



This is The Gibson Assembly song, written by the Cambridge iGEM team 2010. If you can't see it, want it in a higher resolution or just want the link to share, find it here.

The technique they are describing is the "Gibson Assembly" which is a fairly quick and painless way of joining two bits of DNA. In more sciency terms, it works by using PCR to make genes with large overlaps (40bp) at the end. You add a Master Mix to the fragments, incubate for one hour, then just transform into whatever cell you're using. For more details of how it works go here, for the recipe of the Master Mix and detailed protocol go here, and for a program to help you design Gibson Primers, go here.

The video was just made for fun, and took less than a week to put together (not counting the time to write the words!). Filming was done mostly over one day, using one camera and a slightly broken tripod, just using spaces in the lab and the gel room. The green-screen sections were done by throwing a green table cloth over some poster display boards. The music was recorded seperately, and I think each instrument was recorded seperately as well, to get the sound balance right. It was all carried out by about nine undergraduates (and one lab rat!) and massively confused most of the supervisors.

I'm already starting to see some replies and responses to this, the most popular ones seem to be variations on the "why aren't you doing any real work!?!" and "How do you have the time with all your Science!" And given that there's been a lot of kurfluffle in the blogosphere lately about science, time spent doing science and the connection of both with Passion for science I suppose I should address this...

The undergrads who made this video had been working all summer, pretty much starting two weeks after term finished. They worked weekends. They worked evenings. At the time of doing the video, they'd worked several weeks over their stipend and had long ago stopped being paid.

Now their term has started, and they are still coming in to work, in between lectures, in between their own projects. They also have to prepare a presentation and a poster over the next month, all in between their term work. And terms here are manic; eight weeks of non-stop craziness that it's hard to fit anything into.

It was just a quick few days of Fun, in between the Science. :D

Friend or foe? How the immune system copes with the gut microbiotica

The job of the human immune system is to destroy pathogens. Using a combination of quick, immediate responses (the innate immune system) and long-term memory (the adaptive immune system) in humans the cells of the immune system are perfectly primed to seek out any cells that are Other (i.e not Self) and kill them.

Which leads to a slight problem, because rather a lot of the cells within your body are 'Other' cells, and their existence is vital to your health. Within your stomach, and your respiratory tract, live a number of commensal bacteria, friendly and harmless bugs that can survive quite happily inside you and help to fight against incoming pathogenic bacteria. Stripping away the bacteria in the gut (i.e by going on a course of very strong antibiotics) leads to all kinds of problems including digestive problems and, once the antibiotics have finished, increased risk of disease-causing bacteria invading the now bacteria-free stomach.

In fact several notable yoghurt making companies are making a lot of money by selling you drinks with bacteria in them. They reassure you that the bacteria aren't dangerous, which is all well and good, but they never quite explain why the ingestion of many bacteria doesn't cause your immune system to have a panic attack.

A new review in Nature looks at the interactions between the gut microbiome and the immune system. The 'gut microbiome' is the collection of bacteria that start colonising the inside of your intestines soon after birth, both from your mother, and from the general environment. It's helpful here to remember that technically your intestinal tract isn't actually inside your body. There's an open tube right the way from your mouth to your arse (for want of a better word...) so the body has a tendency to treat bacteria living there in similar ways to the bacteria living on your skin, by using barriers to keep them out.

However there still is a trade off. The cells that make up the intestinal walls still need to be able to respond to bacteria, and the commensal bacteria still need to be contained. A non-regulated population of bacteria will simply keep growing until all available space is filled (and all nutrients eaten), and this does not happen within the gut.

Starting with the innate immune system which works by recognising molecules found in all pathogens (called PAMPs) these are recognised by human cells using receptors called TLRs (Toll-like receptors - long story) and lead to a signalling cascade that result in a huge number of cytokines and other inflammatory agents being released to kill the bacteria. In the gut this wouldn't just lead to the massive slaughter of the microbiome, but also to a huge amount of damage to the surrounding human cells. Enough exposure to microbial elements such as lipopolysacharrides can downregulate this response; the lipopolysacharrides (which are in the bacteria cell wall) down-regulate one of the key components of the signalling system, a molecule called IRAK1. This prevents the cell from mounting a response to the bacteria. For those that want scientific details, check out the diagram below (image from the reference):


The adaptive immune system is more complex. In normal situations it works by taking a small sample of the bacteria back to the lymph nodes and preparing a specific immune response against it. Special immune cells (B cells) are then made which will kill the specific bacteria, with the help of T cells, which also act as a memory of the threat and the correct response. The B cells are then sent to the point of infection and secrete antibodies which clump the bacteria into groups and recruit other factors to kill them.

This still mostly happens in the case of the gut microbiome, the B cells release the antibody IgA which diffuses out into the intestinal tract and traps the bacteria in the mucus layer. However the bacteria are able to strike back, not by targeting the B cells, but the T cells. There are many different forms of T cells, and by secreting certain chemicals the bacteria can encourage the formation of T-regulatory cells which encourage tolerance towards both commensal bacteria and molecules in food.

So it seems to be not so much a relationship of mututal tolerence and understanding, but more like a sort of uneasy standoff. Bacteria are still being killed to stop them spreading, but are holding off the immune systeme enough to maintain a steady population. In return, the immune system is still there and active, but not active enough to cause any serious damage to either the microbiotica or the surrounding human cells.

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Cerf-Bensussan N, & Gaboriau-Routhiau V (2010). The immune system and the gut microbiota: friends or foes? Nature reviews. Immunology, 10 (10), 735-44 PMID: 20865020

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Bacterial Fossils

Studying ancient bacteria can be a little difficult. Despite having wonderfully complex internal biochemistry and fascinating ecological interactions they are essentially, when you get down to it, a little soggy bag. Little soggy bags do not preserve all that well; when bacteria die they just break apart, and are often eaten by other scavenging bacteria which see them as just free nutrients.

However some bacteria do fossilise, leaving behind perminant records of their existance. One of the ones that does it the best is cyanobacteria, which can form fossils in two ways. Firstly by forming little calcified shells around themselves as a product of increasing the dissolved carbon dioxide levels inside the cell:

Cyanobacteria fossils - from Berkeley university page

Secondly cyanobacteria can group together with algae to form large layered mat-like structures called stromatolites. Slicing these very thinly reveals tiny cyanobacteria fossils, caught between the layers. There are stromatolytes in Australia that have been alive and growing (very slowly) for millions of years, despite looking faintly uninspiring.

Maybe not quite as impressive as dinosaurs...

While no other bacteria form fossils as nicely preserved as the cyanobacteria, they are capable of leaving behind visible remnants of their existence. The polypeptides of the bacterial cell wall (along with cytoplasm and some secreted lipids) can, under certain conditions, act as nucleation sites for minerals. This eventually leads to the organic cell being replaced by a little mineral cast of the bacteria. There is the possibility for quite a few artifacts with this (artifact being the scientific word for "result caused by the preservation process rather than the bacteria") the most common one being the creation of an artificial nucleus structure. As the bacteria degrades the cytoplasm tends to clump, and the crystallisation of minerals around a cytoplasm clot can in some cases create a structure that looks similar to the structure formed by a nucleus.

Endolithic bacteria that live in rocks can leave behind tiny canals in the rock surface that they bore into. These are a lot harder to find and interpretation is usually helped by the discovery of nearby alive endolithic bacteria. Bacteria have also been found trapped and mumified inside tree resin, a la the Jurassic park mosquito.

Once you start getting larger organisms the bacteria have a brand new niche to exploit. Fossilised bones sometimes show the results of a bacterial infection; while the bacteria themselves are not being preserved their presence is still seen in the fossil record.

Unfortunately although these glimpses are really interesting their also kind of frustrating from a biochemical point of view. They provide clues as to the lifestyles and processes within the bacteria but they are such tiny clues. Past biochemical processes are more often found inside the actual bacteria, by looking at clues in the genome and the genomes of related bacteria, than they are in the remains the bacteria leave behind.

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Perri, E., & Tucker, M. (2007). Bacterial fossils and microbial dolomite in Triassic stromatolites Geology, 35 (3) DOI: 10.1130/G23354A.1

Westall, F. (2001). Early Archean fossil bacteria and biofilms in hydrothermally-influenced sediments from the Barberton greenstone belt, South Africa Precambrian Research, 106 (1-2), 93-116 DOI: 10.1016/S0301-9268(00)00127-3

Guest Post - Survival of the fittest?

I'm very excited about this post, which is a guest post from my sister! She's an undergrad doing biochemistry at Bristol University, and she's currently taking a year working in a research laboratory as part of her degree. She's working with Plasmodium at the moment (which is the little protist that causes malaria) but has sent me a bacteria-related post because she knows me, and she knows my blog and who doesn't love bacteria?

Post - survival of the fittest?

Bacteria have always been very adaptable when it comes to surviving evolutionary stressful situations, such as exposure to antibiotics. Usually some form of mutation will arise leading to the creation of resistant strains of bacteria. These will be selected for via ‘natural selection’ processes and go on to replicate to produce a whole population of resistant bacteria that are able to survive.

However new research looked at a colony of wild type E.coli bacteria in a bioreactor under exposure to increasing levels of the antibiotic norfloxacin and found that no more than 60% of growth was inhibited to maintain a sizable population. The resistance levels of the population as a whole, and of 12 random individuals, was checked every day and it was found that they did not correspond to one another.

The majority of the individual isolates were less resistant than the population as a whole but there was one mutant found that was highly resistant. By isolating the supernatant from the high resistance individual, and conducting gel electrophoresis to separate out the intracellular components, a protein was found that was produced in very high numbers.

This was the enzyme tryptophanase which has the main job of breaking down tryptophan to ammonia, pyruvate and indole. Experiments were done to show that the third molecule, Indole, provided an obvious survival benefit under antibiotic conditions. It upregulated multi-drug efflux pumps which helped in the physical export of the drug and it also had a role in activating various oxidative stress protective mechanisms. The mass production of Indole by the highly resistant mutant allows more vunerable cells in the surrounding area to survive.

a) no antibiotic stress, bacteria naturally produce indole.
b) dead and dying bacteria fail to produce indole
c) mutant appears and supplies indole at a fitness cost to itself.

The resistant bacteria is therefore not selfishly replicating to outgrow the rest of the population but, in helping others to survive, is enduring a fitness cost of its own by mass producing Indole.

This experiment was also carried out using various different antibiotics and the same bacterial altruism was found to exist. The survival of the weaker bacteria does have some advantages as it allows further exploration of mutations that could be even more beneficial to the population. Also, it keeps the opportunity for the bacteria to return to their original state if the stress is temporary, rather than keeping up the energetically wasteful production of antibiotic resistance genes.

So, bacteria working as a team to ensure not just temporary survival but long term advantages for the whole population. Not just survival of the fittest.

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Lee HH, Molla MN, Cantor CR, & Collins JJ (2010). Bacterial charity work leads to population-wide resistance. Nature, 467 (7311), 82-5 PMID: 20811456

I for one welcome our glowing overlords...

I've already shown you pictures of the glowing bacterial lightbulbs, but in terms of glowing products the cambridge iGEM team is going from strength to strength. To start with the most amazing, they've made an oxygen activated bacterial bubble-lamp:



(It can be found here if the above does not work)

Maybe it's not the most efficient thing to read by, but it's an impressive level of brightness and it turns off relatively (biologically speaking!) fast. It's simply a large measuring cylinder with a load of bacterial broth inside it, and a tube to blow air through. Even shaking lets enough oxygen through to start turning on the light.

Another thing they've been doing is playing around with images on 24-well plates. For those who haven't used them, 24-well plates are usually run on a plate-reader which reads samples from every well, used mostly (in our lab) for overnight assays or (as I should be doing tomorrow) as a glorified spectrometer, measuring a range of absorbances over different wavelengths.

The iGEM team are using them to make nerdy pictures. This is my favourite:

The thing I like about the 24-well-plate pictures is that their different. Painting on plates is awesome buts it's been done before a couple of times, and the glowing pixel-pictures just look new and fresh and exciting.

There's probably going to be a bit of speculation as regards this of the "but how useful is it" type. And rest assured the iGEM team are thinking of that but at the moment I'm happy to just enjoy the fact that we have glowing pixelated space invaders sitting on the bench.

Glowing pixelated space invaders!

This is why I went into science :p

Bacteria that tear themselves apart

Bacterial cell division is one of those fairly well studied areas, where time and much study has come forth with a nice standard model. One of the main proteins involved is FtsZ, which seperates one bacteria into two by forming a ring of protein around the middle of the bacteria and tightening it shut as shown below (figure from Nature paper):

Until quite recently it was thought that this was pretty much the only way to get bacteria to divide, until 1999, when the sequences of two Chlamydia species turned out not to contain genes for FtsZ. As Chlamydia are intracellular parasites (which I covered in more detail here) it was at first thought that they might be using some host proteins to complete the cell division, but after the discovery of an FtsZ-less free living archaea, and several more bacteria, it became apparent that the FtsZ-centric model of cell division (shown diagrammatically above) wasn't covering the behaviour of all bacteria.

In the archaeal species (in fact the entire archaeal kingdom Crenarchaea) the cell division was found to be based on a completely different cytoskeletal system. By screening for genes that were turned on at the onset of cell division, a three-gene operon was found to be involved. These genes coded for homologues of eukaryote vesicle trafficking proteins and their regulators, and it was suggested that they formed curved filaments which could pinch off sections of the membrane, forming new archaea. Although the method is similar, the 3D structure of the archaeal proteins is very different to that of FtsZ; the two proteins are not related, but have been coerced into doing the same job.

As well as finding bacteria without FtsZ, it was also discovered that taking a strain of Mycoplasma genitalium and removing the FtsZ didn't stop cell division and in fact showed the same growth kinetics as the wild type. The division mechanism in this case relied on the fact that Mycoplasma move by adhering to a surface and pulling their way along it (in a lab this will be on a glass or plastic surface). To pull their way forward they use a 'terminal organelle', a little protrusion that attaches to the surface and pulls the cell along (figure from the reference).

The diagram above shows Mycoplasma without FtsZ undergoing cell division. You can clearly see not one, but two little terminal organelles at either end of the long stretched cell. What's happening is that in the absence of proper organised proteins to sort out cell division the bacteria has taken matters into its own hands, and sent two terminal organelles determinedly heading off in opposite directions. The bacteria is literally tearing itself apart, splitting into two by ripping in half and letting the membrane close up behind.

It has been suggested that this might be an older method of cell division, used before FtsZ entered the Mycoplasma. It's certainly a lot more brutal than FtsZ-mediated division, and the bacteria has to spend a lot more time in stationary phase recovering from it. As this method only works in bacteria that can attach and hang onto surfaces, it is unlikely to be use by the Chlamydia species (the mechanism for their cell division is still unknown, although some work has been done with L-form bacteria). In the archaeal species it may even be the other way around, that the new filamentous system evolved to be even more efficient that FtsZ in certain species, and so the FtsZ has been dropped entirely.

All of this builds up a picture of just how diverse even simple systems like cell division can be within the bacterial kingdom. And, in my mind at least, is a compelling argument for not just working with model organisms...

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Erickson, H., & Osawa, M. (2010). MicroCommentary: Cell division without FtsZ - a variety of redundant mechanisms Molecular Microbiology DOI: 10.1111/j.1365-2958.2010.07321.x

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...now in red

Got back from the conference last night, absolutely shattered. I had a great time, and the presentation was really well recieved, everyone seemed to love our coloured bacteria and it was my first time doing a conference presentation to Grad students. I met some new friends, and got the chance to visit Venice on the way back.

While I was away, red happened!

I'm still holding out for the glowing green myself...

To find out more visit the Cambridge iGEM team wiki.

I have a lot of stuff to catch up on, but if anything particularly amazing has happened on the internet while I've been away (other than Bora moving house - which I know about already and wish him the best of luck) drop me a note or stick it in the comments.

Bacterial Lightbulbs

I'm off at a conference in Italy this week, where I'll be doing a presentation about the iGEM work from last year along with some of the follow-up work I've been doing this summer. I'm really looking forward to it, but it does mean that I've got things to concentrate on other than blogging at the moment. I might get the last SGM post out on the weekend but I really need to try and spend that time with my fiancé rather than my science, as I feel I've been neglecting him a bit over the last week to try and get this presentation written.

So in the mean time, have a bacterial lightbulb:

This was made by this years Cambridge iGEM team (their website can be found here) by growing bacteria on agar, mashing up the agar in a little pot and growing again overnight with liquid medium poured over. Some of the resulting bacterial/agar glowing mush was then put into this lightbulb pendant (brought off ebay) to produce a little glowing bacterial lightbulb. Even better the little clumps of agar look a bit like sort of glowing crystals, which gives it a wonderfully 'science'-y effect.

The lightbulb used DNA from Vibrio fischeri, a bacteria that lives symbiotically with squid. The team acquired plasmids containing this gene controlled by the lux operon, and simply cut away the control, meaning the gene is now constitutively expressed (in the presence of oxygen) and glows away happily without any kind of exterior control. Eventually the lightbulb above will start to fade as the bacteria die and require more nutrients, but it's been sitting in the lab for a day now and is still fairly bright.

They've gotten some wonderfully spooky pictures. I don't know if it's just my imagination, or if I'm expecting this of biological systems, but it all looks faintly green to me. Green glowing things are probably the ultimate achievement in science. As well as their current white glow, they are also starting to get some other colours too, ultimately I believe their hoping for green, blue, orange red and yellow.

I'm really impressed with all their work, considering it's the efforts of nine students over a few months. They've got another month to go before they present it at MIT in Boston, which will require frantic powerpoint slides, and (I've been lead to believe) some potential animation work.

See more photos here, visit their wiki here, and follow them on Twitter here.