Small Things Considered

Tit-for-Tat: A Bacterial Counterattack System

2013-05-20T04:00:00-07:00

by Spencer Scott & John De Friel

Schematic diagram of a type VI secretion system by Y. M. Cully, C. Cambillau, and E. Cascales. The lower bilayer membrane is the attacker’s, the upper one the host’s. Notice the complex structure of the Type VI secretory apparatus. Source.

Microbial ecology may be a young field but it is well understood already that there is a broad spectrum of interactions between bacterial species, ranging from cooperative to competitive. In a recent paper researchers from John Mekalanos’ lab further characterized a recently discovered mechanism for inter-cell communication. This system, called the Type VI secretion system (T6SS), is a multi-protein complex native to many bacterial strains and structurally and functionally similar to a bacteriophage tail. The T6SS system is unique in that it is used as a weapon for injecting toxic proteins into the cytoplasm not only of animal host cells but also of neighboring bacterial cells by propelling its components through the neighbors’ membrane. The toxic effector proteins, Tse1 and Tse3, are peptidoglycan-degrading enzymes that can cause cell lysis in the absence of antitoxin proteins. For reviews, click here and here.

The Type VI Secretion System Differs in Two Species

To elucidate how and when a cell decides to inject a neighboring cell with its T6SS, these workers studied its behavior in Vibrio cholerae and Pseudomonas aeruginosa. In V. cholerae, the T6SS seems to shoot off at random, constantly showing up in different areas on the cell, which endows it with high pathogenicity and with the ability to kill off many other species of cells. In this light, V. cholerae can be seen as a sort of Yosemite Sam, a character renowned for his excessive and poorly aimed shooting. The analogy to this character and to Batman (see below) was thought up by Robert Cooper and appeared in his fine blog at Science 2.0.

On the other hand, in P. aeruginosa, T6SS often shows up in a cell only in one place exactly corresponding to the T6SS activity of an adjacent cell, something the group dubbed as “dueling activity.” Instead of the haphazard random firing of V. cholerae, P. aeruginosa seems to duel with other nearby cells, so that when one cell fires its T6SS, the other cell would fire right back in the same spot. Thus, P. aeruginosa has a more sophisticated T6SS response system that will fire only in a spatially-targeted fashion and only in response to being attacked first. Because of this more refined offensive strategy (at least with respect to Vibrio), P. aeruginosa can be likened to Batman, a figure that classically attacks aggressors only. The paper unknowingly continues this analogy by showing that P. aeruginosa will happily coexist with non-aggressive cells even though it has the capability to destroy them, just like our hero Batman.

Who Attacks Whom?

Yosemite Sam. Source.

The study began by characterizing the “Yosemite Sam” firing of the wild-type Vibrio cholerae. To appreciate the chaotic firing nature of the aggressive V. cholerae, we suggest watching the paper’s video abstract. The group then focused on P. aeruginosa alone. They noticed that one sister cell would fire its T6SS, the neighboring cell would fire back after a short delay. However, the P. aeruginosa makes an antitoxin to the injected toxic protein, so it can survive attacks from sister cells.

When V. cholerae and P. aeruginosa were co-cultured, the overly aggressive V. cholerae were killed effectively by the P. aeruginosa whereas the P. aeruginosa proved invulnerable to the Vibrio’s attacks. The authors speculate that P. aeruginosa’s immunity to V. cholerae may be due to the impermeability of its outer-membrane since it can survive attacks from V. cholerae even if its own T6SS system is knocked out and has no way of offensively defending itself. The toxin protein P. aeruginosa secretes is called Tse, for which Vibrio doesn’t have an antitoxin. However, even without these proteins (using tse1-3 null mutants) P. aeruginosa could still kill the vibrios. This is likely due to the mere puncturing of the cell, which ruins the structural integrity of its outer membrane.

Is Pseudomonas Like Batman?

Batman. Source.

To test if Pseudomonas is simply a cold-blooded killer, these researchers mixed and grew it together with a non-aggressive E. coli (-T6SS) strain. Through microscopy, they showed that E. coli is killed very slowly, if at all, by the righteous P. aeruginosa. In the same vein, they created a non-aggressive V. cholerae strain by knocking out the T6SS, and mixing it with P. aeruginosa. This knockout proved to be very beneficial in that these mutants were killed at rates ~100 times slower than the wild type vibrios. Its T6SS system is so specific that even when P. aeruginosa was mixed with both the passive vibrio mutants and the Yosemite Sam wild-type vibrios, the aggressive strain is killed but the cooperative strain is spared. Pseudomonas is clearly Batman-like, showing mercy to those who do it no harm.

Lastly, the group made sure that this system was not specific to V. cholerae by growing T6SS+ Acinetobacter baylyi cells together with P. aeruginosa. Once again the P. aeruginosa effectively and efficiently killed the aggressive A. baylyi, proving that it is capable of responding to a variety of T6SS+ organisms. In short, P. aeruginosa can carry out coordinated counter-attacks to a spectrum of aggressive T6SS+ cells. The attack is spatially localized and used as a response to aggression. It is also parsimonious, in that P. aeruginosa saves its ammo solely for counterattacks, as if using its powers only to subdue aggressors.

An Offensive Weapon Can Be Defensive

There are several important conclusions to draw from these different uses of the T6SS system. Essentially, as the authors state “V. cholerae uses the [T6SS] as an offensive weapon, whereas P. aeruginosa uses the organelle as a defensive weapon.” This has a strong evolutionary implication since, as the paper states, biofilms composed of diverse but cooperative bacterial species likely have more growth potential than biofilms composed of a single bacterial species. See here and here. Thus, P. aeruginosa’s ability to discriminate between friend and foe may evolutionarily reflect the adage “don’t bite the hand that feeds you.” This suggests that P. aeruginosa has developed a way to coexist with other bacterial species as long as they are not aggressive. On the other hand, its ability to counterattack an aggressive bacterial species demonstrates a “tit-for-tat” evolutionary strategy. If you are a non-aggressive bacterium, P. aeruginosa is a great choice to create a symbiotic relationship with. It would be easy to create a biofilm with the P. aeruginosa and provide it with nutrients in exchange for protection from predatory bacteria like V. cholerae. And you thought that bacteria live a simple life.

Spencer and John are Bioengineering graduate students participating in the 2013 UCSD/SDSU Joint Doctoral Integrative Microbiology graduate course.

Basler M, Ho BT, & Mekalanos JJ (2013). Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions. Cell, 152 (4), 884-94 PMID: 23415234

Pictures Considered #4. Koch’s Development of Early InstaGram Positive Photography

2013-05-16T04:00:00-07:00

by Daniel P. Haeusser

Figure 1A. Koch’s photograph of B. anthracis, one of several photomicrographs in his 1877 paper, the earliest published bacteria photos. Source.

Robert Koch is one of the key figures in early bacteriology, helping develop culture techniques (e.g. solid media), critical reasoning (e.g. Koch’s postulates), and disease etiology (e.g. cholera and tuberculosis). He also published the first photomicrographs of bacteria (Figure 1A) in his 1877 paper Verfahren zur Untersuchung, zum Conservieren und Photographiren der Bakterien.

Discontent with communicating microscopic observations with hand-drawn illustrations, Koch pioneered the photography of bacteria. On suitable days, Koch would set up to shoot outdoors. In his 1877 publication Koch explains how to take photographs outside through a basic microscope:

“Clean all the lenses, screw them in completely, and place the illuminating mirror on the sunny side of the microscope. With a dark cloth over your head, look through the ground glass and adjust the light and focus the specimen… Once the image is in focus… go inside to prepare the photographic plates. In the darkroom…remove a clean glass plate with forceps and pour over its surface the iodized collodion solution, making sure the film spreads evenly and completely. Once the collodion film is ready, close the darkroom door and carefully lower the plate into the silver bath… Allow it to drain and put it in the cassette… Go back outdoors to the photomicrographic apparatus. Remove the black cloth…and check to be certain that the proper image is still in focus… Then carefully place the cassette, being careful not to move anything. After the exposure… push the slide back in the cassette, remove the cassette from the microscope, and cover the microscope again with the black cloth. This whole procedure must be done quickly! Run back to the darkroom with the closed cassette, develop the plate, and fix the negative. If the photographic image is not completely sharp, or if there are imperfections in the emulsion… it is necessary to repeat the whole process, since nothing is more disheartening in the photographic technique than to try to make prints from unsatisfactory negatives.¹

Quite a process! Following this protocol, Koch reports it took at least three hours to obtain four to six good pictures. Note that this time does not include setting up the equipment, preparing the bacterial sample, or freshly mixing the photographic chemicals. Yet with skill, patience, and perseverance during his early studies on anthrax, Koch obtained photos of the Gram-positive B. anthracis in a quality that rivals images taken today. Indeed, as an exercise of curiosity, members of my department² took an Instagram picture of B. anthracis using a cell phone camera. The image took seconds to capture with modern technology, yet doesn’t come close to rivaling Koch’s.

Figure 1B. A 1982 stamp from Zimbabwe celebrating the centennial of Koch’s discovery of M. tuberculosis (DPH personal collection).

Koch’s realization of bacterial photography was not merely a pretty picture. The communication of accurate images of bacterial shape was vital to the understanding that multiple species of bacteria existed, and Koch’s practice in imaging relatively easy-to-cultivate organisms like B. anthracis allowed his eventual rapid success in discovering the extremely fastidious M. tuberculosis in diseased tissue. It is this attention to detail and scientific resolve that led Koch to the discoveries that have immortalized him in scientific, medical, and indeed general human history (Figure 1B).

Notes:

¹Brock, Thomas D. (1999) Robert Koch: A Life in Medicine and Bacteriology. ASM Press, p. 61.
²Thanks are given to Katie McCallum for her Instagram account and to Lori Horton, Malik Raynor, and Michelle Swick of the Kohler Lab for the microscopy equipment and B. anthracis sample.

Fecal Transplants in the “Good Old Days”

2013-05-13T08:00:00-07:00

by Stanley Falkow

I had a conversation with some colleagues last week about “personalized medicine,” which has been transformed now into the term “precision medicine.” The conversation revolved around what to do about the perceived effects of antibiotic treatment on the microbiota of individuals. How does one treat a patient without disrupting their microbiota? Do we create new classes of antimicrobials that target only a precise pathogen? I opined that I thought the day was coming when all individuals might have the microbiota from each anatomic site preserved so that it could be reconstituted after some catastrophic disruption caused by antimicrobial therapy for an infection, transplantation, surgery etc. The topic of fecal transplantation and how successful it has been for the treatment of intractable Clostridium difficle infection then came up. Would fecal reconstitution really work?

I answered truthfully that I did not know, but my experience many years ago led me to believe it would. One of the people in this conversation, John Mekalanos, no stranger to stools, asked when I participated in a fecal transplantation study. It occurs to me that my experience in this study it might be of interest, or at least titillate, those who read this blog.

I was a 23-year-old medical technologist (MT, ASCP) in 1957 working as a journeyman bacteriologist in several clinical laboratories in Rhode Island and Massachusetts. It was a time when the Staphylococcus aureus 80/81 phage type was raising a specter of uncontrolled hospital infections. It was a time when large doses of antibiotics were administered to patients pre-operatively and continued until they were discharged some days later. Many of these patients reported to their physicians that they suffered from diarrhea, flatulence, indigestion and generally felt terrible after their surgery, though the operation was deemed a success. This was before anyone knew about C. difficle, of course, but antibiotic-associated diarrhea was known even in those days. One of the internists I knew well came to talk with me. I should point out that in the late 1950s many (most) of the physicians were frequent visitors to the bacteriology laboratory because they wanted to look at the Gram stains and the cultures obtained from their patients. The physician in question, who I will simply call Dr. S, thought after examining and talking to patients who had not “felt right” after their surgery had suffered from the aftereffects of the antibiotics that had been given them to sterilize their bowel flora before surgery. The feces of many of these patients would yield no growth on blood agar plates and MacConkey agar for days after their surgery. (We didn’t do anaerobic cultures in those days though). The stools were even odorless. Few stools can make that claim. S thought that their normal flora had been disrupted by the antibiotics. ‘Healthy bowels, and regularity made a happy patient”, he said.

Empty gelatin capsules, ready for filling. Source.

Dr S thought it would be prudent to ask patients to bring in a stool specimen when they came to the hospital for their surgery. He said to me, “Now, Stan, how do we get it back into them?” We decided that gelatin capsules from the pharmacy might do the trick. The pharmacies at that time still made a good deal of their own formulary. We set up a protocol. Stools were obtained from the patients immediately after admission. I would transfer the stool as quickly as possible into 12 large gelatin capsules,. This was a messy and not a precise or enthusiastic process on my part. I would wash the capsules in water and rinse in a dilute solution of mercuric chloride to disinfect the outer surface of the capsule, and then the capsules were rinsed again and put in a small ice cream carton and put in the refrigerator labeled only with the patient’s initials.

Upon discharge, Dr. S and one other physician who became a convert to this “protocol” would obtain the capsules from me. They would tell the patient to keep them refrigerated, to take 2 twice a day until they were all consumed. At least Dr. S told them, “Eat lots of salad.” This uncontrolled trial continued for some months, and, according to the anecdotal reports of Dr. S and his colleague Dr. B was quite successful in comparison to the patients of other physicians who did not have the benefit of the autogenous fecal sandwich. I don’t recall that we ever thought about the ethics of this. It was a time before informed consent. I’m pretty sure, however, that the esthetics of this practice was understood and that the patients in question never knew the contents of the capsules they ingested.

The chief hospital administrator discovered what was up. He confronted me and exclaimed, “Falkow, is it true you’ve been feeding the patients s**t!” He used the Anglo-Saxon phrase for feces. I responded: Yes I had been a participant in a clinical study that involved the patients ingesting their own feces. You’re fired! was the reply, although Dr. S came to my rescue. I was rehired two days later. Thus, the “experiment” came to an abrupt end. I left in June, 1958 to study for my PhD with C. A. Stuart and Seymour Lederberg at Brown University.

Now I am not going to claim that I knew that feeding patients their own feces after intense antibiotic therapy would be beneficial. Dr. S was sure it was the case based on his years of clinical experience. I understood the point that the indigenous flora was important. I had examined hundreds of stool specimens from sick and well people for too long. I routinely Gram-stained fecal samples and examined them in a wet mount. I didn’t have deep sequencing but I could discern differences in the flora of individuals. One fecal flora did not reflect all. I understood this even better after I met Rene Dubos and Russell Schaedler at the Armed Forces Epidemiology Board in the early 1960s and even more when I read the wonderful book by Theodor Rosebury, Life on Man.

My experience presaged the current excitement and exciting information that has deluged us in the past few years about the wonders of the human microbiota. The understanding and the appreciation for the sanctity of the “normal flora,” however, is not a new thing. Fecal transplants, of a sort, were practiced some 50 years ago because of the recognized untoward effects of antibiotic therapy. Mekalanos after hearing this story said: It once was "Eat s**t and die!" Maybe now it will be "Eat s**t and live!”

This experience also reminds me of something I have learned over the years. Experiences that occur during experiments or facts learned in a seminar or read in a paper have a way of reappearing, often decades later, with new meaning.

Stanley Falkow is the Robert W. and Vivian K. Cahill Professor Emeritus of Microbiology and Immunology and Medicine in the Department of Microbiology and Immunology at Stanford University.

Talmudic Question #98

2013-05-09T04:00:00-07:00

What do you think is the single best criterion for telling an endosymbiont of a eukaryotic cell from an organelle?

Post Script

I must come clean and confess that I formulated this question after having read a most stimulating review by John McCutcheon and Nancy Moran that touches on this subject. Some of the responses you provided are related to considerations in this article. Read and enjoy!

-Elio

No Bacterium Is An Island

2013-05-06T04:00:00-07:00

by S. Marvin Friedman

P. aeruginosa colonies showing the characteristic green color that is due to the production of pyocyanin. Source: Gloria Delisle, Microbe Library, ASM.

To paraphrase an old adage, no bacterium is an island. Indeed, bacteria in nature exist as polymicrobial communities where interactions between individuals influence activities of the entire population. This is especially true of pathogenic bacteria, although it has been mostly ignored because we frequently isolate a single species from an infection site and prescribe antibiotic therapy based upon this information. A recent paper by Korgaonkar and coworkers highlights that this practice is somewhat akin to burying ones head in the sand and results in an incomplete picture of the infection dynamics. By studying co-infection systems, these researchers discovered interesting details of how synergy with neighboring organisms can contribute to a pathogen’s virulence.

Quorum Sensing and Virulence Factors

Gene PA0601 is required for NAG and peptidoglycan sensing in P. aeruginosa. (A) Pyocyanin, (B) elastase, and (C) PQS levels produced by WT and mutant P. aeruginosa in the presence of no inducer (succinate), NAG, or peptidoglycan. Source.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that often colonizes chronic wounds along with other Gram-positive bacteria. Together, they exert a synergistic effect. Previous work from that laboratory found that production of the virulence factor pyocyanin (the pigment that gives the colonies of this organism their characteristic green color) is enhanced when P. aeruginosa is exposed to cell wall fragments shed by co-infecting Gram-positive bacteria. In fact, the N-acetylglucosamine (NAG) moiety of the cell wall’s peptidoglycan was shown to be necessary and sufficient for enhanced pyocyanin formation. To study the genetics of this interesting phenomenon, the researchers first isolated a mutant that produces normal levels of pyocyanin in the absence of NAG and not more pyocyanin in its presence. Using transposon inactivation, they found such a mutant with an insertion at gene PA0601. This gene encodes a two-component response regulator with sequence homology to the Lux family of quorum sensing regulators. Once again, the themes of virulence and quorum sensing converge.

Several other virulence factors of P. aeruginosa are co-regulated with pyocyanin production, including the extracellular protease elastase. As expected, in the wild type but not in the PA0601 mutant, elastase production was also enhanced by NAG and peptidoglycan. The autoinducer for this kind of quorum sensing is the Pseudomonas Quinolone Signal (PQS or 2-heptyl-3-hydroxy-4(1H)-quinolone). PQS is known to be an anti-staphylococcal agent, and so are pyocyanin and elastase. The researchers hypothesized that, when stimulated by NAG, the increase in the levels of pyocyanin and elastase is due to enhanced production of PQS. Sure enough, the level of PQS was threefold higher in cells grown with NAG and peptidoglycan. It all fits: quorum sensing becomes a surveillance mechanism for Pseudomonas to detect its bacterial neighbors and respond by producing antimicrobial factors.

Does Quorum Sensing Matter In Infection?

GlcNAc/peptidoglycan sensing enhances P. aeruginosa virulence in polymicrobial infections. Survival curves of (A) antibiotic-untreated Drosophila after infection with WT P. aeruginosa (PA14), the PA0601 mutant (PA0601−), and the genetically complemented PA0601 mutant (PA0601− complement); (B) antibiotic-treated flies infected with P. aeruginosa WT or the PA0601 mutant; and (C) antibiotic-treated flies infected with WT P. aeruginosa or the PA0601 mutant in the presence and absence of peptidoglycan. Source.

The authors turned to an in vivo model of infection, namely infected Drosophila flies. Here, ingested P. aeruginosa colonize the flies’ crop, eventually causing their death. With wild type (WT) bacteria, 50% of the flies died by 2 days. Flies fed the PA0601 mutant lived significantly longer (50% were still alive by 4 days). The PA0601 mutant was able to colonize and persist in the fly’s crop, so that was not the reason for its decreased lethality. The Drosophila crop is colonized by large numbers of Gram-positive bacteria and thus the authors hypothesized that it would naturally contain peptidoglycan shed by its Gram-positive population. To test this, flies were fed a mixture of antibiotics (ampicillin, vancomycin, and erythromycin) designed to kill the native Gram-positive but not Gram-negative iota. Cultures made from the crop showed that this treatment was effective. Killing was now delayed whether the flies were infected with the WT or the PA0601 mutant. Equally tantalizing is the finding that peptidoglycan sensing resulted in a decrease of the size of the Gram-positive biota. Indeed, the Gram-positive population within the fly crop was reduced about 1,000-fold when infected with WT but much less so in flies that had ingested the PA0601 mutant. So, P. aeruginosa surveys the microbial community in its vicinity and both degrades its competitors and enhances its virulence to the host.

Was the delayed killing of antibiotic-treated flies due to a lack of peptidoglycan in the fly crop? Sure enough, feeding peptidoglycan abolished this effect in the WT, but not in the PA0601 mutant. So, does the enhancement of PQS and thus PQS-controlled virulence factors by peptidoglycan play a role in vivo? To test this, the authors extracted RNA from crops colonized by P. aeruginosa and, using reverse transcriptase PCR, measured the expression of pqsA, the first gene in the PQS regulon. pqsA levels were reduced about threefold in antibiotic-treated flies compared to controls. Feeding peptidoglycan to antibiotic-treated flies restored transcription levels of psqA transcripts to those in the controls. Furthermore, these effects required transport of NAG, suggesting that it is the component of peptidoglycan sensed.

Surveillance and Co-infection

In order to expand these observations to a vertebrate system, the authors turned to a murine chronic wound infection model. Surgical wounds infected with monocultures of P. aeruginosa, the PA6061 mutant, or Staphylococcus aureus, showed about the same final growth yields. Coinfections of WT P. aeruginosa and S. aureus initiated using a 1:1 ratio were highly enriched for P. aeruginosa after four days (the median value for the ratio of the two was 110:1). On the other hand, co-culture infections of the PA0601 mutant were enriched for S. aureus. Counts of P. aeruginosa in wild type and the PA6061 mutant co-cultures did not change significantly, indicating that the enhanced S. aureus numbers were not due to increased persistence of WT P. aeruginosa but rather to its ability to recognize peptidoglycan and thus increase production of antimicrobials during co-culture in vivo.

The results presented in this paper clearly show that in a polymicrobial environment, commensal Gram-positive bacteria can potentiate the virulence of a Gram-negative opportunistic pathogen by shedding cell wall fragments, which enhances virulence factor production via a QS system. As an aside, it’s curious that both some bacteria and the host recognize the presence of invading bacteria through their shedding of cell wall fragments. But then, peptidoglycan (or its fragments) is a good choice for a calling card, being that this compound is quite unique in biochemistry and is restricted to bacteria.

These findings suggest that therapeutic strategies targeting Gram-positive bacteria may be helpful for treating P. aeruginosa-dominated polymicrobial infections, including those of chronic wounds. Or, to say the least, it emphasizes that the polymicrobial nature of bacterial infections must always be carefully considered.

S. Marvin Friedman is Professor Emeritus, Department of Biological Sciences, Hunter College of CUNY, New York City and an Associate Blogger for Small Things Considered.

Korgaonkar A, Trivedi U, Rumbaugh KP, & Whiteley M (2013). Community surveillance enhances Pseudomonas aeruginosa virulence during polymicrobial infection. Proceedings of the National Academy of Sciences of the United States of America, 110 (3), 1059-64 PMID: 23277552

Little Known Glomalin, a Key Protein in Soils

2013-05-02T04:00:00-07:00

by Elio

A visible portion of the rhizosphere. Source.

If you had heard of glomalin, you are a better person than I am. Until a couple of months ago I wasn’t aware of its existence, which is close to sinful: it happens to be a very abundant protein in the soil rhizosphere, playing a key role in the soil’s mechanical properties and as repository of soil carbon. Glomalin is a glycoprotein (although the “glyco-“ may be overused here, as biochemical analyses suggest that it contains little in the way of sugars) that binds together silt, sand, or clay soil particles. By ‘supergluing’ the small, loose particles, this gooey protein makes larger granules or aggregates and protect the soils from the eroding forces of winds and water. So, where does glomalin come from? It is thought to be made by fungi, more specifically by members of the arbuscular mycorrhizal fungi, the Glomales (hence the name ‘glomalin’). The hyphae of these fungi synthesize glomalin as part of their stress response. They coat their outer surface with the protein to make a protective waxy coat that keeps the water and nutrients inside the cells. The glomalin coating also makes the fungal strings sticky so they bind soil particles, thus creating an protective ‘armor’ against environmental insults and microbial predators. Most importantly, the fungal “string bags” make soil aggregates. This improves water infiltration and retention in the soils and gas exchange, which makes them more fertile. For more information on glomalin click here.

Filaments and spores of an arbuscular mycorrhizal fungus stained with fluorescent antibodies against glomalin. Photo by Sarah Wright. Source.

Some interesting facts about glomalin hint already at its global significance. Glomalin can contribute to as much as 27% of the soil carbon, especially in the temperate zone. By gluing soil particles together, this protein also helps retain other carbon-containing compounds and protects them from decomposing as well. Glomalin easily beats out humic acids, the soluble and insoluble organic substances generated during plant decay that were thought to be the main contributors to soil carbon. It is also extremely tough, lasting an estimated 7 to 42 years, depending on soil conditions. Glomalin was first isolated as recently as 1996 by Sara Wright, a chemist at USDA’s Agricultural Research Service. To extract it, she had to heat the soil at 121°C for one hour at a pH of 8.0. This separate d the glomalin from other gunky material in the soil . But “milder” conditions, using SDS and phenol at room temperature, can now be used. For an article on glomalin chemistry, click here.

The aggregating effect of glomalin improves the soil fertility, so farmers test the soil texture or “tilth” with their hands. Source.

Finally, glomalin is what gives soils their physical condition, what farmers call its “tilth ” (aka, the “feel” that experienced farmers look for when letting soil flow through their fingers). And although studies on glomalin are in their infancy, there is compelling evidence that it plays a role in enhancing agricultural productivity. It is also a major carbon sink, so we need to start talking about it when discussing global climate change. Time we learned more about it, right?

The Art of Microbial Alchemy

2013-04-29T04:00:00-07:00

by Gemma Reguera

“The Great Work of the Metal Lover” display, by Brown and Kashefi (Michigan State University) (top image). Included is the aesthetically-engaging, original glass chemostat used to grow the microbe, a metal manifold for gas distribution, and a heated glass copper column that removes any traces of oxygen from the gas supply. Cupriavidus biofilms and the gold grains they produce are visible at the bottom of the vessel (bottom image). Source.

In 2001, Kashefi and collaborators published an article in Applied and Environmental Microbiology reporting the surprising finding that several iron-reducing microbes can use gold as an electron acceptor for their respiration. These microbial alchemists included both mesophilic and thermophilic bacteria as well as hyperthermophilic archaea. The beauty of this process is that the oxidized form of gold provided to the microbes, Au(III), is soluble, whereas its reduced form, Au(0), is insoluble. Hence, the microbes respire soluble gold and precipitate it as gold nanoparticles on their outer surface plus, in the case of the Gram-negative bacteria, in the periplasmic space as well. These studies provided the first experimental evidence supporting the role of microbes in the formation of gold deposits in both hydrothermal and cooler environments, thus challenging the prevailing view that gold mineralization was an abiotic process.

Since then, the list of microbes with the ability to precipitate gold has grown. One of them, the Gram-negative bacterium Cupriavidus metallidurans, is an especially abundant component of the so-called gold nugget microbiota, i.e., the bacterial biofilms associated with gold grains. Elio covered this story previously in our blog. This bacterium grows at room temperature and its mechanism for gold biomineralization is one of the best characterized. For this reason, when artist Brown asked Kashefi, both at Michigan State University, to help him design an exhibit that allowed the audience to witness the process of microbial alchemy, Kashefi regarded Cupriavidus asthe microbe of choice.

The greatest challenge was microbiological in nature. They sought a culture system that would produce gold nuggets that were big enough to be seen with the naked eye. This required growing Cupriavidus at high concentrations of gold. But gold, like many other soluble heavy metals lacking biological roles, is toxic to microbes. It traverses the outer membrane of Gram-negative bacteria and then is reductively precipitated by low-potential electron donors present in the periplasmic space, which disrupts cell envelope homeostasis and vital cellular functions. It can also cross the cytoplasmic membrane, triggering oxidative stress and inhibiting the functioning of essential enzymes in the cytoplasm. Cupriavidus detoxifies the Au(III)-complexes that accumulate in its cytoplasm by reductively precipitating them as inert gold-sulfur (Au[I]-S) nanoparticles. Nevertheless, Cupriavidus can tolerate only µM concentrations of Au(III) in solution, and as a result the gold nanoparticles it produces are microscopic. Since the planned display required visible gold nuggets, the team’s microbiologist Kashefi grew Cupriavidus in a chemostat for months, adaptively evolving it to gradually grow at higher concentrations of gold until reaching 1.5 mM, a 30-fold increase from the originally-tolerated 50 µM.

A scanning electron micrograph shows the Cupriavidus cells surrounded by a biofilm matrix with encrusted gold deposits. The deposits were then colorized with gold leaf generated with the very same gold produced by the microbes. Source.

From this undertaking grew the “The Great Work of the Metal Lover”, a display composed of a portable, bench-scale system capable of performing microbial alchemy before an audience. Within this set up, the bacteria grow ‘live’ at room temperature and anaerobically, using hydrogen gas as an electron donor and Au(III) (provided in the medium as gold chloride) as an electron acceptor. As the bacteria respire, they precipitate the soluble gold as colloidal gold particles, whose accumulation turns the growth medium a lovely purple. Beauty is also to be found at the bottom of the culture vessel where purple biofilms and encrusted gold grains accumulate. These biofilms and gold deposits were captured in scanning electron micrographs. For an artistic flourish, they collected gold nuggets from several chemostat cultures, then melted them together to obtain enough material for Brown to create 24K gold leaf. He then enhanced the micrographs by highlighting the gold deposits using the gold precipitated by the microbes themselves. What this scientist-artist duo achieved goes beyond the blending of art and science. This is a visual feast for the intellect and a terrific vehicle for conveying the artistry of the microbiological world to the public. Bravo!

Gemma is associate professor in the Department of Microbiology and Molecular Genetics, Michigan State University and an Associate Blogger at STC.

Kashefi K, Tor JM, Nevin KP, & Lovley DR (2001). Reductive precipitation of gold by dissimilatory Fe(III)-reducing bacteria and archaea. Applied and environmental microbiology, 67 (7), 3275-9 PMID: 11425752

A Good Defense Is Worth Stealing

2013-04-25T04:00:00-07:00

by Merry Youle

Add phage ICP1 to the list. Source.

One widely-used tactic for defense against phage and other mobile genetic elements is to deploy a CRISPR-Cas system (click here and here) to recognize and chop them into pieces. Based on sequenced genomes, 60% of Bacteria and 90% of Archaea have the wherewithal to dispatch invaders this way. But phages also have to protect themselves against enemies, including other mobile elements. Knowing a good thing when they see it—and they have seen it from the receiving end often—some phages have stolen the entire CRISPR-Cas structure and use it to inactivate genetic elements that would interfere with their replication.

What sorts of genetic elements challenge phage supremacy? Some bacteria harbor chromosomal islands that, like prophages, excise and replicate when induced by cellular stress or damage. When phage infection is what triggers the activation, the island is classified as a PICI, a phage-inducible chromosomal island. The ungrateful PICI sometimes then proceeds to interfere with phage replication. One subset of these PICIs includes the SaPIs, Staphylococcus aureus pathogenicity islands, that I introduced here and wrote more about here.

Researchers observed that a PICI-like element (PLE) in Vibrio cholerae is activated by infection by a vibriophage (ICP1, here called simply the phage). PLE then blocks phage replication by some unknown mechanism. But this phage fights back with its own CRISPR-Cas system. Although CRISPR loci and Cas genes had been detected earlier in phage genomes and metagenomes, this paper is the first to document phage survival courtesy of a fully functional, phage-encoded CRISPR-Cas apparatus. For an introduction to CRISPRs, I again refer you to my earlier posts here and here. Briefly, a functioning bacterial CRISPR locus contains short sequences (spacers) that were acquired from the DNA of some invading genetic element, usually a phage or a plasmid. The RNA transcripts from the CRISPR region are processed into short CRISPR-RNAs, one per spacer. Each CRISPR-RNA recognizes the invader from which the spacer came and targets it for destruction by the Cas nucleases. With this vibriophage, it is the phage “invader” that carries the CRISPR and uses it to attack a mobile element resident in the host chromosome.

EM of V. cholerae phage ICP1, a myophage isolated from stool samples from cholera patients. Bar = 100 nm. Source.

The CRISPR locus of the vibriophage investigated here has two spacers that are identical to different regions within the PLE element in the host chromosome. Either one of those spacers is sufficient to protect the phage. However, if the perfect match between that spacer and the PLE is disrupted by mutations in either sequence, the PLE wins and the phage cannot replicate. But there is an adaptive dimension to CRISPR-Cas systems in Bacteria and Archaea. When challenged by a new invader, one never encountered before, these organisms acquire a new spacer from that element, add it to their existing CRISPR locus, and then use it to defend against that same invader. Is this phage as adept? Further experiments demonstrated that this vibriophage can indeed acquire new spacers when needed. The proof? I wrote above that phage without a PLE-matching spacer could not replicate in a host carrying the PLE, but in actuality a few “phage escape mutants” can (efficiency of plaquing could be as high as 3.5 × 10–5). When ten such escapees were sequenced, all ten had a new spacer added to their CRISPR locus, a spacer targeting the PLE.

In some parts of the world, this V. cholerae phage is a recognized ally, helping to control cholera outbreaks as they go about their daily routine of slaughtering bacteria. From our point of view, the PLE is a villain in cahoots with its V. cholerae host, providing its host with immunity against phage infection. Enlisting these vibriophages in our defense might help prevent cholera. With billions of years experience, the ingenious phages are a seemingly endless source of strategies that are, in turn, worth stealing.

Seed KD, Lazinski DW, Calderwood SB, & Camilli A (2013). A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity. Nature, 494 (7438), 489-91 PMID: 23446421

TWiM #55: In the Copper Room

2013-04-25T04:00:00-07:00

Hosts: Vincent Racaniello, Michael Schmidt, and Elio Schaechter.

Vincent, Elio and Michael discuss the finding that copper surfaces reduce microbial burden and hospital-acquired infections in the intensive care unit.

Click to play current episode. (58.5 MB .mp3, 85 minutes)

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Send your microbiology questions and comments (email or mp3 file) to twim@twiv.tv, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twim.

E. coli Cells Face FACS and Get Back into Shape

2013-04-22T04:00:00-07:00

by Kimberly K. Busiek

Figure 1: The importance of size and shape to the animal kingdom. (A) A well-adapted giraffe enjoys the fruits of its evolutionary labor. (B) Beak variations among Darwin’s finches. (C) A bonobo uses his opposable thumb to dig for termites.

There’s no question that variation in size and shape has conferred selective advantages over the course of evolutionary time. One of the most obvious examples is the long neck and legs of the giraffe, which allow it to snatch foliage that is unreachable by vertically challenged competitors. The variable beak shapes and sizes of Darwin’s finches represent the diverse tool set that evolved when only certain food sources became available. And the appearance of the opposable thumb, a simple change in hand shape, undoubtedly influenced the course of human history.

Size And Shape On A Smaller Scale

More recently, microbiologists have appreciated the selective power of size and shape among bacteria. For example, a temporary ban on cell division in uropathogenic Escherichia coli allows the long, filamentous cells to avoid phagocytosis in the host. Helicobacter pylori depends on its strikingly helical shape to establish residence in the host’s stomach. In both cases, we are beginning to understand the molecular mechanisms dictating these adaptations in size and shape. However, these studies have also cast a glaring light on how little we still know about how bacteria acquire and maintain their distinctive morphology. Should we call cell shape the forgotten phenotype?

The Need For Speed

How to begin tackling this question experimentally? As molecular microbiologists, our first inclination is to suggest a genetic screen or selection to isolate mutants with odd shapes. But this approach is complicated for two reasons. First, screening for shape mutants often requires microscopic observation of hundreds or thousands of cells, which requires a lot of time and patience. Second, aberrantly shaped bacteria often show little effect on their growth rate or survival in the lab, which requires complex and clever selection conditions.

With these problems in mind, members of Kevin Young’s laboratory developed an automated approach to distinguish normal-shaped E. coli cells from odd-shaped ones. This screening method requires fixing and fluorescent staining of cells, then running them through a flow cytometer capable of detecting the shape of cells streaming single file past a laser beam. Although this technique is a vast improvement over manual screening procedures, Young’s laboratory recently tweaked the process further, forgoing the staining step and using live cells rather than dead, fixed ones.

Figure 2: Comparison of normal (A) and abnormal shaped (Δpbp4,5,7; B) E. coli cells by phase contrast microscopy. Cells were treated with aztreonam, an antibiotic that inhibits cell division and therefore emphasizes shape differences. Source.

How, do you ask, are the cells sorted without a fluorescent dye? It turns out that the forward scatter of light, which is recorded by a detector in line with the laser beam, is different between normal and odd-shaped cells. As proof of principle, this laboratory compared the light scatter of normal, rod shaped E. coli cells to abnormally shaped ones that lack penicillin binding proteins (PBPs) 4 and 7. These are endopeptidases that cleave crosslinks in the cell wall and influence cell shape. The authors also used mutants in PBP5, a D,D-carboxypeptidase that removes the terminal D-alanine from side chains in the peptidoglycan layer and plays a major role in determining normal cell shape. The forward scatter of the shape-defective cells becomes skewed to the right in the scatter plot compared to the normally shaped cells, indicating that differences in light scatter between cells of different shapes even when the cells are not fluorescent can be detected.

Shape-shifting

Figure 3: Scatter plots of normal (A) and abnormally (B) shaped E. coli cells subjected to flow cytometry. The selection gate used to enrich the suppressors is shown as the grey box labeled “P1.” Source.

Once they confirmed that flow cytometry is capable of distinguishing differences in the shape of these strains, the researchers used this technique to isolate suppressors of the abnormally shaped cells. Here, suppressors are mutants that where the normal shape is restored. To find such suppressors, they used the flow cytometer once again, but this time as a device for cell sorting without the use of fluorescence or CS (“FACS without the FS”). Using FACS one can collect (not just enumerate) a subpopulation of cells based on its fluorescence or, in the case of CS, its light scatter. Suppressors of abnormal cell shape were selected by performing CS on aberrantly shaped cells but collecting only those cells that exhibited a forward light scatter similar to normally shaped cells (designated as “P1” in Figure 2). This subpopulation was then subjected to repeated rounds of growth and further CS until the distribution of light scattering among the once aberrant cells closely resembled the profile of normal cells. Of the isolated colonies selected from this population of potential suppressors, ~50% exhibit near normal light scattering profiles. Microscopic observation also confirms that these isolates were of normal shape. In total, four suppressor mutants were isolated from three different shape-deficient strains. Additionally, through directed mutagenesis, these workers also found that peptidoglycan endopeptidases AmpH and MepA appear to negatively impact cell shape in at least one of the suppressor strains, indicating that other endopeptidases may also play a role in cell shape.

The Young team will fully characterize the isolated suppressors at a later date, but they note that the mutations are not in or near the genes encoding known penicillin binding proteins. This bit of information demonstrates that the FACS enrichment procedure may indeed fulfill its desired role of helping discover novel pathways for shape generation and maintenance. These new cell-shape determinants could serve as attractive targets for drugs that inhibit the morphologic changes need for optimal virulence of pathogens, such as E. coli or H. pylori, and further prove that size (and shape) truly does matter.

Kimberly Busiek is a Ph.D. candidate in the Microbiology and Molecular Genetics program at the University of Texas Health Science Center at Houston. Like associate blogger Dr. Daniel Haeusser, she is a member of Dr. William Margolin’s laboratory where her thesis research focuses on the molecular mechanisms of cell division in the bacterium Escherichia coli.

Laubacher ME, Melquist AL, Chandramohan L, & Young KD (2013). Cell sorting enriches Escherichia coli mutants that rely on peptidoglycan endopeptidases to suppress highly aberrant morphologies. Journal of bacteriology, 195 (4), 855-66 PMID: 23243305

Pictures Considered #3. How Do You Know There Is a Nucleoid?

2013-04-18T04:00:00-07:00

by Elio

A time-lapse series of E. coli growing on agar containing 20% gelatin. The time is shown in the lower left corner. The contrast in these photos was reversed to make it easier to see the nucleoids. In actuality, the nucleoids look pale and the cytoplasm dark. Source.

What is more commonplace than saying that prokaryotic cells possess a nucleoid? It is implicit in the term prokaryote itself. Still, it was not shown definitively until the 1940s that bacteria and archaea have such differentiated structures made up of condensed DNA. It was the careful work of “bacterial cytologists” (as they were then called) and especially that of the eminent Carl Robinow that furnished convincing evidence for the nucleoid’s existence. Bacterial cells, then and now, were commonly prepared for microscopic examination by fixing with heat and staining with basic dyes. Such dyes stain more intense than acid ones because bacteria are replete with acidic groups from the RNA in the ribosomes. So abundant are these RNAs that these dyes stain the cells uniformly, and the nucleoids are not apparent. To reveal the nucleoids, cells were treated with ribonuclease or with dilute acids that selectively degrade the RNA and then stained. The DNA-containing nucleoids retain the dye and are now easily seen. When thin sections of bacterial cells could be examined under the electron microscope, analogous structures were seen, thus providing additional evidence that bacterial nucleoids exist. And yet, a small lingering doubt remained.Both of these methods looked at chemically treated (“fixed”), dead bacteria. Could this lethal treatment create artifacts regarding the observed “nucleoids”?

The point was settled in 1956 when Mason and Powelson clearly showed nucleoids in living cells. The authors resorted to a simple trick to make the nucleoids visible without dyes. They rightly guessed that the nucleoids and the cytoplasm, having different macromolecule densities, would have different refractive indexes. In order to be able to see this difference under a phase contrast microscope, they simply needed to grow the bacteria in a medium whose refractive index approximated that of the cells themselves. When they did this, nucleoids were evident.

To produce such a medium, these researchers added gelatin, a high molecular weight substance that would not increase the osmotic pressure enough to hinder growth. The nucleoids of living, growing bacteria can now be readily made out under a phase contrast microscope. Being able to see these intriguing structures in living cells removed any residual doubts about their existence.Those “simple” bacteria really do have internal structure! Once again, a definitive picture was worth a thousand suppositions.

Whose Planet Is It Anyway?

2013-04-15T04:00:00-07:00

by Elio

This is the title my friend Fred Neidhardt recently used for a talk, and a good question it is. I suppose that most microbiologists and the readers of this blog would split the answer down the middle, the biomass of this planet and the chemical transactions therein being about half microbial, half everything else. However, it’s safe to say that most people, many scientists included, are unaware of the colossal importance of the microbial half, not only in biology and medicine but in geology, meteorology, and in our Earth’s habitability. This state of affairs should not be unexpected, given that we have only became aware of much of this during the last few decades. I lived roughly the first half of my life carrying only a vague notion of the global importance of the microbial world. But now we know, and the word needs to go out. A measure of microbial literacy is required for anyone to understand the workings of our living planet.

(Top) Upper atmospheric oxygen concentration, as a percent of current levels, plotted against geological time. (bottom) Phylogenetic history of life on Earth, scaled to match the oxygen timeline. Note that the origin of the eukaryotes and the subsequent diversification of animals both correspond to periods of increasing atmospheric oxygen. Source.

Through the years, many influential writers have endeavored to convey the global influence of microbes to scientists and non-scientists alike. We can now add to these efforts a new contribution that speaks to scientists of all spheres, but especially to other biologists. It was recently published as a Perspective in PNAS, a most appropriate venue. Entitled Animals in a bacterial world, a new imperative for the life sciences, it is authored by 26 scientists whose names are bracketed by those of Margaret McFall-Ngai and Jennifer Wernergreen. It deals specifically with the role of microbes in the lives of animals. While interactions with plants and the inanimate environment are not included, this seems a fitting focus given the anthropocentric interest of most readers. The other stories are for another day, to include the viruses, the most numerous of all players and which interact with all other living things.

Perhaps you think that in this blog I am preaching to the choir, and admittedly I am. But the sermon provided is superb and deserves sharing. The authors traverse the whole animal kingdom, from humans to fruit flies to shrimp to squid to sponges to protists, with stops in between, offering up fascinating examples of symbiotic relationships, some well known, others less so. I will single out a few that especially caught my fancy. A choanoflagellate, belonging to the oldest of animal phyla, initiates colony formation in response to bacterial signals. (might this speak of how multicellularity arose?). There is a shrimp whose embryos are protected from a fungus by the 2,3-indolinediole made by Alteromonas bacteria on board. A Bacteroides found in the guts of Japanese persons has genes for degrading the seaweeds in their diet, probably acquired from a marine bacterium. An alga protects itself by producing compounds (furanones) that mimic quorum-signaling molecules, thereby blocking communications between invading bacteria. And so on. But don't think for a minute that this is just a parade of astonishing stories. The authors use each example to elucidate a particular category of interactions, thus infusing the work with deeper meaning. Reading this article will sharpen one’s insight. For those enticed to follow up any story, the authors provide a list of well-chosen citations in the supplemental material.

A phylogeny of choanoflagellates and selected animals, annotated to indicate the evolution of characters particularly relevant to interactions with bacteria. (Right) Interactions between bacteria and eukaryotes, corresponding to the phylogeny. Bacteria are prey, sources of metabolites, inducers of development in symbiosis (morphogenesis) and in larval settlement (environmental cues), and activators of immune systems. Source.

I could stop here, but can’t resist listing other exciting material found here that I should have known but didn’t.

The fossil record tells us that early animals (in the Ediacaran) grazed on dense bacterial assemblages and that burrowing animals originated together with bacterial mats.
A majority of the genes that animals carry descend from bacteria and archaea, or protists.
Human-associated bacteria exchange genes at a rate that is 25-fold higher than those not living in host-associated host environments.
Up to one third of an animal’s small molecules in the blood (its metabolome) are of microbial origin.
Segmented filamentous bacteria with a reduced genome that live in the gut of some mammals are critical for the maturation of the immune system.
At the bottom of the sea, bacteria feeding on carcasses make noxious metabolites that repel animal scavengers 10,000 times their size.

Nested ecological interactions of animals and bacteria and their underlying metabolic bases. (A) A forest canopy insect illustrates the cascading effects of animal-bacterial interactions across multiple spatial scales. Bacterial symbionts (Left), residing in the gut (Center Left), are essential to nutritional success of insect species (Center Right) in tropical forest canopies (Right), where they often make up a majority of animal biomass. (B) Diversity of energy metabolism in bacteria and animals. Animals can ferment and aerobically respire but are unable to perform the vast diversity of other, ecologically vital, energy-harvesting processes. Beyond phototrophy, which they share with plants, bacteria can also contribute to primary production by using inorganic energy sources (lithotrophy) to fix CO2. Animals are directly or indirectly dependent on bacteria for extracting energy and cycling biomolecules, whereas animals actively contribute to bacterial productivity through bioturbation, nutrient provisioning, and as habitats for colonization and shelter. Source.

The authors pose some great questions: How have bacteria facilitated the origin and evolution of animals? How do animals and bacteria affect each other’s genomes? How does normal animal development depend on bacterial partners? How is homeostasis maintained between animals and their symbionts? How can ecological approaches deepen our understanding of the multiple levels of animal–bacterial interaction? They point out that the answers to these questions are relevant to all biologists and beyond.

Progress is undoubtedly being made along the “it’s time to accept that microbes are a big deal” front. The huge effort designed to understand the human microbiome and how it changes between people in different states of health and disease has certainly caught the public’s eye. With the promise of personalized medicine, the microbiome of each person will be part of everyone’s medical history. As the Romans said: Suum cuique (to each his own). The Earth Microbiome Project will follow, although where it will take us is for the future to tell us. We must thank this happy state of affairs in good part to the development of the various ‘ omics’. (By the way, if you find the term –omics grating, what do you think of subsuming them under the term polyomics?)

But for now, the task of changing the general perception of microbes in the scheme of things is still ongoing. Voicing a concern is one thing; delivering the message with such a formidable voice is another. I am grateful to the authors who have made such a commendable effort and there by have contributed to both our understanding and our enjoyment.

McFall-Ngai, M., Hadfield, M., Bosch, T., Carey, H., Domazet-Loso, T., Douglas, A., Dubilier, N., Eberl, G., Fukami, T., Gilbert, S., Hentschel, U., King, N., Kjelleberg, S., Knoll, A., Kremer, N., Mazmanian, S., Metcalf, J., Nealson, K., Pierce, N., Rawls, J., Reid, A., Ruby, E., Rumpho, M., Sanders, J., Tautz, D., & Wernegreen, J. (2013). Animals in a bacterial world, a new imperative for the life sciences Proceedings of the National Academy of Sciences, 110 (9), 3229-3236 DOI: 10.1073/pnas.1218525110