Guarding Microbial diversity - SGM series

This is the first of the Spring 2011 SGM series; where I steal random topics from the Society for General Microbiology Spring Conference and write about them in my blog. It should be noted that I am in no way affiliated to the Society, I'm just currently not rich or scientific enough to go to their conferences. (next year...)

I was quite intrigued when I saw this topic, as microbial diversity has always seemed to me to be a little bit like rats. Interesting to the people that study them, irritating and potentially hazardous to those who don't, but not really in need of any special protection. Bacteria evolve quickly, and share DNA easily, forming many, many diverse species capable of occupying a wide variety of niches.

However while bacteria are indeed very diverse and happy to remain so, the challenge comes in cataloguing all of that diversity. New bacteria need to be examined, named, placed in a taxonomic group, and preferably stored so that if anyone has a particular urge to work on (say) a specific type of hydrogen-eating swamp bacteria, they can find a sample of it and do that work.

What I hadn't realised was that there are quite a few places that are designed to store bacterial cultures. One such place is the DSMZ collection in Germany which boasts over 20,000 cultures of assorted microorganisms. These are stored as dried samples, not alive yet easily able to resuscitate. For a small fee, you can order a sample from them, together with instructions as to how to bring it back to life and culture it within a laboratory. Without storage facilities such as these, it's easy to see how interesting new bacteria would simply get lost, due to freezer melt-downs in labs, or people discarding the wrong samples.

It's not just the finding and the storing of bacteria that are vital in order to maintain scientific knowledge of their diversity, you've also got to name the things. After all, an unlabelled catalogue is no use at all. And with the name comes characterisation - a whole list of the properties and behaviour of the bacteria down to as much biochemical information as is feasible. The speed and accuracy of full-genome sequencing does make this a lot easier, but there are still many properties that depend on more than just the genes. A bacterium might possess the gene for (say) iron metabolism, but that doesn't mean it uses it all the time, or indeed at all. Knowing the genome sequence also makes it a lot easier to quickly place a bacterium into an existing group or species. Although bacteria do share DNA between each other, recently acquired DNA can usually be distinguished from the core genes that mark the species.

UK strains can be acquired through the Health Protection Agency, which is an aggregation of four previously separate culture collections. The bacterial arm of it has around 5000 different bacterial cultures. I did do a quick check as to whether I could get a small sample of S. erythraea using my Debit Card, but you need to be officially registered before they start handing out the bacteria!

It's quite strange to think that these talks are actually taking place up in the north of the country while I'm writing this. Next year though, I'm aiming not just to turn up, but actually bring a poster with me, to show off some of my own work.

New SGM series!

The Society for General Microbiology runs regular conferences that concentrate exclusively on the world of the very small. Although I am currently not a member :( I have been in the past, and plan to be again next year!

The conferences are always very good, and last august I bemoaned not being able to make one. This spring I am also not able to attend as I've only just come back from my honeymoon, and in the general scheme of work-life balance it would be a very sad and hardcore worker who would put an actual wedding second to marriage.

The last SGM conference that I missed, I decided to do a nerdy little one-women conference of my own, here at Lab Rat. And it worked so well that this time I'm going to try and do it again. One post every two days, covering the following topics in no particular order:

Seeing the cell through the “eyes” of the virus

Guarding microbial diversity

Vaccines

Insect symbiosis

Life at Zero Growth Rate

Maths & microbes - heh that'll be fun!

Social evolution in micro-organisms

Seven topics, which should take me two weeks to get through. It'll be a good way to get my self back into blogging and back into the exciting world of bacteria. It should be an interesting fortnight!

...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.

Bacteria vesicles - SGM series

The SGM autumn conference is now over - thanks to everyone who tweeted it so people like me could catch up on events without actually going. I've just got two more topics of my own little personal blog-conference to go, and this one is going to be on bacterial vesicles rather than secondary metabolism because it suddenly struck me that I don't actually know much about outer membrane vesicles, and this might be a good opportunity to explore them.

So this is the penultimate post in my SGM topic series: Bacterial vesicles.

The first thing to note about them is that they only form in Gram negative bacteria, which have an outer membrane covering a small glycopeptide layer (Gram positive bacteria have no outer membrane and a very large glycopeptide layer). The top layer simple peels off into a little vesicle, taking periplasmic proteins with it, as shown below (diagram from the reference):

The mechanism for vesicle formation is largely unknown, but it is found in both pathogenic and non-pathogenic strains of bacteria, and used for several different purposes. In pathogenic bacteria the vesicles often contain virulence factors, which can destroy or damage host cells. in the wild, they may also bind to or destroy other bacteria. In less-virulent strains they have been shown to act as a method of removing misformed or unwanted proteins from the periplasmic space (the space between the two membranes). They can also play a part in antibiotic resistance, it's not yet certain how but my guess is that they pump the antibiotic into the periplasmic space then vesicle it off to stop it just diffusing back in again.

When first discovered, the vesicles were thought to be a by-product of bacterial death, after all, why else would little bits of membrane with bacterial proteins inside be found floating around a large colony? However work done on pathogenic bacteria (which get more funding) and biopsies of infected tissues showed the vesicles playing an important part in infection. They are produced during the stationary phase of growth, the same period when bacteria start to produce most of their virulence factors and secondary metabolites. In an infected organism, this phase is after the bacteria has set off an inflammation reaction, and once it has multiplied in the site of infection.

Factors that affect the formation of vesicles include oxygen stress, the availability of iron (finding a regular iron source inside human bodies is a regular problem for bacteria) and the composition of the outer membrane (suggesting that at least some of the mechanism is mechanical). It is a ubiquitous process carried out across a range of Gram negative species.

As well as being used offensively, some vesicles were also shown to carry DNA between bacteria, although it's not at all clear how, or how the DNA gets into the periplasm in the first place. P. aeruginosa are also capible of transferring antibiotic resistant enzmyes between bacterial cell using the vesicles. This is not totally an act of complete altruism, as P. aeruginosa carries out much of it's infectious cycle as a biofilm, which requires lots of cells to form.

As I said, the actually mechanism for the formation of the vesicles is not yet established, so there's probably quite a lot of work to do with imaging their formation, genetics to find out any genes involved, and a mixture of genetics and protein work to discover more about what goes inside the vesicles. It looks like an interesting area of research, with the potential for some quite amazing imagery-work, and I look forward to reading more about it.

---

Kuehn MJ, & Kesty NC (2005). Bacterial outer membrane vesicles and the host-pathogen interaction. Genes & development, 19 (22), 2645-55 PMID: 16291643

Studying Streptococcus - SGM series

The fourth post now in the SGM series, and this one focuses on Streptococci. Streptococci are a genus of spherical Gram-positive bacteria containing both pathogenic and harmless strains, including the flesh-eating bacteria (which cause the delightfully named necrotizing fasciitis) as well as bacteria responsible for making swiss cheese. Commensally they are found on many parts of the human body, including the mouth, skin, intestine, and upper respiratory tract .

Streptococcus - growth and division leads to long chains of bacteria (image from lenntech)

It's quite a broad topic which allowed plenty of speakers to address their favourite issues with these bugs, but as well as discussions of the virulence factors, biofilm properties and various different intracellular survival properties of the Streptococcus there were also some talks covering new research mechanisms. Rather than focusing on the properties of the bacteria, these talks were about new methods used to study them.

The one that jumped out at me the most was about using Bioluminescent imaging to track a Strep infection. This appealed to me because the iGEM team next door are working on Bioluminescence so it's a word I've heard a lot over the last eight weeks. By adding bioluminescent bacteria to a mouse model, the course of the infection can be tracked over several weeks (using small animal imagine machines it can be tracked in the same mouse). This provides a far better understanding of the pathogenesis of the bacteria; how it spreads through the body and at what point it is most infectious.

The process of using bioluminescence to track diseases (image from the reference).

Using luminescence to study disease progressions isn't a new idea, but the use of whole animal scanning mechanisms now means that fewer animals have to be sacrificed in order for the study to done. The luminescent tissue does not have to be extracted, and the more natural disease progression can be followed.

Other methods explored included the by-now predictable whole genome study analysis to organise the different types and virulence levels of a Streptococcus suis which leads to meningitis in piglets. Comparative genome hybridization studies allow many genomes to be compared at once, giving a better idea of the differences and similarities between them. This helps to separate the strains into serotypes (different groups), and to compare the differences that lead to virulence. Genome comparison work was also being done for Streptococcus equi species which cause infections in horses.

In other news (pretend that was a smooth transition!) the latest Carnival of Molecular Biology is out over at Thoughtomics. There are some brilliant articles covering the intra-cellular happenings of organisms from bacteria to frogs to Tibetans. If you've ever wondered about noisy bacteria, zombie enzymes or what micro-RNA is, go take a look and visit the submissions.

---

Timothy C. Doyle, Stacy M. Burns, Christopher H. Contag (2004). In vivo bioluminescence imaging for integrated studies of infection Cellular Microbiology, 303-317 DOI: 10.1111/j.1462-5822.2004.00378.x

How bacteria die - SGM series

This is the second post of my SGM conference series and the topic is Microbial Death. I was very interested in this one as a topic, because the mechanisms that lead to bacterial death aren't something I've covered so much. It's generally assumed that antibiotics screw up whatever they target such that the bacteria can no longer survive, and when they aren't around the bacteria just keep dividing.

There were two talks concerning antibiotics in bacterial death, the first addressing a theory that's been bandied about for a while (and which I've already written about) that antibiotics don't really kill the cell by acting on their target. Instead, they just lead to sufficient damage to set off a series of death events within the bacteria themselves, a common pathway for bacterial cell destruction (first reference).

I think if I could have chosen any one talk to watch it would have been that talk, actually given by Kohanski whose been working on the stuff. I think there would have been some interesting questions as well, as this is somewhat controversial research.

The other antibiotics talk covered something I'd never heard about; the ability of some antibiotics in certain cases to prevent bacterial death. Work done on Microbacterium turberculosis - which causes TB and a related strain (Microbacterium bovis) showed that when in stationary phase (i.e the bacteria were not growing and dividing) the addition of antibiotics that usually kill only growing cells helped to aid cell survival. Antibiotics that targeted both growing and non growing cells did not have this effect. The reason for this is not clear, however comparing transcriptomes between cells both with and without antibiotics showed a difference in protein production on addition of antibiotics. These antibiotics are in someway helping to turn on genes for survival, which are keeping the stationary phase bacteria alive.

Another interesting talk was about the regulation of mutagenesis in bacteria, another idea I love. It's based on the observation that as bacteria start to get stressed they go into a sort of massive meltdown, leading to lots of genetic mutations being generated. It's been suggested that rather than this being a side-effect of the surrounding stress, this is actually a deliberate ploy by the bacteria to give themselves a last ditch attempt at getting out of a stressful situation.

Unlike multicellular organisms, bacteria have no surrounding restraints on their mutation rate - with the exception of bacteria in aggregates the only thing a bacteria will harm by changing it's DNA is itself. This gives bacteria a lot more genetic plasticity. Added to this, changing DNA is one of the main ways bacteria go about improving themselves, and adapting to new conditions. Changing the DNA by wholescale random mutagenesis is a bit extreme, but if you're in a stressful situation anyway it might be worth a shot.

Studies for this have mostly been done on E. coli, usually a lab strain, so I'm not sure how much they translate into bacteria in the wild, which might be better adapted at coping with stress situations, or, given that experimental bacteria are in a privileged nutritional environment, it might just be too risky for wild bacteria to start messing around with their genome. Also there's no concrete mechanism been found for it yet, so increased mutagenesis producing different phenotypes in times of stress may just be a happy byproduct of the usual genetic craziness that goes on when a cell dies.

These two theories both don't have as much supporting science as they could do, but they are new ideas which are still being worked on, and I really like them both.

---
Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, & Collins JJ (2007). A common mechanism of cellular death induced by bactericidal antibiotics. Cell, 130 (5), 797-810 PMID: 17803904

Ivana Bjedov, Olivier Tenaillon, Bénédicte Gérard, Valeria Souza, Erick Denamur, Miroslav Radman, François Taddei, Ivan Matic (2003). Stress-Induced Mutagenesis in Bacteria Science, 1404-1409 DOI: 10.1126/science.1082240

---
Follow me on Twitter!

Getting OMPs to the membrane - SGM series

This is the first post of my SGM conference series: I'm going to try and write about seven topics from the Society for General Microbiology September conference over the course of two weeks. The first topic I'm looking at is Protein Folding and Misfolding which consisted of thirteen presentations covering various aspects of protein folding in bacteria, fungi and yeast. As a quick background: when proteins are synthesized they are constructed as long chains of amino-acids which then need to fold up into the correct shape.

This may not sound terribly interesting at first, but it proves problematic for proteins that are in awkward places, for example in the outer membrane of Gram negative bacteria. These outer membrane proteins (OMPs) not only have to fold up correctly inside the membrane but they have to actually get to the outer membrane In Gram negative bacteria, this means first getting through the inner membrane, then across the peptidoglycan layer between the membranes, and finally half way through the outer membrane in order to coil up correctly inside it.

Gram negative cell membrane

The type of proteins found inside the outer membrane are usually B-barrel proteins, so called because they contain lots of protein folds known as a B-sheets, which can wrap up to form a channel shape as shown in the example on the right. Each blue arrow is a single B-sheet and these fold specifically to form pore-like structures which in the case of porins make a little hole through the membrane.

Transport of B-barrel OMPs accross the inner membrane is achieved by synthesising them with a signal sequence attached to one end. This signal sequence is recognised by proteins on the inner membrane and ATP energy is used to pump the proteins accross the inner membrane and into the periplasm (the space between the two membranes). Once in the periplasm they bind to little chaperone proteins which carry them safely to the complex responsible for folding them correctly into the outer membrane, the rather awesomely named BAM complex.As an aside the chaperones do have to get the OMPs there fairly promptly as there are proteases that float around in the periplasmic space and degrade any proteins that don't get incorporated into the outer membrane quick enough.

One of the key proteins in the BAM complex is BamA as knocking it out results in a lot of unfolded OMPs in the periplasm (and probably a field day for the proteases). BamA consists of two major components, a B-barrel domain which anchors it into the outer membrane, and five "polypeptide transport-associated" domains, shortened to POTRA by someone who didn't like three-lettered acronyms. The POTRA domains do what they say, they are associated with the transport of proteins (polypeptides).

It's still a little uncertain quite how the BAM complex works but a couple of the presentations on the topic were convering it, including work done on changing the genes between different bacterial species. All Gram-negative bacteria have a BamA gene, however taking the BamA gene from one bacteria and putting it into another does not end happily unless it's done between two very close species. Closer research with chimeric proteins (i.e proteins that are half from one bacteria and half from another) shows that this only applies to the POTRA domains. The anchoring B-barrel can be switched between several different species, but the POTRA domain is very species specific.

Another interesting thing to address is how BamA gets itself into the outer membrane. One of the periplasmic chaperone proteins, Skp, is thought to be involved in this process, and it was found that when the outer membrane was negatively charged Skp is involved in inserting BamA into the membrane, whereas when the negative charge is removed Skp inhibits BamA folding and insertion. Negative charge is caused by an increase in the phosphatidylglycerol content in the membrane. I found that idea quite exciting as it implies that the bacteria can control where they want the BAM complex to go. The idea of membranes forming "lipid rafts" with certain components that organise where proteins are held is not a new one, and the BAM complex forming in specific places in order to create the correct outer membrane protein concentration is one that appeals to me.

They may just be single little cells with no true nucleus, but they are capible of a lot of control over their intracellular processes!

---
Knowles TJ, Scott-Tucker A, Overduin M, & Henderson IR (2009). Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nature reviews. Microbiology, 7 (3), 206-14 PMID: 19182809

Johnson, A., & Jensen, R. (2004). Barreling through the membrane Nature Structural & Molecular Biology, 11 (2), 113-114 DOI: 10.1038/nsmb0204-113

---

Follow me on Twitter!

Unfortunate change of plans

So...anyone following me on Twitter probably saw me getting all excited when I signed up to go to the Society of General Microbiology conference a couple of months ago. The conference is next week, but unfortunately I won't be at it, which is a pity because I was all set to blog about it and everything.

There are two main reasons that I can't make it:

1-Time. I'm just finishing off my summer project and starting to need/get exciting results. Also I'm sorting out my wedding, trying to get a PhD organised, and heading off to another more work-related conference later this month. Taking four days off would be slightly overindulgent at this point.

2-Money. I do not have any. As an undergraduate SGM member I can't get a travel grant from them (graduates only) and as I've graduated the university isn't about to give me any money. So I have to pay to transport myself up there, and for somewhere to sleep for four nights. Train fares are expensive nowadays and even the cheapest B-and-B I had planned came to £180. Quite frankly if I had £180 I'd use it to clear my overdraft.

HOWEVER - all is not lost. As I registered for going to the conference (for free - the perks of being an undergraduate member) they sent me a PDF of the abstracts for each session. So, over the next two weeks I'm going to have a rather sad and nerdy little single-person conference of my own on my blog. I'm going to try and get a post up every other day (so seven posts over two weeks) covering one topic at the conference with each post.

I've got a choice of seventeen, but here are the topics I'll probably aim to cover as they're the ones I most wanted to see:

Metals and Microbes

Streptococci

Acid Stress

Microbial Death

New Insights into Secondary Metabolism

Extremophiles

Protein Folding and Misfolding

(and if I have time, Microbial Models of Human Diseases)

Before anyone starts feeling too sorry for me about missing this I should point out that a) I have another conference I'm going to this month b) that conference is fully funded and c) that conference is in Italy...

And there will be many more conferences in my life, I'm sure!

America!

I was hoping to get another paper-analysis post in before I left, but I ran out of time. I'm off to America tomorrow morning (*early* tomorrow morning) for a synthetic biology conference. I get the feeling it's going to be utterly mad, and leave me completely exhausted by the time I get back (on Tuesday evening).

Expect some residual synthetic biology stuff when I get back! I'm hoping to scribble down enough for a post while I'm there, and type it up when I get back. I could bring my laptop along, but I'm trying to keep my luggage down to hand-luggage and I suspect I wouldn't have the time. Also, I'm not quite sure of the etiquette of conference-blogging. Some of this stuff might have publishing-potential but not yet been constructed into a paper, and I don't want to accidentally 'out' someones research.

As a quick teaser, here's a picture of what me and my fellow summer-project lab rats will be taking about. All the pigments were made in E. coli:

Living without a cell wall...

A cell wall is one of the most important features bacterial cells possess. They provide a barrier against the harsh conditions of the outside world, as well as helping the cell maintain its shape and integrity. They are vital for nutrition uptake, and for cell and chromosomal division.

They are also, however, the main point of attack for other competing organisms, and for the human body when under attack. There are numerous antibiotics that direct against the cell wall. It is thought therefore that some cells have adapted to live in the body without a cell wall, their innards kept inside by merely a small lipid membrane.

But how do they survive? How do they replicate? And, most importantly, how on earth do you study them in a lab. If you take the cell wall off a bacteria under laboratory conditions it turns inside out. And then explodes. It certainly doesn't stay in any kind of workable state.

Recently though (very recently) the Center for Bacterial Cell Biology in Newcastle have found a way to grow bacteria (Bacillus subtilis to be exact) without a surrounding cell wall. The mutation is quite simple to make, and by adjusting the outside conditions to prevent the cells being damaged, they managed to grow colonies of cells with no cell wall at all, and keep them alive to study.

One of the most interesting things about these cells was their division mechanism. In normal bacterial cells, division depends on the cell wall as an anchoring point to hold the chromosomal DNA while it divides, and then control the lengthening and splitting of the cell, as shown in the diagram below (from here):

How do cells without a cell wall manage to divide? In order to find out, the group at Newcastle took little movies of their cells, following them as they grew and developed. The movie isn't in the paper, but there are a series of stills from it, showing a single cell growing and dividing, and following a very different pattern of division than usually seen in bacterial cells, or in any cells:

Image taken from reference one: link

Instead of splitting into two in an organised manner, the cell blobs out to form a long strand, which then breaks up into many little pieces, each containing a copy of the cell DNA. The usual proteins needed for organised division (in particular FtsZ) are not required, the cell is using a totally different system.

What is even more interesting, is that this looks very similar to a system proposed by Ting F. Zhu and Jack W. Szostak for how the very first forms of proto-life might divide, back when life consisted of not much but a small membrane with a twisted DNA coil inside. Working totally indepentantly, their work was examining the growth and division of simple loops of lipid membrane. They would form one, and make it grow by adding micelles, little circles of membrane. They found that as they added them, the cell would eventually start elongating and, when agitated, split up into little blobs, which could then grow and divide in a very similar manner:

Image taken from reference two: link

This looks strikingly similar too the images of the dividing bacteria shown above. In both cases the membrane stretches out and then splits up again into little circles. The only change the proto-life would have to make to the physical behaviour of the membrane would be to make sure that copies of the DNA got packaged inside each little circle.

This makes the work done at the centre at Newcastle even more exciting. Not only are they developing systems to study and explore bacteria that are immune to a wide variety of antibiotics, they are also helping to explore how the earliest forms of life might have survived and propagated. This provides a glimpse into a world before even bacteria had evolved, and does being to light just how highly sophisticated and complex bacteria are, compared to their membranous blob-like ancestors.

---

Leaver, M., Domínguez-Cuevas, P., Coxhead, J., Daniel, R., & Errington, J. (2009). Life without a wall or division machine in Bacillus subtilis Nature, 460 (7254), 538-538 DOI: 10.1038/nature08232

Zhu TF, & Szostak JW (2009). Coupled Growth and Division of Model Protocell Membranes. Journal of the American Chemical Society PMID: 19323552

On Conferencing

I spent most of last week at a conference. The Society of General Microbiology conference to be precise, up in Harrogate. It was my first conference, so I was pretty excited about it, and it certainly lived up to expectations. It was fun, exciting, and I learnt a whole lot of stuff. Some of it was even about science.

But the most amazing thing? Julian Davies was there. The same Julian Davies I spent the last blog post ranting about; with the papers about how sub-inhibitory antibiotics acted as signalling molecules. Best of all, I had a chance to talk to him as well. After the initial slight embarrassment of me being slightly uncertain of how to introduce myself (mostly I was desperately attempting not to gush as him in an 'I've-read-all-your-papers-and-omg-they're-amazing' way) I finally talked a little about my research.

He actually seemed interested! It turns out all his work has been on soil bacteria, so he was quite interested in my work, which was on an antibiotic-producing bacteria. We chatted for a bit, then someone else came up and I politely scarpered out the way feeling a little wobbly around the knees. Gushing mostly avoided. Serious scientific talk achieved :) It was a good feeling.

One thing I didn't realise about conferences was just how much the evenings play a part. During the day there are talks and lectures and poster displays and promotional stalls for various companies that sell scientific equipment. So there is nothing official planned for the evenings (I even brought some revision along for then. hah. Like that happened). But the evenings, it turns out, are all about going out with the people you've met at the conference; getting to know people, talking with them, sharing experiences and ideas and having the most amazing conversations about scientific things with people who pretty much think along the same lines as you. And nobody rolls their eyes and tries to change the subject. It was amazing.

So that was my first conference. Hopefully, it will be the first of many.