Bacteria that use antibiotics...for food!

Antibiotic resistance is by now a well-known phenomenon. Resistance is carried in both antibiotic producing bacteria to protect themselves from their own weaponry, and the soil bacteria they attack, in an attempt to defend themselves. The sudden influx of pharmaceutical antibiotics has encouraged the spread of resistance to human pathogenic strains, leading to the so-called 'superbugs' seen in the media such as MRSA and vancomycin-resistant C. difficile.

However researchers at Harvard found that not only are some bacteria able to neutralise the threat of antibiotic resistance, they actually use antibiotics as a food source. Not only that, but they were capable of using antibiotics as the sole carbon source. The table below (taken from the reference at the end of the post) shows the survival of bacteria on antibiotics using samples from three different types of soil, Farmland (F), Urban (U) and Pristine (P - soil from non urban areas with minimal human contact for 100 years):

The antibiotics used include natural, synthetic and semi-synthetic molecules, all all of which could be used by bacterial species as a carbon source. Even more interestingly (or alarmingly) the antibiotics were at concentrations of 1g/litre, 50 times higher than the concentration normally used to test for resistance.

The 'pristine' soil is the one that the researchers found the most interesting, as the general expectation was that this area would contain fewer antibiotic-eating bacteria, having had minimal interaction with people and pharmaceutical antibiotics. However the data showed no noticeable difference, despite not being in contact with human-designed antibiotics, the bacteria are meeting plenty of bacterial-based antibiotics, and adapting to use them for food.

The big question of course is Will it Spread? Around the quarters of the isolated strains belonged to orders containing clinically relevant strains such as Salmonella and E. coli, meaning that hypothetically at least antibiotic consumption should be able to spread. On the other hand, actual consumption of antibiotics is unlikely to provide a greater evolutionary advantage than just resistance, and will confer a larger metabolic load on the bacteria. Although the pathways of antibiotic metabolism have not yet been fully determined, the first few steps seem to be similar to well-known resistance mechanisms (particularly in penicillin consumption). One conclusion, therefore, is that only part of the metabolic pathway would be (or already has been) passed on to pathogenic organisms, enough to provide resistance without placing unnecessary metabolic burdens on the cell.

Hat tip to Byte Size Biology for alerting me to the paper.

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Dantas, G., Sommer, M., Oluwasegun, R., & Church, G. (2008). Bacteria Subsisting on Antibiotics Science, 320 (5872), 100-103 DOI: 10.1126/science.1155157

Changing Projects

My synthetic biology project has pretty much ended now, bar the handover. I've got a little more lab work to do (still have one more restriction site in the MelA gene, and I'll have a bit to do when my vio DNA arrives) but the majority of work in that direction is over...I now have a week of safety talks to prepare me for my next project: back safely in the field of bacteria-antibiotic interactions.

I've enjoyed this project. It's been fun, I've got to meet new people, and I've learnt a lot of new and very useful techniques, particularly involved in genetic manipulation (ligation, restriction, PCR etc). I've also learnt something very important. That wherever the winding road of life may take me, it is unlikely to take me very far in the direction of synthetic biology.

It's an interesting and very exciting field, it's just not one I feel I could survive a project in. These ten weeks have been long enough, now that the novelty has worn off, I'm beginning to realise that this just isn't the area of science I'm interested in. I like exploring bacteria, how they work, what they do, how they interact with the world around them. Synthetic bacteria doesn't really cover that; it uses bacteria, sure, but only as DNA-expressing chassis for carefully constructed molecular circuits. Circuits just don't hold my interest for the length required for an in-depth project.

I can see how it could be an interesting field, for engineers becoming excited in the natural world, or biologists who suddenly realise they have a passion for circuitry and building biological machines. But not for nerdy little microbiologists who get far too excited about how bacteria behave in the worlds they inhabit, how they deal with the dangers and the changes and the constraints of the physical world.

I can't wait to get into my new lab. A whole week of safety talks is going to be...so... irritating...

Bacterial Hunting Strategies

Like every other form of life, bacteria need nutrients to survive. In the laboratory, these can be provided on agar plates at the perfect balance for growth and propagation. In the wild, however, nutrients seldom float around uneaten, which is why many bacteria have evolved to be predators, using a variety of strategies to seek out, destroy, and consume their prey: any other bacteria incapable of defending themselves.

There are a number of ways to eat other bacteria, and probably the simplest is phagocytosis, shown to the right (image from the free dictionary). It's a relatively easy system, involving nothing more than the ability to warp the cell membrane in response to binding, and release degrading enzymes once the prey has been captured. However, it does rely on the prey being smaller than you...and not forming complex multicellular structures such as biofilms, or fruiting bodies.

A second method is to parasitise, to crawl into the cell wall of your prey and destroy it from the inside out. This is the method used by the small bacterium Bdellovibrio bacteriovous, which attacks a large number of other bacteria (only Gram negatives though) and grows inside them, eventually destroying them. Its life cycle is shown below:This image is taken from the Nunez Group homepage, which contains a lot more information (and some beautiful pictures) about this method of predation.

The third option for bacteria is to use chemical warfare, release a large number of cell-destroying enzymes and then eat up the debris. This is the strategy of my Streptomyces, which excrete antibiotics capable of destroying a whole range of different bacteria. It is still not totally certain whether they do this for food, or simply to remove predators or potential rivals for space, but either way it's an effective method of killing bacteria which has been exploited by the pharmaceutical industry since Flemming first marketed the idea.

The final major strategy is to hunt. Bacteria do not all exist as solitary blobs in isolation, many species are able to form semi-multicellular structures that can move together, grow together, form spore-producing fruiting bodies and, in the case of Myxococcus, hunt together. Myxococcus xanthus is the model organism for this, capable of forming large swarms of bacteria that can swarm towards prey and then destroy it.

The image on the left (taken from the first reference below) shows a single M. xanthus bacteria (the long thin bacteria highlighted with an arrow) approaching and killing a round coccus bacteria. As soon as the xanthus touches its prey, it releases hydrolytic enzymes which destroy it, producing nutrients for the xanthus to then consume. Unlike Streptomyces (which can't move) these bacteria can form large colonies, which move forward together into an area colonised by prey, forming rippling shapes which can be seen on plates. Although xanthus are perfectly capable of hunting on their own, the large rippling group allows them to disrupt structures such as biofilms, giving more access to prey.

Gathering together in large groups also allows differentiation and division of labour within the group. While consuming prey, the M. xanthus tend to form two distinct subpopulations; bacteria near to the prey will be feeders, forming the characteristic rippling pattern. Behind them, bacteria in the less-nutrient rich area will begin to aggregate and form fruiting bodies which, if conditions suddenly turn bad (i.e all the food runs out) can sporulate, ensuring survival of the population.

It's a fascinating little world; hunters, predators, parasites, and a whole world of physical challenges to get through (swimming through water for bacteria is similar to moving through treacle for a human). The pressure and challenge of surviving in such a world has produced a whole mass of different shapes, sizes and strategies, from single celled packets of explosive chemicals to larger and more complex multicellular assemblies.

However good people get at killing bacteria, other bacteria will always be able to do it better. And that is why why study them.

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Berleman JE, & Kirby JR (2009). Deciphering the hunting strategy of a bacterial wolfpack. FEMS microbiology reviews, 33 (5), 942-57 PMID: 19519767

Scientia Pro Publica - 12th Edition

Welcome to the twelfth edition of scientia pro publica (science for the public) hosted here on Lab Rat. This is a blog carnival, designed to collect some of the most interesting posts on anything scientifically minded, written for people to understand and enjoy.

There's a wide selection for this edition, ranging in size from protein molecules within the cell to giant floating piles of trash in the sea. Being a microbiologist, of course, I'm going to start with the smallest and work up...

At the level of the very small:

We have an explanation of how Ritalin works, from Scicurious, and a look at the competition faced by sperm from Kelsey. There's also a lone little physical post about how to determine the charges on sticky-tape, at A Posteriori.


At the level of the slightly bigger:

Moving up to animal-sized things; there's a review of shore birds from DC birding blog, a good scientific look at the various myths surrounding chameleon colours at Ionion Enchantment and a great exploration of urban wildlife by Reconciliation Ecology.


At the evolutionary level:

In a wonderful example of the scientific method of working we have a post from Eric Johnson discussing laboratory work that shows evidence for the breakdown of the selfish gene theory, and then another post from Bob O'Hara saying that it doesn't. There's also a post by Cubic deconstructing an article written by David Stone about what makes a 'Darwinian'.

Closer to home - at the level of people:

Technically I suppose I should have dumped humans in with the rest of the eukaryotes, but there's enough exclusive posts about them to form a separate group. Dr Shock looks at whether Salvador Dali suffered from a mental illness, while Greg Laden examines the phenomenon of phantom touches. There's also a guest post at DermMatters about the importance of clinical photos, and why it's sometimes a good idea to take your own, as well as a glimpse back into the body-snatching era (the more dubious face of clinical anatomy) by Providentia.

At the level of society:

Two posts about using science in the court: a look at the importance of forensic evidence from Suzanne Smith, and Radio Frequency Identification from Adrienna Carlson. There's also a great post from A Blog Around the Clock, looking at scientific reporting, in the specific case of a giant pile of trash floating around in the Pacific.

And finally, if you have the need for more science blogging, the Online Universities Weblog has a list of the top 100 Science Professor's Blogs.

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.

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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

The Importance of Fairy Stories

I was reading around the posts at ScienceBlogs, when I came across this one by Bioephemera which, while talking about a recent google-doodle took a quick jokey look at the respective merits of Hans Christian Ørsted and Hans Christian Andersen. The following quote from The Guardian about the issue was produced: "while there's nothing wrong with fairy stories, they haven't contributed much to the development of electric motors."

I couldn't resist it. Put "Discuss" on the end and it's practically an HPS (history and philosophy of science) essay. (I won't answer it in essay form, because I haven't got the time to plan out a nice long essay right now, but I will damn well be discussing it)

"While there's nothing wrong with fairy stories, they haven't contributed much to the development of electric motors." Discuss.

My main problem with this statement is that it seems to see science (and technological development, which I am lumping in the same field here) as an isolated process, remote and aloof from the rest of human life and development. Science, according to the guardian, progresses by scientific-minded people doing scientifically relating things and coming up with greater and better ways of achieving useful things, such as the electric motor. These scientists would be important and serious men (well...lets face it they probably were thinking of men) working away through detailed experimentation on serious topics, a far distance away from the whimsical and childish world of little stories.

Scientists, whatever Hollywood tries to insist, are people too. They grow up as children, hearing the same stories and tales and getting the same cultural and emotional baggage from the society around them. And science itself develops within that society, affected by it, changed by it and to a certain extent controlled by it as well. Ørsted grew up listening to the same kind of stories as Anderson, the only difference was that he didn't write them down.

In fact Hans Christian Andersen and fairy stories is a particularly bad example of things-that-do-not-affect-science, because Anderson wasn't just making these stories up. He was taking stories that were already being told. Folk-tales rather than fairy-tales, and folk-tales are crucial to human development. In a way, they are cultural development, especially in small communities where not very much writing occurs. They're how you teach your children, how you pass messages across, how you define what is acceptable and what isn't. How, in fact, you lay down the very rules and laws by which your society develops by, rules from which science is not exempt.

A few hundred years ago we had the Magician's Apprentice, adapted from a fairy-tale that makes it clear what happens if you mess with things you don't understand. The Victorian era brought forth the gothic novel Frankenstein, with a fairly similar message (among others). And now, in England, people protest against GM crops, for pretty much the same reason. I'm pretty sure there was at least one headline with the words "Frankenstein Fruit" in it. People have the stories in their heads, and stories are very powerful things to get rid of.

They told me when I was writing up my presentation for my project "make it a story". People understand stories, they understand things through stories. They develop, change, and form cultures, mostly based around stories. And science cannot be separated from the culture that surrounds it. Nor can it even remain strictly "scientific". Kekulé 'discovered' the ring structure of benzene after falling asleep and dreaming about a snake eating it's own tail. Science progresses through humans and humans progress through stories.

And, well, there's a reason the 'geeky-scientist' exists as a stereotype. We *like* fantasy, and science fiction, and other stories of other worlds. If you bring a child up telling them stories of fantastic places, and then bring them up slightly further by showing them the inside of a cell, they'll be hooked. It's a magical place, with magical rules, where everything moves and acts differently and, best of all, it really exists and you can get paid for exploring it.

On the face of it science may seem a long way from 'The Princess and the Pea' (although maybe not too far away from 'The Emperor's New Clothes'...) But these are the stories that western scientists and western science have grown up on, taking them, using them, being influenced by them. Without the stories, without the cultural background and development, the electric motor would have been a lot longer in arriving, and the rest of science would have been far, far slower. You cannot separate development into "that achieved by surrounding culture" and "that achieved by scientific and technological development", they're all far too tangled up in each other for that too be possible.

[There's even a book about physics and philosophy called 'The Emperor's New Mind'. You can't take the stories out of people.]

Probably more a 'graphic novelette'

I was reading through all the bioephemera archives that I missed through being hideously busy and was motivated to find something pretty and artistic connected with Synthetic biology (if my lovely gene designs weren't pretty enough...) Somewhere at the beginning of our course we were sent a link to a quick comic explaining synthetic biology, which I didn't bother with but which the engineers in our course were incredibly pleased with, and found really useful.

I went back and took a look at it last night. And actually, it's really good:The full story can be found here.

And it's actually in a nature paper! Which means it is most definitely not just a childish 'comic' and deserves to be taken seriously, like any other graphic novel.

The Lab Rat guide to DNA Synthesis

The structure of DNA is a double helix of two sugar-phosphate backbones joined by hydrogen-bonds between nitrogenous bases, as shown below:

Image from Bioinformaticsweb.org

The letters of the DNA code come from the bases; adenine (A), thymine (T), guanine (G) and cytosine (C). They code for amino-acids, which make up proteins, in groups of threes, i.e GCC codes for alanine, GGA codes for glycine etc.

Each base, along with the associated sugar and phosphate, forms its own little subunit. Joining these together in the correct order can code for any protein you want. As a Lab Rat I don't know very much about this process, except that I send the sequence off and get back a little vial full of DNA (or a stab containing the bacteria that have my DNA held on a separate plasmid). So what I'm writing here is just what I've managed to find out about the process - it might not reflect the most up-to-date method used in the top sequencing companies, but it's a plausible way to make DNA.

There are two main types of DNA synthesis. Firstly there's small sequence oligonucleotide (aka small-bit-of-DNA) synthesis, to make primers and things. Secondly there's whole gene synthesis, which deals with larger sections of DNA. As whole gene synthesis mostly involves sticking together little bits of DNA, I'm mostly going to focus on small oligonucleotide synthesis.

The basic process involves sticking the growing DNA strand to a solid support and then just washing the next DNA base through, over and over again. This is pretty much automated nowadays, so you just program a robot to do it. The supports used are mostly either Controlled Pore Glass or macroporous polystyrene (plastic with small holes to select for size, allowing the salts and bases to be washed away before the larger DNA molecule is eluted). Both of them covalently attach to the end of the DNA chain, holding it in place as the nucleotides are washed through.

In their natural state, however, nucleotides are not very reactive, so special modified versions are used. Large bulky groups such as DMT and cyanoethyl are used to block the ends of the bases and the phosphorous linkages, to stop them reacting or participating in reactions.

The first base is then attached to the support and the DMT group (attached to the bottom of the base - the five sided ring) cleaved off with an alkaline wash. The next subunit is then activated before being added to the support. This involves adding tetrazole, which cleaves off the three big rings shown on the left, making the subunit more reactive. The activated subunit is then washed through the column, where it can react with, and bind too, the preceding base.

Once all the bases have been added in the correct order the mixture is purified, to isolate the required sequence. This is done by desalting, usually with chromatography, to produce the final product.

In case anyone was wondering, the robot/machine/computer used for synthesis looks like this:

Image taken from monash university website.

Cell wall under attack - bacterial response to antibiotics

I took a quick break away from synthetic biology and DNA synthesis research the other day, to dive back into my happy little world of antibiotic research, in preparation for my new project in October. I'll be working with Streptomyces bacteria again, which after a whole summer of E. coli I'm quite looking forward to. What I'll be doing with them is examining the response of the cell wall to antibiotics.

The bacterial cell wall is made up of glycopeptide molecules (sugars and proteins joined together) and surrounds the whole cell. Without it, bacteria swiftly loose their integrity and salt-balance across the membrane, which is why many antibiotics target the cell wall in order to kill bacteria. Both for antibiotic resistance, and for surviving conditions that could damage the cell wall, bacteria have a system of monitoring the state of the cell membrane and responding quickly to any changes.

The system that was discovered in Streptomyces coelicolor (which I'll be working on) was named the sigE system, and consisted of an operon (string of genes) encoding four genes:

SigE encodes for a sigma-factor, a protein used in bacteria to switch on certain sets of genes. The cseA codes for a cell membrane lipoprotein, possibly used in a sensor system, while cseB and C are a two-component signalling system (very common in bacteria). CseC is a sensor (a histadine protein-kinase sensor for those who are interested) while cseB is the response regulator, acting out a response when it receives a signal from cseC.

And now...the science :)

In order to test that this operon was involved in cell membrane responses to antibiotics the lab carried out a variety of experiments, all producing evidence that lead towards this conclusion. The main experiments were as follows:

1. Removing the sigE operon and placing it on a separate plasmid, that activated resistance to Kanamycin. The bacteria were then plated on agar containing antibiotics and challenged with a kanamycin disk. Cell wall attacking antibiotics induced kanamycin, whereas antibiotics that attacked (say) the ribosome didn't.
2. Keeping the sigE in its original chromosomal context, the group then challenged it with different concentrations of vancomycin (an antibiotic which attacks bacterial cell walls). They then measured the level of the sigE operon proteins being produced in the cell. Higher concentrations of vancomycin, lead to more proteins.
3. Going back to the sigE-kanamycin resistant protein, they tried knocking out the sigE promoter, effectively switching all these genes off. The effect seen previously disappeared.
4. Leaving the lab, they then did some computational work, scanning the database to see what genes the sigE sigma-factor actually switched on. They found a group of 12 genes, all of which coded for cell-wall synthesis enzymes.

All of this leads up to some pretty conclusive evidence - in case of cell wall damage, the sigE operon is switched on. The interesting thing is, is that this isn't just a response to antibiotics either. It is highly unlikely that the system is able to respond to every different cell-wall destroying antibiotic, instead, the response is triggered by cell-wall intermediates, or degradation products that signal "Help - cell wall is being destroyed!" and switch on the sigE response, which produces proteins to mend it again.

But there are still a lot of unanswered questions. What is the cseC actually sensing? What is the exact purpose of the cseA? Why produce both a sigma-factor and a heafty response pathway? Which intermediates are used for activating? And, most importantly, can we hijack this somehow to kill bacteria?

I can't wait to get to work with it :D

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Hutchings, M., Hong, H., Leibovitz, E., Sutcliffe, I., & Buttner, M. (2006). The E Cell Envelope Stress Response of Streptomyces coelicolor Is Influenced by a Novel Lipoprotein, CseA Journal of Bacteriology, 188 (20), 7222-7229 DOI: 10.1128/JB.00818-06

Hong, H., Paget, M., & Buttner, M. (2002). A signal transduction system in Streptomyces coelicolor that activates the expression of a putative cell wall glycan operon in response to vancomycin and other cell wall-specific antibiotics Molecular Microbiology, 44 (5), 1199-1211 DOI: 10.1046/j.1365-2958.2002.02960.x

Jacobs C, Frère JM, & Normark S (1997). Cytosolic intermediates for cell wall biosynthesis and degradation control inducible beta-lactam resistance in gram-negative bacteria. Cell, 88 (6), 823-32 PMID: 9118225

Sequencing vs. synthesis

Sequencing = the process of finding out what base-pairs a piece of DNA consists of (AAAGGGAAA etc)

Synthesis = the process of actually making the DNA from separate base pairs.

In my last post I got the two processes chronically mixed up. The links lead to a description of DNA sequencing, whereas the process I describe doing with my DNA is preparing them for DNA synthesis.

I was going to edit the post but I couldn't find any meaningful way to do so, without either deleting the information or deleting the links, both of which contain (hopefully) quite interesting science.

I will write a post about synthesis at some point.