The Painted Lady butterfly, Vanessa cardui. Picture taken in Ename, Belgium by Tim Bekaert (July 12, 2005).

Until fairly recently, it wasn’t known exactly how these butterflies migrated. They appeared in the UK around spring-time and then promptly vanished in the autumn. In order to track them a project was set up in 2009, involving systematic surveys, observation through citizen science projects, and the use of high altitude insect-monitoring radars.

What they found was that when the Painted Lady migrated back down south, the butterflies travel at incredibly high altitudes, up to 500m up. By using favourable winds, they can reach speeds of up to 30mph, which for a butterfly is fairly zooming along. They’d never been seen previously because people weren’t monitoring butterflies at such high altitudes. Butterflies such as Red Admirals were seen and recorded travelling southwards, but the Painted Ladies were so high up they were able to vanish unnoticed.

Painted Ladies aren’t the only butterflies that carry out these multi-generational migrations. The most famous migrating butterflies are probably the Monarch butterflies in America, which takes four generations to migrate from Canada and Mexico. Once again, these butterflies aren’t using memories, or even passed on knowledge of the migration routes to find their way. Each generation must work it out alone with the help of genetics and instinct.

Monarch butterflies resting on a tree mid-migration. Image by Brocken Inaglory, credit link below.

I find these multi-generational migrations truly fascinating; the idea that these tiny little creatures, with brains approximately the size of a pin-head, are capible of following migratory patterns laid down many generations ago. It’s like a butterfly generation-spaceship, with each individual seeing only a small fraction of the distance and scenery of the full journey.

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Brocken Inaglory images

Reference: Stefanescu, C., Páramo, F., Åkesson, S., Alarcón, M., Ávila, A., Brereton, T., Carnicer, J., Cassar, L. F., Fox, R., Heliölä, J., Hill, J. K., Hirneisen, N., Kjellén, N., Kühn, E., Kuussaari, M., Leskinen, M., Liechti, F., Musche, M., Regan, E. C., Reynolds, D. R., Roy, D. B., Ryrholm, N., Schmaljohann, H., Settele, J., Thomas, C. D., van Swaay, C. and Chapman, J. W. (2013), Multi-generational long-distance migration of insects: studying the painted lady butterfly in the Western Palaearctic. Ecography, 36: 474–486. doi: 10.1111/j.1600-0587.2012.07738.x

In order to eat, therefore, the internal bacteria must find ways of stealing and sequestering nutrients from the infected cells. A recent paper from, PLoS Pathogens (reference 1) shows how Salmonella infecting rat cells manage to find enough nutrients to grow and develop.

Salmonella typhimurium. Photo: Volker Brinkmann, Max Planck Institute for Infection Biology, Berlin, Germany. Image from reference 2.

In order to explore what pathways the bacteria could use to intake nutrients, the researchers used both computational and in vitro experiments to look at proteins and genes suitable for metabolism. What they found was that following an infection the Salmonella was able to mobilise a large section of its genome in order to carry out metabolic reactions.

Rather than just concentrating on using one particular metabolite from the host cell (i.e having lots of pathways to metabolise glucose) the Salmonella was able to exploit a diverse range of host molecules, without preferring one to the other. Adding either glucose or mannitol to established Salmonella colonies caused an increase in growth. Figure A below shows a schematic of the nutrients being added to the cells, while figure B shows the results. The more CFU (colony forming units) found, the more growth was seen:

White circles = low concentration glucose. Black circles = high concentration glucose. Grey circles = glucose + mannitol (both low concentration). Image from reference 1

Further experiments, combined with the computational data, indicated that the Salmonella bacteria use a wide range of chemically diverse nutrients inside the host cell in order to grow; including different lipids, carbohydrates, amino acids, nucleosides, and various pro-vitamins. This does mean that the Salmonella requires a large number of genes to deal with nutrition, but on the plus side, once it gets into the cell it’s far more likely to grow and survive with any small amount of food it can scavenge.

Why does Salmonella require such a large range of different metabolic pathways while other intracellular bacteria (including E.coli) are happier to rely on far few? One potential explanation given in the paper is due to the conditions in which Salmonella lives inside the host cell. In order to protect itself from attacks from the cells defence system, the bacteria stays safely wrapped up in a vacuole inside the cell. While this does stop the cell destroying it, it may make it harder for the bacteria to cannibalise available nutrients, meaning it has to have the capacity to utilise whatever it can get hold of.

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For more details on how Salmonella invades the host cell, along with a great animation, go here.

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Reference 1: Steeb B, Claudi B, Burton NA, Tienz P, Schmidt A, et al. (2013) Parallel Exploitation of Diverse Host Nutrients Enhances Salmonella Virulence. PLoS Pathog 9(4): e1003301. doi:10.1371/journal.ppat.1003301

Reference 2: (2005) A Novel Data-Mining Approach Systematically Links Genes to Traits. PLoS Biol 3(5): e166. doi:10.1371/journal.pbio.0030166

I’m currently 15 and a half weeks pregnant!

In the UK there is a fairly good and well thought-out system to make sure pregnant women get all the necessary checks and scans. The first scan is usually at 12 weeks, once the fetus has developed far enough to actually resemble something human. For me and my husband however, there were a combination of factors (uncertainty about conception date, unfortunate bleeding) that meant we ended up with three scans before the 12-week mark giving us a nice little progression of how the fetus was developing and allowing us to bore people with baby photos before we even had a baby.

So I thought I’d share.

First scan: 7 weeks

As it’s not immediately obvious what’s going on in the scan, I have circled the developing embryo in yellow. At this stage, even the most excited and generous interpretation of the scan can’t make out much more than a fuzzy grey blob. The dark area surrounding the blob is my womb, and the fuzzy little speech-bubble off to the right is the yolk sac, which nourishes the embryo for the first few weeks.

The most exciting thing was that even at this early stage it had a heartbeat! We could just about see one area of the fuzzy grey blob turning from lighter to darker at a pretty rapid pace.

First NHS Scan: Week 9

As the first scan we’d got was private, the NHS still didn’t know how far developed our little embryo was, so our first official scan was a bit early. This scan is a lot easier to read, but once again I’ve circled the important bit in yellow, and the black area surrounding it is the womb.

At this stage, the embryo has something that’s a bit like a head on the left hand side and you can just about make out four little stumpy blobs pushing up from the main body. These were little pod-like proto-arms and legs and it was easier to make them out in the scan because they were moving! The poddy-arms were wriggling around and the embryo as a whole was rolling back and forward.

The official scan: Week 12

Despite the fact that we now knew for certain there was something in there, and that it seemed reasonably happy, the official scan was still a pretty exciting thing. After 12 weeks most of the major organs have developed, the embryo officially becomes a fetus, the likelihood of miscarriage goes right down and, most excitingly, you’re allowed to tell everyone about the pregnancy.

By the third scan, the fetus is looking less like a pod-creature from Mars and recognizably human. The head is on the left and arms and legs can be seen sticking up out of the body. We thought the previous scan had showed a fairly active young thing but in this one the fetus was all over the place; rolling around, waving its arms and legs, and doing cartwheels in my womb. It’s still too early to tell if it’s male or female, that exciting bit of news will be revealed at the next scan.

So how will the baby affect the blog? I do want to keep a bacterial and biochemical focus, but there may be the odd baby post as scans or exciting information come in. Probably the biggest change is that I’ll be dropping down from four posts a month to two/three just to give myself a bit more time and breathing space.

Now the first trimester is over and I am once again capable of basic functions like eating and remaining awake, I do want to get back into regular blogging! So more items of bacterial interest will be coming soon…

I haven’t been on holiday for a while, so for this issue I thought I’d take a trip around the world, looking in on all the exciting research and work being done in the field of evolution. There are some great posts here, from some wonderful bloggers, so go take a look!

Journey Start: Europe

I’m based in the UK, so will start the journey as close to home as possible. At The Mermaid’s Tale Anne Buchanan explores Robert MacFarlane’s description of Scua off the coast of Scotland, and how their behaviour may have evolved, as well as a post on the 100th anniversary of the famous “Piltdown Man” hoax from a gravel pit in the sound of England. There are some thoughts and comments on Gilbert White’s review of the natural history of Selbourne at Evolving Thoughts along with views on his use of the word ‘instinct’ to describe natural behaviour.

Moving down through Europe, from a conference in Venice, Italy, comes a discussion about the great wrinkled finger debate from a paper by Stephen J. Gould (no relation).  Heading out to Sweden, we have research from the University of Uppsala on the genetic changes that turn a wolf into a dog. In a subject very dear to my heart, researchers have also found that the European diamondback moth has evolved resistance to several insecticides and may provide exciting information about how insects adapt to their host plants.

Middle East and Africa

Before heading into Africa, there’s time to take just a brief stop-off into the Atlantic Ocean, to do some whale-watching, and ponder what size whales should be in a post from NeuroDojo. On the mainland of Africa, we can go back in time to the Middle Permian era, where gigantic reptiles roamed over the earth including the gorgonopsid, a reptile with some mammal-like characteristics in a post from the wonderful Fins to Feet blog. Slightly further forward in time from that, Kathy Orlinsky wonders about the climbing abilities of ancient hominins.

Asia

Heading out into Asia  the Beijing Normal University gives us the first piece of microbiological research featured in this carnival looking at how bacteria cope with phage and antibiotic challenges. The post author (guest poster Dr. Levi Morran) confirms his view of what I’ve always suspected; the apocalypse will be bacterial.

Australasia

Not strictly set in Australia, but over at The Loom Carl Zimmer talks about the origin of venom and why it is that some animals can be so deadly. I’ve always associated dangerous and poisonous animals with Australia myself, but of course they exist all over the world.

South America

Stopping by at the Federal University of Rio de Janeiro, there’s some wonderful research done on the mathematical modelling of Evolution by  Gregory Chaitin, whose book is reviewed at Synthetic Daisies: Metabiology and the Evolutionary Proof. There’s also a fascinating post from Victor Frankel on evolution around the Isthmus of Panama, that tiny stretch of land where North and South America join.

North America

From the Isthmus of Panama up to the final continent of this round-the-world trip: North America. In Utah we find two researchers coming up with some rather … interesting explanations as to the origins of fists, which T. Ryan Gregory dissects while cautioning about Just So stories. From the sunny Bahamas comes a story of adaptive radiation of some very beautiful fish by Noah Matton. At the BEACON centre in Michigan State University some more bacterial reserach is being carried out, student Mike Wiser is working on a long-term bacterial growth study, looking at bacterial evolution and mutation through hundreds and thousands of generations. There’s also a post about the work of Niles Eldredge in using networks rather than trees to create phylogenetic diagrams.

Back Home Again

Just because I’ve been around the world doesn’t mean I’ve seen everything there is to see; there are still plenty more marvels out there and not all of them revolve around animals, or even science. Marc Cadotte has a book review of Edmund Russell’s Evolutionary History which examines how humans have shaped the evolutionary landscape, while David Morrison has a truly epic post discussing the false analogies between anthrology and biology, which concludes that the genotype model is not always the best model to apply to non-biological disciplines. And finally, there’s some more bacterial stuff waiting for me, a podcast interview of Dr. White discussing the bacteria H. ducreyi and cultural evolution.

That brings an end to this edition of the Carnival of Evolution. If you’d like to see more, please visit the carnival on facebook, follow it on twitter and visit the blog which contains all the old editions. If you’re writing your own blog posts with an evolutionary theme, please go submit them for the next issue, which will be out on the 1st of March.

Diagram of the membrane that surrounds human cells. The two layers of phospholipids can be seen (blue and while spheres with the lipid tails pointing inwards) studded with bright red proteins. The yellow blobs within the phospholipid layer are cholesterol. Image from the National Institute of Standards and Tchnology - link below

There are two ways cells can get hold of the cholesterol needed for the membranes, by using food sources containing low-density lipoproteins (LDL), or by synthesising it within the cell. Defects in the cholesterol synthesis pathway can increase the likelihood of the cell breaking down through apoptosis or due to oxidative stress. Around 20-25% of the cell membrane is made up of cholesterol in mammalian cells.

Despite the above diagram, the phosolipid molecules are not rigidly stuck in place within the cell membrane, as long as they keep the phosphate facing outwards and the tails inwards both they and the steroids can travel around the membrane. This means that some areas will gather clumps of cholesterol, known as lipid rafts, which play important roles in cell signalling, membrane shape, and of course, bacterial invasion. Many bacteria target these lipid rafts when looking for places to attach onto human cells, and they act as the first point of cellular invasion.

Researchers found that limiting the amount of cholesterol in the mammalian cell membrane (by blocking the internal cholesterol synthesis pathway) led to far less effective invasion of bacteria and bacterial toxins. The diagram below shows an electron micrograph of mouse tissue, in the one on the left the cells cannot make cholesterol and in the one on the right the cells have normal cholesterol-making activity. Little black arrows show where the toxins produced by the cholera bacteria have been taken up by the cells.

Scale bar = 500 nm. Image from reference 1.

Only 9% of −cholesterol cells contained 10 or more toxin-containing vacuoles, compared to 80% of the +cholesterol cells.

Repeating the assay shown above with different bacterial strains revealed that the bacteria C. burnetii also require cholesterol to enter the cells,  while Salmonella typhimurium and Chlamydia trachomatis enter both cholesterol and non-cholesterol containing cells at the same rate. While lipid rafts are required for cell entry by some bacteria, it seems that others do not seem to rely on them.

(A). The number of internalized C. trachomatis was unchanged between −cholesterol and +cholesterol calls. In contrast, internalization of C. burnetii was decreased by 87% (p = 0.0009) in −cholesterol calls. (B). Wild type S. Typhimurium and a mutant without the Salmonella toxins invaded −cholesterol and +cholesterol cells with equal efficiency. Image from reference 1.

The researchers suggest that as well as affecting bacterial cell attachment to the cell surface, the cholesterol may also be vital for the uptake of certain bacteria and their internal transport. It may therefore be possible that the cholesterol is not only important for helping bacteria enter the cells, but also for their further growth and development inside the host cell.

The particularly interesting thing about this research was the method used to remove cholesterol from the cells. Because it is such an important membrane component, chemical methods tend to drastically alter the shape of the cells which causes more problems for bacteria trying to get in. For this paper, the researchers instead targeted the cholesterol synthesis pathway, removing the final enzyme. This system therefore allows a cholesterol-free environment to be explored without causing any significant changes to the cell membrane integrity.

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Credit link for image 1

Reference 1: Gilk SD, Cockrell DC, Luterbach C, Hansen B, Knodler LA, et al. (2013) Bacterial Colonization of Host Cells in the Absence of Cholesterol. PLoS Pathog 9(1): e1003107. doi:10.1371/journal.ppat.1003107

A simple outline of the process of photosynthesis, showing the light reactions and the Calvin cycle. By Daniel Mayer, credit link below.

Turning carbon dioxide into sugar may sound fairly magical, but it becomes a more conceivable when you consider that both carbon dioxide (CO2) and glucose (C6H12O6) contain roughly the same sort of elements. The Calvin cycle just adds on all the extra elements required. Having said that, the ‘just’ is still a fairly major task, requiring different enzymes all working in the correct order.

The carbon dioxide molecules diffuse into the cells through small holes in the underside of the leaf. The first enzyme that picks them up is called Rubisco. Despite sounding like a small corporate venture, Rubisco is actually one of the most important enzymes in the world. Without Rubisco, plants would not be able to make sugars, which means that animals would not be able to survive on plants.

Rubisco catalysis the connection of the small molecule ribulose-1.5-bisphosphate phosphate (RuBP) to carbon dioxide – therefore fixing the inorganic CO2 as an organic molecule. RuBP contains 5 carbons as well as oxygen, hydrogen and phosphate and it bonds to the CO2 to create a 6 carbon molecule. This promptly splits into two small 3 carbon molecules as shown in the reaction scheme below:

RuBP reacting with carbon dioxide. This reaction also requires water molecules. Image credit below.

These two 3 carbon molecules then go through a series of reduction stages, during which they react with the ATP (energy molecule) and NADPH (reducing molecule) that were produced during the light reactions of photosynthesis. Even though the Calvin cycle doesn’t require any light itself, it is completely reliant on molecules created by the light-reactions. This stage creates two molecules of the 3-carbon “glyceraldehyde 3-phosphate” – which can be turned into useful plant sugars by further reactions.

In order to continue running the Calvin cycle, and the reason that it is a cycle rather than just a process, the Rubisco must be recycled in order to go and pick up new carbon dioxide molecules. To do this also requires molecules of glyceraldehyde-3-phosphate – which are modified and then joined together to re-form the RuBP. The final result of all this is that for every 3 rounds of the cycle three molecules of RuBP go in, 3 RuBP come out, and one new glyceraldehyde 3-phosphate is made.

When put like that it might seem like a lot of effort for very little, in reality it’s a very stable and important cycle. As the components of the cycle are all recycled, the Rubisco can just keep picking up carbon dioxide and shooting out sugars, turning an inorganic gas into an energy molecule useful for life.

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Credit link for image 1

Credit link for image 2

Featured Image by  Jon Sullivan. More of his awesome photos can be found here.

The first draft of this was incorrectly posted too early and contained some of my place-holder notation and Unchecked Mathematics. Apologies to all who followed a broken link, and thanks to the commentators for alerting me to the issue (it slipped my mind that I’d set it to publish).

The rod shaped Clostridium difficile bacteria, image from the Centers for Disease Control and Prevention, part of the United States Department of Health and Human Services.

C. difficile produces two main toxic molecules, labelled TcdA and TcdB. These toxins lead to fluid secretion, inflammation, and colonic tissue damage associated with the bacterial infection. Both of the toxins get into human cells and disrupt the internal structure, causing them to look round and blobby under a microscope. Both toxins are capable of activating apoptosis – the internal pathway used by cells to kill themselves, however some studies have shown that when cells were exposed to high concentrations of TcdB they tended to just destroy themselves (necrosis) without using the apoptotic pathway.

Normal cells on the left, cells treated with TcdB in the middle showing the rounded phenotype. Cells treated with high concentrations of TcdB on the right showing necrosis (cell death). Image from reference.

Before it is sent out of the bacterial cell to cause its damage, the TcdB molecule is processed inside the cell. This processing releases the catalytic effector part of the TcdB molecule which can then damage the host cell. By knocking out the genes responsible for this processing, the researchers were able to create TcdB mutants – unprocessed forms of the TcdB. They then tested both the normal wild-type and the unprocessed TcdB to see what affect they had on human cells (using both standard HeLa cells, and cells derived from the human colon). Surprisingly, both forms of TcdB were able to kill the human cells. This suggests that the internal processing is not required for TcdB necrosis.

Graph showing cell viability at increasing concentrations of TcdB for five different unprocessed mutants and wild type. Image from reference.

The researchers suggest that while low concentrations of TcdB have a “cytopathic effect” i.e they damage the cells leading to apoptosis, high concentrations of TcdB lead to a “cytotoxic effect” killing the cells off without causing the controlled cell death of apoptosis. Having shown the effects in culture the researchers then explored whether the same effect would be seen in actual tissues, using pig colon tissue. No significant difference was shown between the wild-type and unprocessed mutant; in the case of this toxin, internal processing is not required to cause cell damage.

Although the results might seem negative when put in those terms, it’s very important for pathology research to explore the effects of cell toxins, and to rule out areas where future research would not be useful for revealing anti-bacterial therapies. In this case, any antibiotic that targets the processing of TcdB would not be of use for therapy, although it would prevent the molecule being processed it would not stop the bacteria from causing damage.

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Reference:  Chumbler NM, Farrow MA, Lapierre LA, Franklin JL, Haslam D, et al. (2012)Clostridium difficile Toxin B Causes Epithelial Cell Necrosis through an Autoprocessing-Independent Mechanism. PLoS Pathog 8(12): e1003072. doi:10.1371/journal.ppat.1003072

In a previous post I wrote about how two-component systems evolved in bacteria while dying out in animals, so for this week I thought I’d give a recent example of work on a two-component system in Pseudomonas aeruginosa. P. aeruginosa is a dangerous human pathogen that is particularly notorious for forming biofilms – a large bacterial city, consisting of lots of bacteria all held together within a sticky mess.

P. Aeruginosa from wikimedia commons.

P.  aeruginosa has a large number of two-component systems, many of which are involved in managing the transition from the free-floating planktonic state to the colony-based community lifestyles of the biofilm. In particular the researchers were looking at a system labelled “PprAB” where PprA is the sensor (which recieves the input) and PprB is the reciever (which creates the output by turning on certain genes).

Activating this PprAB system switches the bacteria from free-living single cells to a large multi-cellular biofilm. PprB activation is also associated with increased susceptibility to antibiotics and reduced virulence. As most biofilms are incredibly antibiotic resistant and usually increase virulence, the researchers decided to take a closer look at the PprAB system and what it was activating.

In order to do this, the researchers used a mutant strain of the bacteria labelled PprBK which constantly expressed PprB. They compared the growth of this strain to a normal wild type bacteria labelled PAO1. They found that the cells constantly expressing PprB (the PprBK strain) formed biofilms a lot faster than the wild type,with cells clumping together after just one day:

Biofilm formation after one day - wild-type cells on the left, PprB expressing cells on the right, which are already starting to clump! Image from the reference.

They also tested the virulence of the two strains in human HeLa cells using an LDH release assay. The more LDH is released, the more the human cells are damaged and dying. The graph below shows the effects of both strains on cell death over the course of three hours, with the wild-type PAO1 far more virulent and leading to more cell death:

Statistical differences evaluated using a t-test (*:<0.05, **:<0.0.01, ***:<0.001). Image from the reference below.

Another observation that the researchers made was that this biofilm is more susceptible to the antibiotic tobramycin. Several of the genes that the PprAB system activates are used to increase the permeability of the cell membrane (to allow signal molecules in so all the cells can talk to each other and form a biofilm at the same time) and to change the composition of the cell matrix. it looks like trying to form a biofilm quickly makes each cell more permeable, allowing antibiotic molecules easier access inside. The speed of biofilm formation may also be related to the loss of virulence - rather than forming a coordinated attack, the cells are just trying to build a city as quickly as possible.

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Reference: de Bentzmann S, Giraud C, Bernard CS, Calderon V, Ewald F, et al. (2012) Unique Biofilm Signature, Drug Susceptibility and Decreased Virulence in Drosophila through thePseudomonas aeruginosa Two-Component System PprAB. PLoS Pathog 8(11): e1003052. doi:10.1371/journal.ppat.1003052

It wasn’t until later that I found out what the pH scale actually meant, and when I did, it blew my mind completely. I’d always assumed that it either stood for two words or was named after someone. I did sometimes wonder why it was written like that (small p, capital H), but never enough to seriously ask about it.

For starters, that H isn’t a H. The reason it’s capitalised is because it’s the symbol for hydrogen.

The p isn’t a p either, it’s a letter used as a shorthand for a mathematical operation. To be specific, the operation “-log10″. What I’d been assuming were the letters pH was actually a scientific formula, -log10 of the concentration of hydrogen ions:

pH = – log₁₀[H+]

With that in mind, the pH scale made a lot more sense as a measure of acidity. Acids have a few different definitions, but overall they are substances that can generate hydrogen ions when in a solution. pH is a way of showing how many hydrogen ions they release without having to count up all the ions. pH also has no units, which is very useful for people like me who find units confusing.

In order to be acidic then, a substance must contain hydrogen, in a form that can be released into water. Substances such as CH4 (methane) are not acidic as all four hydrogens are bound very tightly to the carbon and are not going anywhere. CH4 has a neutral pH, around 7. On the other hand, substances such as hydrochloric acid, HCl, are held together by polar ionic bonds and when placed into water the hydrogen will break away to form hydrogen ions, making the liquid acidic. HCl therefore has a very low pH and is a very strong acid.

Weak acids, with pH 5 or 6 are slightly more complex. These are formed when a compound can release hydrogen ions, but only very weakly. Examples are often organic compounds, which while they have many hydrogens in their chemical structure, very few of these can break away in solution. Acetic acid (HC₂H₃O₂) is a weak acid, as only the hydrogen at the front of the equation that can dissociate, and it is not hugely energetically favourable for it to do so.

The pH scale, with a list of substances at each pH. Acids at the top, bases at the bottom. Image credit below.

I do like the fact that “pH” has an actual chemical and mathematical meaning, rather than just being a random set of letters. Although the mathematics of acidity can get a little complex the further into chemistry you go, the -log10[H+] is still the major definition of both acidity and alkalinity.

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Credit link for image 2

The components of the two-component signalling system. Picture (c) me.

The picture above shows this process happening. The ‘communication’ of the message from the sensor to the responder, as shown by the coloured arrows, is carried out by transferring phosphate molecules. The signal interacting with the sensor causes the sensor to autophosphorylate (phosphorylate itself) and then pass the phosphate molecule onto the responder to trigger the response. The letters “H” and “D” are the actual amino-acids being phosphorylated; Histadine and Aspartate.

Although Two-Component Systems (TCS) are found in all three superkingdoms of life (archaea, bacteria and eukaryotes) they are suspiciously absent from the animal kingdom. Plants have them, as do fungi and several protazoa, but they just aren’t present in animals. For this reason they’ve been looked into as potential antibiotic targets as knocking out the Two-Component Systems of most bacteria is fatal.

Why don’t animals use TCS? To answer this you have to start looking at the evolution of the system itself, because despite being nominally present in eukaryotes such as plants and fungi, TCS are used very differently. Bacteria use TCS for sensing a wide variety of signals; stress, metabolism, nutrient regulation, chemotaxis, pathogen-host interactions etc. In eukaryotes on the other hand they are used sparingly; for ethylene responses and photosensitivity in plants and osmoregulation in fungi and slime moulds.

Bacteria (especially soil bacteria which have a lot of environment to sense) can contain up to 50 TCS although many internal parasite bacteria contain a lot fewer. The maximum number found in archaea is around 20 and they are even scarer in eukaryotes with only one in bakers yeast (Saccharomyces cerevisiae – one sensor kinase and three response regulators). None have yet been found in any animal genomes, or in the protist genomes as far as I know (although it is possible recent protist research may have unearthed a few)

Comparing the TCS genes of bacteria, archaea and eukaryotes leads to the interesting conclusion that the bacterial and eukaryotic systems are far more closely related than the archaeal, and in fact are thought to be monophyletic (all evolved from a single common ancestor). In contrast, the archaeal TCS appear to be polyphyletic (several ancestors) and some archaea lack TCS entirely. It’s therefore thought that TCS originated in bacteria and spread by horizontal gene transfer to both archaea and eukaryotes. As horizontal gene transfer relies on DNA moving from one species to another, no further transfer to eukaryotes could occur after they developed larger cells with a nuclear membrane. In eukaryotes very little further diversification took place, whereas the bacterial TCS diversified widely, and occasionally passed new systems back to the archaea. I’ve tried to show this in the diagram below:

The passage of genes for two-component-systems through the three superkingdoms of life

The diagram above attempts to show the movement of the TCS genes through the three superkingdoms of life. Red arrows show the horizontal transfer (straight arrows) and gene duplication (curved arrows) of TCS genes. No horizontal gene transfer can take place in eukaryotes after the nuclear membrane (well….it can do but is very, very rare) although gene duplication may still have occurred.

The eukaryotic superkingdom appears not to have contained very many of these TCS genes to start with, and the animal kingdom may just have lost the very few it possessed. This makes sense from the point of view of cellular control because while TCS are very useful in bacteria with their small genome and independent lifestyles, it’s less clear how useful they are in eukaryotes as a whole. Introducing a membrane around the nucleus makes it harder for proteins to get in and bind to the DNA, and in a large complicated cell it’s harder for a simple two-component system to sense what’s going on. Added to which, cells inside a multicellular organism don’t really need to sense what’s going on, they get told what’s going on by the surrounding cells and circulating hormones.

Whatever the reason though it is clear that despite this system being vital for bacteria it isn’t used widely, or most likely at all, in animals. Research into this would be particularly useful against opportunistic pathogens which tend to have a large selection of two-component systems to allow them to adapt to different lifestyles depending on the conditions of their immediate environment.

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Kristin K. Koretke , Andrei N. Lupas , Patrick V. Warren , Martin Rosenberg , and James R. Brown (2000). Evolution of Two-Component Signal Transduction Mol Biol Evol, 17, 1956-1970

Wolanin PM, Thomason PA, & Stock JB (2002). Histidine protein kinases: key signal transducers outside the animal kingdom. Genome biology, 3 (10) PMID: 12372152

Everything on earth is made up of combinations of different elements – all of which can be found on the periodic table. Considering that the periodic table contains 118 elements it seems a pity that organic life tends to feature only five or six of those elements in any vast quantities. The main one being carbon.

As well as being the main element in organic matter, carbon atoms are the only element in both graphite and diamond. Image credit below.

It would be impossible for life on earth to exist without carbon. Carbon is the main component of sugars, proteins, fats, DNA, muscle tissue, pretty much everything in your body. The reason carbon is so special is down to the electron configuration of the individual atoms. Electrons exist in concentric ‘shells’ around the central nucleus and carbon has four electrons in its outermost shell. As the most stable thing for an atom to have is eight electrons, this means that each carbon can form four bonds with surrounding atoms.

Carbon forming four bonds with four hydrogen atoms.

Each bond in the above molecule is formed by the sharing of two electrons; one from the carbon and one from the hydrogen. The ability to form four bonds isn’t restricted to carbon though, it’s a property of every atom with four outer electrons, including silicon, tin and lead. What’s special about carbon, and the reason that silicon-based lifeforms are restricted to science fiction (and lead-based lifeforms are hardly ever mentioned) is that it can form double-bonds which share more than one electron with another atom, as shown below:

Each carbon has formed four bonds (four lines coming out of each C) but two of the bonds are with the same molecule.

Why is carbon able to do this while silicon can’t? Although the bonds are all drawn as straight lines in the diagram above, in real life not all bonds are equal. The double bond consists of two different types of bond. Each bond is made up of two electron orbitals (one from each atom) which have overlapped. The easiest way to think of an orbital without getting into some serious physics is as a blurry sort of zone in which a fast-moving electron is most likely to be whizzing about. When two orbitals overlap, you have double the space which two electrons can whiz around in.

The single bond is formed by two circular orbitals overlapping and surrounding both atoms:

Two spherical orbitals overlap and share electrons and electron-space. The carbon nucleus is in the centre of each sphere.

The second bond is formed slightly differently. The electrons that form these bonds are not in a spherical orbital around the nucleus, they are in oval-shaped orbitals that protrude above and below the nucleus. When they overlap the bond forms above and below the first bond, as shown in the diagram:

The first bond is shown by the line surrounded by the fuzzy green plane. Image credit below

So why can carbon and not silicon manage this double-bond trick? The answer lies in the size. Carbon is the smallest of all the atoms with four outermost electrons, which means that the electrons in the above-and-below orbitals are close enough to overlap and form that second bond. For silicon however, there are more electron orbitals in the way, the entire atom is bigger, and it is almost impossible for the outer orbitals to get close enough to form a double bond. This is why carbon dioxide is is a small gaseous molecule consisting of two oxygens both forming a double bond with a single carbon while silicon dioxide is a massive behemoth of a molecule made of huge numbers of alternating oxygen and silicon atoms and is more commonly known as sand.

Silicon dioxide (sand) on the left, carbon dioxide on the right

You can just about get silicon-silicon double bonds if you try hard, but they are fairly unstable and will take any chance they can to loose that double-bond in favour of forming another single one. Carbon-carbon double bonds on the other hand form naturally and easily, and are crucial for every living organism on earth. If there were to be silicon-based lifeforms, the sheer chemistry of their atoms means that they would have to be built along very different lines to life on earth.

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Although milk is exclusively a mammalian production, some birds, such as pigeons, penguins and flamingos, produce a milk-like substance which provides similar benefits to their young. Both female and the male pigeons produce it in their crop, and like mammalian milk production is controlled by the hormone prolactin. It contains protein (60%), fat (32–36%), carbohydrate (1–3%), minerals (calcium, potassium, sodium and phosphorus), IgA antibodies (important for the immune system) and assorted non-pathogenic bacteria.

Two pigeons in the UK. Photo by Mr SG from wikimedia commons (credit link below)

In order to explore the benefits of pigeon milk on the young, researchers fed the milk to baby chickens to see how they benefited from it. They found that after 7 days, the milk-fed chickens were around 12.5% heavier than the non-milk-fed control group and were also expressing different genes in the gut associated lyphoid tissue (where the immune system and the gut come into contact). Milk fed chickens had a much greater expression of the immunoglobulin IgA than the non milk fed, as shown in the graph below:

Differences in IgA expresion in the pigeon milk chickens "PM-fed" and the control group. Image from reference 1 below. *p=0.033

As well as affecting the immune system, feeding pigeon milk to the chickens also changed the composition of bacteria within the chicken cecum (a part of the gut). Pigeon milk fed chickens had a much greater bacterial diversity, at both phylum and genus levels. Unsurprisingly, milk fed chickens contained bacteria found within the pigeon milk (originally produced to go inside little pigeons) although not all of the bacteria present in pigeon milk had managed to survive in the chickens.

The reason chickens were used for this experiment rather than pigeons (which would seem more natural) is that pigeon milk contains so many benefits required by baby pigeons that they end up dead or very sickly if you try to bring them up without it. The fact that pigeon milk provides significant growth advantages for chickens is also interesting for the farming industries. Nobody is particularly fussed about making bigger pigeons, but bigger chickens can lead to more money, although the current lack of formula pigeon-milk means that this is not a viable strategy for chicken farming.

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Credit link for image 1

Reference 1: Gillespie MJ, Stanley D, Chen H, Donald JA, Nicholas KR, et al. (2012) Functional Similarities between Pigeon ‘Milk’ and Mammalian Milk: Induction of Immune Gene Expression and Modification of the Microbiota. PLoS ONE 7(10): e48363. doi:10.1371/journal.pone.0048363

The respiratory disease in question is COPD, which stands for chronic obstructive pulmonary disorder. It’s caused primarily by smoke getting into the lungs, from tobacco or industrial processes, and leads to narrowed airways and overproduction of mucus. It’s not really curable, although many medications exist to help people live with it, and to slow down the progression of the disease.

Healthy lung and a lung with COPD - from the National Heart Lung and Blood Institute, credit below.

In order to explore which bacteria were present in healthy smokers, COPD patients, and people who had never smoked, researchers used massively parallel pyrosequencing. This technique allows the bacteria to be identified by their DNA without the need for producing bacterial cultures, so even ‘unculturable’ bacteria can be identified.

In the smokers, never-smoked, and patients with mild COPD a far wider range of bacterial diversity was found than in patients with severe COPD who tended to have a much smaller number of different bacterial species. There were no specific bacterial species common for each group, the major difference appeared to be in diversity. The most commonly found bacteria in the healthy subjects included Pseudomonas, Streptococcus, Prevotella and Fusobacterium which the researchers suggested may make up a core lung microbiome.

As a secondary analysis, the researchers also looked at bacterial populations in very specific areas of the lung, and found that there was no homology of bacterial species throughout the whole lung. In some cases one small microenvironment in the lung could be exclusively populated by one form of bacteria. Samples from different places in the lungs could therefore result in a very different view of the bacterial microflora in the lungs.

Lung biopsy showing normal lung tissue - from the Columbia University Medical Centre, credit link below

Although this research is fascinating, it is important to note that despite differences seen the microflora cannot be used as a diagnostic for COPD as there were no exclusive differences seen in this small sample size between diseased and healthy lung flora. The small patches of different microflora may suggest areas that are more prone to developing COPD and other respiratory diseases, in the same way that patches of different skin bacterial flora can be more prone to dermatitis and skin diseases.

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Credit link for image 1

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Reference: Erb-Downward JR, Thompson DL, Han MK, Freeman CM, McCloskey L, et al. (2011) Analysis of the Lung Microbiome in the “Healthy” Smoker and in COPD. PLoS ONE 6(2): e16384. doi:10.1371/journal.pone.0016384

There 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 they manage within the confines of a single cell, and I love that you can go into a bacterial cell fairly easily and just mess around with the 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. I’ve covered how natural throat bacteria can help destroy dangerous pathogens such as MRSA Staph aureus so in this post 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 (henceforth shortened to C. jejuni) is that it’s very fussy about the amount of oxygen it’s in. C. jejuni is microaerophilic, which means it needs oxygen, but only small amounts,. If you give it too much all the cells will die. This problem was solved in my old lab by using tightly sealed containers and special packs of … stuff … which were put inside the containers to create the right conditions. This raises an important question; if the bacteria have to be coddled and protected just to culture on a plate in the lab then how on earth do they survive and grow on the surface of chicken meat?

A recent study (reference below) found that the C. jejuni were being aided by surrounding bacteria. The picture below shows both C. jejuni and a bacteria called Pseudomonas putida in close interaction, with long fibre-like structures connecting them. No one 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 C. jejuni is the more slender and slightly spiral shaped bacterium 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 P. putida was required for C. jejuni survival – a range of different C. jejuni strains were grown in both the presence and absence of the supporting P. putida to see how long they could survive in completely aerobic conditions. The results are almost hilarious, without the help of the P. putida bacteria the Campylobacter just die, really quickly:

Image taken from the reference below

Figure A (top) shows the C. jejuni with P. putida grown as well, Figure B (beneath) shows the C. jejuni grown alone. The graphs show the count of living cells over time, and it’s clear that without the help of P. putida the C. jejuni dies much quicker. Interestingly it was found that the interaction between particular strains of both C. jejuni and P. putida was fairly specific as well. As you can see in the graph above, only three of the C. jejuni strains have survived past 50 hours with the help of this particular P. putida, although they surive longer than with no help at all.

As P. putida are aerobic, the most likely explanation for how they are helping is that they create a microenvironment of decreased oxygen within their immediate surroundings. This is the kind of environment that it is thought C. jejuni will naturally migrate to. It’s interesting to consider that this might be less of a mutual helping relationship and more of a seriously exploitative one, with the C. jejuni swarming as quickly as possible towards the environment created by the P. putida and then wrapping them all up in a sticky mesh to stop them moving away.

This special relationship is not applicable for all C. jejuni, in other environments such as in humans and in chicken poo the C. jejuni 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 C. jejuni) but this is another reminder that despite laboratory conditions bacteria do not just exist in isolation. They inhabit a whole tiny world, with challenges of its own, surrounded by other bacteria that change their environment both for better and for worse.

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Reference: 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

Copper can cycle between two different ionic forms: Cu⁺ and Cu²⁺. Its most important use is as an electron carrier for the process of creating the energy-rich molecule ATP. Although it is vital to the cell in small amounts, in large quantities it can become toxic. By ‘large quantities’ we are talking about greater than one atom per cell, so anyone suggesting that consuming or being close to large quantities of copper is beneficial for your health is probably wrong.

Copper mineral. Consumption not recommended. Image credit below.

Large amounts of copper are toxic for pretty much all living cells, which can be exploited by the human immune system to fight off invading bacteria. When macrophages (the white blood cells that surround and break down invading bacteria) are activated and engulf a bacteria they start to accumulate copper ions inside the cell, in particular in the part of the cell that has just engulfed the bacteria. As a response, many bacteria have increased resistance to copper ions, and those that have lost this resistance are more susceptible to being broken down by the macrophages. While large amounts of copper are indeed bad for your cells, inadequate copper levels can compromise the immune system and make people more likely to suffer from bacterial infection.

As bacterial cells are much smaller and more compact than human cells, they tend to keep all their copper-containing enzymes very close to the cell membrane, to prevent them causing unwanted redox reactions inside the cell. When presented with elevated copper levels, the bacteria can turn on a group of genes which produce proteins capable of shuttling copper out of the cell. These are the copper resistance genes. As a large number of pathogenic bacteria contain these copper-resistant genes, and as there are very few copper-containing enzymes in bacteria, it is likely that an excess rather than a deficit of copper is a problem for bacteria inside the human body.

If you do feel you need more copper in your life, these are the foods you should be eating! From the Agricultural Research Service, the research agency of the United States Department of Agriculture, credit below.

The use of copper as a strategy against bacteria may have important clinical implications. While using copper as a therapeutic strategy would be potentially dangerous for the host, targeting bacterial copper resistance may provide help for the macrophages and immune system cells to clear the bacteria naturally. Developing a way to compromise the bacteria by reducing their copper resistance could also be used along with antibiotic therapy in order to increase the effectiveness of the antibiotic.

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Credit link for image 1

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Reference: Festa RA, Thiele DJ (2012) Copper at the Front Line of the Host-Pathogen Battle. PLoS Pathog 8(9): e1002887. doi:10.1371/journal.ppat.1002887

For those who have not yet entered the matrix, cystitis is a bacterial urinary tract or bladder infection. The main symptoms are a strong and desperate desire to pee coupled with pee that feels like burning acid, which is not a happy combination. If left for too long, eventually you start to pee blood which is completely terrifying the first time it happens. It tends to occur when bacteria from the outside world make their way into the urethra (the little pipe where pee comes out) and given that women have far shorter urethras than men, it can happen to them fairly frequently. Nobody is quite sure what leads to an attack, or why some people get it far more often than others, but anecdotal evidence suggests a strong correlation with sexual activity, personal hygiene, and/or dehydration.

This is your bladder on cystitis. The purple dots are the white blood cells that cause inflammation. Image credit below.

There isn’t a huge amount of research on cystitis, which is why going to the doctor tends to result in not much more than vague murmurings about cranberries, loose underwear and drinking plenty of water. (As an aside, it wasn’t until my third attack that I was given the only piece of really useful anti-cystitis advice I’ve ever received: pee after sex). So I’m always interested in new papers that come out discussing it. In particular there’s a really interesting paper in PLoS Pathogens at the moment (reference below) detailing the life cycle of the bacteria that cause cystitis.

The most common bacterial cause of cystitis is E. coli, because there tends to be quite a lot of it hanging around on the body. Because of this, the body usually has quite an efficient innate immune response that keeps the bacteria from getting anywhere near the tissues or bloodstream, but for some reason in cystitis this response simple isn’t effective. This may be genetic factors in some women, for others there are clear physical reasons (such as a strangely formed urinary tract, or a problem with the kidneys) and is definitely helped by the fact that the bladder is a sort of water reservoir, where bacteria can settle down and flourish.

Bacterial colonies on the surface of a hot spring. Generally bacteria like bodies of warm water. Credit link below.

The first challenge the bacteria face is getting up the urinary tract in the first place. True, it’s fairly short in women, but it’s still a piece of human tissue protected with white-blood cells and with a regular streams of water running through it. Pathogenic bacteria have a wide range of adhesive molecules and other binding agents, which, when they bind to a surface, cause the bacterium to change from a floating blob to a blob capable of crawling along a surface. The particular E. coli associated with cystitis has a whole host of other molecules to help with binding to urinary tract tissue, although it is not yet clear which are directly associated with the infection in humans (research has been done mainly on mice). The E. coli is also able to bind and sense urine, and uses the rush of urine flow as a signal to cling tightly to the tissue where it’s attached.

Once they get into the bladder, the E. coli are then internalised by the host-cells in order to destroy them. While most of the bacteria will be killed in this way, others actually start to form little biofilm-like clusters inside the cells. This highly organised little bacterial city is surrounded by a sticky mess of proteins and sugars, which protect it from the cell trying to kill it.

Remains of a Staphylococcus infection from a catheter. The stringy gooey bits between the spherical bacteria are the remains of the biofilm that would have surrounded the entire colony. Image credit below

This is where the story starts turning into horror sci-fi. Remember this is all happening inside a human bladder.

The biofilm starts to take over the cell it’s growing in, engulfing the nucleus and filling the cytoplasm. It gets so large that the cell actually starts to bulge inwards, into the bladder space. This large bulging cell then starts to extrude bacterial filaments that grow outwards from the surface, latch onto surrounding cells, and start infecting them as well. Not only are the bacteria starting to take over the cells in the bladder, they also suppress the answering human immune system by suppressing the production of cytokines (small immune system signalling molecules) and encouraging the production of IDO, a molecule which tells the immune system that enough cytokines have been produced and they don’t need to make any more. By breaking down communication channels within the immune system, the bacteria can evade attack.

The problem with the outer tissue layer of the bladder though, is that it’s continually being shed through exfoiliation, which makes it a rather unsafe place to have a bacterial colony. For longer lasting survival, the bacteria can burrow down to the underlying basal epithelium and surround themselves with a protective network of actin molecules. These bacteria are resistant to antibiotic attack, and can simply lie dormant for several months. The mechanisms by which it stays like this, or by which it initiates recurrent cystitis, are still a mystery.

One of the main problems with recurrent cystitis, from the point of view of the sufferer, is that each bout of infection leaves the tissues inflamed and, even after healing, more likely to succumb to an infection again. Like asthma, diabetes, and various other long-term diseases and symptoms, it’s not something that can be cured, it’s something that has to be lived with.

If you do suffer from cystitis, the internet contains many sources of advice, all of which tend to list the same sort of things. The methods I’ve found most work for me are: peeing after sex, drinking 4/5 pints of water a day and taking bicarbonate of soda if I start to feel twinges. Despite what they tell you, there is no real evidence for the cranberries.

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Credit for image 1

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Reference: Jorgensen I, Seed PC (2012) How to Make It in the Urinary Tract: A Tutorial by Escherichia coli. PLoS Pathog 8(10): e1002907. doi:10.1371/journal.ppat.1002907

Flesh Eating Bacteria Can Infect Anyone – What You Should Know

What is it?

Necrotizing fasciitis, commonly known as flesh eating bacteria, infects various layers of the skin. In most cases, an immunocompromised individual, such as a smoker, drug addict, diabetic, or cancer patient is most vulnerable, although healthy individuals are also susceptible. A recent surge of media coverage for this disease has brought about widespread awareness – and here are some things you should know.

High magnification micrograph of necrotizing fasciitis. H&E stain - the purple sections are bacteria. Image credit below.

How does it infect?

The causal agent can be one of many infectious bacteria, including Staphylococcus aureus, Streptococcus pyogenes, Clostridum perfringens, or Aeromonas hydrophila. These organisms can be part of normal human skin microflora or found in common settings such as a freshwater environment. Entry through just a small or even non-apparent cut may be enough to induce inflammation and subsequently, tissue necrosis. A common misconception comes from the name “flesh eating bacteria” itself, as the bacteria isn’t actually damaging the tissue, rather the toxins they leave behind. If left untreated, the toxins eventually spread to the rest of the body and can cause death through sepsis. It’s not uncommon for victims to undergo amputations in order to prevent systemic spread of the bacteria.

How do I treat and prevent it?

The most effective preventative measure would be to thoroughly wash your wound. Antibiotics are effective for treatment, but most doctors and victims don’t think twice about a minor cut and begin a regimen soon enough. It is only when the victim experiences excruciating pain from the infection when physicians begin to realize it is flesh eating bacteria, at which point it may be too late. Antibiotics would rid the bacteria from the body, but they do little to eliminate the toxins left behind, which continue to produce devastating effects.

NeutroPhase, a new wound cleanser recently cleared by the FDA, was used by Dr. John Crew and his nurses Randell Varilla and Thomas Allandale Rocas III, when one of his patients, Lori Madsen, fell and scraped her elbow. Dr. Crew overheard the screams of Ms. Madsen and after reviewing her symptoms, decided to use NeutroPhase in combination with negative pressure wound therapy. Ms. Madsen was discharged from the hospital within a few weeks.

The wound cleanser is a solution of pure hypochlorous acid, the same compound released during oxidative burst when white blood cells engulf foreign particles as part of the body’s immune response system. The product has recently been approved for treatment of chronic non-healing wounds such as venous, pressure, stasis, and diabetic foot ulcers as well as post-surgical and burn wounds.

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Credit link for the image

The research isolated a range of interesting compounds, labelled GP-1 to GP-12. These compounds were tested for their ability to kill the bacteria Burkholderia pseudomalia, a naturally occurring tropical/sub-tropical bacteria. Time-kill assays were performed, measuring how many bacteria both compounds killed over time, and showing a decrease in the number of bacteria after 24 hours treatment with the compounds. They also tested the compounds in mice infected with the B. pseudomalia strains, looking at the number of bacteria found in the spleen and lungs following treatment.

This crystal structure shows the drug candidate molecule GP-12 binding to one of its cellular targets, Gyrase-B, from pathogenic E. faecalis. (c) Trius Therapeutics, Inc.

To get a further picture of how these compounds were carrying out their antibiotic activity, the company also carried out research on the pharmokinetics of the drug, i.e how it works within a body. In order to discover how stable the compounds were (i.e whether they just break down inside a body) they were incubated with animal or human liver microsomes and appropriate co-factors at 37 degrees C for up to 60 min. To study the behaviour of the compounds within a body, plasma and tissue were collected from animals treated with the drug compounds, to see where they ended up, and how they behaved in the blood. The results showed compatible binding to blood proteins in both mice and human samples, which is important as it means that further mouse research is more likely to produce results relevant for humans. The drug dispersed well throughout the body, particularly appearing in the liver and kidneys (not surprising as they tend to excrete antibiotics) and only minimal amounts appearing briefly in the brain, which is also good.

The final part of the research involved testing the drugs ability to destroy pathogens in mouse models. The researchers tested the response of GP1-8 on two important clinical strains; S. pneumoniae (a Gram positive bacteria) and E. coli (a Gram negative bacteria). Excitingly, the results showed that infections from both Gram negative and Gram positive bacteria could be cleared – infections ranging from bacteria in the lung (in the case of S. pneumoniae) and in the kidneys and thigh muscle (in the case of E. coli).

There’s plenty more research to be done before these compounds start moving into actual clinical trials, but it is interesting to see the potential for new broad-spectrum antibiotics – i.e compounds that can attack a range of bacteria rather than just a few isolated species. The great thing about industrial research is that with the money and infrastructure behind the research, there is the potential for interesting compounds to be shunted through to clinical trials very efficiently, resulting in the eventual production of new pharmaceutical products.

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Thanks to Trius Therapeutics for letting me have a sneaky peak at their posters and research. Further details of their research can be found here. Please note that I am not in any way economically affiliated with this company.

We are happy to be presented as the first team from Argentina to participate it the iGEM competition.

As the first of many teams to come we have a huge responsibility and getting started was a hard job: we had to start the lab from scratch, make connections and look for sponsors most of whom had never heard about the competition. But we did it all with great confidence and we’ve found a lot of support from scientists working in Synthetic Biology and former iGEM teams. Fortunately, we got support from several organizations, including our university, UNU-Biolac and Argentina’s Science, Innovation and Production Department (MINCYT), which supported us both with funding and trust.

Image from iGEM Team Buenos Aires

In April 2012, our university held the first Latin American EMBO Global Exchange Lecture Course which at the same time was the first Latin American gathering of people interested in Synthetic Biology. Here, students from the region were invited to learn and share their thoughts. We had lecturers from invited speakers, including several of the first iGEM founders. The response was amazing, the university received students coming from Mexico to Chile and the outcomes were fabulous: Latin America is a growing region and a great promise for this upcoming discipline. We are certain that many local problems can be solved through Synthetic Biology. Furthermore, the free exchange of knowledge and technology, which are the basis of the competition, are invaluable.

In this spirit, we translated the concept of community build up into our project, where we aimed to create a community of microorganisms, in which each member is in a defined and stable proportion. If we succeed, this could be used as a standard tool in lab and industry, defining a new layer of modularity. The design of the system is based on a “Synthetic symbiosis”, in which each member of the community produces and secretes aminoacids the other members need to survive. By regulating the amount of aminoacid secreted, we could in principle control the proportions of each strain.

The coculture of strains with His and Trp auxotrophies. Image from iGEM Team Buenos Aires

Our goal is to be able to control the proportion of each strain in the coculture, just as species interactions are naturally balanced in ecosystems and we used a biobrick (shown in the image below) in order to control the export of Trp. The Trojan is a class of peptide with translocating properties that would be capable of carrying the signal (His or Trp) across the plasma membrane for secretion and into the plasma membrane of the target cell (namely, the other strain).

The Biobrick designed by the iGEM Buenos Aires team

Possible applications of our project are endless: food industry, optimization of bioreactors, bioremediation and alternative energy source generation, among others. It would also become a very useful tool for academia and medicine studies since it would allow, for example, the study of biopathways by isolating the different steps in different strains or the study of the interaction of gene products that would otherwise not fit into a single organism. And more, the innovation of our proposal is that we are introducing a new level of modularity since our main unit is the actual engineered organism and our system is the whole community.

As part of our human practices, we have related to Garage Lab, a group of Argentinian social entrepreneurs that are keen on technology and informatics. Together we plan on using the heavy metals detectors that can be constructed with the iGEM kit for the assessment of the Rio de la Plata pollution, a big environmental problem here in Buenos Aires. Work will continue after the Jamborees – this is just the start!

For more information about the team’s project, go visit their wiki page or their iGEM webpage.

A great example of this appeared in PLoS Pathogens this month (reference 1), concerning the bacteria Wolbachia. These bacteria infect insects and other arthropods and are much beloved of journalists (well, compared to other insect bacteria at least) because one of their effects is to stop insects producing male offspring (so only female survive to pass on the bacterial genome), which gives journalists the opportunity to write silly headlines.

An electon micrograph of an insect cell, with three Wolbachia bacteria inside (the large circular blobs with white lines surrounding them). Image from reference 2.

As well as passing from females onto their offspring, Wolbachia can also be transmitted horizontally, that is between insects in the same generation. In its normal host the Wolbachia is not hugely damaging (apart from removing all males from the population) but when transmitted to a new species it causes various unpleasant nervous system complications, often leading to death. Clearly, the bacteria are more virulent when they encounter a new species. However when the bacterial infection was closely examined, it was found that infected individuals of both species contained the same number of bacteria. It wasn’t just that the new species couldn’t respond to the infection, it was in fact the way they responded that was doing the damage.

As it turns out, the reason Wolbachia are more dangerous in new species isn’t because the bacteria go wild in the unexplored territory, rather it’s because the new host doesn’t know how to deal with them. The insects that are used to dealing with the presence of the bacteria have developed ways to contain the infection, or just tolerate it. New species however, tend to panic, particularly as the bacteria tend to congregate in the gonads (sex organs) and the central nervous system, which even insects understand are bad places to have bacteria.

As the bacteria are found inside cells, the best way for an insect immune system to get rid of them, is by destroying the cells that house the bacteria. Which, as previously mentioned, are mainly the gonads and the central nervous system. When the Wolbachia get into a new species, the first response of the insect is to quickly and efficiently destroy any cells which have bacteria inside them. As a consequence the unfortunate insect basically destroys its own brain, leading to various unpleasant symptoms and death.

The carpenter ant, Camponotus pennsylvanicus. Many species of Camponotus are infected with Wolbachia. Image from reference 3

Even in insects, the immune system is vital to defend animals from bacterial, fungal, and viral attacks. However it’s fascinating to see the cases where the immune system (even ‘primitave’ immune systems that consist of nothing more than infected cells quickly being removed) can lead to issues by over-reacting to a threat. The best response to the Wolbachia is for the insects to learn to deal with it, rather than to attempt to launch counter-attacks which can be damaging for the animal as a whole.

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Reference 1:Le Clec’h W, Braquart-Varnier C, Raimond M, Ferdy JB, Bouchon D, & Sicard M (2012). High virulence of wolbachia after host switching: when autophagy hurts. PLoS pathogens, 8 (8) PMID: 22876183

Reference 2: (2004) Genome Sequence of the Intracellular Bacterium Wolbachia. PLoS Biol 2(3): e76. doi:10.1371/journal.pbio.0020076

Reference 3: Wernegreen JJ (2004) Endosymbiosis: Lessons in Conflict Resolution. PLoS Biol 2(3): e68. doi:10.1371/journal.pbio.0020068

The differences between RNA (left) and DNA (right). RNA is also found as a double helix, a triple helix and a strange looped thing. Image from wikimedia commons, credit link below.

Given its power to act as an intermediary between DNA and protiens, which are two of the most important macromolecules within the cell, RNA has a huge number of jobs to do. One of those jobs is to regulate which parts of the DNA are making proteins. Not all of the DNA in the cell is being used all the time, and small pieces of RNA have the ability to show the cell which parts of the DNA need to be working at any one time.

In the case of the bacteria Streptococcus pneumoniae, the small RNAs can turn on the parts of the DNA required to make the bacteria virulent. It seems that the bacteria uses very specific RNA fragments to turn on different genes at different stages in its virulence cycle. At each stage, a specific set of small RNAs will be produced in order to control gene expression. A recent paper from PLoS Pathogens (reference below) carried out three sets of experiments to show this. Firstly, they sequenced the entire genome of the Streptococcus in order to find the sections that looked similar to other bacterial small RNAs. Once identified (they found around 89!) they specifically removed the ability of the cells to make the some of the small RNAs to see the effect that had on bacterial virulence. Finally, they looked for the targets of these RNAs, to find out which parts of the DNA expression they were actually affecting.

Streptococcus pneumoniae from the Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #262. Credit link below.

To explore the effect of removing the small RNA sections they looked at a measurement called the “competitive index” which involved infecting mice with both the wild-type and mutated bacteria (bacteria without the small RNAs) and seeing how well they compared when in competition with each other. A competitive index of one means that equal amounts of wild-type and mutant bacteria were found, less than one means that more wild-type bacteria were found and greater than one means that more of the mutated bacteria were found. As expected, in almost all individual cases the mutants performed worse than the wild-type in infection, some performing significantly badly.

Results from reference 1 below

The graph above shows the effect of removing certain sRNA from bacteria infecting the blood. Each point represents a single individual, with the lines showing the average of the strain. The scale is logarithmic, which means that while strain F5 was around 5 times worse than the wild-type on average, strain R12 was over 100 times worse. What’s also interesting is that one individual with the F5 mutation performed better than the wild type, although two of them also performed much, much worse. The paper explored the fate of mutations on virulence in the nose/throat area and the lung, with mutants performing worse in all cases.

Finding the target sites of the small RNAs was more complex, as each one appeared to both up-regulate (turn on the production) and down-regulate a large number of proteins. To explore this, the researchers carried out Northern Blots, which leave a stain for every protein produced inside the cell, for both the mutants and the wild type and then compared the similarities and differences. The graph below shows the huge differences in proteins controlled, and suggests that each small RNA has a large number of effects within the cell, controlling a range of responses including DNA repair, synthesis of nucleotides, and virulence.

The number of upregulated and downregulated proteins found for each mutation. Image from the reference below

Using RNA to send important messages to the genome is an advantageous strategy for bacteria, as it uses less energy than creating proteins to do the job, and requires less DNA to store the information. RNAs can either be very specific, or focus on multiple targets, allowing them to have very defined roles in controlling large genetic changes, such as the onset of bacterial virulence. The main task the researchers have now, is to work out the precise function of all these RNAs, and which genes they tweak to create the pathogenic bacterial cell.

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Credit link for image 1

Credit link for image 2 (Link to the CDCs Public Health Image Library)

Reference 1: Mann B, van Opijnen T, Wang J, Obert C, Wang YD, Carter R, McGoldrick DJ, Ridout G, Camilli A, Tuomanen EI, & Rosch JW (2012). Control of Virulence by Small RNAs in Streptococcus pneumoniae. PLoS pathogens, 8 (7) PMID: 22807675

One of the ones I was most proud of spotting over the weekend, was the Wall butterfly, Lasiommata megera, which is getting rather rare in England. Along the south coast of Dorset I saw a good three of them, as well as large numbers of Gatekeepers, which were everywhere.

A male wall butterfly, photo by Jörg Hempel via wikimedia commons. Credit link below.

I found that the most recognisable feature about these butterflies was the large dark band across the top wing. My first thought was that it was a fritillary, because I’ve never seen a fritillary before, but no fritillaries have the round circular spots. This butterfly has a pattern that seems halfway between a fritillary and a gatekeeper, making it recognisable as a wall butterfly.

Those dark bands aren’t just for show. Only the males have them (the females just have lighter bands, I don’t think I saw any females) and they are scent bands, used to attract mates. It’s fairly easy to see as all the wall butterflies I saw seemed determined to pose on the gravel path, wings outstretched. Unlike the white butterflies (which just do not keep still) these ones were happy to sit with their wings open while I thumbed desperately through my field guide to work out what they were, getting distracted by pictures of fritillaries.

A silver washed fritillary. All fritillaries have a similar sort of pattern but they do not have the dark black bands or the eye-spots of the wall butterfly. Photo by Zeynel Cebeci via wikimeida commons, credit link below.

Despite the ones I saw on the south coast, the wall butterfly is still rare in Britain. It’s found most easily around the coast of England and Wales and a few spots in Ireland. The butterfly is found most often around May and June, with a slight dip in numbers in July and then again around August-October time. Eggs are laid over the winter. The butterfly prefers hot and sunny conditions, so it was lucky there was a heat-wave this weekend!

I also saw a peacock butterfly, a speckled wood and a marbled white, all of which were beautiful but slightly less rare. There were also several whites of some variety, but they wouldn’t stop moving long enough for me to identify exactly which kind of whites they were. I was really hoping to see some blues along the coast, but a lot of them are even rarer and a lot harder to distinguish.

Your regularly scheduled bacteria will return next week.

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Credit link for image 1

Credit link for image 2

The chemical structure of salinomycin, a polyketide antibiotic. Credit link below.

Polyketides don’t just exist in bacteria, they are also present in plants, fungi, protists and even some animals. They are most commonly found in organisms that are sharing mutualistic relationships, such as colonies of sponges, and the symbiotic bacteria of insects. This may be due to their usefulness as a signalling molecule. In particular lichenised fungi, i.e fungi sharing a relationship with algae or green-bacteria (cyanobacteria) contained a large number of polyketides. When examined closer however, it was found that only 10% of these were related to plant polyketides, the rest were more unique.

As polyketides are modular genes, it is relatively easy to evolve new ones. Accidental duplication of sections of the genomes can add in a bunch of new modules, which can then diversify to new and exciting purposes. If one gene adds a -COOH group, and another adds a -CH3, duplication of those genes means that your molecule suddenly has two -COOH groups and two -CH3 groups and a bunch of new properties as a molecule. While many of the new fungal polyketides will have evolved within the fungi, it is also strongly suggested that a group of them originally came from bacteria. The genes for making polyketides are very close to genes called “mobile elements”, bits of DNA that are particularly good at jumping in and out of cells. Also the codons (the code by while DNA is turned into protiens) of the polyketides genes more closely resemble those of bacteria than of other fungi or plants.

A tree in Sicily covered in different types of lichen, credit below

For anyone interested in taxonomy there are some interesting clade diagrams in the paper (reference below). What they seem to show is that a group of polyketide genes from bacteria were picked up by the fungi, and the evolved within it to form new groups of polyketides unique to lichenised fungi. These genes are not in all types of lichenised fungi, suggesting that they have been lost from species where they are not needed. As polyketide genes tend to be quite large and bulky (at least for bacteria, it might not matter so much inside the larger eukaryote genomes!) it does make sense that they would be quietly dropped if found not to be useful.

It’s not unlikely for fungi to be stealing bits of the bacterial genome either. Fungi and bacteria occupy many of the same niches in nature and can live in very close semi-mutualistic partnerships. There’s even fossil evidence to suggest ancient lichen-like organisms formed by a symbiotic interaction between fungi and cyanobacteria. While it is tempting to think that the fungi took the polyketide genes from the cyanobacteria they were next to, the genes seem to be more similar to those of soil bacteria rather than cyanobacteria. Like any other organism though, lichen do not exist as isolated entities but surrounded by soil, dirt, parasites, other fungi and other bacteria. Which will include a healthy dollop of soil bacteria in close proximity to the fungi, certainly close enough to be swapping genes around.

As an overall picture therefore, it looks like the fungi picked up a group of very useful signalling and defence molecules from soil bacteria. Within the fungi, these genes then continued to evolve and develop, to form a fungal-specific and very useful little group of genes.

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Credit link for image 1

Credit link for image 2

Schmitt I, & Lumbsch HT (2009). Ancient horizontal gene transfer from bacteria enhances biosynthetic capabilities of fungi. PloS one, 4 (2) PMID: 19212443

A fluorescent micrograph capturing the presence of bacteria (shown as green) on the surface of an emerging Arabidopsis lateral root (plant nuclei shown in blue). Image created by Sarah Lebeis and reposted here with permission. Credit below.

As an (ex)-biochemist I’m used to the idea of studying plant-microbe interactions by taking one plant and one microbe and seeing what chemicals they produce, so I was fascinated by recent research from the University of North Carolina that looked at entire microbial ecosystems. The researchers went outside, collected two types of soil from different locations and grew samples of the plant Arabidopsis in each one. They then collected soil that had grown around the roots and looked at the bacterial species within that soil, as well as the bacterial species growing within the root itself.

Collaboration with a NextGen sequencing team (the Department of Energy’s Joint Genome Institute) allowed them to identify the different bacterial species present. They found that a subset of all the bacteria in the soil were found clustered around the roots, while an even smaller subset were allowed inside. Looking for similarities between the bacteria from each plant revealed a core microbiome, a set of specific bacterial species that the plant was attracting from the soil towards itself.

Arabidopsis thaliana - the plant most commonly used by plant-scientists. Credit below.

It was also found, however, that as well as this core microbiome, the pattern of bacteria that the plant recruited varied depending on the type of soil. Dr Dangl, the lead researcher, suggested that this might be for nutritional reasons – plants often use bacteria to help provide their nutritional requirements and in different soil types different bacteria may be more useful. This points to a fascinating interaction between the plants and the bacteria, a secret underground community of mutual benefit.

This interaction isn’t just a highly interesting story, it also has huge potential for further research. Arabidopsis is relatively easy to genetically manipulate, removing certain genes from the plant and seeing how long these interactions last could start to show how these interactions form. Dr Dangl comes from a background of working with plant immune systems so another potentially interesting area is how the plant distinguishes between the bacteria that it wants to keep in and around its roots, and those it wants to get rid of.

As plants collect bacteria for nutritional reasons it might also be possible to tweak those interactions to support plant growth in nutrient-poor soils. By manipulating the plant microbiome rather than the plant itself there is the potential to promote growth and development in areas where plants may find it difficult to thrive.

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Credit for image 1: Published in Lundberg, D.S., Lebeis, S.L., Paredes, Yourstone, S., Gehring, J., Malfatti, S., Tremblay J., Engelbrektson A., Kunin V., Glavina del Rio, T., Edgar., R.C., Eickhorst, T., Ley, R. E., Hugenholtz, P., Tringe, S.G., and Dangl, J.L. 2012. Nature 488: 86-90.

Credit link for image 2

Thanks to Dr Dangl for agreeing to an interview.

Participating in this cross-network blogging festival is nature.com’s Soapbox Science blog, Scitable’s Student Voices blog and bloggers from SciLogs.com, SciLogs.de, Scitable and Scientific American’s Blog Network. Join us as we explore the diverse interpretations of beginnings – from scientific examples such as stem cells to first time experiences such as publishing your first paper. You can also follow and contribute to the conversations on social media by using the #BeginScights hashtag.

The first signs of life on earth appeared about 4.5 Ga (1 Ga is an American billion, ie. 10⁹ years) ago. It’s not yet completely certain exactly how this life arose; hot volcanic mineral springs have been suggested, as have the more traditional lightning-struck primordial soups and (rather wonderfully) radioactive beaches. At any rate something happened which lead to a little membrane-bound ball with internal nucleic acids which, crucially, could replicate…

I've never been convinced by the lightening explanation myself, but it would have been a wonderfully dramatic moment!

And then it was all over really, bar the evolution.

The atmosphere back then was very different, little oxygen and an abundance of carbon dioxide with plenty of methane being released into the atmosphere once the first life forms started eking out an entropy-defying existence. In order to get energy to power cellular processes you need to set up redox pathways, which involve cycles of electron donors and acceptors. The main electron donors around at the time were H2, H2S and CH4 and the main acceptor probably nitrogenous. Water, the electron donor used for photosynthesis, was around in abundance, but none of the little proto-life-blobs quite had the energy required to split it (or the physical proteins required back then either) so it mostly stayed unused.

Carbon dioxide levels went down, methane levels went up and the planet warmed up a little due to global warming. Things stayed like that for a billion years or so (1 Ga) and then something quite special happened, something that would have mindblowingly devastating affects on the life surrounding it.

Photosynthesis. The process by which carbon dioxide is fixed into usable sugars by the splitting of a water molecule. The process of photosynthesis produces oxygen, which is highly dangerous for cells; it can screw up the internal redox potential, create dangerous free-radicals and precipitate ions out into soluble forms. This means that from the point of view of every other organism the newly-evolved photosynthetic blobs were floating around spewing toxic gas into the atmosphere.

Floating green blobs of DEATH. Image from wikimedia commons.

The arrival of this new resource (oxygen) lead to a change in the way organisms respired as well. Up until what is sometimes called the Great Oxidation Event most respiration was anoxic, probably similar to anaerobic respiration, or fermentation,  in anaerobic bacteria around today. This process, while enough t0 keep life going, is around sixteen times less efficient than aerobic respiration. The proto-bacteria that managed to use the oxygen would therefore have gained a major energy boost.

This energy boost allowed the oxygen-using bacteria to go forth and multiply, leaving the anoxic bacteria clinging to the few environmental niches where no oxygen could penetrate. Some of these oxygen-using bacteria were swallowed up by larger cells who then used them as specialised intracellular breathing compartments. The bacteria became mitochondria, and the cells with mitochondria grew bigger and formed more intracellular compartments. They became eukaryotic cells, the kind of cells that all multicellular animals are made from.

So even now, when you breath, it’s ancient bacteria inside your cells that process the oxygen. The only part of the human cell that does oxidative-respiration is the mitochondria. Sure, the human part of the cell can produce small amounts of energy in the cytoplasm, but then the whole process is shuttled into the mitochondria in order to get the massive oxygen energy boost.

One biochemical trick that evolved around two billion years ago to take advantage of oxygen is still being used for respiration by all multicellular life on earth.

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This post was largely taken from a previous one, over at my old blog.