Field of Science

Biofilms and Bioshields

This post was chosen as an Editor's Selection for ResearchBlogging.orgExisting as a bacteria is tough, especially on your own. For a pathogenic strain it's even worse, not only do they face the challenges of the environment, but the human body is full of cells whose main task within the body is to seek out and destroy them. For this reason many bacteria in the body tend to stick together to form multi-cellular-like biofilm structures which give them a better chance at surviving the body's antibacterial defenses.

Biofilm formation, diagram taken from Davies lab website

Pseudomonas aeruginosa is an opportunistic human pathogen that can form biofilms and is often used to study biofilm formation. It can survive in soil, on the skin, and on a variety of surfaces, including hospital equipment such as catheters. It tends to infect humans who are already ill, and can colonise the urinary tract, kidneys and lungs, the latter making it a major secondary complication in cystic fibrosis.

One of the defenses against lung bacteria are polymorphic neutrophilic leukocytes (a type of bacteria-eating white blood cell, hereafter referred to as PMNs). These produce a variety of antibacteria chemicals, which break down invading species. However they are not able to destroy the P. aeruginosa biofilms. This isn't just because the biofilm is too complex to break into, it's due to a specific chemical; rhamnolipid. Remove the rhamnolipid from the bacteria and the PMNs can happily munch through the biofilm. This was shown with in vivo mouse studies, animals infected with bacteria unable to produce rhamnolipid contained twice as many viable PMNs in their lungs.

However rhamnolipids are not expressed all the time. They are quite potent chemicals after all, and could disrupt the surface of the cells that the P. aeruginosa are trying to grow on. They are only expressed in the presence of PMNs, as shown in the graph: (figure adapted from the reference below).

The dots on the very left show wild-type P. aeruginosa cells exposed to PMNs. Next, just the wild type cells, without any PMN exposure. These produce a lot less rhamnolipid. The final two results are taken from cells which are unable to produce any rhamnolipid, due to a gene knockout.

One of the most interesting things about the rhamnolipids is that they are not secreted outside the biofilm. Rather than release them into the surrounding environment the bacteria holds onto them, using them as a shield to coat the biofilm and protect it from PMN attack. Microscopy studies show that this is where the PMNs are killed as well, on the outside surface of the biofilm. This allows the action of a potentially destructive attack chemical to be carefully controlled. The rhamnolipid is only produced by the bacteria when a specific threat is present, and will then only act as a defense against cells that specifically try to attack the biofilm (microscopy figure taken from the reference below):


The biofilm is stained pink, while the surrounding PMN cells are stained blue, showing a clear dividing line between the bacteria and the surrounding attacking cells. The blue stains DNA, rather than cells, so it is the nuclei of the cells that are visible. In the top left of the picture, near the biofilm, some fuzzy blue lines can be seen, rather than the better defined blue blobs; these are DNA from PMNs that have been lysed and killed by the rhamnolipids.

The importance of this work lies in the fact that antibiotics don't really work on biofilms. Biofilms are made up of many layers of bacteria, packed deep in a peptidoglycan matrix. The antibiotic can kill off bacteria near the top of the biofilm, but can't penetrate deep enough to kill all the bacteria near the bottom. As these bacteria are exposed to a lower concentration of antibiotic (i.e not enough to kill them) they are also a lot more likely to develop resistance.

Researching interactions with the immune system therefore provides more information about new potential mechanisms for removing the bacterial infection. This is especially important for diseases like P. aeruginosa which hang around in hospitals, and are a major risk of secondary infection.

---

Alhede M, Bjarnsholt T, Jensen PØ, Phipps RK, Moser C, Christophersen L, Christensen LD, van Gennip M, Parsek M, Høiby N, Rasmussen TB, & Givskov M (2009). Pseudomonas aeruginosa recognizes and responds aggressively to the presence of polymorphonuclear leukocytes. Microbiology (Reading, England), 155 (Pt 11), 3500-8 PMID: 19643762

Endosymbiosis - a big tangled mess of algae

ResearchBlogging.orgNext term I'm taking a short course on plastid evolution (e.g chloroplasts), as it was the choice that came closest to my beloved bacteria. While I hold no great love for the inner workings of multicellular creatures, I'm forced to admit that there is something quite special about eukaryotic cells. They're full of little compartments, closed off organelles and selfishly horded genomes, including bacterial genomes within their mitochondria and chloroplasts. Also Psi Wavefunction writes about plastid-containing things a lot, and has convinced me that while they'll never be bacteria, they are pretty amazing in their own right.

The theory of endosymbiosis is that mitochondria and plastids (pigmented organelles such as chloroplasts) were once free-living bacteria-type organisms, which were engulfed by larger cells. Rather than being subsequently digested, these organisms managed to survive inside the larger cells, providing energy for them in return for a safe place to stay:
Diagram 'borrowed' from last years lecture notes

However this state of affairs immediately creates a problem. Not one of space, or resources, but of nuclei. Two nuclei are now present in the same organism, creating problems of control. The plastid cannot simply divide, replicate and produce energy whenever it wants, and the same is true of the surrounding cell, the two must work together, which means putting at least some of their genes under the same system of central control.

What usually happens is that over time the internal plasid's genes migrate to the nucleus of the surrounding organism (although apparently some protists will hold fairly epic genomic battles about who ends up with the majority of the genome). Genomic analysis shows the plastid or mitochondrian genes sitting happily in the nucleus, although the organelle will retain some of its genes in it's own little chromosome, probably for the same reason that the USA remains a federal government.

Doing further genetic analysis however, particularly on the chromalveolates (a large group of protists which include, among others, the red-tide producing dinoflagellates and the photosynthesising marine diatoms) shows that the story is not quite so clear cut. The chromalveolates are believed to all originate from an ancestor containing an engulfed red-algae plastid for photosynthesis. Analysis of the genome of the diatome P. tricornutums does indeed show red algal genes, however it also shows genes from green algae. In fact, large-scale phylogenetic analysis of algae and diatoms revealed over 1700 green algae genes in the P. tricornutums nucleus, outnumbering the red algae genes.

How did they get there?

One theory presented is that this diatom has in fact had two endosymbiotic events in its past; a green algae that later somehow disappeared or was lost, and then the red algae. However as there are many different branches of the chromoveolates containing varying amounts of red/green algae material this seems to make a rather large assumption about the ease of endosymbiosis...it sounds like algae are being absorbed and lost surprisingly easily, and quickly. The discovery that some chromoveolates without any plastids also have plastid genes in their nucleus doesn't help matters. How are the genes getting in, and is multiple rounds of endosymbiosis, followed by subsequent plastid loss really a realistic answer?

There are other explanations, although as yet no real answers. Green algae are not the most well-sampled of organisms, and the genomic record is distinctly patchy. This makes it a lot harder to determine where genes truly come from. Also there is the matter of horizontal gene transfer. Before being engulfed by the chromalveolates, the algae would have been able to share genes in much the same way bacteria do. The green algae genes might have got into the red algae before they were engulfed.

Horizontal gene transfer can also occur in some ciliates, which may explain the presence of plastid genes in their nucleus, despite the fact that they don't have any plastids. Ciliates often form symbiotic relationships with algae. This means that they will often come into contact with lysed algae, and may have been able to pick up genetic material from them. There also may be a certain 'background' of algae-like genes which in reality have nothing to do with algae. Some plastid-like genes have been found in amoeba, which really don't have any reason to have them, so it might just be an artifact.

Even so, the evolution and origin of plastids is clearly a wonderfully convoluted and undetermined area. Away from the weirdness of algae-containing protists there are still many questions to be answered. In the plant model-organism arabidopsis there is still a fascinating interplay between the genomes of the chloroplast and the nucleus. The chloroplast uses several nuclear genes that it never even supplied in the first place, it seems to have hijacked some of the nuclear genes for it's own purposes. The arabidopsis nucleus has returned the favour, with less than half of the genes supplied by the chloroplast being used for chloroplast-related purposes.

I can't wait to start studying it. (Especially because it means the endlessly boring set of 'techniques' lectures will finally be over.)

---

Elias M, & Archibald JM (2009). Sizing up the genomic footprint of endosymbiosis. BioEssays : news and reviews in molecular, cellular and developmental biology, 31 (12), 1273-1279 PMID: 19921698

Second Generation Biofuels

ResearchBlogging.orgAs part of my course last year, I wrote an extended long essay concerning the use of bacteria in biorefineries. As I've had a very lazy weekend (and to celebrate crossing the hundred post mark) I've decided to reproduce some of it here. More may be forthcoming at some point, depending on the laziness of my weekends.

Second Generation Biofuels

Second generation biofuels consist of lignocellulose material, which is broken down into simple sugars via enzymatic reactions and then fermented to produce ethanol. As lignocelluloses can be found in inedible plant matter (e.g corn husks, rice stems, and wheat stalks) they have the advantage that, unlike first generation biofuels, their utilisation does not compete with food production.

The three main components of lignocelluloses material are celluloses, hemicelluloses and lignin. These cannot be fermented directly and must therefore be broken down:The pre-treatment of the biomass is necessary both to remove lignin (although effective ligninases have been found in white-rot fungi, their rate of product turnover in bacteria is still too slow to be commercially successful) and to partially break down the cellulose to allow easier digestion by microbial processes. As the pre-treatment consists of harsh chemical processes, it would be advantageous within a biorefinery to use microbes which are able to withstand high temperatures and low pHs. For example, the cloning of thermostable cellulases into Trichoderma reesei allows a higher hydrolysis temperature compared to commercial Trichoderma enzyme, reducing the energy needed to cool the system after pretreatment with steam. The ability to save energy in this way could have a large economic impact, making the biorefinery more commercially feasible.

Currently one of the most popular microorganisms for use in lignocellulose biofuel production is Clostridium thermocellum which has an optimum temperature of around 60°C and also contains a cellulosome; a multi-protein cellulose-degrading complex attached to the bacterial cell wall. Cellulosomes are found in several bacteria, both Gram negative and positive, although they can differ in their structure and organisation (particularly of the cohesins and dockerins).
As potentially the entire process of ethanol production from lignocelluloses could be carried out by the microbes within a fermentor, the use of second generation biofuels in biorefineries has generated a lot of interest. The three main economic obstacles are the high processing costs, the narrow margin between biomass and fuel prices, and the large capital investment needed to initiate a cellulosic biorefinery. This could however, be overcome by increasing the potential for the production of high-value goods alongside the biofuel, either by adding pathways for the production of oleochemicals or bioplastics to the fermenting bacteria, or by utilising the lignin. This would provide the biorefinery with a greater capital return.

---

Gilbert, H. (2007). Cellulosomes: microbial nanomachines that display plasticity in quaternary structure Molecular Microbiology, 63 (6), 1568-1576 DOI: 10.1111/j.1365-2958.2007.05640.x

Blumer-Schuette, S., Kataeva, I., Westpheling, J., Adams, M., & Kelly, R. (2008). Extremely thermophilic microorganisms for biomass conversion: status and prospects Current Opinion in Biotechnology, 19 (3), 210-217 DOI: 10.1016/j.copbio.2008.04.007

Zhang, Y. (2005). Cellulose utilization by Clostridium thermocellum: Bioenergetics and hydrolysis product assimilation Proceedings of the National Academy of Sciences, 102 (20), 7321-7325 DOI: 10.1073/pnas.0408734102

Motility of Cancer Cells

ResearchBlogging.orgCancer is a disease of multicellular organisms. In order to become multicellular, a certain amount of control needs to be exerted over each individual cell, cells can no longer move around, grow, and divide when they want too. Instead they must obey signals from the surrounding environment (including their fellow cells) which tell them what to do. Cancer, like anarchy, is what happens when the control breaks down, and individual cells start growing and dividing regardless.

Uncontrolled growth leads to a neoplasm, a large mass of abnormal tissue. These can be benign, and merely exist in the body without causing too many problems, or they can start to become cancerous, invading surrounding tissues, and sometimes entering the bloodstream and spreading to further locations within the body.

(Because of this, cancer is primarily a disease of deterministic multicellular organisms. Plants and other non-determinists can get tumours, but tend not to be so badly affected by them, as they are constantly growing anyway)

In order to break away from the neoplasm and spread the disease cancer cells must gain motility. Studying how cancer cells move can be difficult in vivo because the conventional method of immuno-histology (which involves taking slices out of a tumour during development then fixing and staining them) prevent movement all together. Newer work has been done using Intravital imaging (shown diagrammatically on the rather cute little picture on the right) , where a fluorescently-labelled tumour is generated in an animal and then observed while the animal is anaesthetised. This gives a perfect in vivo image of what is actually happening inside the living tumour cell, in these images you can see cells moving in real time, and examine how they act under the effects of internal mutations and changes in external conditions.

One of the things that this type of imaging revealed was that most of the cells in a tumour don't move (less than 0.1% tumour cells in vivo/hour). Furthermore, there were two types of movement. Firstly, individual cells, that darted around on their own, fairly quickly and in all directions. Secondly large clumps of cells, that moved relatively slowly, but in the same direction with a more ordered internal microtubular structure.

Single celled movementTwo types of movement were first described; mesenchymal and amoeboid. As the main difference between them lies in the speed and number of direction changes it has been suggested that the distinction may be an artifact of different experimental conditions, rather than actual physical difference. The movement (which has been studied extensively in mesenchymal cells as they can be stuck down onto 2D surfaces) is initiated by signals to receptor tyrosine kinases. These turn on the small G-protein, Rac which, among other things, activates Arp2/3 proteins, which control the nucleation of actin subunits. In the diagram above the actin is shown in red, and it is used to generate movement of the cell. As the cell is forcing it's way through a thick external extracellular matrix (EMC) it also secretes proteases and MMPs to break this down, allowing easier forward movement.

Collective cell movement
(All images are taken from the reference below, and I've included the key for those with an interest in how eukaryote cells hold themselves together)

Collective motility is the movement of whole groups of cells, either in clusters or chains. These cells can move between tissues, spreading the tumour (in particular they can get into the lymph system) but they are not normally found in the bloodstream. Interestingly, collective motility is seen when the conditions for single-cell motility have been blocked, suggesting that the rapid-moving single cells are a transient stage in order to get the tumour into the bloodstream. Once it finds a new environment, it reverts back to the less-motile stage, with large clumps of tissue still able to (very slowly) manoeuvre themselves into nearby tissues.

---

SAHAI, E. (2005). Mechanisms of cancer cell invasion Current Opinion in Genetics & Development, 15 (1), 87-96 DOI: 10.1016/j.gde.2004.12.002

Trends in bacterial signalling pathways

ResearchBlogging.orgBacterial (and archaeal) signalling systems are remarkably similar to eukaryotic ones. As well as the typical and well described two-component signalling systems (a histadine-kinase sensor which senses a signal and passes this on to a response-regulator) bacteria also contain multi-component systems, for both inter- and intra-cellular signalling.

All cells constantly monitor their external and internal environment in order to effectively exploit their surroundings. Bacteria are no different, and all contain a variety of different signal transduction pathways in order to do this. The distribution of these transduction pathways varies between bacteria, based not just on phylogeny but lifestyle and environment as well. Galperin's study (reference below) looked at the distribution of bacterial signalling systems over 167 different genomes (including some archaea) to examine if there were any overall trends.

The first trend he found was that the number of signal transduction pathways in a genome increases with genome size:
Image taken from reference (1) below
The graph above shows log[protein number] on the y-axis plotted against log[genome size] on the x-axis. There are exceptions, but the overall trend is that the two have a clear linear relationship: the more genes a bacteria has, the more of them it will dedicate to monitoring its internal and external surroundings.

The second trend, which perhaps is not quite such a surprising find, is that the number of signal transduction pathways depends markedly on the surrounding environment. Ruminant bacteria (with a few exceptions) have smaller and simpler transduction pathways, with fewer external signals being reported. In contrast, facultative pathogens, which have diverse ecological niches, will have far more systems for sensing their surroundings. This makes sense, as faculative anaerobes need to respond and react to the different environments in which they find themselves. Ruminant bacteria, which live their whole lives in the gut of ruminant mammals (such as sheep and cattle), only can survive in one environment, so have very little need for sophisticated sensor-response systems.

It was also found that gram negative bacteria have a larger number of signalling systems than gram positive. This may be due to the fact that gram negative bacteria are motile, and therefore need to monitor what kind of environment they are moving into, and how their internal conditions are reacting to the change. It may also have something to do with the difference in cell wall structure. Gram negative cells have two cell membranes, with a glycoprotein layer in the middle (Gram positives are just glycoprotein all the way up) and are therefore more likely to have signalling systems with more than two componants, as the signal in some cases must be transmitted first through the outer membrane, and then through the inner.

As well as looking at responses to extracellular conditions, the study also examined intracellular signalling pathways, the bacterial sensing of its internal conditions. Intracellular pathways followed different trends to extracellular ones, for example free-living archaea were found to have fewer extracellular signaling transduction pathways, but more intracellular ones. Cyanobacteria were found to have the largest number of intracellular pathways. A potential reason for this is that cyanobacteria are the only bacteria that produce oxygen, rather than trying to keep it outside the cell. Oxygen is dangerous, it produces reactive species and affects the overall redox balance inside the cell. Cyanobacteria therefore may need more signalling pathways to keep a close eye on the oxygen inside it's cell.

The study also shows that, despite the evolutionary similarity to eukaryotic systems, there are some unique features within bacterial signalling systems (especially the lesser explored internal cell signalling). Many of these are shared by pathogenic bacteria, offering the potential for another target for bacteriocidal drugs, or at the very least drugs that prevent the growth and invasion of bacteria in the body, allowing the immune system time to safely remove them.

---

1)Galperin MY (2005). A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts. BMC microbiology, 5 PMID: 15955239

Lab etiquette and priorities...

I've been blogging quite a lot about actual science lately (which is good) so I think I'm excused one quick blog post about being a scientist, or in my case, being a Lab Rat. The thing is that I don't rank particularly highly in the lab; I'm doing a one year project, with not a huge amount of significance and in terms of funding I'm pretty sure the ants underneath the floorboards get paid more to be here than I do. And while I personally like to think I'm doing something useful for the lab I'm in, they'd probably be reasonably grateful if I served my time without causing any major damage, or using up too many resources.

So what do I do when (as happened just now) someone asks me if they can use the hood for the afternoon? And in a way that suggests that they kind of really need to use the hood that afternoon...

(Hood = laminar flow hood, which is a sterile working space).

What I did was to internally rearrange my schedule inside my head, to free up space for them. But what if that hadn't been an option? What if I'd been doing an infinitely-long colony pick or something? Who, in that situation, has the priority?

The PostDoc with their relevant research, or the Lab Rat who, by an accident of bureaucratic shuffling, happens to actually be in the lab that owns the hood? (there's about three labs in the space upstairs and we tend to share equipment)

I quickly finished up everything I needed to do right then, and then told the aforementioned PostDoc that she could use the hood whenever. Except...I still have more work to do in there this evening. And she hasn't actually started work in there yet. I don't want to start my work in case she suddenly appears, but I have no idea how long it is polite to wait before getting naggy at someone. Especially when that someone is a) older than me b) doing more important work for the lab and c) bringing more money in for the lab.

I have decided to deal with this problem in my usual way of dealing with all problems, by scuttling away and hoping it disappears in it's own time if I stop looking at it. Which is why I'm currently sitting downstairs in the dry lab using the computer and consoling myself with vending machine chocolate bars. I can't really go up until either she's finished with the hood or my E. coli plates dry, and they won't for a while because I accidentally massively over-poured one of them.

On the plus side, I discovered why my transformations didn't work yesterday. I plated them out on the wrong antibiotics and they all died. In my current state of mind, I can't help but find this kindof hilarious.

Amphibian Skin

ResearchBlogging.orgI decided to take a break from bacteria today and decided it might be fun to just choose something totally random to write about. Taking a daring leap into the unknown I decided not only to try and find out somthing about multicellular creatures, but about those multicellular creatures about which I know the least: amphibians.
The above picture of an axelotl may have had something to do with my decision. Note the similarity to a pokemon that has accidently wandered into Star Wars.

There are three main orders of amphibians; salamanders and newts, toads and frogs, and caecilians; the blind legless ones that live at the bottom of caves. They are cold blooded and, unlike many other multicellulared animals, they don't regard the outside environment as completely seperated from the inner. They can exchange both water and oxygen through their skin, in fact some salamanders exchange all of their oxygen in this way, and thus don't have any gils or lungs at all.

Skin is therefore a very exceptional organ in salamanders, as it is used for fluid balance, respiration and the transport of essential ions, as well as the more traditional uses of protection and sensing. Possibly because of this, it has it's own protection system against infection. Amphibians have both an adaptive and innate immune system, but in addition to this they have granular glands under the dermis layer of the skin that release antimicrobial peptides in response to stress. Peptide release is stimulated by the adrenergic receptors, so any circumstance of shock of pressure results in an extra layer of protective peptides over the skin surface. They are quite potent as well, providing potential protection from bacteria, fungi, protazoa and even viruses.

As cold-blooded creatures have a slower reacting immune system, this quick, automatic and generic response to stressful conditions provides important protection for the skin, which is vital for maintaining internal homeostasis. And as well as peptide-releasng glands, they also have pigment granules under the skin, which give them bright colours and mean they can change between colours depending on environmental conditions or what they want to communicate.
Amphibians have such beautiful colours...why do they always make dinosaurs blotchy khaki!

There is however a downside to using your skin to take things up from the environment. Water isn't the only thing that gets through permeable skin, chemicals dissolved in the water can as well, which can be fatal if the chemical in question is a herbicide such as atrazine or glyphosate. Chemical contamination may be one reason (and there are, no doubt, many others) for the dramatic decline in the number of amphibians over recent years. Apparently conservationists are majorly concerned about this.

And they don't seem to get as much press as endangered mammals either. Which is a pity because that axelotl does look quite sweet. And amphibians are the only living proof we have left to remind us that the dinosaurs could have had bright-coloured polkadots:
Imagine those colours....on a velociraptor!
---

Rollins-Smith, L. (2005). Antimicrobial Peptide Defenses in Amphibian Skin Integrative and Comparative Biology, 45 (1), 137-142 DOI: 10.1093/icb/45.1.137

Quaranta A, Bellantuono V, Cassano G, & Lippe C (2009). Why amphibians are more sensitive than mammals to xenobiotics. PloS one, 4 (11) PMID: 19888346

Idea Space

One of the things about working in a research lab, is that it's very easy to get lost in the little world that you're currently working in. Day to day research really is little-picture stuff; while you know that your work has bigger implications, and you drag them out the filing cabinet every time you want to write a grant application, you don't often have the time, or the inclination, to just sit down and think about where your work could go.

This is why scientists need friends. And I discovered over the summer that this especially applies to friends who are also art and design students. Because while you're busy squinting at gels and trying to convince yourself you have a band 2kb long they are getting excited at the fact that you have purple bacteria, actual purple bacteria, and they're thinking of all the amazing things you can do with that.

I'm still recovering from jet lag a little, so I'll try to put this in context just using pictures. This is what I see:
And this is what they see...

Scatalog picture from the E.chromi design team, more details here

Once you've stopped sniggering at the fact that it is a case of coloured poo, it starts to dawn that this is actually a very elegant system for searching for intestinal problems. taking bacteria that turn different colours in response to different conditions can result in a full spectrum (as it were) of the conditions in your stomach, just from looking at your poo. And doctors generally do look at poo to check how a patient is doing, this just gives a clearer picture.

And once you start thinking about it, there are a huge number of applications for coloured bacteria. Here's a few I've thought up over the course of the last few weeks:
  • Putting the colours into spores (e.g from B. subtilis) could give you little dots of colour: bacterial pixels
  • Industrial fermentors use bacteria and yeast. Adding conditional-dependant colours could allow you to check the conditions (i.e temperature, pH) without needing monitoring equipment, the bacteria just tell you themselves.
  • Bacterial pigments for the pigment industry in general, there is a whole range of different colours in nature, you could make them into paint/dyes/etc just with a fermentor.
  • Moving bacterial art. Bacteria swarm in the direction of food sources. Swarming coloured bacteria would be awesome, they'd look like that bit near the end of 2001:A Space Odyssey where he does the trippy planet landing.
  • And of course, environment monitoring. Get lead sensing bacteria, drop possibly-contaminated water on them, leave in the incubator overnight and if the thing turns bright red the water isn't safe. Easy, convenient, and potentially quite cheep.
There's probably many more, whole worlds of idea-space for potential applications. They're just a little hard to see when you're standing two inches away from a gel, squinting through an ethidium bromide visor.

This is a massive shoutout to the two design students (who know who they are!) who helped me and my fellow lab rats retain our sanity over the holidays. You took our humble experiments and took them in such wonderful, marvelous directions, and at the end of it all you managed to get a case of coloured poo past Heathrow Airport security. And to all design students anywhere who are working with science; trust me, we need you. :)