The Times Microbial

Speculation Surrounding Sporulation in the Mycobacteria

noreply@blogger.com (Tim Sampson) — Tue, 06 Apr 2010 12:00:00 +0000

by Tim

The Mycobacteria are quite the unique genus; not quite Gram-positive due to their waxy mycolic acids on their outer surface, but certainly not Gram-negative as they do not have an outer lipid bilayer. (Although, there are some interesting micrographs showing a structural feature that does look a lot like a typical Gram-negative outer membrane on the surface of Mycobacter ia.) Much slower growing than the average bacteria studied in the lab and not so easily manipulated genetically (though see here and here for some recent advances), studying this important group of pathogens challenges a researcher's patience for results. (Imagine waiting a month or more for a colony to grow to see if a simple transformation was successful!)

Neveretheless, the study of these bacteria is vital as the Mycobacteria represent one of the largest, global public health concerns. Over 1/3 of the world's population is thought to be infected with Mycobacterium tuberculosis, and a growing percentage of this population is exceedingly drug resistant. During infection, the bacterial population often enters a latent state, making antibiotic treatment (and diagnosis) difficult.

In an effort to study this latent phase, a research group happened upon what appeared to be textbook endospores in very late stationary phase cultures of Mycobacterium marinum, a common model for acute Mycobacteria infection. As they closely examined cell morphology over long periods of time (2 months), they began to see forespore formation, and subsequent endospores. Utilizing TEM, the distinct outer coat and cortex of the spores could be identified in the M. marinum population.

These researchers analyzed rRNA from the sporulating cells and identified it as M. marinum, ruling out contamination issues. Also, they demonstrated heat tolerance, malachite green staining, and the presence of dipicolinic acid an important compound necessary for heat resistance in most spore-forming species. Furthermore, their bioinformatic analysis revealed the presence of homologs of genes utilized in sporulation within the M. marinum genome. Taken all together, the authors hypothesize that M. marinum forms spores, and posit that these spores may be how Mycobacteria stay dormant in a host.

However, here is where the speculation steps in...

A group of rather prominent US labs in the field attempted to replicate M. marinum sporulation in their labs, and were unable to demonstrate sporulation by any of the methods used by the original researchers. Furthermore, in an attempt to see if spores were present during latent M. marinum infection as the original authors postulated, they attempted to isolate heat resistant cells from infected frogs. Although they could recover Bacillus spores from spiked tissue samples, they could never isolate M. marinum.

Additionally, the authors demonstrate that the majority of the genes picked out by the original researchers as being sporulation homologs in Mycobacteria, are actually rather universal Gram-positive genes, with homologs present in a wide variety of species, without being used for sporulation. Some of these genes identified by the original group are also not necessary for sporulation, even in Bacillus sp. Finally, neither group of researchers could identify a group of coat proteins, in M. marinum, that are necessary for spore formation in Bacillus.

The lack of this set of coat proteins could be explained by the hypothesis that M. marinum forms its coat differently. This would be a fine rationale, however the spores the original researchers imaged looked identical to those found in B. subtilis. This is of note, particularly because even within the Bacillus genus (with similar coat proteins), spores between species are highly dissimilar.

With this set of data, the second set of researchers conclude that M. marinum does not form spores, and that the simplest solution is that the original authors were looking at Bacillus sp. spores, and not those of M. marinum.

Looking at these two articles, what do you think? (It's time to stop reading articles that don't pertain to my thesis proposal and get back to studying for quals!)

Sources:
Ghosh, J., Larsson, P., Singh, B., Pettersson, B., Islam, N., Sarkar, S., Dasgupta, S., & Kirsebom, L. (2009). Sporulation in mycobacteria Proceedings of the National Academy of Sciences, 106 (26), 10781-10786 DOI: 10.1073/pnas.0904104106

Traag, B., Driks, A., Stragier, P., Bitter, W., Broussard, G., Hatfull, G., Chu, F., Adams, K., Ramakrishnan, L., & Losick, R. (2009). Do mycobacteria produce endospores? Proceedings of the National Academy of Sciences, 107 (2), 878-881 DOI: 10.1073/pnas.0911299107

Other Articles of Interest:
A Fatty Acid Synthetase is Necessary for Active TB Infection
Recombineering: A Practical Application of Phage Biology
Prokaryotes Can Do Geometry, And Even Have Their Own Protractor

Help me Win a MiniGrant!

noreply@blogger.com (Tim Sampson) — Fri, 26 Mar 2010 18:40:00 +0000

BenchFly.com is sponsoring a mini-grant competition. The winner(s) receive $500 towards the pursuit of their particular project.

Proposals have 100 words or less to convince readers (including non-microbiologists) to vote for the project. You can vote for mine through Facebook at this link.

I've included my proposal here to catch your interest:

Bacterial Super-Ninjas: Staying in the Shadows:
Like a ninja on a mission, Francisella tularensis is a silent, deadly bacterial pathogen. Despite being weaponized and a recognized bioterrorism agent, little is known about how Francisella escapes immune detection. We have isolated a Francisella protein that blocks the immune system’s ability to sound alarms during infection. Preliminary evidence suggests that it is used as a projectile weapon, directly attacking immune communication lines. This grant will allow us to examine how this protein is fired from Francisella into our immune cells, rendering them mute. Importantly, this represents a novel target for vaccine development against this bacterial assassin.

Here is the voting link again: Bacterial Super-Ninjas: Staying in the Shadows

If you have any more questions, such as specifics to what I will do with the funds if I win or a more technical outline of my project, feel free to e-mail me!

You can vote once per day, so feel free to continue voting until the contest ends in May!

Thank you so much!

A Role for the Host-Microbe Interface in Obesity

noreply@blogger.com (Tim Sampson) — Mon, 08 Mar 2010 13:30:00 +0000

by Tim

Our bodies are home to 10 times more bacterial cells than human cells. With such sheer numbers, we have developed an intricate balance with the mutualists living on our skin and in our guts. Their mere presence is vital for protection from pathogenic species; but at the same time, our immune system must keep their numbers in check to prevent overgrowth.

Those bacteria within our guts perform important roles in fermenting carbohydrates and producing essential nutrients like vitamin K and biotin. As such, it is not too difficult for us to realize the importance of our gut flora in our health. I do not even need to mention the countless mainstream (and not so mainstream) probiotic supplements available in grocery stores.

Over the last few years, there have been a number of studies demonstrating the importance of gut flora in proper metabolic function, where dysfunction yields obesity. One article published in Nature in 2006, described a microbiome sequence comparison of obese vs. lean mice, finding the ratios of different phyla between the two mice strains. A few complementary studies have been performed since with the same result: the metabolism of the microbes in the gut is directly linked to the host's metabolic health.

This raises the question, how is gut flora composition determined? While studying up on some innate immunology literature for my qualifying exam, I came across this fantastic article set for publication in Science describing the role of the innate immune system in regulating those microbes necessary for proper metabolic function.

The authors describe the metabolic dysfunction of Toll-like Receptor 5 knockout mice. TLR5 is a receptor present on innate immune cells and intestinal epithelium and is responsible for sensing bacterial flagellin, subsequently priming cells for an immune response. Interestingly, mice deficient in TLR5 show drastic metabolic dysfunction; the authors go to a great extent characterizing this phenotype. Everything from fat pad size, to blood glucose, insulin, cholesterol and triglyceride levels are significantly increased in the mutants.

Furthermore, when the gut flora of the TLR5 knockout mice are depleted by antibiotics, the metabolic dysfunction clears and the mice's metabolism returns to wild-type levels. To top it off, the gut flora of TLR5 knockout mice were removed and introduced to wild-type germ-free mice. The introduced gut flora induced metabolic dysfunction in these wild-type mice identical to that of the TLR5 knockouts.

Unlike previous studies which saw different ratios of bacterial populations between obese and lean mice (ie. %Firmicutes vs %Bacteroidetes), this study found that instead specific species were present or absent in the TLR5 knockouts, indicating a role for TLR5 in maintaining specific characteristics in population of gut microbes.

This is the first study, to my knowledge, which equates mucosal immune surveillance with a metabolic phenotype. Furthermore, it strengthens the argument that the complex interactions we have with our bacterial symbiotes are vital to our physiology. The microbes around us do more than just cause disease or ferment our cheese, but are also key in allowing us as an organism to function.

Source:
Vijay-Kumar, M., Aitken, J., Carvalho, F., Cullender, T., Mwangi, S., Srinivasan, S., Sitaraman, S., Knight, R., Ley, R., & Gewirtz, A. (2010). Metabolic Syndrome and Altered Gut Microbiota in Mice Lacking Toll-Like Receptor 5 Science DOI: 10.1126/science.1179721

Other Articles of Interest:
How Helicobacter Gets Around
A Home for the Bugs in Our Appendix
A MAP to Crohn's Disease: Revisiting Koch's Postulates
Altruism in Bacteria: Allowing Yourself to Die for the Good of the Species

Noisy and Bistable Gene Expression: Hedging Your Bets

noreply@blogger.com (Tim Sampson) — Fri, 05 Mar 2010 14:00:00 +0000

by Tim

This is Part 2 of a two part series. Part 1 can be found here.

Often times resistance to antibiotics has a genetic basis. That is, a bacteria encodes a protein that functions to export, degrade, or otherwise block the function of a given antibiotic compound. However in the last few years, the observation of non inherited antibiotic resistance has come into view. One of my favorite articles describing the phenomenon is one by Nathalie Balaban et al, in Science back in 2004, particularly due to her super cool use of microfluidics and single cell imaging.

Following treatment with an antibiotic, a bacterial population doesn't completely die off, but instead shows at first a drastic decrease in viable counts followed by a much slower rate of death. Those cells isolated at later time points are not resistant to the antibiotic, and when grown to full populations show the same sensitive phenotype. These cells are termed "persistors" and are a form of non-genetic resistance to antibiotic treatment.

Many of us know that some antibiotics require actively growing bacteria to be effective. Particularly, the use of the beta-lactams which are only effective when a cell is growing it's peptidoglycan, such as during replication. However, Balaban shows us that even in an actively growing population of identical cells in identical environments (microfluidic chambers), there is a very small subset of cells which are not growing. These cells aren't defective, but rather are in a paused growth cycle, allowing them to be resistant to ampicillin at that moment in time. When these cells restart their growth, their progeny are still sensitive. Again, a key point in demonstrating that this form of resistance is non heritable.

Very likely, genetic noise, as described previously, plays a role in determining the cell's fate. However, the more important point here (in my humble opinion) is that this is a beautiful example of a population that is "hedging its bets."

By having two sub populations: a majority that is actively growing, and a small percentage that is paused, that identical set of genes in both populations is capable of either 1) exploiting the current resources at the risk of antibiotic death or 2) taking a short pause in growth allowing antibiotic resistance at that time, at the risk of losing out on some resources. A risk, but with large rewards.

Each has their consequences, but together allow for a dynamic population without changes in genetic content (hence the non inherited resistance). Other great examples of bet-hedging include: sporulation and cannibalism in Bacillus, and the induction of inflammation by Salmonella.

Source:
Nathalie Q. Balaban, Jack Merrin, Remy Chait, Lukasz Kowalik, Stanislas Leibler (2004). Bacterial Persistence as a Phenotypic Switch Science, 305 (5690), 1622-1625 : http://www.sciencemag.org/cgi/content/full/305/5690/1622

Other Articles of Interest:
Noisy and Bistable Gene Expression: Why Genes and Environment Aren't Everything
Altruism in Bacteria: Allowing Yourself to Die for the Good of the Species
Antibiotic Treatment: Increasing the Rate of Genetic Exchange

Personal Note:
Due to my ever-looming qualifying examination (April 30th), updates to The Times Microbial may be rarer than usual this semester. I hope to get back to full swing, perhaps with some surprises, this Spring.

Not All Mice Are Created Equal When it Comes to Gonorrhea

noreply@blogger.com (Tim Sampson) — Mon, 28 Dec 2009 14:00:00 +0000

by E. Ohneck

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection, gonorrhea, is a Gram-negative diplococcus and an obligate human pathogen. An estimated 800,000 cases of gonorrhea occur each year in the United States (1). The most common sites of infection are the cervix and the male urethra, and symptomatic infection is characterized by a purulent exudate composed of numerous polymorphonuclear leukocytes (PMNs) containing intracellular gonococci (3).

The development of whole model systems to study N. gonorrhoeae infection is important, as several different cell types are involved in host response. Currently, infection in males is examined through urethral inoculation of male volunteers, and results indicate a strong innate immune response characterized by induction of proinflammatory cytokines (3). Due to the risk of serious complications and the frequency of asymptomatic infection in women, such volunteer infection models cannot be utilized for female infection, and studies are currently limited to tissue culture cells and a developing 17β estradiol-treated BALB/c mouse model (3).

To better characterize the innate immune response to gonococcal infection in BALB/c mice, and to determine if response to infection varies among different inbred mouse strains, Packiam et al analyzed the ability of BALB/c, C57BL/6, and C3H/HeN mice to support vaginal colonization by N. gonorrhoeae and mount an inflammatory response. Their results show the establishment of a productive infection in BALB/c mice characterized by a large influx of PMNs and the induction of proinflammatory cytokines, including TNFα, IL-6, and MIP-2, a homolog of human IL-83.

C57BL/6 mice, however, while able to support colonization by N. gonorrhoeae to the same levels as BALB/c mice, did not mount an inflammatory response, and thus might provide a model for asymptomatic infection (3). Finally, C3H/HeN mice were inherently resistant to N. gonorrhoeae by a mechanism other than an overwhelming innate response, as shown by their inability to support colonization and a lack of PMN influx upon infection (3). Taken together, these results suggest an import role for host genetic background in determining susceptibility to N. gonorrhoeae.

This phenomenon is not restricted to N. gonorrhoeae. In fact, variation in vulnerability to specific pathogens among different inbred mouse strains has been observed for numerous bacteria, including Mycobacterium tuberculosis, Salmonella typhimurium, and Legionella pneumophila (2).

These results, therefore, raise several important questions. What genetic factors mediate susceptibility to specific pathogens in otherwise healthy individuals? What bacterial systems and components are involved in determining host genetic background-specific susceptibility? How can we exploit these host-pathogen relationships in the development of more efficient therapies and vaccines?

Bacterial disease is often studied from two distinct angles: microbial mechanisms of pathogenesis and virulence, and host defenses against microbial invaders. Currently, however, more emphasis is being placed on examining the host-pathogen interface, and studies such as this elucidate the demand for the breakdown of the barrier between microbiology and immunology. We cannot fully understand microbial disease mechanisms without taking into consideration host, pathogen, and the complex interactions that mediate their relationship.

Sources:
1. CDC. Increase in fluoroquinolone-resistant Neisseria gonorrhoeae among men who have sex with men—United States, 2003, and revised recommendations for gonorrhea treatment, 2004. MMWR Morb Mortal Wkly Rep 2004; 53: 335 – 338.

2. Kramnik I and Boyartchuk V. Immunity to intracellular pathogens as a complex genetic train. Curr. Opion. Microbiol. 2002; 5:111 – 117.

3. Packiam M, Veit SJ, Anderson DJ, Ingalls RR, & Jerse AE (2010). Mouse strain-dependent differences in susceptibility to Neisseria gonorrhoeae infection and induction of innate immune responses. Infection and immunity, 78 (1), 433-40 PMID: 19901062

Other Articles of Interest:
Out With the Bad: Efflux in Klebsiella
Nitric Oxide Synthase Isn't Just Used By Our White Blood Cells
A Home for the Bugs in Our Appendix

Noisy and Bistable Gene Expression: Why Genes and Environment Aren't Everything

noreply@blogger.com (Tim Sampson) — Mon, 21 Dec 2009 14:00:00 +0000

by Tim

Classically, a cell's phenotype was thought to be a product of its genetic background and its environment. All changes within a cell would be due to the cell's genetic capability to react to the environmental changes happening around them. However, as we begin looking more in depth at cell populations at the single cell level, we are finding that this paradigm isn't always true.

In this two part series, I want to examine how genetically identical cells in equal environments can undergo very different developmental changes. Specifically, we will look at the induction of competence in Bacillus subtilis and the development of persister cells in E. coli. In both of these cases, the cells are genetically and environmentally identical. But in each case, a subset of the population undergoes a drastically different developmental change.

As Bacillus begins to enter stationary phase, a subpopulation of cells begins to become competent, allowing a small percentage of cells to take up DNA from the environment. This process is activated by the ComK protein, which also activates its own expression in a positive feedback loop. As ComK levels increase, a threshold is hit, leading to a rapid increase in ComK production, and a subpopulation of cells begin to enter competence. This leads to the question as to the mechanism which allows only a subset of cells to enter competence.

Back in 2007, the Dubnau Lab at UMDNJ published a paper in Science discussing how very slight variations, or noise, in the amount of comK mRNA could lead to certain cells becoming competent, while others remained vegetative. The authors used fluorescent in situ hybridization to measure the exact amount of comK mRNA in individual cells. They found that in early stationary phase the number of comK mRNAs in the total population increased from 0.7 to 1 per cell. As the population average increased ever so slightly, a subpopulation began switching to competence. Thus, as the average was shifted, a small subset was well-enough above the mean to hit the ComK threshold and enter competence. Noise in gene expression dictated which cells became competent.

Those cell to cell variations of comK expression could be due to intrinsic noise, events such as mRNA decay or transcription initiation rates, or extrinsic noise such as the concentration of polymerases or transcription factors. To test which of these contributed to comK variability, the authors again used the in situ hybridization technique to measure comK mRNA as well as a control mRNA under a comK promoter. Extrinsic variations should affect both mRNAs equally, while intrinsic variations would only be acting on a single locus. Counting the number of mRNAs immediately before the induction of competence, they found that the number of each mRNA type was uncorrelated, indicating that random intrinsic noise was responsible for the variations in comK expression.

So what does this all mean?

In essence, we must first understand that when we see a population with our favorite gene (OFG) expressed at level "X," really "X" just represents the mean expression of the population, and that per cell, the expression of this gene falls somewhere in a normal curve around "X." Cells with slightly higher or lower levels of OFG can end up having large effects on phenotypic outcomes, particularly if OFG is a regulator.

Importantly, it shows that not all phenotypic outcomes are entirely the sum of genetics and environment, but can be due to completely random events within the cell.

It also raises the question as to why the whole population would only want a subset to become competent. Perhaps it is a form of the bacteria hedging their bets, allowing only a small percentage to take a great risk (i.e. bringing in harmful DNA, delaying sporulation) to receive great reward (new genetic information to outcompete others), while the remaining population continues laissez faire. But this can be a topic for next week when we take a look at bet-hedging in persister cells.

Source:
Maamar H, Raj A, & Dubnau D (2007). Noise in gene expression determines cell fate in Bacillus subtilis. Science (New York, N.Y.), 317 (5837), 526-9 PMID: 17569828

Other Articles of Interest:

Altruism in Bacteria? Allowing Yourself to Die for the Good of The Species

Prokaryotes Can Do Geometry, and Even Have Their Own Protractor

Throwing the Clutch (not the Brake) on a Bacterial Flagella

Antiobiotic Treatment: Increasing the Rates of Genetic Exchange

Site Announcements

noreply@blogger.com (Tim Sampson) — Wed, 16 Dec 2009 13:30:00 +0000

1) As you can see, Blogging for Bacteriophages has now become "The Times Microbial" @ Phagehunter.Org. After some long thought, I decided to change the name in an effort to address the fact that the site covers material in the microbial world beyond just bacteriophage biology. This, along with the layout changes, are part of a larger transformation that will take place VERY slowly over the next year or so.

2) Articles that are written on peer-reviewed research (which make up the vast majority of the posts here) are aggregated at the site ResearchBlogging.org. Relatively recently, their editors have begun selecting notable articles as "Editor's Selections" each week. I am proud to announce that two of our articles here have been selected thus far. These are denoted by the "Editor's Selection" badge, as well as, a post tag to allow quick access to the best of the articles published here.

3) As always if you have any questions or comments, send me a message. Feedback is always appreciated.

Thanks!

Nitric Oxide Synthase Isn't Just Used by Our White Blood Cells...

noreply@blogger.com (Tim Sampson) — Fri, 11 Dec 2009 16:20:00 +0000

Phagehunter.org is proud to have J. Kandler, a microbiology graduate student at Emory University, present to us this interesting post on a defense feature common in our immune system, but being utilized by bacteria as well.

After Halloween, I came across this spooky article in Science describing yet another way bacteria are dodging antibiotics. Don’t worry, there’s a silver lining! Gusarov and his colleagues may have found a new target for the antibiotic industry, bacterial nitric oxide synthase (bNOS).

It turns out bNOS is present in numerous Gram-positive species, along with some Actinobacteria and even a member of the Archaea (Natronomonas pharaonis, a resident of pH 11 soda lakes in Kenya and Egypt that likes its NaCl to the tune of 3.5 M). While most of the organisms cited in the paper are nonpathogenic, there are a few notable nasties you might recognize, including Bacillus anthracis (anthrax) and Staphylococcus aureus (MRSA). Though less extravagantly equipped than its eukaryotic cousins, bNOS is still able to produce NO/NO+ with the help of cellular reductases. The common reason for bNOS in all these species remains elusive, but Gusarov may be onto something with his antibiotic-killer theory.

Using three different but similar antimicrobials (acriflavine [ACR], pyocyanin [PYO] and cefuroxime [CEF]), Gusarov demonstrates that bNOS provides two important survival mechanisms: 1) direct detoxification of some antimicrobials the and 2) destruction of reactive oxygen species. He performed a screen of B. subtilis growth rates against 21 different antimicrobials and chose 3. The first two are both highly hydrophobic and contain conjugated 6-membered rings, making them likely DNA intercalators. However, while ACR is a man-made drug that fights sleeping sickness, PYO is a natural bacteriocin produced by Pseudomonas aeruginosa and seems to be a natural weapon against competitors in the soil. The third, a cephalosporin, is a modified version of the well-known beta-lactam group of antibiotics and was developed in the 1970’s in response to growing concern over beta-lactamases.

Gusarov moves on to show that ACR—but neither PYO nor CEF—is directly neutralized by NO/NO+, specifically at its dangerous arylamino groups. The other, and far more important, function of bNOS is revealed when Gusarov starts monitoring the levels of superoxide dismutase (SodA) transcription in a B. subtilis nos null mutant. Bacteria without bNOS fail to boost transcription of sodA in late log phase, rendering them helpless against the onslaught of radicals and oxidizers present in stationary phase. As a result, mutant B. subtilis reaches stationary phase at ~60% of the cell density reached by WT.

Taken together, these data cast bNOS as a major factor in the cellular response to reactive oxygen species, with a minor talent for direct NO/NO+ neutralization of antimicrobials. Perhaps the original role of bNOS was to help prepare the cell for the stressful conditions of stationary phase and keep up with competitors, but now it seems that the protein can double as a shield against a plethora of antimicrobials. If we are able to find a bNOS inhibitor in the future, it might help decrease bacterial load in non-Gram-negative infections by supplementing conventional treatments, in the style of augmentin. Furthermore, given the high involvement of ROS in the body’s innate immune response, taking out bNOS could make our natural defenses all the more potent.

Source
Gusarov I, Shatalin K, Starodubtseva M, & Nudler E (2009). Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics. Science (New York, N.Y.), 325 (5946), 1380-4 PMID: 19745150

Other Articles of Interest
Out With the Bad: Efflux in Klebsiella pneumoniae
Antibiotic Treatment: Increasing the Rates of Genetic Exchange
Utilizing Natural Killers: Phage Based Antimicrobials

Last Month in the Blogs (#2)

noreply@blogger.com (Tim Sampson) — Wed, 09 Dec 2009 12:30:00 +0000

In case you missed them:

Hiroshi Nikaido, at Small Things Considered, tells us about the limitations of lysogeny broth (LB media, often a misnomer for "Luria Broth" or "Luria-Bertani media"). A highly interesting read, it points out that most of the large peptides in LB are unusable by cells, leading to a lower cell density than optimal. Differences in salt content, as well as the presence of bile salts, cause changes in the cell envelope as well as activates specific efflux pumps, which would normally not be active.

A. J. Cann, at MicrobiologyBytes, writes a review on an article demonstrating that bacterial infection within the gut can drive proliferation of intestinal stem cells, which can lead to cancer in organisms with a genetic predisposition. A good read to go along with the other articles on gut bacteria that I've brought up lately.

And finally, to continue our gut-bacteria discussions, at BiteSizeBio, Suzzane goes point by point on food-borne pathogens. The ones that we should watch out for, and how they get their in the first place. If you find this article interesting, you may find this one on how bacteria stay protected in vesicles on greens interesting as well.

Enjoy!

And as always, questions, comments, suggestions, etc. are more than appreciated!

How Helicobacter Gets Around

noreply@blogger.com (Tim Sampson) — Sat, 05 Dec 2009 22:50:00 +0000

Helicobacter pylori is the only bacterium (that I know of) that is capable of colonizing the rather intimidating environment of the human stomach. A very low pH, and a thick viscous and elastic mucus, make for a difficult niche to inhabit. But for a bacterium, the payoffs are huge: a constant supply for nutrients, no other prokaryotic competition, and little interaction with the immune system.

It is well described that to survive the low pH, Helicobacter utilizes a urease system. Taking in urea present in the stomach, the bacterium creates and secretes ammonia, raising the pH to nearly neutral. It is also known that to escape the lower pH of the stomach lumen, and to successfully colonize, Helicobacter must move through the thick gastric mucus lining the stomach into gastric pits within the stomach epithelium. The question that this phenomenon raises is how the bacterium is able to be motile through the very thick, viscous, and elastic mucus.

Some ways Helicobacter could solve this problem include: very strong flagellar power using brute force to tunnel through the mucus, or via secretion of mucus degrading enzymes to breakdown the mucus making it more liquid. It is known that as the pH of gastric mucus is raised, the gel-like structure becomes less organized and more liquid. Relatively recently, a study in the Proceedings of the National Academy of Sciences, has directly connected the raise in pH due to Helicobacter urease, to motility and liquidity.

Using techniques in rheology that are well beyond my expertise, the authors show that in the presence of both urea and Helicobacter, low pH mucus becomes both less viscous and elastic, and more liquid as the pH raises. However, without urea, Helicobacter is unable to transform the mucus from a gel-like structure.

Next, using live cell imaging techniques, the authors demonstrated that Helicobacter cannot be motile in raised pH mucus gel, without urea, but can be highly motile in mucus solution at the same pH. These two findings indicated that motility was a direct function of the structure of the surrounding mucus, which was a direct function of the pH. Finally, using a dye that fluoresces at more neutral pH, the authors were able to show that at the exact point that the pH is raised by Helicobacter, the mucus becomes more liquid, and the cells become motile.

These findings show that Helicobacter uses the same urease system to both change the pH to a more hospitable level, while at the same time alter the structure of mucus to allow motility to the more protected gastric pits for colonization.

Source:
Celli, J., Turner, B., Afdhal, N., Keates, S., Ghiran, I., Kelly, C., Ewoldt, R., McKinley, G., So, P., Erramilli, S., & Bansil, R. (2009). Helicobacter pylori moves through mucus by reducing mucin viscoelasticity Proceedings of the National Academy of Sciences, 106 (34), 14321-14326 DOI: 10.1073/pnas.0903438106

Other Articles of Interest:
A Home for the Bugs in our Appendix
A MAP to Crohn's Disease; Revisiting Koch's Postulates
Altruism in Bacteria? Allowing Yourself to Die for the Good of the Species
Where the Wild Microbes Are: A New Theory on How Pathogens Survive Food Processing

Out With the Bad: Efflux in Klebsiella pneumoniae

noreply@blogger.com (Tim Sampson) — Wed, 25 Nov 2009 02:33:00 +0000

Phagehunter.org is proud to announce an article by guest author, E. Ohneck, of Emory University, as she discusses the importance of efflux pumps in bacterial systems.

The Resistance-Nodulation-Division (RND) family of efflux pumps is widely utilized, especially among Gram-negative bacteria, for the export of a diverse array of antimicrobial agents from the bacterial interior, making these efflux systems important in multidrug resistance (1). RND family pumps are composed of three parts: a transporter protein in the inner cytoplasmic membrane, an outer membrane protein channel, and an accessory protein that connects the two (1). The AcrAB-TolC efflux pump is one such efflux system found in multiple Gram-negative bacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (1).

In their recent study, presented in a paper entitled “Klebsiella pneumoniae AcrAB efflux pump contributes to antimicrobial resistance and virulence,” Padilla et al confirm a role in multidrug resistance for the AcrAB-TolC efflux pump of K. pneumoniae, a highly prevalent nosocomial enterobacterial pathogen responsible for both urinary tract infections and bacterial pneumonia.

Through the use of knockout strains deficient in AcrB, the transporter protein component of the pump, the authors show the importance of AcrAB-TolC in resistance to β-lactams, aminoglycosides, quinolones, and other antibiotics (2). Interestingly, these AcrB knockouts were also significantly more susceptible to human antimicrobial peptides and were less able to colonize mice lungs and cause pneumonia, effects which were shown to be attributable to AcrAB-TolC deficiency (2).

These results have several important implications. First, like other RND family efflux pumps, the AcrAB-TolC efflux pump likely contributes significantly to the multidrug resistant phenotype observed in some K. pneumoniae strains, due to its broad antimicrobial substrate specificity (2). Secondly, AcrAB-TolC may also mediate resistance to host antimicrobial peptides (2). To date, this phenomenon has been established for relatively few efflux pumps, one of the best characterized examples being MtrCDE-mediated resistance to the host antimicrobial peptide LL-37 in Neisseria gonorrhoeae (3). The ability of bacterial efflux systems to pump out host-derived antimicrobial agents provides pathogens with an important mechanism of protection against one of the first-line defenses of the innate immune system.

Taken together, these conclusions raise the concern that antibiotic treatment can select not only for increasingly antibiotic resistant strains, but also for strains better able to resist the host immune defenses, and thus better able to cause disease. Clearly, efflux-mediated resistance to host antimicrobial compounds and its relation to antibiotic resistance and pathogenesis is an area that demands further study in order to develop efficient methods of combating the increasing number of multidrug resistant bacteria.

Sources:
1. Piddock LJV. Multidrug-resistance efflux pumps—not just for resistance. Nat Rev Microbiol. 2006 Aug 4(8): 629 – 36.

2. Padilla E, Llobet E, Doménech-Sánchez A, Martínez-Martínez L, Bengoechea JA, & Albertí S (2009). Klebsiella pneumoniae AcrAB efflux pump contributes to antimicrobial resistance and virulence. Antimicrobial agents and chemotherapy PMID: 19858254

3. 3. Shafer WM, Qu X, Waring AJ, Lehrer RI. Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1829-33.

Other Articles of Interest:
The Origins of Antibiotic Resistance
Wild Bacteria that eat Our Antibiotics? Of Course!
Antibiotic Treatment: Increasing the Rate of Genetic Exchange

A Home for the Bugs in Our Appendix

noreply@blogger.com (Tim Sampson) — Tue, 17 Nov 2009 13:00:00 +0000

The appendix has long been considered a useless, vestigial organ, primarily based on the fact that it can be removed with no visibly harmful effects on the appendectomy recipient and that it is rather susceptible to severe inflammation. In fact, many theories have been proposed for its ancient purpose, ranging from being a place to allow plant matter to ferment to being a locale for crushed bones to be dissolved.

However, recent evidence posits that the appendix plays a crucial role in maintaining important gut symbionts. A recent report by Smith et al compares the structural features of the appendix across various mammalian species, and uses this comparison to further elucidate the function the appendix.

It has been known for a few years that the lymph tissue in the appendix has played an important role in the immune-regulation of gut symbionts. The tissues in the appendix actively promote and support biofilm growth with various secreted effectors to a significantly greater degree than the rest of the gut.

Structurally, the appendix is in relative isolation from the rest of the gut, bounded by a rather constricted opening and narrow lumen. This, coupled with the above observations, suggest a role for the appendix in maintaining important gut symbionts during in an immune response to gut pathogens, or to the diarrheal effects of certain pathogens. Due to the unique structure of the appendix, symbionts within this structure would be more likely to survive such conditions and be capable of re-colonizing the gut following pathogen clearance.

The authors show, through comparative structural analysis, that in many mammals the structure of the appendix is similar, with differences occurring due primarily to the greater gut structure (a product of the mammals diet). Furthermore, they show that some animals may have lost the appendix, but that in other clades, its presence has been maintained for tens of millions of years.

Ultimately, these observations show that the appendix is far from useless and vestigial. Though this does raise questions about those individuals who have had their appendix removed. Is there a higher incidence of post-antibiotic treatment Clostridium difficle infections, or inflammatory bowel diseases? Or are appendix-less individuals more susceptible to adverse reactions and longer recovery times following diarrheal illnesses like norovirus, Salmonella, or cholera infections?

Citation:
Smith HF, Fisher RE, Everett ML, Thomas AD, Bollinger RR, & Parker W (2009). Comparative anatomy and phylogenetic distribution of the mammalian cecal appendix. Journal of evolutionary biology, 22 (10), 1984-99 PMID: 19678866

Other Articles of Interest:
A MAP to Crohn's Disease; Revisiting Koch's Postulates
Altruism in Bacteria? Allowing Yourself to Die for the Good of the Species

Listing of Graduate Research Fellowships

noreply@blogger.com (Tim Sampson) — Thu, 15 Oct 2009 15:41:00 +0000

Taking another break from writing on current work microbiology, to bring a list of current graduate research fellowships. This list is not inclusive, but lists some of the more popular and prestigious. Feel free to add any that I may have missed!

1) National Science Foundation Graduate Research Fellowship (NSF GRFP)

Who: Senior undergraduates and 1st/2nd year graduate students in all science, engineering, math and technology fields covered under the NSF mission statement.

What: $30,000 a year stipend for 3 years, plus $10,500 towards tuition and $1,000 for travel

When: Applications are due the first week in November

Where: Research is performed at the accredited institute of your choice

Why: "Fellows share in the prestige and opportunities that become available when they are selected. [...] NSF Fellows are anticipated to become knowledge experts who can contribute significantly to research, teaching, and innovations in science and engineering. These individuals are crucial in maintaining and advancing the nation's technological infrastructure and national security as well as contributing to the economic well-being of society at large."

How: NSF GRFP Online Application

2) National Defense Science & Engineering Graduate Fellowship (DoD NDSEG)Who: Students early in their graduate education, in most science, math, and engineering fields.

What: $30,500 stipend, increasing yearly for 3 years, plus full tuition and fees coverage, and $1,000 towards health insurance.

When: Applications are due the first week in January.

Where: Research is performed at the accredited institute of your choice.

Why: "The [NDSEG] is a highly competitive, portable fellowship [...][which] confers high honors upon its recipients. The [DoD] is committed to increasing the number and quality of our nation's scientists and engineers."

How: DoD NDSEG Fellowship Online Application

3) Science, Mathematics & Research for Transformation Scholarship (SMART)

Who: Undergraduates and graduate students at ALL levels of education in most science, math, and engineering fields.

What: Full tuition and fees, $25,000 to $41,000 stipend per year, $1,000 towards books, $1,200 towards health insurance, guaranteed summer internships and employment following graduation. (Both mandatory for an equal amount of time spent on fellowship, in school)

When: December 15th

Where: Research is performed at an accredited institution of your choice, summer internships and post-graduation employment are performed at a DoD facility chosen upon award receipt.

Why: "To support undergraduate and graduate students pursuing degrees in science, technology, engineering, and mathematics disciplines. The program aims to increase the number of civilian scientists working at DoD laboratories."

How: Smart Scholarship Online Application

4) The Office of Science Graduate Fellowship Program

Who: Senior undergraduates and 1st/2nd year graduate students in science, math, or engineering, in a discipline covered by the DOE / Office of Science

What: $35,000 stipend a year for 3 years, $10,500 towards tuition and fees, $5,000 research stipend per year for laboratory supplies or travel, travel and lodging to the annual DOE Research Conference.

When: Applications are due November 30th

Where: Research is performed at any accredited institution.

Why: "To support outstanding students to pursue graduate training in basic research areas of physics, biology, chemistry, mathematics, engineering, computational sciences, and environmental sciences relevant to the Office of Science and to encourage the development of the next generation scientific and technical talent in the U.S."

How: Office of Science Graduate Fellowship Online Application

5) The Hertz Foundation Applied Science Fellowships

Who: Senior undergraduates, and early level graduate students in all applied sciences

What: Either $31,000 for 9 months plus full tuition both renewable for up to 5 years, OR $36,000 for 9 months plus full tuition both for 2 years, then $3,500 supplement for 3 years while under another fellowship.

When: October 30th

Where: Only at one of the institution's tenable schools

Why: "[To] build America's capacity for innovation by nurturing remarkable applied scientists and engineers who show the most promise to change the world."

How: Hertz Foundation Online Application

This Month in the Blogs (#1?)

noreply@blogger.com (Tim Sampson) — Sun, 27 Sep 2009 15:35:00 +0000

In case you missed them:

Merry Youle, at Small Things Considered, writes up a beautiful piece on the direct connections of microbes to the multicellular world. She walks us through some recent work highlighting the connections between parasitic wasps, the aphids they lay eggs within, the bacteria that colonize the aphids, and ultimately to the phage that lysogenize those bacteria. Truly remarkable!

Urinary tract infections are one of the most common infections in the country, costing around $2.4 billion dollars a year. Alan Cann over at MicrobiologyBytes discuss the first ventures into vaccine development for this rather uncomfortable and widespread disease.

I was lucky enough to start my research career by isolating and characterizing a novel bacteriophage of the Mycobacteria. This lead to some interesting findings, including the first description of putative transposons in these particular phages(which we published in this month's Microbiology). Phagehunting, however, is not restricted to only Graham Hatfull's lab, where I did my undergraduate work. The Howard Hughes Medical Institute has funded curriculum development to bring this research experience to colleges across the country. John Dennehy, at The EvilutionaryBiologist, talks about his new Phage Hunting course at Queen's College.

And finally Nick Oswald, at BiteSize Bio, tells us to simmer down when our experiments give us results other than what we expected. Instead, we should concentrate on making sure we are asking the right question.

Enjoy!

And as always, questions, comments, suggestions, etc. are more than appreciated!

The Next Steps in Synthetic Biology

noreply@blogger.com (Tim Sampson) — Thu, 24 Sep 2009 13:15:00 +0000

The advent of genome sequencing and analysis, coupled with the relative simplicity of DNA synthesis, has given rise to the field of synthetic biology. Described in 1974 by Waclaw Szybalski, the practice of synthetic biology would include the ability to "devise new control elements and add these new modules to the existing genomes or build up wholly new genomes."

More than 20 years later, this description is being realized. With the great minds and funds of the Venter Institute, we have seen the development of a completely synthetic genome and whole genome transplantation. The genome of Mycoplasma genitalium (at ~580Kbp) was synthesized primarily in vitro before being pieced together in Saccharomyces cereviseae. The whole genome of M. mycoides was transplanted into M. genitalium, changing the metabolism, physiology, structure, and subsequently the species of the recipient.

Now, this group has taken this technology one step further. The synthetic genome they pieced together in yeast, must be isolated and transplanted into a donor cell; thus completing the construction of a synthetic, replicating organism.

In a soon-to-be published article in Science, the group describes a method for modifying the complete bacterial genome while in yeast, and then transferring the modified genome back into the original cell. This process allows bacterial genomes to be modified using the well-described genetic systems in yeast, before being introduced. Thus, new possibilities are open for bacteria that once had little to no genetic tools available.

The authors emphasize this development as it directly relates to Mycoplasma biology. They focus on the fact that Mycoplasma and related species now have an entire repertoire of manipulations available to them, however, the ramifications of this experiment certainly do not escape them and should not escape us.

This is the first example of a bacterial cell being created and engineered completely outside the cell itself. Although the recipient cell began with all the necessary physical components for life, this group synthetically created a new organism to control and change those components. This is the closest we have come to synthesizing an organism from scratch.

Some may say this is playing God; however, it will certainly have a positive impact on the development of biofuels, environmental remediation, and chemical synthesis on an industrial scale.

Citation:
Lartigue, C., Vashee, S., Algire, M., Chuang, R., Benders, G., Ma, L., Noskov, V., Denisova, E., Gibson, D., Assad-Garcia, N., Alperovich, N., Thomas, D., Merryman, C., Hutchison, C., Smith, H., Venter, J., & Glass, J. (2009). Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast Science DOI: 10.1126/science.1173759

Other Articles of Interest:
Phage + Metal = Battery?
Free Hydrogen -- Algal Biofuel Prodution

Prokaryotes Can Do Geometry, and Even Have Their Own Protractor

noreply@blogger.com (Tim Sampson) — Thu, 17 Sep 2009 21:49:00 +0000

Despite what once was a popular opinion, bacterial cells are not mere sacks of enzymes. Rather, we are discovering that they are highly structured. (Although, this probably should have been expected) Bacterial cells have to know where their poles are located in order to create such structures as E. coli’s chemotactic nose, Caulobacter’s stalk, and polar flagella. Cells must also be able to recognize where their midpoints are located in order to divide or differentiate properly. Furthermore, we are finding that the bacterial chromosome has a distinct physical arrangement, which can ultimately determine gene expression.

In order to attain such organization, bacteria must have ways to pinpoint proteins to these specific locations. One way they may do this is through curvature sensing. As we can easily see, in rod-shaped cells, poles tend to have a larger curvature than the midsection. In some cases, curvature can differ via the direction, not just magnitude. In the case we will look at today, done by the Richard Losick lab, a protein recognizes the positive curvature of a forming forespore.

SpoVM (“Spo-Five-Em”) is a Bacillus protein that localizes to the outer forespore membrane during the beautiful process of sporulation. Despite being a membrane protein produced in the mother cell, SpoVM is present only on the forespore and does not appear at all in the greater cell membrane. Therefore, this raises the obvious question of how such recognition occurs.

The first (and most probable) method is that known as “diffuse and capture.” The idea is that SpoVM localizes to the cell membrane and then diffuses through to the connected membrane of the developing forespore. Once here, a forespore-produced membrane protein captures the diffusing SpoVM. To test this, the Losick lab prevented SpoVM production until after the forespore engulfment. This way, there would be no contiguous membrane from the mother cell to forespore for SpoVM to diffuse through.

The result was that SpoVM still localized only to the forespore, despite a lack of continuous membrane. Since this ruled out diffusion, SpoVM must have another method for such a specific localization. Rather than attempting to address whether a second protein attracts SpoVM (a question that would be slightly difficult to address) the researchers thought that localization might occur due to the specific curvature of the forespore. A logical thought, since the forespore is the only positively curved membrane structure in the Bacillus cell.

If true, then SpoVM should localize to any membrane with positive curvature without regard to the membrane’s original source, location, or other proteins present. In fact, this is what occurs. SpoVM localizes to any positively curved membrane surface. This was shown in vivo with mutant Bacillus that formed internal vesicles, along with mutants of E. coli and Saccharomyces cerevisae that have the same phenotype. Furthermore, in a completely cell free system, SpoVM shows the same localization to small membrane vesicles. Mutants of SpoVM can also be isolated that show a non-discriminatory phenotype and will localize to membranes without regards to their curvature. This shows that the ability to recognize positive membrane curvature is inherent in the protein itself.

Adding to these data, SpoVM appears to have specificity not just for positive membrane curvature, but also for curves with a diameter of 1 micron or less. This is about the same as a Bacillus forespore.

What is the lesson here? Bacteria have unique ways to examine their surroundings and their selves to allow for proper cellular organization. Although membranes with positive curvature within a bacterial cell are rare (photosynthetic vesicles and forespores), negative curvature is obviously not a rare phenomenon. Perhaps similar mechanisms maybe used to identify cellular poles based on degree of negative curvature. There is even a possibility that curvature-sensing molecules are used in eukaryotes to identify various organelles within the cell.

Citation
Ramamurthi, K., Lecuyer, S., Stone, H., & Losick, R. (2009). Geometric Cue for Protein Localization in a Bacterium Science, 323 (5919), 1354-1357 DOI: 10.1126/science.1169218

Other Articles of Interest

Throwing the Clutch (Not the Brake) on Bacterial Flagella

Extracellular Membrane Vesicles in Bacteria: Taking Quorum Sensing in a New Direction

Throwing the Clutch (not the Brake) on a Bacterial Flagella

noreply@blogger.com (Tim Sampson) — Fri, 15 May 2009 19:16:00 +0000

For most my of readers, it is common knowledge that bacteria are more than just singled celled entities; and instead bacteria are complex organisms capable of undergoing large-scale, multicellular activities. Of particular interest to many current microbiologists, is the development of biofilms.

These multicellular structures are likely how many bacteria exist in the environment, and are implemented in a variety of diseases. Taken alone, biofilms are fascinating, but I have a keen interest in understanding exactly how a bacterial cell decides it is time to create a biofilm. (In reality, I have a keen interest in how a bacterial cell decides it is time to do anything, but that is beside the point)

While studying the switch between motility and biofilm development in Bacillus subtilis, Daniel Kearns of Indiana University, wanted to know the status of the flagella during the switch.

Mutants of the biofilm regulator sinR are constitutively in a biofilm state and are nonmotile. However, flagellar genes are still expressed and flagella are still produced. The question then became what is preventing the flagella from moving while in a biofilm.

The first idea would be that the extracellular matrix is physically inhibiting the movement of the flagella. By knocking out a gene required for matrix production (epsH), Kearns saw that the flagella became free, but were unpowered.

Then, as any good geneticist, he screened for mutants that were able to suppress the non-motility phenotype of a sinR, epsH double mutant. All of these mutants mapped to a putative glycosyltransferase (epsE) within the matrix operon. Furthermore, expression of epsE alone was sufficient to inhibit flagellar motion, and known conserved glycosyltransferase residues were not required for inhibition.

This brings up the questions: Where is epsE acting? and Is it a brake (completely stopping all flagellar movement) or a clutch (preventing active rotation)?

By selecting for supressors of redundant epsE, the group found that all suppressors mapped to the fliG gene; a gene known to encode the transduction motor between the proton pump (motAB) and flagellar basal body. So, somehow epsE acts to inhibit the motor.

Furthermore, upon examining flagellar motion (in some awesome movies available here) Kearns showed that the flagella were not braked, that is, the flagella were still capable to rotate freely (and in fact did), however all rotation was due to Brownian motion. This implies that the flagella were disconnected from the motor, rather than stopped completely.

So, epsE is acting as a clutch to disconnect the flagellar basal body from the motor. This actually makes quite a bit of sense. If the cell no longer requires flagellar motion as it went into biofilms, it could do a variety of techniques. One is that it could shut off gene expression for the flagella. However, it would take many generations before its progeny were non-motile and the flagellar apparatus was diluted out. Another is that it could put a brake into the flagella and prevent motion entirely. But, this would cause lots of cell envelope stress while in a biofilm. Brownian motion alone could potentially tear the cell apart.

This leaves the cell with the clutch option. Capable of stopping flagellar motion quickly while keeping the cell envelope intact. Due to its high homology to glycosyltransferases, epsE likely represents an example of a duplicative evolution; whereby a duplication of an enzyme leads to the capability to take one copy of said enzyme and "play with it" to create new functions. In this case a sugar-acting enzyme has become a structural protein in the flagellar apparatus.

Source:
Blair, K., Turner, L., Winkelman, J., Berg, H., & Kearns, D. (2008). A Molecular Clutch Disables Flagella in the Bacillus subtilis Biofilm Science, 320 (5883), 1636-1638 DOI: 10.1126/science.1157877

Other Articles of Interst:
Extracellular Membrane Vesicles in Bacteria: Taking Quorum Sensing in New Directions
Altruism in Bacteria: Allowing Yourself to Die for the Good of the Species
Where the Wild Microbes Are: A New Theory in How Pathogens Survive Food Processing

Phage + Metal = Battery?

noreply@blogger.com (Tim Sampson) — Sat, 09 May 2009 16:49:00 +0000

By now, many people have read about Angela Belcher, a professor at MIT, and her lab's recent developments in the use of bacteriophages as a component of batteries. Having had a very distinct privilege to hear her speak yesterday, I wish to share what I have learned.

In a broad sense, the goal of her lab is to give inorganic compounds (batteries, medical devices, solar cells, etc), "genetic intelligence." That is, to give the power of evolutionary adaptation and self-correction to inanimate objects. Life evolved the ability to perfectly use the ions and metals present in its environment, things like calcium, silica, etc. However, she wants to know what happens when we allow life to evolve in the presence of technologically important compounds, like gold, silver, aluminum, platinum, etc.

One of the original goals was to develop a biological system that could recognize and mark atomic scale cracks in layered materials. She set to do this using a phage library. The phage, M13, is capable of having many of its parts replaced with random gene sequences, allowing us to add in peptides that allow recognition for any particle of our choosing. (A concept referred to as "phage display" and has been used for lots of detection assays)

She selected for attachment proteins that allowed the phage to attach to atomic scale cracks in the alloy used in engine blocks and computer parts. You can then propagate, mutate, and select for phages that have tail fibers with the strongest possible affinity for the substrate of your choosing. This was very successful, and from my impression, is being scaled up to allow identification of these atomic cracks in engine blocks, airplane wings, and helicopter blades. (She is now figuring out how to mesh the identification of the cracks via phage, to self-healing properties via metal nucleation)

She then found that the proteins in the phage body could be altered as well. Using similar techniques she found that metal ions could be nucleated in both poly crystalline and mono crystalline structures to the phage body. Thus allowing the creation of nanotubes (with a phage inside). By altering ratios of metal ions added, she can create very specifically composed alloys. Importantly, all of this is done at STP, with a rare exception (Ag-Pt tubes for fuel cells) requiring 80C temperatures. She can also rid the system of organic material by heating to >100C, but keep the inorganic structures intact.

Using phages with affinities for cobalt oxide, lithium, these scientists managed to create a functional battery that is only on the order of nanometers in thickness. Paper thin batteries that have the capacity and power to replace automotive batteries now. The batteries are capable of being charged and recharged numerous times without losing power or capacity. (This was a problem at first). They are very fast to produce (<6hrs)>
If you can think of any application for nanowire like phage nucleation of metals, her team is already working on it. This battery concept is definitely going to be an important milestone in development of new energy storage, usage, and production.

Normally when we think of applications of microbial genetic systems, we think of human health and perhaps fermentations. Now, we can truly see the power when genetics are applied to technologically challenging engineering applications.

Source:
Lee, Y., Yi, H., Kim, W., Kang, K., Yun, D., Strano, M., Ceder, G., & Belcher, A. (2009). Fabricating Genetically Engineered High-Power Lithium Ion Batteries Using Multiple Virus Genes Science DOI: 10.1126/science.1171541

Other Articles of Interest:
Utilizing Natural Killers: Phage-Based Antimicrobials
Evolution of Phage Capsid and Genome Size
How Far Do Those Phages Stretch?
Phages with Horns? What's Next?
I Got You Phage

Blogging for Bacteriophages is Back

noreply@blogger.com (Tim Sampson) — Sat, 09 May 2009 16:09:00 +0000

After a rather long sabbatical, Blogging for Bacteriophages is ready to start reporting the exciting and interesting news in the world of microbiology.

The layout is finally optimized. Comments are fully functional. Blogger has also introduced "reaction boxes" to allow you to give feedback with just one click.

Most importantly, I finally have time to devote to publishing again. A realistic goal is 2 articles each month, with a hopeful goal of 4 per month. As always, I would love input from others in the field, especially other graduate students.

I am looking forward to sharing some fun and ground breaking science, as well as interesting experiences I have had over the past few months. I have been privilaged to listen to some fantastic science coming from notable speakers, such as James Watson, Matthew Meselson, Angela Belcher, Pete Greenberg, and many others.

Here's to a new start!

Antibiotic Treatment: Increasing the Rates of Genetic Exchange

noreply@blogger.com (Tim Sampson) — Mon, 15 Dec 2008 23:26:00 +0000

The dangers of single antibiotic treatment are well known and well established. It is common knowledge that improper use of antibiotics can lead to the development of resistant strains in which the antibiotic was created to be used against.

The development of these resistances in a population is most often thought to be due to direct selective pressure. That is, those cells containing mutations that resist the antibiotic, or those that contain a protein to export or degrade the antibiotic, are heavily selected for. Because of this, as bacterial populations grow resistant, treatment includes higher levels of antibiotic or utilizing multiple types of antibiotics.

However, there is a 2006 study, published in Science, which shows that antibiotic treatment does more than "merely" select for resistant strains. Instead, when Streptococcus pneumoniae detects one of a variety of antibiotics, it activates it's competence regulon, thus turning on its ability to be transformed.

To me, this finding is drastic. What this means is that not only do we push direct selection for resitant strains, but we are also stimulating gene aquisition. The ramification of which, means that sensitive strains that detect a low level of antibiotic are now more capable of becoming resistant by aquiring genes via transformation.

This finding stemmed from the hypothesis that since both transformation and DNA repair (sometimes due to antibiotic damage) require similar (the same) recombination enzymes. The authors went on to show that competence was activated in sub-lethal ranges of antibiotics at around 200 minutes post treatment.

The antibiotics that were shown to induce competence, cross many classification boundries. The list includes: mitomycin C, norfloxacin, kanamycin, streptomycin, levofloxacin, moxifloxacin, and norfloxacin. So, mainly the quinolones and the aminoglycosides. (To note: The quorum sensing molecule discussed in the membrane vesicle article, is a quinilone. Perhaps a connection here to competence?)

Those that did not induce competence include: ampicillin, cefotaxime, nalidixic acid, erythromycin, tetracycline, novobiocin, rifampicin, and vancomycin.

The authors propose a few hypotheses on how these are acting to induce the competence regulon, including chromosomal arrest and ppGpp levels, but nothing concrete.

The bottom line is that antibiotic treatment has the ability to increase the rate of gene transfer by activating competence genes (at least in Streptococcus), thus increasing the rate of resistance in a population, compared to direct selective pressures alone.

Source:

Prudhomme, M. (2006). Antibiotic Stress Induces Genetic Transformability in the Human Pathogen Streptococcus pneumoniae Science, 313 (5783), 89-92 DOI: 10.1126/science.1127912

Other Articles of Interest:
Extracellular Membrane Vesicles in Bacteria: Taking Quorum Sensing in New Directions
Utilizing Natural Killers: Phage-Based Antimicrobials
The Origins of Antibiotic Resistance
Wild Bacteria That Eat Our Antibiotics? Of Course!

Update

noreply@blogger.com (Tim Sampson) — Thu, 11 Dec 2008 01:44:00 +0000

Comments are still down....the host site is apparently having trouble with comments across the board. I will have this fixed as soon as I can.

Overall though, I am quite pleased with the new layout. This is looking like how I am going to keep it, for the most part. Small changes may appear over the next few weeks.

I am in the midst of final exams, however I will be back to science posting by the weekend. I have some thoughts itching to be written.

Hope you enjoy the new look and feel. As always, feel free to e-mail me comments, questions, and concerns!

*****Update 13 Dec 08******
I'm fooling around with the site code....so the site may undergo drastic changes in short periods of time. The host site is giving me troubles with comments still and with changing view sizes.

Future Changes

noreply@blogger.com (Tim Sampson) — Thu, 04 Dec 2008 18:57:00 +0000

I hope all of you had an enjoyable Thanksgiving holiday!

Blogging for Bacteriophages has seen a rapid increase in traffic over the last two months. Page views jumped dramatically this fall (Nearly 60% from the summer averages and 130% from the spring)

So thank you! It delights me to no end that there are individuals who take the time to peruse my site. It is funny to think that this started as a study mechanism for me, and turned into a wonderful little hobby that others are benefitting from.

In order to make this site easier to read, explore, and interact with, I am in the process of reformatting the site. Hopefully this will go easily, and may even be finished by the end of today. (Though I can give no promises)

Most importantly, I can not stress this enough, I would greatly enjoy feedback on the articles here. Feedback on everything from the science itself, to how I communicate the science, to new topics, etc. This is ever important to me. So, to facilitate this, I plan on opening up the comments section. I hope that with the great increase in readership, this comments section will be used.

I am also interested in having guest posts, with an interest in hearing from fellow graduate students in the field. I can not offer payment, although free advertising is available. I would also consider sending prints of some of my Phage Artwork in return for articles used.

Thank you!

Extracellular Membrane Vesicles in Bacteria: Taking Quorum Sensing in New Directions

noreply@blogger.com (Tim Sampson) — Mon, 24 Nov 2008 01:46:00 +0000

Bacterial quorum sensing is not a new phenomenon by any means. Although the term has only recently come into the existence, the concept has been around for decades.

In 1964,Tomasz and Hotchkiss, out of Rockefeller, demonstrated the presence of a macromolecule responsible for induction of competence in pneumococcus when the cells reached a specific growth point in mid to late log phase.
This concept has been vastly expanded upon and the ramifications of such diffusible communication molecules are vast. Everything from biofilm formation to stringent responses to bioluminescence have been shown to be controlled by quorum (or diffusion) sensing molecules.

Commonly, quorum sensing molecules are lactones, small peptides, or small lipids. It is thought that these small molecules are secreted by the cell and diffuse through the aqueous environment where they can interact with other cells. This is all well and good, IF the molecule of interest is hydrophillic and freely diffusible. But researchers are finding that many quorum sensing molecules are NOT hydrophillic, and instead very hydrophobic, such as a quinilone with a long chain fatty acid attached. One such example of this, is the Pseudomonas aeruginosa molecule called PQS.

How can a molecule be used as an extracellular signal, if it can not diffuse freely? Dr. Marvin Whitely and colleagues have shown that PQS is able to promote the formation of membrane vesicles off the outer Pseudomonas membrane. Pseudomonas is known to naturally produce these vesicles, however PQS directly induces changes in the lipid membrane to form such vesicles.

Furthermore, these vesicles have the strong potential to be able to capture a variety of macromolecules that are in the vicinity of the membrane bleb. A prime example would be the beta-lactamases that are in the periplasmic space. But we could imagine other molecules being packaged up and delivered.

Molecules that are packaged could be delivered to the same cells in the population, or to foreign cells of different species, perhaps even to humans during pathogenesis. The possibilities are endless as to what these membrane vesicles could be providing instructions for.

One very important piece of data that is missing from this model, is that researchers have yet to observe membrane vesicles of one cell fusing with another cell. This would provide solid evidence that these vesicles could, in fact, deliver signals to other cells.

I had the privilege to have Dr. Whitely as a guest lecturer recently. He has some very interesting work on going in his lab, including the amazing creation of bacterial lobster traps. This work is not yet published, so I should not say anymore than that. However, once it is in the public domain, it has the potential to revolutionize the study of small bacterial populations.

Sources:
1) Tomasz and Hotchkiss. "Regulation of the transformability of Pneumococcal cultures by macromolecular cell products." PNAS (1964). 51, 3. p480

2)Mashburn, L., & Whiteley, M. (2005). Membrane vesicles traffic signals and facilitate group activities in a prokaryote Nature, 437 (7057), 422-425 DOI: 10.1038/nature03925

Utilizing Natural Killers: Phage-Based Antimicrobials

noreply@blogger.com (Tim Sampson) — Fri, 14 Nov 2008 02:30:00 +0000

My last article on the origins of antibiotic resistance perked my interest in the current thinking of how we scientists are planning on overcoming this challange. The two answers that most people will consider are 1) develop new chemical analogs of current antibiotic compounds, and 2) discover novel compounds that act as antimicrobials.

It is this second concept that appeals most to me. However, it raises the question, "Where do we look for novel antimicrobial compounds?"

Some are taking a step back to the days of Felming and looking everywhere: fungal isolates, plant extracts, bacterial products, etc. However, we can not forget the natural-born killers of bacteria. . . thier phages.

Bacteriophages have been coevolving with thier hosts since the dawn of time. As such, I would imagine they know a thing or two about killing a bacterial cell. Phage proteins interact with those of their host to modify and shutdown various functions. By taping into these types of interactions, we could exploit phage proteins to develop ways to attack the host. Furthermore, with the number and diversity of the phage world, there is great potential that each phage attacks the host in a slightly different fashion.

So now the question becomes, how do we find which proteins of the bacteriophage which function as bacteriocidal or bacteriostatic molecules?

The screen is relatively simple. Ask which phage genes, when inducibly expressed in the host, kill the host cell. Researchers have already performed this type of assay in Staphylococcus, and more recently, in the Mycobacteria.

Utilizing an acetimide-inducible promoter system, 3 phage genes (from temperate mycobacteriophage L5) were discovered to have toxic effects on the host, Mycobacterium smegmatis. Further characterization of these genes can find the specific host targets on which they act. Subsequently, small molecules could be developed and screened to act in the same location.

This method could allow the discovery of new drug targets and the drugs which access them. It is fast assay that uses phages in a clever and indirect way to fight disease.

Sources:

1) Liu et al. (2004). "Antimicrobial drug discovery through bacteriophage genomics". Nat Biotechnol 22, 185-191.

2)Rybniker, J., Plum, G., Robinson, N., Small, P., & Hartmann, P. (2008). Identification of three cytotoxic early proteins of mycobacteriophage L5 leading to growth inhibition in Mycobacterium smegmatis Microbiology, 154 (8), 2304-2314 DOI: 10.1099/mic.0.2008/017004-0p>

Other articles of interest
The Origins of Antibiotic Resistance
I'll Have My Bacteria Extra-CRISPR

It's Gonna Be There (The E. Coli Song)

noreply@blogger.com (Tim Sampson) — Thu, 23 Oct 2008 21:04:00 +0000

I'm back again with a dorky song. Written originally years ago for undergraduate Microbiology Lab, this song was in response to the many outbreaks of E. coli (specifically those occuring in spinach and other produce).

I hope you find this song as enjoyable as I do.
I would love to hear some feedback on this and the other "performances" I have posted under the "Phage Fun" heading. Feel free to e-mail me at the address on the left.

It's Gonna Be There (The E. Coli Song) To the tune of Blessid Union of Souls' "I Wanna Be There"

Lyrics
Don't you let that burger fall
I really hope you washed your hands
Invisible to eyes, that means small
Is that too hard to understand.

You got to clean everything
Or it will be filled with coliforms
And pretty soon, someone may be dead.

Chorus
It's gonna be there when you zip your fly
It's gonna be there when you rub your eyes
It'll cramp your stomach, cause internal pain
It'll make your intestines go insane.
It's gonna be on beef, whether or steak or ground
And in leafy greens it has been found
You better cook your food
You better wash your hands
Cause E. coli's on the lamb

You'd never know that it was there
But it has been there all the time
And if it had its way, it stay in cows on farms
But it loves bad hygeine and your grime

Cause you got dirt on yourself
When you leave the toilet room
Though the sign says, "Employees Wash Your Hands"

Chorus

And if its got blood cells to kill
Well you'd have to take some pills
And with some time, your kidneys may fail

It's gonna be there when you zip your fly
It's gonna be there you don't have to try
Gram negative and anaerobic too
And it lives inside of me and you

It's gonna be there on your burger
It's gonna be there on your knife
It'll cramp your stomach, cause internal pain
It'll make your intestines go insane.

Other Songs to Listen To:
The Brillant Dance of the Starvation Response
The Ballad of the Virus (They're Everywhere)
I've Got You Phage