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June 3, 2010
02:58 PM
2,921 views

Antibiotics and Synthetic Biology

by Lab Rat in Lab Rat

The model for bacterial death by antibiotics was fairly simply until recently. Antibiotics work by targeting a certain area of the bacteria; beta-lactams target the cell wall, Rifamycins target RNA synthesis, tetracyclins inhibit protein synthesis etc. The theory was that by inhibiting these processes, a certain vital function within the bacteria would be stopped, leading to its death.However due to research done by Kohanski (references below) the story is looking a bit more complicated. Looking at three different classes of antibiotics they found that no matter what the site of action, all the antibiotics induced hydroxyl radicals. This was in bactericidal drugs, which actually kill bacteria, rather than bacteristatic ones (which just prevent cell growth). They also demonstrated that this mechanism of hydroxyl radical production was the end product of a chain of reactions involving damage to the TCA cycle (aka the Krebs cycle - which is a major part of respiration) which lead to damage to iron-sulphur clusters and subsequent production of the DNA-damaging hydroxyl radicals. This is shown diagramatically below, and this first paper was covered by Jim at Mental Indigestion with some great follow-up comments and discussion.They've recently put out a review (second reference below) of which I find the most exciting parts are the two little extra-information boxes. One of them covers drug synergy and the second covers synthetic biology, both of which I'm getting increasingly more interested in.Drug synergyOne of the most useful things about modelling drug actions is it can help to show which drugs would work most effectively in pairs. Using two drugs together can have many potential effects; it can make the treatment more effective, sometimes is can make the treatment less effective and of course some can be dangerous for the patient. Work on drug synergy showed that aminoglycoside antibiotics (which affect RNA synthesis) become more affective when given simultaneously with B-lactam antibiotics (which lead to cell wall breakdown) as the increased cell wall breakdown helps the aminoglycosides to get inside the cell. Conversely, drugs that inhibit protein synthesis are less effective when given at the same time as drugs which inhibit DNA synthesis as making it harder to synthesise proteins from sub-optimal DNA actually makes the cell more able to survive.These interactions will affect the dosage of drugs used during synergistic treatments, and it is hoped that using two different types of antibiotics at low doses might be more healthy for the patient, and might help to combat against antibacterial resistance to one of the drugs.Synthetic BiologyAnother interesting concept the paper brings attention too is the potential use of synthetic biology to aid in both the study and application of antibiotic-related death systems. By using synthetic genes to disrupt or alter the proposed antibiotic network novel drug targets could be discovered. If turned into a high-throughput system this would be far more useful than the current screening system which tests for a potential drugs interaction with a target, rather than the ability of this interaction to lead to cell death. Synthetic genes can be delivered into the bacterial cell via bacteriophages. Adding a synthetic gene into a bacteriophage for bacteria cell delivery has been attempted successfully before when they were used to enhance E. coli cell death by delivering genes for proteins that disrupted the DNA-repair system within the bacteria. This allowed faster and more effective killing of the bacteria at lower doses of antibiotic.At a time when bacteria are fast becoming resistant to even the front line jobs, research that suggests novel ways of killing bacteria can produce some very useful outcomes. Using combinations of drugs at lower concentrations, or aiding antibiotics by introducing them along with synthetic genes in bacteriophages allows an increased shelf-life of the drugs that we currently possess as well as providing potential systems to aid the discovery of new antibiotics.---Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, & Collins JJ (2007). A common mechanism of cellular death induced by bactericidal antibiotics. Cell, 130 (5), 797-810 PMID: 17803904Kohanski MA, Dwyer DJ, & Collins JJ (2010). How antibiotics kill bacteria: from targets to networks. Nature reviews. Microbiology, 8 (6), 423-35 PMID: 20440275---Follow me on Twitter!... Read more »

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

Kohanski MA, Dwyer DJ, & Collins JJ. (2010) How antibiotics kill bacteria: from targets to networks. Nature reviews. Microbiology, 8(6), 423-35. PMID: 20440275

September 28, 2009
04:44 PM
985 views

Bacterial Hunting Strategies

by Lab Rat in Lab Rat

Methods of bacterial predation, including a short exploration of the 'hunting' behaviour seen in Myxococcus xanthus.... Read more »

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

November 16, 2010
02:47 PM
928 views

Storing DNA

by Lab Rat in Lab Rat

DNA is one of the most important components of the cell. In eukaryote cells (i.e the cells of humans and plants) it is stored inside a nucleus that keeps it safe and away from dangerous things like free radicals produced by the metabolic reactions of the cell. In bacterial cells the DNA isn't nearly as well protected, but the main bulk of the bacterial chromosome (excluding the little floating plasmids) is all kept together in a bundle usually referred to as a nucleoid. However the DNA in cells is rather long which means that in order to package it into a small space it needs to be coiled up. Eukaryotic cells do this by using proteins called Histones, which coil the DNA around them, and can also signal which genes the cell should be turning into proteins.Image from wikimediaBecause eukaryotes cells have so much DNA they need to keep it all tightly coiled up, with little molecular tags on the histones to remind them which bits are needed reguarly, and which bits can be mostly ignored. Prokaryotes on the the other hand don't need quite as much control, most of their DNA is going to be used most of the time, but it still needs to be packaged up to fit inside the cell.The cells do this using by using the unbelieveably eukaryot-ist named "histone-like proteins" such as H-NS which is found in E. coli and related bacteria. Although their precise mechanisms are not quite clear, they have been shown to play a clear role in the coiling of the DNA and maintaining nuceolide stability. They also (like their eukaryote counterpart) help to control gene expression as well as chromatin structure.In most cases, the H-NS functions as a transcription repressor merely through its physical presence wrapped around DNA - it's harder to transcribe DNA (the first step for protein production) when it's all wrapped up tightly in balls. Some genes though, seem to be activated when associated with H-NS, as it can affect the stability of the mRNA transcript. These are usually genes associated with stress responses; in fact many of the genes that are affected by H-NS are linked to the stress response or changes in the environmental conditions, such as high or low temperature, high osmolarity, changes in pH or oxygen concentration.Quite how the H-NS controls these parts of the DNA is not entirely clear. Unlike histones, which are positively charged to stabilise the negative charge on the DNA, H-NS proteins are neutral. Although the domain of the molecule that binds to the DNA has been identified, it is not yet certain which parts of these are vital, or how they interact with the DNA molecule. One thing that is clear however is that H-NS are capible of forming dimers, and probably carry out much of their task as a dimer of two joined molecules.Domains of an H-NS molecule, from the reference. The oligomerization domain binds to a fellow H-NS while the DNA binding domain attaches to DNA.Whatever the binding mechanism is, it is unrelated to the actual sequence of the DNA, as H-NS can bind to many different regions of the gene, regardless of sequence. Until recently the H-NS molecules were thought to have no post-translational modifications (unlike histones which are often decorated in molecular markers to indicate which kind of gene they are on) but some have recently been found to be marked with poly-3-hydroxybutyrate, a small lipid molecule. The reason for this is unclear, but it does raise some exciting implications for H-NS control of gene transcription.When it comes to controling DNA expression, it's clear that in both eukaryotes and prokaryotes, the scaffold proteins that hold the DNA coiled up act as more than just a scaffold. Instead they are involved in a substantial amount of the control of DNA expression, often working closely with other control proteins to ensure the correct genes are turned into proteins.---Schröder O, & Wagner R (2002). The bacterial regulatory protein H-NS--a versatile modulator of nucleic acid structures. Biological chemistry, 383 (6), 945-60 PMID: 12222684---Follow me on Twitter!... Read more »

Schröder O, & Wagner R. (2002) The bacterial regulatory protein H-NS--a versatile modulator of nucleic acid structures. Biological chemistry, 383(6), 945-60. PMID: 12222684

March 1, 2011
06:04 AM
927 views

Signals for Infection

by Lab Rat in Lab Rat

Neisseria meningitidis is a bacteria which lives in the throats of around 30% of the human population. In most cases it causes no problems at all and just exists as a normal part of the throat microbial flora. In some patients however it can start to colonise the bloodstream and brain, leading to cases of septicemia and meningitis which are highly dangerous and can be fatal.The invasion starts with individual bacteria, which adhere to the epithelial cells that cover the inside of the throat. They then start to divide and proliferate to form large aggregated colonies. Within these colonies they are connected to each other, and to the epithelial cells, by protrusions from the bacterial cell surface called pili which are shown below for a wild-type (i.e un-genetically modified) Neisseria meningitidis:Image taken from the reference below. The arrow points to one of the pili, and the insert shows a close-up of it.These pili are often modified by the attachment of small molecules to the pili proteins, including the molecule phosphoglycerol (shown on the right for those interested in structure). To test the effects of the addition of phosphoglycerol, the researchers found which gene caused the addition of this molecule onto the pili (the pptB gene), and removed it from the cell. Without the pptB gene there was still the same number of pili around the cell, but they were not clumping together as much. Instead of the thick fibres seen in the wild type above (caused by large bundles of pili) only little stringy fibres were seen. These thin spindly fibres show that without the addition of phosphoglycerol, the pili cannot clump together.This is important medically as Type IV pili bundle formation and N. meningitidis aggregation for infection are linked. Interestingly it was not the aggregation that was affected by removing the phosphoglyerol but the ability of individual bacteria to leave the aggregate to infect other parts of the body. In wild-type bacteria, the pptB gene is strongly activated only after several rounds of division within the aggregate, so it looks like the addition of phosphoglycerol acts as a switch, communicating to the bacteria that enough of them have aggregated and it is now time to leave. If the pptB is activated due to large numbers of bacteria it could act as a communication of the population density - signalling to the individual bacteria that the current location is far too crowded, and it has better chances of survival if it leaves.---Chamot-Rooke J, Mikaty G, Malosse C, Soyer M, Dumont A, Gault J, Imhaus AF, Martin P, Trellet M, Clary G, Chafey P, Camoin L, Nilges M, Nassif X, & Duménil G (2011). Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination. Science (New York, N.Y.), 331 (6018), 778-82 PMID: 21311024---Follow me on Twitter!

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Chamot-Rooke J, Mikaty G, Malosse C, Soyer M, Dumont A, Gault J, Imhaus AF, Martin P, Trellet M, Clary G.... (2011) Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination. Science (New York, N.Y.), 331(6018), 778-82. PMID: 21311024

September 14, 2009
12:48 AM
925 views

Living without a cell wall...

by Lab Rat in Lab Rat

Exploring work done on L-form baacilis subtilis (without cell walls) and how this provides clues to how early life might have grown and propagated.... Read more »

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

April 22, 2011
05:30 AM
911 views

Life at zero growth rate - SGM series

by Lab Rat in Lab Rat

This is the third post in my latest SGM series.One of the first topics that I learnt in Biology was that there are two types of things; living things, and dead things. Living things are given a whole host of distinguishing characteristics (growth, reproduction and, my favourite, irritability) where as dead things are defined as everything else. Biology was usually defined as the study of living things.As I grew older, I found that there were many complications to this neat little classification. Viruses - which are neither fully living, nor properly dead. A whole organism can be dead, despite the fact that many of its cells are still alive (how alive is a freshly killed animal? Or the flowers in a vase?). And of course what is for me the most intriguing case, that of dormant bacteria.Dormancy is an odd state to be in. A dormant organism shows none of the signs of being alive. It does not eat, grow or divide (although some very basic metabolic processes may still continue). It shows no response to any outside stimulus, and can often be placed in conditions that would lead the living organism to perish, such as extremes of temperature and pressure. Yet somehow just one simple stimulus can cause this previously dead looking organism to spring magically back into life.Bacteria are not the only things that can go dormant. Someanimals can as well, the most famous example being tardigrades -the thing shown on the right that looks a bit like a plushie made by Tim Burton (image from wikimedia commons). Yeast are well-known for forming dormant spores, and it can be argued that a seed is technically a dormant plant, just waiting for water to be added to bring it back to life.One of the most medically important dormant bacteria is Mycobacterium tuberculosis which infects humans and leads to TB. One of the reasons for its pathogenicity is that they can go dormant, both outside the body (which makes them hard to shift from a hospital) and inside the body, after the primary infection (which makes them even harder to shift from inside a human body).Although the latent cells can remain within the body for many years, sometimes never coming back from dormancy at all, ideally there should be some signal to bring them back to life. These signals are known as "resuscitation-promoting factors" or RFPs. These RFPs are required for virulence, and to bring the bacteria back from dormancy, but are not necessary for the growth and proliferation of cultures in the lab.Within human tissues, and throughout the cycle of the disease, you can track these RFPs to try and get an insight into what the bacteria is up too, and when it may move from latent periods to periods of active growth. As well as being useful for tracking the course of infection, this might also have therapeutic implications. If you can convince the bacteria not to come out of dormancy then you have an infection state that might not be completely curable but is at least controllable.How organisms survive in a state of dormancy, and indeed how they ever come out of it, is a subject I find really fascinating. I'm unlikely to ever get to do much research on it (because as fascinating as it might be screwing around with my little bugs till they do what I want is endlessly more fun) but I'll probably have a good few more posts writing about it and exploring how it works.---Kana BD, Gordhan BG, Downing KJ, Sung N, Vostroktunova G, Machowski EE, Tsenova L, Young M, Kaprelyants A, Kaplan G, & Mizrahi V (2008). The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro. Molecular microbiology, 67 (3), 672-84 PMID: 18186793Davies AP, Dhillon AP, Young M, Henderson B, McHugh TD, & Gillespie SH (2008). Resuscitation-promoting factors are expressed in Mycobacterium tuberculosis-infected human tissue. Tuberculosis (Edinburgh, Scotland), 88 (5), 462-8 PMID: 18440866

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Kana BD, Gordhan BG, Downing KJ, Sung N, Vostroktunova G, Machowski EE, Tsenova L, Young M, Kaprelyants A, Kaplan G.... (2008) The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro. Molecular microbiology, 67(3), 672-84. PMID: 18186793

Davies AP, Dhillon AP, Young M, Henderson B, McHugh TD, & Gillespie SH. (2008) Resuscitation-promoting factors are expressed in Mycobacterium tuberculosis-infected human tissue. Tuberculosis (Edinburgh, Scotland), 88(5), 462-8. PMID: 18440866

February 21, 2011
01:33 PM
884 views

Multicellular signalling

by Lab Rat in Lab Rat

I like studying bacteria. I find them fascinating, wonderful little creatures, able to do as much (and often more!) with a single cell as other organisms need whole multicellular bodies to achieve. I like exploring the places bacteria live, the things they can do, the ways they manage to exploit practically every niche on earth, and of course most importantly how I can exploit them.But not everyone loves bacteria, and at heart I am a biochemist which means, among other things, that I get to teach younger biochemists. This means I do occasionally find myself venturing uncertainly into the world of the multicellular and while doing so recently I found an interesting paper on cell signalling (reference below) which I thought I would share.All cells need to be able to communicate, but while bacteria know that everyone they communicate with is a competitor, multicellular organisms have cells that need to be able to cooperate in a strange and slightly twisted form of cellular-communism. Each cell needs to know when it can divide (usualy never), when to grow, when to release chemicals and, ultimatly,when to sacrifice itself for the Greater Good.Cellular communication is mostly a chemical affair, with small molecules called ligands being sent from one cell to another and recognised by receptors on the cell surface. These receptors can take many forms, but one of the more common ones is the form of a seven-transmembrane spanning receptor, so called because it goes through the membrane seven times:Picture (c)me and my dodgy art skills. The protein is in blue, the membrane in pink, and the ligand bound on the outer cell surface is the red blob.Binding of a ligand causes a conformational change in the whole structure, most importantly in that long intracellular tail shown above. This can then activate other molecules inside the cell, with the end result that a specific gene is turned on or off. In the classical model of this process the intracellular tail interacted with a little molecule called the G protein which carried the message through to the genome. Another protein that featured in this model was B-arrestin, which was thought to desensitise the receptor and the G-protein by re-setting it back to its original state, i.e switching the thing off. This model is shown below:Picture (c) me. This is a simplified diagram, in 'reality' there are a lot more different proteins involved, but these are the main ones, and the important ones for this paper.New evidence is coming to light which modifies this model. Firstly, it's been found that the B-arrestin does more than just switch off the G-protein, it is also capible of sending its own signals, through a cascade of different proteins. Both the G protein and the B-arrestin can be used to pass on the message sent by the ligand. Secondly, it's been found that these two proteins are not activated equally, a bias can be displayed, sending the signal through one of these two intermediate proteins; either the G protein, or the B-agonist or a mixture of the two. This bias can be either due to the properties of the receptor, or those of the ligand binding to it. Experimentally you can generate a bias by altering either the receptor or the ligand to prefer binding to the B-agonist, and you can plot these on mathematical-looking graphs.You can tell this is a biology graph because there are no actual numbers, just vague concepts :p (c) me.The actual physiological effects of this are only starting to be explored, as it introduces an extra level of complexity to intracellular control. The use of several different ligands, all with varying degrees of bias at the same receptor, could produce more subtle cellular output responses. Within a multicellular organism, the better your intracellular communication is, the more likely your organism is to grow happily and survive.---Rajagopal S, Rajagopal K, & Lefkowitz RJ (2010). Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nature reviews. Drug discovery, 9 (5), 373-86 PMID: 20431569---Follow me on Twitter!

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Rajagopal S, Rajagopal K, & Lefkowitz RJ. (2010) Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nature reviews. Drug discovery, 9(5), 373-86. PMID: 20431569

August 17, 2010
05:01 PM
876 views

Hitchhiking through the nervous system

by Lab Rat in Lab Rat

I while ago I wrote a post about how virus's get from the outside of the cell to the interior of the nucleus and found that virus particles are able to hitchhike on the cells internal transport systems. I was quite interested therefore to find a paper in Nature Reviews (reference below) that revealed that not only do virus's latch on to host proteins to travel around inside the cell, they also use host extracellular processes for travelling around the body. And outside the cell it's not just virus's either, bacterial toxins need transport systems too, unlike whole bacteria they can't move around under their own power.One place that the body wants to protect particularly well against infection is the central nervous system. It provides this protection by surrounding it with a wall of tightly sealed endothelial cells known as the blood-brain barrier. However despite this the body itself still need to get some things into the CNS; small molecules such as glucose and oxygen as well as larger cells of the immune system. These immune system cells provide the first sneaky point of entry; virus's such as HIV can hitch a ride inside these cells and get into the central nervous system that way. This is the equivalent of hiding in a truck to avoid border patrols.However some virus's and toxins use an even more sneaky method, dressing up as a border-patrol guard and simply walking in. Throughout the blood brain barrier there are long neuronal projections that connect the central nervous system to peripheral organs. A picture of one of these cells is shown below:Like all cells, this contains the transport molecules Kinesin and Dynein, which virus's can latch onto in order to transport themselves through the cell (see earlier post here). Once they get inside the cell, the cell's own proteins will carry the virus particles all the way through it, and into the central nervous system. However first it has to get inside the cell, through the little blue blob at the bottom (in the diagram above it's highlighted with a little dotted square).As well as receiving chemical signals for electrical impulses (that make the neuron function as a nerve) the blue blob also contains various different receptors capable of engulfing and uptaking small molecules, including those used to signal some neural impulses. This means that there are a range of chemical receptors on that blue blob which allow the uptake of molecules, and you can probably tell where this is headed...The diagram above is the intramolecular equivalent of Han Solo dressed as a Stormtrooper wandering into the Death Star. By changing its outer coat enough to mimic the proteins that are usually taken up by the cell the Herpesvirus can attach to the outer membrane and then be absorbed into the cell. Once inside, it can latch onto the dynein and get a free pass all the way into the nucleus (and neurons are pretty long so it is a bit of a journey). Poliovirus and rabies can also carry out this trick (at the neuromuscular junction for anyone interested) along with the bacterial botulinum toxin, which gets taken up by synaptic vesicles and essentially kills the end of the nerve, which can either lead to instant death or a scarily smooth robot-plastic forehead, depending what context you take it.I always find it quite spooky to think of my body in that way, as a huge maze of intracellular processes, being negotiated, infected and protected by tiny substances outside of my conscious control. I think that's another reason I find cellular biology so fascinating, by studying it we gain control (or if not control at least an understanding) of these detailed processes that we would not normally be able to influence.---Salinas S, Schiavo G, & Kremer EJ (2010). A hitchhiker's guide to the nervous system: the complex journey of viruses and toxins. Nature reviews. Microbiology, 8 (9), 645-55 PMID: 20706281... Read more »

Salinas S, Schiavo G, & Kremer EJ. (2010) A hitchhiker's guide to the nervous system: the complex journey of viruses and toxins. Nature reviews. Microbiology, 8(9), 645-55. PMID: 20706281

July 17, 2010
02:53 PM
855 views

How viruses hijack cellular transport systems

by Lab Rat in Lab Rat

Even in the world of the very small, there are significant differences in size. A eukaryote cell (i.e a human cell) for example is relatively big, in microscopic terms. Most other things that interact with the cell at the microscopic level, are far smaller than it, such as bateria, viruses and signalling molecules.A virus isn't much more than a small capsule of proteins with a little bit of DNA inside. Once it gets inside a eukaryote cell, it's very much in the position of a small child wandering into a big city. In front of it lies the vast interior of the cell, full of reactions, enzymes, proteins scurrying too and fro, mRNA being translated, proteins being folded and other busy bustling cellular processes. Surrounding it are large organelles (larger than the virus particle!) with strange and mysterious procedures going on inside them...That sort of view, stretching across to both horizonsFrom here, the virus has to make its way to the nucleus, pushing its way through the crowded and complex cellular interior without being spotted as an intruder. Fortunately it has some help here, because it's facing the same problem faced by every molecule and organelle already in the cell. Transport mechanisms are already in place so that things can move around the large intracellular space with relative ease. The viruses simply hijack these transport systems and get a free ride all the way too the nucleus.Work on the herpes simplex virus helped to produce a model of how the viral particles move around the cell. After entering the cell through the cell surface membrane, the virus is picked up by dynein which carries it along microtubes towards the nucleus. The microtubules form a network within the cell (like train rails) which the dynein motors along (using ATP energy). This is shown pictorally below:Dynein moves in one direction along the microtubule while kinesin moves in the other direction. Together they move molecules all around the cell.Once at the nucleus, the viral DNA enters through the nuclear membrane and is replicated inside the nucleus (entering the nucleus is a critical step for DNA-viruses; for those viruses that contain RNA this step is not so vital). The replicated DNA then comes back out of the nucleus and is transcribed into protein in the cytoplasm, which leads to the formation of new viral particles. These new viruses then have to travel back down the microtubule (carried by kinesin) to the outer membrane of the cell where they can be released into the surrounding environment and go on to infect more cells.One interesting question is what exactly the dynein (and kenesin) bind to on the virus cell surface. As well as being an interesting point, answering this comes with the usual funding bait that if you find how viruses move inside the cell you may be able to find ways of stopping them from moving which would leave them at a severe disadvantage. To examine this the virus was isolated and the parts of the surrounding protein coat that bound to cellular factors further separated. These separated capsid proteins were then tested for their ability to bind to mammalian intracellular proteins. They found that several of the capsid proteins could bind to important transporter molecules, and furthermore that several different transporter molecules could sometimes bind to the same capsid protein.Drawing showing the site of attachment of the motor transport proteins to the (green) virus capsule. The other end of the motor proteins is used to move along the microtubule.As I'm in a fairly syntheticly-biological mood, I couldn't help but notice the mention at the end of the paper that this could have implications beyond virus treatment or vaccinations. The ability to create a little molecule that the cell can carry to the nucleus could have implications for both future genetic treatments and nanotechnology. The ability to get a little capsule of treatment right to the nucleus of cells could even have the potential for treating cancer cells, as it utilizes the cells own transport mechanisms to deliver treatment to the intracellular place it is needed.---Kerstin Radtke, Daniela Kieneke, André Wolfstein, Kathrin Michael, Walter Steffen, Tim Scholz, Axel Karger, Beate Sodeik (2010). Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures PLoS Patholgens, 6 (7) : e1000991---Follow me on Twitter!... Read more »

Kerstin Radtke, Daniela Kieneke, André Wolfstein, Kathrin Michael, Walter Steffen, Tim Scholz, Axel Karger, Beate Sodeik. (2010) Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures. PLoS Patholgens, 6(7). info:/e1000991

August 20, 2009
04:45 AM
835 views

Cell wall under attack - bacterial response to antibiotics

by Lab Rat in Lab Rat

The response of Strep. ceolicolor to cell-wall attacking antibiotics.... Read more »

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

April 27, 2011
12:04 PM
828 views

Social Evolution in Bacteria - SGM series

by Lab Rat in Lab Rat

This is the fourth post in my latest SGM series.

The social behaviour of bacteria is something that I get very excited about. From the wolf-pack hunting strategies of Myxococcus xanthus to the terminal differentiation of cyanobacteria, it's something that I never get tired of writing about. As well as providing interesting quirks of bacterial behaviour, living within a colony also gives new scope for exploring the evolution of bacteria; not just as single entities but as a fully functioning social group.
One of the differences of living within a social colony as opposed to alone means that altruistic-type behaviour has to be adopted. Bacteria living within a biofilm need to excrete the sticky goo that holds the biofilm together, which is problematic because synthesising and secreting goo takes up a lot of energy. So within this colony, there will be 'cheaters' - those bacteria that live in the surrounding goo produced by others, while making none themselves.

A bacterial biofilm, showing individual bacteria in green. Image taken from the FEI website, shown there courtesy of Paul Gunning, Smith & Nephew
As with all colonies, cheating might benefit the individual but has no benefit for the colony as a whole. Too many cheaters and there won't be any biofilm. And recently an even more subtle form of cheating has been shown within the biofilms of the bacteria Pseudomonas aeruginosa, with bacteria that don't just refuse to make vital sticky chemicals, but also abstain from the entire process of forming a biofilm.
Bacteria use a complex communication system called quorum sensing in order to determine how many other bacteria they are surrounded by. Once enough bacteria are present, all signalling their existence, the biofilm will start to form. However some bacteria isolated from the biofilm were shown not to be taking part in any quorum sensing at all. Quorum sensing appears to be quite a burden for a growing cell - cells with the quorum sensing genes knocked out tend to grow a lot faster that the socially conscious cells that allow biofilms to form.
The paper that goes through this (reference one) highlights it as a form of social cheating, with bacteria avoiding quorum sensing to benefit themselves while mooching off the quorum sensing behaviour of others. I'm not entirely certain that this is the case though. It may just be an good example of job allocation within the bacterial society. Clearly not all bacteria are required to be continually quorum sensing, so why should they all have to? Would it not be more sensible to have some exempt from that task, so that they can concentrate on growing, dividing, and spreading the colony? This may be more a case of tax-breaks than of benefit-cheats.
Social evolution doesn't just take place within species, but also between them, and like every other organism bacteria are in a constant state of coevolution with both their 'prey' and their predators. Most predator-prey interactions take long periods of time to study, but the beauty of bacteria is that you can go through several generations in the course of one week's growth. Studies of the bacteria Pseudomonas fluorescens and its bacteriophage parasite showed that both the bacteria and the bacteriophage evolved far quicker when interacting together than they did when competing against a non-changing opponent.

Bacteriophage surrounding a bacteria. Image from wikimedia commons
'Evolve' here means that the bacteria and the bacteriophage showed a greater change in their genetic makeup, and a greater genetic divergence from bacteria not pitted against the phages. Unsurprisingly, the genes that changed the most were those involved in host-phage interaction. This study (reference 2) is also a great example of the usefulness of whole genome sequencing. Whole populations of bacteria and phage were allowed to evolve both together and separately and then just sent away for sequencing with the results analysed at the end.
You really can't be an anti-evolutionist while studying bacteria. They just do it so damn quickly and often you can see it happening.
---Sandoz, K., Mitzimberg, S., & Schuster, M. (2007). From the Cover: Social cheating in Pseudomonas aeruginosa quorum sensing Proceedings of the National Academy of Sciences, 104 (40), 15876-15881 DOI: 10.1073/pnas.0705653104

Paterson S, Vogwill T, Buckling A, Benmayor R, Spiers AJ, Thomson NR, Quail M, Smith F, Walker D, Libberton B, Fenton A, Hall N, & Brockhurst MA (2010). Antagonistic coevolution accelerates molecular evolution. Nature, 464 (7286), 275-8 PMID: 20182425
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Sandoz, K., Mitzimberg, S., & Schuster, M. (2007) From the Cover: Social cheating in Pseudomonas aeruginosa quorum sensing. Proceedings of the National Academy of Sciences, 104(40), 15876-15881. DOI: 10.1073/pnas.0705653104

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

August 31, 2010
02:09 PM
811 views

Getting OMPs to the membrane - SGM series

by Lab Rat in Lab Rat

This is the first post of my SGM conference series: I'm going to try and write about seven topics from the Society for General Microbiology September conference over the course of two weeks. The first topic I'm looking at is Protein Folding and Misfolding which consisted of thirteen presentations covering various aspects of protein folding in bacteria, fungi and yeast. As a quick background: when proteins are synthesized they are constructed as long chains of amino-acids which then need to fold up into the correct shape.This may not sound terribly interesting at first, but it proves problematic for proteins that are in awkward places, for example in the outer membrane of Gram negative bacteria. These outer membrane proteins (OMPs) not only have to fold up correctly inside the membrane but they have to actually get to the outer membrane In Gram negative bacteria, this means first getting through the inner membrane, then across the peptidoglycan layer between the membranes, and finally half way through the outer membrane in order to coil up correctly inside it.Gram negative cell membraneThe type of proteins found inside the outer membrane are usually B-barrel proteins, so called because they contain lots of protein folds known as a B-sheets, which can wrap up to form a channel shape as shown in the example on the right. Each blue arrow is a single B-sheet and these fold specifically to form pore-like structures which in the case of porins make a little hole through the membrane.Transport of B-barrel OMPs accross the inner membrane is achieved by synthesising them with a signal sequence attached to one end. This signal sequence is recognised by proteins on the inner membrane and ATP energy is used to pump the proteins accross the inner membrane and into the periplasm (the space between the two membranes). Once in the periplasm they bind to little chaperone proteins which carry them safely to the complex responsible for folding them correctly into the outer membrane, the rather awesomely named BAM complex.As an aside the chaperones do have to get the OMPs there fairly promptly as there are proteases that float around in the periplasmic space and degrade any proteins that don't get incorporated into the outer membrane quick enough.One of the key proteins in the BAM complex is BamA as knocking it out results in a lot of unfolded OMPs in the periplasm (and probably a field day for the proteases). BamA consists of two major components, a B-barrel domain which anchors it into the outer membrane, and five "polypeptide transport-associated" domains, shortened to POTRA by someone who didn't like three-lettered acronyms. The POTRA domains do what they say, they are associated with the transport of proteins (polypeptides).It's still a little uncertain quite how the BAM complex works but a couple of the presentations on the topic were convering it, including work done on changing the genes between different bacterial species. All Gram-negative bacteria have a BamA gene, however taking the BamA gene from one bacteria and putting it into another does not end happily unless it's done between two very close species. Closer research with chimeric proteins (i.e proteins that are half from one bacteria and half from another) shows that this only applies to the POTRA domains. The anchoring B-barrel can be switched between several different species, but the POTRA domain is very species specific.Another interesting thing to address is how BamA gets itself into the outer membrane. One of the periplasmic chaperone proteins, Skp, is thought to be involved in this process, and it was found that when the outer membrane was negatively charged Skp is involved in inserting BamA into the membrane, whereas when the negative charge is removed Skp inhibits BamA folding and insertion. Negative charge is caused by an increase in the phosphatidylglycerol content in the membrane. I found that idea quite exciting as it implies that the bacteria can control where they want the BAM complex to go. The idea of membranes forming "lipid rafts" with certain components that organise where proteins are held is not a new one, and the BAM complex forming in specific places in order to create the correct outer membrane protein concentration is one that appeals to me.They may just be single little cells with no true nucleus, but they are capible of a lot of control over their intracellular processes!---Knowles TJ, Scott-Tucker A, Overduin M, & Henderson IR (2009). Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nature reviews. Microbiology, 7 (3), 206-14 PMID: 19182809Johnson, A., & Jensen, R. (2004). Barreling through the membrane Nature Structural & Molecular Biology, 11 (2), 113-114 DOI: 10.1038/nsmb0204-113---Follow me on Twitter!... Read more »

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

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

September 30, 2009
10:09 AM
804 views

Bacteria that use antibiotics...for food!

by Lab Rat in Lab Rat

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

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

June 14, 2010
04:29 PM
802 views

Colony behaviour and metatranscriptomics

by Lab Rat in Lab Rat

Most places which contain bacteria tend to contain lots of them. In the environment (i.e outside human bodies) bacteria often live in large colonies which can make it difficult to explore their reactions to changing conditions. In the lab, with just one bacteria, information about responses can be obtained by transcriptomics; looking at how the transcriptome changes as the environment does.The transcriptome is the set of all the mRNA within the cell. Unlike the genome, which is the all DNA present within the cell, the transcriptome only reveals those genes which are being turned into proteins. This therefore acts as an indication of the changes in protein production within the cell.My MSPaint skills are improving.For large bacterial colonies however, individual transcriptomic studies aren't much use for finding out the state of the whole colony. Each bacteria within the colony will not only be reacting slightly differently, but will also be experiencing different conditions within the colony depending on where it is within the colony. It's often best, therefore to treat the entire colony as one 'cell' and carry out transcriptome studies on the whole lot. This is metatranscriptomics.Nowadays one of the easiest ways to carry this out is by isolating the RNA and getting it all directly sequenced by Pyrosequencing (which saves the trouble of making microarrays). As well as giving information about the changes within a colony due to environmental factors, it can also show the changes in protein production at different stages in the colony lifecycle such as at the beginning and end of an algal bloom. The integration of new third-generation sequencing methods into this process will make it faster and hopefully allow isolation of the rarer, less abundant transcripts to find more subtle changes in gene expression.This has important implications for things like oceanic cyanobacteria which are involved in carbon sequestration in the oceans. Understanding how changes such as increases in ocean acidity (or decreases in salinity) affects their growth and ability to remove carbon from the atmosphere could have important implications for global warming, and how it can be dealt with.---Gilbert JA, Field D, Huang Y, Edwards R, Li W, Gilna P, & Joint I (2008). Detection of large numbers of novel sequences in the metatranscriptomes of complex marine microbial communities. PloS one, 3 (8) PMID: 18725995... Read more »

Gilbert JA, Field D, Huang Y, Edwards R, Li W, Gilna P, & Joint I. (2008) Detection of large numbers of novel sequences in the metatranscriptomes of complex marine microbial communities. PloS one, 3(8). PMID: 18725995

March 1, 2010
11:00 AM
785 views

Evolving Molecular Machines: The Plant Edition

by Lab Rat in Lab Rat

Over at Thoughtomics, Lucas has a post up about the evolution of mitochondrial import systems. He starts by going back in time two billion years:"Life was well underway at the time, with proto-bacteria already populating the oceans for over hundreds of millions of years. One of the cells alive at the time, swallowed an alpha-proteobacterium. Something remarkable happened: the alpha-proteobacterium did not die but survived in the host cell. Over time, the host and symbiont became to be dependent on each other." That symbiont became a mitochondria.He gets massive brownie point for writing 'proto-bacteria' rather than bacteria, and it is a very remarkable event to have happened. However from the point of view of a plant, it's only half the story, because plants carry two endosymbionts within them: the mitochondria and the chloroplast. Their stories are remarkably similar. After becoming engulfed by the surrounding cell, two major things happened to them: First (and it had to be first otherwise major problems would have arisen!) a protein import mechanism arose, creating more communication between the symbiont and the host and allowing things to pass between them. Second, the symbiont lost bits of its genome, transferring them into the nucleus of the surrounding cell to create the cooperative arrangement seen today:Picture above from the amazing science illustration gallery by California state University. Nucleus is purple, chloroplasts are green, and the mitochondria are orange. Lucas's post covered the evolution of the import mechanism for the mitochondria. I'm going to write about the same thing, but for chloroplasts. After all, the plants already have mitochondria so they can't use exactly the same import process, they have to be able to differentiate between the two.Like mitochondria, the chloroplasts are surrounded by two membranes, and outer membrane and an inner membrane. Two transporters are therefore required to get proteins across. In the mitochondria these are called TOM and TIM (Transport of Outer, and Inner Membrane respectively) and in the chloroplasts they are called TOC and TIC, just to keep things simple (Transport of Outer and Inner Chloroplast membrane). They look fairly similar to TIM and TOM, but recognise different sequences attached to the proteins. While the mitochondrial transport machines recognise sequences that contain a lot of the amino acid arginine and form a specific helical shape, the chloroplast machines (TOC and TIC) recognise sequences rich in serine and proline:TOC and TIC. The proteins of the TOC machine are coloured green, and the TIC machine proteins are all the rest. Diagram from here.One of the questions that Lucas asks in his post is: where did all of these proteins come from? After all, before you have an endosymbiont, you don't need any kind of apparatus to transport proteins into them. Once you start looking closer at the transport machinery it starts looking suspiciously like a rather rushed and last minute job. Different proteins with different functions have been cobbled together, and while there's still a bit of a debate as to whether these proteins came from the surrounding cell or the endosymbiont I suspect that it may be a bit of both. The cell needs to communicate with the little alien inside it, and once the endosymbiont started loosing genes, it needed a way to keep resources coming in.So how do you make a protein importer? What do you assemble it from? Plants had a slightly easier task with the chloroplasts as they already had a perfectly serviceable TIM/TOM transporter present. Looking at the TIC complex, the first two components to come in contact with the imported protein (TIC22 and 20, shown in the diagram above in dark purple and orange) show homology to components of the TIM machinery; TIM23 and 17 for anyone interested in the detail. However TIC22 also has far stronger homologues in cyanobacteria, which means it is likely to be a protein owned by the chloroplast, similar to the proteins owned by the mitochondria that got roped in to help with protein import.The TOC proteins (all green in the diagram above) all appear to have no other function in modern plants other than protein transport. Toc 34 is the GTPase, and as there are many GTPases in cells (used to provide energy) it could have arisen from any one of them. The other TOC proteins are involved in membrane and may have arisen from ancestral membrane receptor proteins, while some components, (including TOC64, not shown above) appear to be rather redundant, as the machinery works perfectly well without them.The research on this is a little sketchy, there are no good solid biochemical means as yet to discover what might of happened somewhere around two billion years ago in order to create a transport mechanism between the cell and it's organelles. There are plenty of different views out there as well, about where the different subunits might have come from. The only thing that seems clear is that like the mitochondrial import system, this was clearly pulled together from bits of old machinery lying around. It had two billion years to get better after all, and reach the efficiency of the modern-day protein import machines.---... Read more »

Gross J, & Bhattacharya D. (2009) Revaluating the evolution of the Toc and Tic protein translocons. Trends in plant science, 14(1), 13-20. PMID: 19042148

July 12, 2010
11:32 AM
773 views

Programming bacteria for search and destroy

by Lab Rat in Lab Rat

As iGEM season is now properly underway, I thought I'd have a look at a synthetic biology paper and found this fairly awesome one about programming bacteria to hunt out and destroy atrazine, a chemical herbicide pollutant. One of the most exciting things about this work was that it didn't just involve bacteria with the ability to remove atrazine from the environment but to actively migrate towards the chemical and then destroy it.The chemical structure of atrazineThe bacteria are controlled using riboswitches - little RNA pieces that can bind directly to a ligand (or signal molecule) and cause a change in gene expression, changes which can include switching on or off the genes involved in cell movement. Atrazine is a good molecule to start with because as well as being a relevant pollutant it also contains plenty of N-H bonds which are good for forming hydrogen-bond interactions with RNA. As well as that it has a well-characterized breakdown pathway, all components of which have been expressed successfully in E. coli.The first stage in creating these seek and destroy bacteria was finding RNA sequences that would bind to atrazine. This was done by attaching the atrazine to a solid support and running bits of RNA past it, to see which ones would bind. They then took these successful binders and tested for riboswitch activity, i.e whether the binding to atrazine caused a conformational change in the RNA that lead to the turning on of a gene. They did this by putting a sequence complimentary to the isolated RNA upstream of the DNA for the CheZ gene, which controls motility in E. coli and then carrying out the selection process shown below:In the absence of atrazine the CheZ is not synthesized and the bacteria stay where they are. When atrazine is added to the plate, the bacteria start to move...Dose-dependent assays were then done on the successful RNA sequences, to characterise the reaction and check that it actually was the atrazine levels that lead to movement rather than some other confounding factor. Sequencing and examination of the binding site also helped to characterize the riboswitch and determine how it was working. The genes for atrazine-consuming ability were then added to the bacteria that moved towards the atrazine, leading to a little search-and destroy module capable of seeking out a dangerous pollutant and removing it from the environment.---Sinha J, Reyes SJ, & Gallivan JP (2010). Reprogramming bacteria to seek and destroy an herbicide. Nature chemical biology, 6 (6), 464-70 PMID: 20453864---Follow me on Twitter!... Read more »

Sinha J, Reyes SJ, & Gallivan JP. (2010) Reprogramming bacteria to seek and destroy an herbicide. Nature chemical biology, 6(6), 464-70. PMID: 20453864

October 18, 2009
12:44 PM
758 views

Protists and their plastids

by Lab Rat in Lab Rat

A quick skim through this blog reveals fairly quickly that I have a slight fixation on bacteria. I like to research them, read about them, and then blog about them, most specifically about their cell walls. However life contains more than just bacteria, and occasionally, strange though it might seem, people write papers about such non-bacterial things, and they end up on my desk with a small post-it attached reminding me that I have a presentation for my supervision group coming up.So for the sake of my supervision, and to prevent myself becoming too scientifically blinkered, I took a quick foray this weekend into the murky world of protists, the strange and wonderful organisms that occupy the taxonomic equivalent of the 'misc.' draw in a filing cabinet. The creatures that are neither plant, nor animal, nor demonstrably bacteria. Many of them are single celled, some of them photosynthesise, and they all seem to occupy little evolved niches of their own, producing proteins with no noticeable homologues in any other branch of life.The paper has the rather terrifying title of : "Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium". And I am not ashamed to admit that I had to go double-check the meaning of several of those words.Dinoflagellates are little organisms that live in water, and mostly look a little like the picture on the right. Many of them are marine organisms, making up a large amount of the photosynthesising biomass in the ocean, and occasionally blooming to form 'red tides', leading to whole sweeps of water turning bright red (possibly occasionally on biblical command). The photosynthetic ones contain chloroplasts, which are wrapped up in three membranes, rather than the usual two. These, like all chloroplasts, contain their own genetic material (known as plastid genes), although unlike plant plastids, they don't seem to contain very many, and those that they do posess are found on little minicircles.What the paper is interested in is whether there are any other genes in the chloroplast which aren't in minicircle form. There are, afterall, only 12 genes encoded on the minicircles, which is a small amount for a plastid. In order to explore this, it uses a characteristic property of the dinflagellate species it's working with. All organisms, when making proteins, make them from an mRNA copy of the genetic code. This mRNA copy tends to have a long string of adenosine residues added to the end, in order to prevent the mRNA getting degraded. This happens in our dinoflagellate species as well, but it doesn't happen to the plastid genes.However instead of getting multiple adenosine repeats the plastid genes get multiple uracil repeats. It's just a different base, but it allows the mRNA made in the nucleus, and the mRNA made by the chloroplast to be separated. You can probe for adenosine enriched and adenosine depleted mRNA as shown on the gel below (A and B show different species). The psbA mRNA is clearly strongly present A+ (adenosine enriched) and therefore codes for a nuclear encoded protein. Conversely, the 23S RNA is A- (adenosine depleted) and is coded for in the chloroplast, from a plastid gene.(Image taken from reference below)The paper selected 300 random poly-uridine mRNAs (A-) and sequenced them to see if they corresponded to genes found in minicircles, or whether they might be plastid genes held in some different architecture. All the A- mRNA corresponded to the 12 genes discovered in the minicircle. They carried out rarefaction analysis to see if their sample size was large enough, apparently it was, in fact 300 clones was way in excess of the amount needed to find a further, non-minicircled-gene.This suggests that minicircles are the only architecture for plastid genes and, importantly, that there really are only 12 genes contained in the chloroplast of the dinoflagellate Lingulodinium. This is a very small number of genes, all the rest have somehow migrated to the nucleus, leaving these 12 behind. And it's still very much an open question about why these have been left behind. The paper, in its discussion section puts forward the possibility of size. The genes that have been left behind all code for some of the longer proteins usually found in chloroplasts, although the paper does have the good grace to admit that that's not the most convincing of arguments.It's worlds away from my little bacteria. But still just as fascinating.---Wang, Y. (2006). Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium Nucleic Acids Research, 34 (2), 613-619 DOI: 10.1093/nar/gkj438... Read more »

Wang, Y. (2006) Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium. Nucleic Acids Research, 34(2), 613-619. DOI: 10.1093/nar/gkj438

November 28, 2010
06:38 AM
755 views

Bacterial comet tails

by Lab Rat in Lab Rat

I haven't worked very much with bacteria that infect humans. Most of my lab work has been done in the fields of either synthetic biology (which works with model organisms) or antibiotic production, which works on soil bacteria that produce the antibiotics. Human bacterial parasites therefore hold the fascination of the slightly exotic, not least because they sometimes do things like this:Figure from"Molecular Biology of the Cell, Fourth Edition"by Alberts et al.I've written before about some of the interesting features of intracellular bacteria, but this is possible one of the more exciting and fun things that they do. The picture above shows a eukaryote (human) cell outlined as a green oblong. Within the cell are lots of invasive bacteria (the red dots) some of which have a beautifully long green 'comet tail' flying out behind them.That comet tail isn't just for show, it is vitally important for movement. The inside of a eukaryote cell is a fairly crowded and busy place, bacteria can't just swim around inside the cell like they would in the wild. Instead they have to rely on physical methods to push them through the cell and like invading virus's (which I wrote about here) they hijack machinery inside the cell to move them around.Virus particles can latch onto the intracellular transportation machinery to hitch a free ride, but bacteria are too big for that. Instead, what most of them do is to produce proteins known as nucleation-promoting factors. These co-opt cellular proteins (the Arp2/3 complex for anyone with a background in actin polymerisation) which form branched actin fibres behind the bacterial cell, pushing it forward. The 'comet tail' pattern seem above, is seen by using a green stain for the structural actin protein, so you can see it forming long fibrous complexes behind the bacteria. These actin tails can move the bacteria wherever they want to go in the cell, and can also help with the invasion of neighbouring cells. This process is shown diagrammatically below:Figure from"Molecular Biology of the Cell, Fourth Edition"by Alberts et al.The actin system is a good one to use, as actin is ubiquitous inside all eukaryotic cells. In normal cell conditions is it used for structural purposes and is vital in cell division. Also important for the pathogenic bacteria (which after all does not want to kill its host straight away) is that none of these important cellular processes are compromised by the bacteria 'borrowing' some of the actin to move around with.Another interesting point is that different intracellular bacteria often produce different types of actin tails. L. monocytogenes and S. flexneri have short, highly crosslinked filaments producing short stubby little tails, whereas Rickettsia species have actin tails that are composed of distinctly longer bundles of unbranched actin filaments. Part of the reason for this is that different bacteria will produce different nucleation-promoting factors and some of the more lazy ones (i.e S. flexneri) don't even bother to do that and just use the host nucleation-promoting factors within the invaded cell! Recent work has shown that Rickettsia on the other hand, doesn't even rely on the host Arp2/3 complex to polymerase the actin and instead relies almost entirely on their own, bacterial, proteins.They truly are beautiful to look at though. Even without all the fancy colour staining:Listeria monocytogenes pushing right at the cell membrane, with actin tail behind. Electron micrograph picture taken from the se reference below---Ray K, Marteyn B, Sansonetti PJ, & Tang CM (2009). Life on the inside: the intracellular lifestyle of cytosolic bacteria. Nature reviews. Microbiology, 7 (5), 333-40 PMID: 19369949Kuo SC, & McGrath JL (2000). Steps and fluctuations of Listeria monocytogenes during actin-based motility. Nature, 407 (6807), 1026-9 PMID: 11069185---Follow me on Twitter!... Read more »

Ray K, Marteyn B, Sansonetti PJ, & Tang CM. (2009) Life on the inside: the intracellular lifestyle of cytosolic bacteria. Nature reviews. Microbiology, 7(5), 333-40. PMID: 19369949

Kuo SC, & McGrath JL. (2000) Steps and fluctuations of Listeria monocytogenes during actin-based motility. Nature, 407(6807), 1026-9. PMID: 11069185

October 28, 2010
08:32 AM
752 views

Bacterial cell division and membrane potential

by Lab Rat in Lab Rat

Bacterial cell division is usually quite a regular business. As I mentioned previously, not all bacteria use the regular FtsZ ring method of dividing, but for those that do division is mostly a matter of lining the right proteins along the middle of the bacteria, and then contracting a little ring of protein (FtsZ) around the centre of the bacteria to split the one cell into two cells.Many of the more critical proteins in the process are membrane-bound, in particular the Min proteins, which in E. coli accumulate at the cells poles and prevent formation of the FtsZ ring anywhere but the centre of the cell: A very simple diagram showing the Min proteins in fuzzy blue at the poles, and the FtsZ ring in red in the centre of the cell. This ring then contracts to create two bacterial cells. So a group of researchers in Newcastle were working on the Min system and looking at it by immobilising the bacteria on slides which were covered in a layer of polylysine. They were finding it very difficult to actually get this localisation of the min proteins, rather than being membrane bound at the poles the protein was all over the place. They did a few more tries and eventually realised that it was the polylysine that was causing the problem. Adding polylysine to cells trying to divide slowed the process right down as it stopped the Min from properly localising.What polylysine does to cells is affect the protein-motive force (pmf), which is used by bacteria to produce energy.Diagram shows a stylised version of protein motive force at the bottom of the bacteria. Ions are pumped through the membrane creating energy in the form of ATPIn order to verify that the crazy-behaving Min proteins were due to the polylysine affecting the pmf they tried using slides covered with other pmf-blockers such as CCCP, which is actually a bacterial poison as it kills the pmf completely (and before people start enquiring about its use as an antibiotic, bear in mind it's also a human poison, for the same reason). The results were clear, when CCCP is absent the Min clusters at the poles of the dividing bacteria, when it's present the Min is diffused throughout the cells:Cells in the absence (on the left) and the presence (on the right) of CCCP. MinD is attached to GFP, which is bright.In the figure above (from the reference) you can see the Min in the poles of the dividing cells on the left and in no particular orientation on the right. Killing the pmf leads to lack of arrangement of the proteins required for cell division. This affect was shown to be independent of the concentration of ATP in the cell, so it's not just that the lack of energy is preventing protein attachment, it's that the Min proteins rely on the correct ion concentration across the membrane in order to attach.The moral of the story: Sometimes things don't work for exciting reasons!(The other moral of the story: always wash your slides :p)---Strahl H, & Hamoen LW (2010). Membrane potential is important for bacterial cell division. Proceedings of the National Academy of Sciences of the United States of America, 107 (27), 12281-6 PMID: 20566861---Follow me on Twitter!... Read more »

Strahl H, & Hamoen LW. (2010) Membrane potential is important for bacterial cell division. Proceedings of the National Academy of Sciences of the United States of America, 107(27), 12281-6. PMID: 20566861

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