Phage Genomics Research Initiative

My school has been selected to participate in the Howard Hughes Medical Institite's Science Education Alliance.

"The SEA’s first project is the National Genomics Research Initiative, a two-part, year-long research course offered by colleges and universities selected through a national competition. The course is aimed exclusively at beginning college students, who make real discoveries by doing research on bacterial viruses, called phage. In the first term, the students isolate colonies of phage from locally collected soil samples. Given the diversity of phage, each one is almost certain to be unique, so the students get to name their newly identified life form. They then spend the rest of the term purifying and characterizing their phage and extracting its DNA.

Between terms, the purified DNA is sent to the Joint Genome Institute-Los Alamos National Laboratory in New Mexico, where it is sequenced. In the second term, the students receive files containing their phage’s DNA sequence. The students then use bioinformatics tools to analyze and annotate the DNA from their phage."

I'll be offering this course to Queens College freshmen next year. It will be real exciting to see what new phages we can isolate from the Long Island soil. Part of the process involves visualizing phage thru electron microscopy (phage are too small to be seen thru ordinary light microscopy).
I've never done this before and am looking forward to seeing phage for the first time. Other professors report that this was the highlight of the course for their students.

"The students told professors teaching the SEA course that the most exciting moment came when they saw a picture of their phage for the first time....As the image of the phage emerged on the computer screen, many students pointed and jumped up and down. One student from Hope College in Michigan called her mom from lab when she saw her phage for the first time. In Findley’s class, the students later had the equivalent of a phage fashion show, and they “oohed” and “aahed” over the phage with the longest tail or darkest head.

The students’ excitement and creativity was also reflected in the names they gave their viruses. The quirky names aren’t the normal staid acronyms often seen in the scientific literature. For example, some students named their phage after the Comedy Central duo of “Colbert” and “Jon Stewart.” Other groups chose “Peaches” and “LRRHood” for Little Red Riding Hood. A student at Spelman College named her phage “Hope” the day after Barack Obama was elected President of the United States."

The hope is that this initiative will encourage college freshmen to consider science as a career.

Fight Infection with Infection

There was a recent article in Popular Science magazine on bacteriophage therapy. Scientists, including d'Herelle the discoverer of phages, have long recognized the value of phage therapy. In fact, the protagonist of Sinclair Lewis's novel Arrowsmith (publ. in 1925) cured the residents of a fictitious Caribbean island of plague using phage.

Despite its early popularity, phage therapy never quite caught on in the West. Most speculate that the arrival of antibiotics precluded their widespread acceptance, except in the former Soviet Union (e.g. Georgia).

The article discusses some of the advantages of phage therapy.

They prey only on bacteria, never human cells, they rarely spread from person to person, and, perhaps most important, bacteria have trouble becoming immune to them. As living organisms, phages are constantly changing and adapting in tandem with their host bacteria to kill them more effectively. Phage therapy could therefore eliminate the vicious cycle in which bacteria evolve resistance to antibiotics, necessitating the development of new, even more powerful drugs, at which point the process begins all over again.

I'm skeptical that phages rarely spread from person to person (but the research on this is minimal if not nonexistent), and bacteria DO become immune. In fact, bacteria frequently win arms races with phage in coevolution experiments.( A good example is the trap cells I used in my virus trap experiments. Several attempts to generate phage able to infect these trap cells have failed). Nonetheless, the article is correct in that, unlike antibiotics, phage evolve. This is a powerful tool to generate new phage variants.

Unfortunately, as the article points out, this precise point makes it difficult for phage treatments to past muster at the FDA.

Although there have been no reports of adverse effects resulting from mutations, phages that don't normally nest inside the human body could potentially swap genes with other phages that do and produce foreign proteins that trigger an immune reaction. And it's impossible to say exactly how a virus might mutate when exposed to different bacteria, says Paul Sullam, a microbiologist at the University of California at San Francisco.

FDA regulation, which some would say is excessive, has slowed phage therapy research in the US.

"People in this country have a right to be incensed that we have a very different situation here than in Europe with regards to phage," says Betty Kutter, a phage researcher at Evergreen State College. "Our whole regulatory environment has been one major thing that has slowed people down."

So where does one go when they have an uncurable infection? The Eliava Institute of of Bacteriophage, Microbiology and Virology.

Randy Wolcott calls Eliava the "mother ship of phage research," a worldwide Mecca for people suffering from antibiotic-resistant infections. Only it doesn't look like the sort of place you'd want to go with a health problem. When Wolcott visited to hunt down alternatives for his patients, the four-story facility bore a closer resemblance to a neglected sanatorium. The walls were unpainted, the rooms were dark, and the equipment looked like museum pieces. "The conditions were abysmal," he says. "Yet the science is amazing."

Perhaps, as Rockefeller's Vincent Fishetti says , the way to go is phage-based therapy.

This distinction might seem arcane to nonbiologists, but in Fischetti's mind, it's a crucial one. While Wolcott sees phages as a major therapeutic coup, Fischetti sees them as merely an intermediate step toward a new generation of even better bacteria-fighters. He contends that the uphill regulatory battle phages face, as well as the risk of mutations, make them too big a gamble for American drug companies. "Phages are going to be a boutique treatment, nothing more," he says. So he is taking an alternative approach, purifying the phage to extract the lysin, the enzyme it uses to dissolve the bacterial cell wall and kill the bacterium. Having observed that lysins were the phages' "active ingredients," Fischetti aims to harvest the lysins from them and turn them into stable antibacterial drugs. If successful, he could accomplish a double feat previously thought impossible: getting the bacteria-fighting benefits of phages to patients, while doing an end run around the regulatory Rube Goldberg machine that researchers like Wolcott face.

Incidentally, I am currently hosting a doctoral student from the Eliava Institute, Sophie Rigvava, who is characterizing the phages of Enterococcus faecalis in my laboratory.

I've posted a few times on phage therapy here, here, here and here.

Photo: Phages [in orange] prey on a lone bacterium, using prong-like proteins to anchor themselves to the cell before they inject their genes into it Lee D. Simon/Photo Researchers

This Week's Citation Classic

John J. Dennehy, Stephen T. Abedon, Paul E. Turner. HOST DENSITY IMPACTS RELATIVE FITNESS OF BACTERIOPHAGE Φ6 GENOTYPES IN STRUCTURED HABITATS. Evolution (OnlineEarly Articles). doi:10.1111/j.1558-5646.2007.00205.x

Citation classic? Ah...not quite. Just kidding. No, this is just my latest published paper, now appearing online early in Evolution. The work stems from a side project I conducted while I was a postdoc in Paul Turner's laboratory at Yale. What originally began as a simple experiment in the spring of 2005 to determine whether the density of hosts in a habitat affected parasite competition quickly snowballed as additional experiments were conducted, data analyses and reanalyses performed, revisions made and resubmitted (3x! The first submission was on 1/31/06), authors added (the estimable Stephen Abedon) and much sweat, angst and time sacrificed. This paper is the fruit of the most difficult effort I've undertaken in my fledgling career. (However, as Homer Simpson might say, "The most difficult effort of your career... SO FAR!").

Here we competed two Phi6 strains over a range of host densities in two separate habitats: in liquid culture and on agar plates. In liquid culture, the results were not surprising. The more fit phage out-competed the less fit phage over all host densities. This result was expected because, in a well-mixed liquid culture, the phages are not spatially limited in their access to hosts, and their net reproduction should be a product of the number of hosts and the reproduction per host. Since the more fit phage produced more babies per host (in the paper, greater burst size), its advantage over the less fit phage should be consistent over all host densities, and the total number of babies produced by both strains should increase with increasing host density (See Fig. 5 below. Note: the slopes are not significantly different despite appearing to converge.). By contrast, in agar, phage dispersal is limited by its ability to diffuse through the viscous agar. This causes a shift from direct competition to indirect competition between the phage strains. That is, in the liquid culture, phage compete globally for hosts, whereas in the agar culture, competition is limited locally. Here the results were surprising; the relative fitness of the less productive phage strain increased with increasing host density (up to a point where it leveled off). (See Fig. 4 below with fitness comparisons to unstructured habitat).
This result caused us considerable consternation and its cause is still under debate. Since what is actually occurring in a growing plaque (i.e., the region on a lawn of bacteria where hosts are infected and lysed) is somewhat of a black box, we can only speculate as to why we observe increasing relative fitness with increasing host density for the less productive strain. One possible explanation, as described in our paper, is:

...bacteria may differ physiologically over space depending on their initial densities. This phenomenon may be attributed to the fact that bacteria form microcolonies on a lawn, and microcolony size depends on initial density (Kaplan et al. 1981). Lower initial inocula lead to larger microcolony sizes. This outcome makes intuitive sense if we assume that microcolonies are spheres that are packed within a constant volume (the top-agar layer). Each microcolony is initiated by a single bacterial cell seeded in the top agar. Thus, if fewer bacteria are seeded, then microcolonies must grow to a larger size in order to attain the same cumulative volume.

Large microcolonies contain relatively fewer outer-surface bacteria with access to oxygen and nutrients, and with relatively unobstructed diffusion of wastes. For these reasons, large microcolonies may contain lower numbers of bacteria that are competent for phage infection. Thus, the final 20% of infections at low initial bacteria densities likely result in reduced burst sizes per cell (due to the larger microcolony size) and, therefore, less particles per plaque. This effect could be substantial with a Pseudomonas host given that it is an obligate aerobe, and that bacteria in the center of Pseudomonas microcolony may be particularly physiologically inappropriate for phage infection. To summarize, greater input of bacteria into a habitat may lead to smaller microcolonies that contain greater numbers of bacteria competent for phage infection, and this may lead to better phage growth.

Why does the less fit strain display even greater increase in phage density as plaques form under greater bacterial densities? We speculate that the presumptive poorer host physiology with larger microcolony size has a greater impact on Φ6M relative to wild type Φ6. Alternatively, Φ6M may be less able to efficiently penetrate into larger microcolonies, resulting in fractionally fewer bacteria infected within the confines of the plaque, rather than fewer phage produced per bacterium infected.

Whatever the cause of the changes in relative fitness, one thing is clear: the strength of selection against the less productive phage strain was reduced at greater host densities. Consequently these genotypes may gain time to adapt to the habitat conditions, and may eventually out-compete the more productive phage strain in evolutionary fitness. This result is relevant in a practical sense with regard to phage therapy, and is consistent with the general finding that biofilms are more resistant to phage or antimicrobial attachment than are planktonic bacterial populations. It may also be relevant to the emergence of infectious diseases where habitat structure and increased host densities permit the persistence of less fit genotypes.

Photo credit: Dennis Bamford. Phi6 adsorbing to Pseudomonas phaseolicola pili.

Phage Hunters

18 freshmen students have enrolled in my Genomics Research Experience course aka Phage Hunters. This course is supported by the Howard Hughes Medical Institute's Science Education Alliance. My students have begun the process of isolating novel Mycobacteriophages by collecting soil samples from the wild and plating them on lawns of Mycobacterium smegmatis, a M. tuberculosis relative. Unlike M. tuberculosis, M. smegmatis is non-pathogenic and is easier to grow and manipulate under experimental conditions. Nonetheless, by virtue of their close phylogenetic relationship, the two bacteria are quite similar in many respects. Thus, M. smegmatis may be an excellent model for deriving treatments against tuberculosis.

Collecting Mycophage is already paying handsome dividends. Albert Einstein College of Medicine Professor William Jacobs isolated a phage he named the Bronx Bomber from soil from his own backyard in the Bronx. With University of Pittsburgh Professor Graham Hatfull, Jacobs characterized this phage in the laboratory. They found that this phage is able to insert itself into the genome of M. smegmatis at a very specific location in the groEL1 gene, thus disabling the gene. One of groEL1's functions is to facilitate the production of biofilms.

Biofilms are extracellular polymeric substances that aid and protect microbes. They allow bacteria to persist in the face of antibiotics. It's estimated that 80% of infections involve biofilm formation. While biofilm formation in tuberculosis has not yet been uneqivocally confirmed, M. tuberculosis does have a groEL1 gene with 90% similarity to that of M. smegmatis.

If the phage is able to infect M. tuberculosis or is mutated to infect M. tuberculosis, it is possible that some day the phage could be used as therapy against tuberculosis. As one of the three primary diseases of poverty, tuberculosis has a devastating impact in the developing world.

Top Photo: Bxb1 is a mycobacteriophage that was originally isolated from Dr. Jacobs' backyard in the Bronx. It is affectionately called "The Bronx Bomber" as it forms large plaques on a plate with lawn of Mycobacterium smegmatis cells (left panel). The Bxb1 phage plaques are characterized with their clear centers surrounded by turbid rings. The turbid rings represent lysogens (i.e. M. smegmatis bacterial cells into which Bxb1 has integrated) of M. smegmatis that are resistant to superinfection with Bxb1 phage. These lysogens are defective in biofilm formation. A transmission electron micrograph of Bxb1 is shown in the right panel. Courtesy of Jordan Kriakov, William R. Jacobs, Jr.

Middle photo: Image shows Mycobacterium smegmatis growing as a biofilm on a liquid surface, with its characteristically textured folds. Courtesy of Anil Ojha, Tom Harper, Graham Hatfull.

What has phage lambda ever done for us?

Murray and Gann published an essay in this month's Current Biology titled "What has phage lambda ever done for us?" Since my current study organism is phage lambda, naturally I was interested. The essay is informative and interesting and, for those who have institutional access, I urge you take a gander at it for your own edification.

Bacteriophage lambda was first discovered in 1951 by Esther Lederberg (wife of Nobelist Joshua Lederberg and probably deserving of a Nobel in her own right, but that's another story). Lambda was immediately interesting to the burgeoning molecular biology community because it propagates by two alternative pathways: lytic and lysogenic. In the lytic cycle, the phage's DNA enters an E. coli cell, induces the cell to produce phage progeny, then subsequently causes the cell to lyse (break open) releasing the contents into the surrounding media. By contrast, the lysogenic cycle is largely harmless, possibly even beneficial, to the cell. Here the phage's DNA enters the cell whereupon it is integrated into the cell's own DNA. As the cell replicates, the phage replicates as well. How cool is that?

Murray and Gann highlight where phage lambda was used as a model organism in many major biological discoveries. Here is a ^not so^ brief listing:

* gene regulation (Lwoff, Jacob and Monod 1961; Roberts 1969; Guarneros and Galindo 1979)

* DNA recognition and cooperative binding (Ptashne 1967)

* genetic fine structure (Benzer 1957)

* messenger RNA (Volkin and Astrachan 1956, 1957)

* acquisition and loss of genes from genomes (Campbell 1959, 1962)

* triplet nature of DNA code (Crick, Barnett, Brenner and Watts-Tobin 1961)

* restriction and modification (Dussoix and Arber 1962)

* DNA and protein are colinear (Sarabhai, Stretton, Brenner and Bolle 1964)

* DNA ligase (Gellert 1967)

* epigenetic gene regulation (Ptashne 2004)

* chaperones and protein folding (Georgeopoulos, Hendrix, Casjens and Kaiser 1973)

* repression and activation (i.e. turning genes on and off, Ptashne 2004)

* molecular basis of DNA recombination (Meselson and Weigle 1959, 1961)

That's quite a list. And it is nowhere near being exhaustive. Phage lambda has proven uniquely malleable in the laboratory and has illuminated large facets of previously hidden knowledge to biologists. Today lambda is still being used in many laboratories, which is testament to its enduring legacy.

The photo depicts Ur-Lambda (the father of all laboratory lambda phages); it's a lambda with LEGS! *ahem* tail fibers. The Electron Microscopy image was taken by Bob Duda at the Pittsburgh Bacteriophage Institute.

Are bacteriophages picky eaters?

I don't often see bacteriophage ecology and evolution papers in the open source literature, but there is a nice one in next month's American Naturalist (occasionally Am Nat selects papers for open access).

The paper by Rick Heineman and colleagues addresses the question of optimal foraging, a body of theory that seeks to explain the food choices of organisms in terms of how they maximize energy intake over time. As a model organism, the authors use the bacteriophage T7, a parasite of Escherichia coli.

Naturally, phages don't "eat" per se, nor do the make conscious "choices", but for the purposes of the theory that's largely irrelevant. We can still model optimal foraging in terms of the evolution of phage host range (i.e. "dietary choices"), provided that phage are able to "discriminate" among possible host types. As the authors write, "Phages have no behavioral plasticity in the usual sense, yet they evolved to make host range choices that qualitatively match optimality predictions."

Heineman et al. found that T7 phage could evolve the ability to discriminate between several host strains. T7wild-type "was independently adapted in two mixes of Escherichia coli strains: C with either B or K12. In both adaptations, C was the permissive host, while the other (B or K12, depending on the adaptation) aborted T7 infections due to deletion of a host gene needed for viral replication. Both adapted phages evolved to largely avoid the nonpermissive host but maintained a high adsorption rate to C." Notably the phage evolved the ability to discriminate via single amino acid substitutions in the tail fiber gene (used to bind host receptors).
To see if discriminating phage could be favored over non-discriminating phage, Heineman et al. used E. coli strains that differed in their resistance to tetracycline with C being Tet resistant and K lacking Tet resistance. Adding tetracycline would alter K into poor quality hosts, but leave C unaffected.

The authors tested whether increasing tetracycline (at levels that disable, but not kill K cells) in the culture media would favor one phage strain over the other. The results showed that T7Choosy
out competed T7wild-type when tetracycline levels were high, but not when they were low. The study has implications for other viruses. As the authors write, "the principles demonstrated here for phages may operate in other viral systems. The main requirement is that a virus that avoids infecting one host (or cell type) will have opportunities to infect a different type. This property may apply to many viruses infecting multicellular hosts with respect to tissue tropisms—differences in the ability to infect various tissues within the body. Viruses within a host are likely to be selected to use some tissues and not others, and the nature of selection on tissue tropism may parallel those found here for phages."

Overall this is a very nice study, with clever use of bacteriophages and bacteria, to test a body of theory that has not commonly been tested with microbes. The only main issue is that there is a lack of a quantitative aspect to the study (which is difficult with microbes), and as such, despite the cleverness of the approach, phages might not be the best arena to test optimal foraging theory. Nonetheless, it is an effective demonstration that organisms that cannot make conscious choices can evolve to prefer some resources over others.

Heineman, R., Springman, R., Bull, J. (2008). Optimal Foraging by Bacteriophages through Host Avoidance.. The American Naturalist, 171(4), E149-E157. DOI: 10.1086/528962

Photo: T7 phage from the The Microbial World.

What I did last summer (and year)....

Earlier in these pages I gave a brief background of what I've been doing for the past year. I've been interested in how differences in mRNA production and differences in holin structure led to variability in lysis timing. Max Delbruck was the first to consider this question; he looked at variation in the number of babies a single phage produces and found a great deal of variation in this important life history trait. Delbruck's method, diluting a suspension of infected bacteria such each dilution contained on average less than one bacterial cell, and then plating all the (hundreds of) dilutions, is especially onerous, and perhaps explains why his work was not followed up.

(i.e. production rate, and observe the changes in Moreover, his work leaves open the questions of what causes the variation and whether it is evolutionarily significant. My work follows up on Delbruck's, except that I chose to look at a different, but closely related life history trait, lysis time. Earlier work has shown that there is a direct correlation between lysis time and burst size: the longer the phage waits to lyse the cell, the more babies it can produce. So it makes a good proxy for burst size, as well as an interesting life history trait in its own right. Plus, we now have the genetic techniques to manipulate the phage's genome and dissect the causes of variation: to wit, the ability to manipulate the holin protein structure and to alter the strength of the promoter that controls holin production. All we need is a way of observing lysis time for individual cells. Enter the microscope-mounted perfusion chamber! Here is the setup:

The way this works is you place a glass cover slip on the bottom, a cell-binding agent (poly-lysine) and some E. coli infected with lysogenic phage, then place another cover slip on top. The cells form a single layer on the bottom cover slip. The whole unit is placed on a heating platform, under a normal light microscope, and tubes are attached to allow a flow of nutrient broth thru the chamber. The heating platform generates a heat spike, inducing the phage to begin the lysis process. With a microscope mounted camera, I filmed the bound cells following the heat spike until they lysed, then recorded the time of lysis for each cell.

Given are the variation in lysis time for the wild type (JJD3), the most variable altered holin sequence genotype (JJD9) and the most variable altered promoter genotype (SYP028). This histogram (below) clearly shows the greater variability (standard deviation, SD, in lysis time for the latter two genotypes.

In general, SD increases with the mean lysis time (below, P = 0.0087). This result is consistent with the idea that, on average, it takes a longer time for a weak holin-holin interaction to attain the critical holin raft size that is necessary for hole formation. Furthermore, the the timing of attainment also varies widely among individual infected cells. That is, we should expect to observe a positive relationship between the mean lysis times and their standard deviations.

Greater promoter strength leads to a shorter lysis time (P = 0.0065). Interestingly, the lysis time variation seems to show a threshold effect. Within a range of promoter strength (between 150 to 250), different mean lysis times showed a similar stochasticity (P = 0.9593). However, once the promoter strength was dropped to a low level, the stochasticity increased dramatically.

Finally I showed an environmental component to lysis time stochasticity by reducing the nutrients provided to the host cells. Expression of the phage holin protein requires the host synthesis machinery, including the RNA polymerases (RNAPs) for transcription, ribosomes for translation, and many other raw materials for protein synthesis and enzymes for modifications. In general, cells growing at a higher rate will have a higher concentration of the synthesis machinery. Here as growth rate increases, the stochasticity (SD) decreases (P = 0.0346), demonstrating that host physiology can greatly influence the outcome of a viral infection.

The commonly invoked causes for cellular stochasticity are the random events of gene transcription, translation, and degradation of the expressed protein. Besides the usual causes, there is another layer of random event for holin hole formation, namely, the association and disassociation of holin raft on the cell membrane. However, in this preliminary study, I was not able to attribute the relative importance of each cause for the observed lysis time stochasticity. If the main cause is due to host biochemistry and physiology, then it would be difficult for the phage to reduce stochasticity. On the other hand, if the main cause is due to the amount of holin production or holin-holin interaction, then it is possible for mutations to change the promoter strength or holin sequence to increase or reduce the level of stochasticity.

Whether the observed lysis time stochasticity is evolutionarily significant remains to be determined; however it is conceivable that selection can favor genotypes with greater or lower levels of phenotypic stochasticity depending on the circumstances. Future experiments will address (1) whether variation in lysis time stochasticity translates into variation in fitness; (2) whether genotypes expressing greater or lower levels of lysis time stochasticity can be selected for; (3) whether similar patterns of stochasticity exist for different phage species.

This Week's Citation Classic

This week's citation classic honors Seymour Benzer who passed away yesterday.

Benzer S. 1955. Fine structure of a genetic region in bacteriophage. PNAS 41(6):344-54.

Benzer began his career as a physicist, but was inspired to switch to molecular biology by Schrodinger's book What is Life and by Max Delbruck. He took a leave of absence from his position at Purdue to pursue research on the structure of the gene, and he never looked back. Biology, bacteriophages in particular, captivated him.

In his classic 1955 PNAS paper, Benzer used the bacteriophage T4 to map the r gene.

Benzer describes his experiments:

I plated some of these r mutants on two different strains of E. coli bacteria. And I had two different strains of K-12 phage, one that was lysogenic and had Lwoff’s lambda phage in it, and one that didn’t. What happened first was that when I plated these r mutants on the plain K-12 strain, instead of making the big plaques they made the little plaques. So they were showing lysis inhibition on that strain. Then, when I plated them on the strain that had the lambda, I got zero plaques. And having been alerted by reading Pontecorvo’s article, I immediately, really instantly, realized…. Well, at first I thought I made a mistake. I thought I had forgot to put the phage on there. Dummkopf, do it again! I did it again and saw the same phenomenon.

Benzer's key insight was that he had stumbled upon a system where he could do genetic mapping by using recombination between r mutants to map the r gene much in the way that Alfred Sturtevant used recombination to map Drosophila. The trouble with using recombination to map genes is that the closer two genes are together, the less likely they will recombine. Thus mapping a single gene was thought to be impossible.

But not so for phage. So many offspring (~100 million) are produced that the odds are that recombination can occur even among two adjacent nucleotides.

Benzer describes the moment:

So I immediately realized—a eureka moment, and they’re all too rare—that this was a system in which I could do very fine genetic mapping. I could take two r mutants, cross them with each other, and take the progeny and put them on this K-12 lambda strain. The r mutants themselves would produce no plaque, but if in any of the progeny there was a crossing-over between these two different mutations, such as to produce a wild-type recombinant that had neither mutation, that would produce a plaque. And that you could put 100 million plaques on one plate. So a quick calculation told me that that was enough, knowing the number of nucleotides in the DNA of the bacteriophage. This was about 1954 or ’55—after the Watson-Crick discovery. So, based on the number of nucleotides in the DNA and the phage, I would have enough resolving power to separate the rII mutations, even if they were just one nucleotide apart.

When r mutants are plated on lysogenic K-12 E. coli, only those that have undergone recombination within the r gene to form wild-type phage will form plaques. The number of plaques on the bacterial lawn indicates how far apart the two nucleotide mutations were on the gene. Thus, Benzer had the tool of exceptionally high resolution; he could to map the r gene down to the nucleotide.

Benzer's results showed conclusively that genes were not indivisible as commonly thought.

Benzer later went on to dissect the nervous system of Drosophila in a third highly productive career.

Even at 86 years of age, Benzer still ran a laboratory at CalTech. A very entertaining oral history interview series with Benzer is available here. Benzer's CV should give us all pause; he was a tremendously accomplished man.

Updates: Larry Moran points out this paper: Adventures in the rII Region and other tributes to Benzer in the blogosphere.

This Week's Citation Classic

This week's citation classic is "The Molecular Biology of Bacterial Viruses" in honor of Gunther Stent, who recently passed away. Stent was one of the unsung heroes of the molecular biology revolution and member of the "phage group" and the RNA Tie Club. Although he was not credited with any major discoveries in molecular biology, "Gunther was part of the intellectual glue that kept this small band of pioneers together".

As one of Max Delbruck's students, he was asked "'Do you want to work on phage?' 'Yes sir,' Stent replied, 'that’s exactly what I want to work on, but could you refresh my memory as to just what phage is actually all about?'"

His classic text on bacteriophages is still frequently consulted in my laboratory and has been a standard reference for phage workers since the 60's.

Stent was remarkably diverse. After receiving his PhD in physical chemistry, he switched to phage biology. Then as the 60's came to a close, Stent became "bored with molecular biology". He switched to studying neurobiology with leeches as model organisms. Later Stent focused on the history and philosophy of science, publishing important works such as "Paradoxes of Free Will".

Stent explained his frequent career changes, "The problem is that I get bored. I see something new, and it becomes exciting for me, so I move on."

His autobiography is "Nazis, Women and Molecular Biology: Memoirs of a Lucky Self-Hater."

This Week's Citation Classic

This week's citation classic is Gunther Stent's Molecular Biology of Bacterial Viruses (WH Freeman, 1963). Stent is a polymath, currently a professor emeriti in the MCB Department at UC: Berkeley. He started his career by obtaining a Ph.D. in Physical Chemistry in 1948 from the University of Illinois, then joined Max Delbruck's lab at the California Institute of Technology, before joining the University of California, Berkeley faculty as an Assistant Research Biochemist in 1953. Stent soon left his mark on fields as diverse as bacterial genetics to DNA structure to neurobiology and even the history and philosophy of science. His published books include, the classic, Phage and the Origins of Molecular Biology (1966, 1992), the textbook, Molecular Genetics: An Introductory Narrative, The Neurobiology of the Leech (1981), Mind from Matter: An Evolutionary Epistemology (1986), and his autobiography, Nazis, Women and Molecular Biology.

Molecular Biology of Bacterial Viruses is part textbook, part memoir, part history, part lab manual and thoroughly entertaining. I still use it and cite it in my own work. Dedicated to Max Delbruck, it opens with a stunning photo of the DNA of phage T2 liberated from the phage's capsid. It proceeds then to discuss phage history, morphology, growth, life cycle, genetics and theory. Anyone with even a cursory interest in phage cannot help being educated and entertained by this book.

Here is a poetic tribute to Gunther Stent on the occasion of his 80th Birthday.

This Week's Citation Classic

d'Herelle, F. 1917. On an invisible microbe antagonistic to the dysentery bacillus. Comptes rendus Acad. Sci. Paris 165: 373-375.

Credit for the discovery of bacteriophages is usually given to Frederick Twort and Felix d'Herelle. I somewhat disagree. While Twort's report preceded d'Herelle's by a year and a half, he was thoroughly misguided about its nature (he thought it was an enzyme and persisted in this view for an unseemly length of time). Moreover, Twort failed to pursue his discovery, did little work of any serious import, and spent much of his career trying to grow viruses on artificial media.

By contrast, d'Herelle understood immediately what he observed, comprehended its implications and set forth a far reaching research program to explore its practical use.

d'Herelle is also a far more interesting character. A high school graduate, self-taught in microbiology, d'Herelle would have been noteworthy for another reason besides discovering bacteriophages: d'Herelle can be credited with originating modern biological pest control through his use of bacterial diseases to control locust infestations.

Here are some of d'Herelle's notable achievements:

-made whiskey from maple syrup
-built a chocolate factory but went out of business
-1st scientific paper "proved" carbon was not an element, but a compound
-made liquor from bananas
-cured coffee rot in Guatemala
-made schnapps from sisal
-destroyed a locust plague in Argentina
-initiated the field of phage therapy

d'Herelle discovered phage while employed at the Pasteur Institute in Paris. In the course of studying bacteria in fecal samples from dysentery patients, he observed that a filterable (i.e. able to pass through a fine filter) microbe caused the complete lysis of of dysentery bacilli cultures. Almost singlehandedly, d'Herelle developed new experimental approaches to study this new microbe and elaborated theories about its nature and role in infectious diseases.

The citation classic I have cited is a marvel of clear thinking, able writing and concise form. It served as the basis not only for d'Herelle's entire subsequent career, but also for much of the 20th century's phage research. d'Herelle's work also presaged experimental evolution, originated Luria and Delbruck's famous Fluctuation Test and anticipated the importance of adaptive evolution. d'Herelle most certainly deserved a Nobel Prize for his discovery. Alexander Fleming received one for discovering penicillin, and that finding did not have a fraction of the import on science as did the discovery of phage. Etienne Wolff, professor on the faculty at Strasbourg, argued that "the existence of a destructive principle of bacteria, which was revealed to be a filterable virus, is very much more important from the general point of view than the discovery of penicillin, a bactericidal substance extracted from an organism....Bacteriophage represent a scientific revolution, while penicillin is a particular case of chemotherapy."

In the 1960s Félix d’Hérelle's name appeared on a list published by the Nobel Foundation of scientists who had been worthy of receiving the Nobel Prize, but did not.

Photo: (left to right) Elena Makashvili, Felix d'Herelle, Georgiy Eliava.

What I did for the last year....

I've just returned from the GRC: Microbial Population Biology conference where I presented a poster on my work for the past year. In a few installments I'd like to reproduce this poster here.

My major question of interest was, "How does molecular stochasticity in the individual cell affect major life history traits?" To address this question, I used the enterobacteriophage lambda strain cI857 as a model. Under normal circumstances, cI857 integrates itself into E. coli's genome where it is passed horizontally to daughter cells. Most of the phage's genome is repressed at this point. However, after a temperature spike, the phage is induced into the lytic cycle. Here the "late" genes are expressed, including the lysis cassette and the genes that make phage babies.The lysis cassette contains four genes that produce five proteins. I'll focus just on two: holin and endolysin. The best available model suggests that holin integrates itself into the host's inner membrane. Over time, the holin concentration in the membrane rises, until it spontaneously condenses into a raft. Subsequently the holin undergoes a conformational change producing a hole in the inner membrane. This permits endolysin to attack and degrade the outer membrane, leading to host cell lysis and the release of phage babies into the surrounding medium. Thus holin, and its rate of production, is the main determinant of lysis time, or in life history theory, generation time. The rate of holin production depends on protein translation from low copy mRNA transcripts. Since a single mRNA transcript can produce multiple holin molecules, small changes in mRNA numbers can have large effects on holin production and, hence, the timing of lysis. My project looked at how differences in mRNA production and differences in holin structure led to variability in lysis timing. I'll discuss these in greater detail in a later post and show some of my data.

A cool animation sequence of the lytic cycle is available here.

Lead photo: Maria Schnos, Institute for Molecular Virology, University of Wisconsin, Madison

Genetic Map of the λ genome from CA Reinhart, W. Kentucky University.

Figure by me.

This Week's Citation Classic

Volkin E & Astrachan L. 1956. Phosphorus incorporation in Escherichia coli ribonucleic acid after infection with bacteriophage T2. Virology 2 (2): 149-161.

After the discovery of DNA structure by Watson and Crick, the way seemed paved to a quick resolution of how proteins were made. Unfortunately, both theory and experiment quickly stalled at an impasse. Data from several labs was extremely difficult to interpret, namely findings that 1) for several bacterial species, DNA bases varied widely, but the cytoplasmic RNA did not (Belozersky & Spirin 1958) and 2) the cytoplasmic RNA was exceptionally stable (Davern & Meselson 1960). These findings seemed to suggest it was impossible for RNA to be a template for protein synthesis.

The problem was that this cytoplasmic RNA was not what later came to be known as messenger RNA (mRNA), but rather a relatively "inert" form of RNA associated with ribosomes (i.e. rRNA)(Note: rRNA is now thought to carry out key reactions during translation).

On Good Friday 1960, Sidney Brenner, Francis Crick and Francois Jacob were hanging out at Brenner's rooms at King's College when they put together some anomalous data and created a theory to explain protein synthesis. These data were 1) Hershey et al.'s (1953) discovery of a small fraction of RNA that was synthesized rapidly just after phage infection, 2) Pardee et al.'s observation of extremely rapid enzyme synthesis following mating between two bacterial types and 3) a report from two relative unknowns, Eliot Volkin and Lazarus Astrachan, from Oak Ridge National Laboratory.

Volkin and Astrachan infected E. coli with phage, then exposed the culture to radioactive ³²P for a few minutes. What they found was a "DNA-like RNA" that did not resemble the previously found RNAs (i.e. rRNA and tRNA) and turned over very rapidly. The main difference was that it had similar bases to the DNA of phage.

	f DNA	labelled f RNA	E. coli DNA	E. coli RNA
A	32	33	25	26
U or T	32	29	25	19
C	18	20	25	25
G	17	18	25	19

What Volkin and Astrachan did not realize is that they discovered the key to solving the protein synthesis mystery.

Crick describes the Good Friday Meeting in What Mad Pursuit, "What the PaJaMo [i.e. Pardee et al. 1959] type of experiment showed was that the ribosomal RNA could not be the message.... Where, then, is the message? At this point Sydney Brenner let out a loud yelp -- he had seen the answer. (So had I, for that matter, though nobody else had). One of the peripheral problems of this confused subject had been a minor species of RNA [found by Volkin and Astrachan] that occurred in E. coli ... [Volkin and Astrachan's] result had hung in midair, surprising but unexplained.... What Sydney had seen was that the Volkin-Astrachan RNA was the messenger RNA for the phage-infected cell... It is difficult to convey two things. One is the sudden flash of enlightenment when the idea was first glimpsed. It was so memorable that I can recall just where Sydney, François, and I were sitting in the room when it happened. The other is the way it cleared away so many of our difficulties. Just a single wrong assumption (that the ribosomal RNA was the messenger RNA) had competely messed up our thinking, so that it appeared as if we were wandering in a dense fog."

Paul Berg, winner of the 1980 Nobel Prize in Chemistry, calls Volkin and Astrachan's research an "unsung but momentous discovery of a fundamental mechanism in genetic chemistry" and a "seminal discovery [that] has never received its proper due."

What happened? Everybody knew of Volkin and Astrachan's findings at the time. In an interview at his Oak Ridge home in late 2003, Volkin recalled his conversation with Sydney Brenner at Cold Spring Harbor Laboratory in New York, where Volkin conducted research on the hot topic of bacterial viruses during the summers in the late 1950s. "I can well remember sitting on the lawn at Cold Spring Harbor and telling Sydney Brenner about our experiments," Volkin says. "I gave a presentation on our RNA research to the group there." In a 1977 issue of Nature, renowned biophysicist T. H. Jukes wrote that in 1956, "I had squeezed my way into a doorway of a packed room to hear a paper by Volkin and Astrachan on DNA-like RNA." (ORNL).

The problem was that Volkin and Astrachan's data was self-admittedly "sloppy", Volkin and Astrachan weren't well known, ORNL wasn't highly regarded and the data simply did not fit with the dominant paradigm at the time. Brenner, Crick and Jacob ended up getting recognized with "discovering" mRNA, while Volkin and Astrachan were never properly credited. Many scientists felt Volkin and Astrachan deserved Nobel recognition.

Belozersky AN & Spirin AS. 1958. A correlation between the compositions of the deoxyribonucleic and ribonucleic acids. Nature 182: 11–112.

Hershey AD, Dixon J & Chase M. 1953.Nucleic acid economy in bacteria infected with bacteriophage T2 .1. Purine and pyrimidine composition. Journal of General Physiology 36 (6): 777-789.

Davern CI & Meselson M. 1960. Molecular conservation of ribonucleic acid during bacterial growth. Journal of Molecular Biology 2: 153.

Pardee AB, Jacob F & Monod J. 1959. Genetic control and cytoplasmic expression of inducibility in the synthesis of Beta-galactosidase by E. coli. Journal of Molecular Biology 1 (2): 165-178.

Photo: One of the early RNA electron micrographs scanned with the vidicon/RTPP system (Jacob Maizel, Bruce Shapiro, and Lewis Lipkin). The sample was adenovirus type 2 messenger RNA. Bruce developed boundary segmenters and boundary shape descriptors that could map electron micrograph data to the secondary structure.

Phage Therapy

Cesar Sanchez put together an excellent post on bacteriophage therapy over at Twisted Bacteria. The highlight includes a BBC documentary that I had previously been unaware of.

Phage therapy is the use of the parasites of bacteria to combat bacterial infections. It was once held in high regard, but use declined with the advent of antibiotics. However, now that antibiotic resistant microbes are becoming more common, scientists in the West are re-exploring phage therapies (some in the East never stopped).

I've posted a few times on phage therapy here and here.

Photo: Phi6 infecting Pseudomonas phaseolicola from Dennis Bamford.

Phage in the News

Science Daily reports on a new paper in Biophysical Journal from Joshua Weitz and team. I met Joshua last winter at the Viral Paradigms Workshop. It was clear then that he was doing some exciting work.

"The researchers modeled the complex gene regulatory dynamics of the lysis-lysogeny switch for lambda phage.... The decision circuit is a race between two pathways and in the case of a single virus, the outcome is biased toward lysis," explained Weitz. "In our model, when multiple viruses infect a given cell, the overall production of regulatory proteins increases. This transient increase is reinforced by a positive feedback loop in the latency pathway, permitting even higher production of lysogenic proteins, and ultimately the latent outcome."

The central idea in the model proposed by Weitz and collaborators is that increases in the overall amount of viral proteins produced from multiple viral genomes can have a dramatic effect on the nonlinear gene networks that control cell fate."

I've previously written about the lysis-lysogeny decision in phage.

I've just downloaded the paper and look forward to reading it closely.

Photo: Phage lambda electron-micrograph. Notice it lacks tail-fibers which is typical of laboratory reared lambda. Wild lambda have tail fibers.

Bacteriophage Lysogeny

There's a great new post over at Microbiology Bytes about bacteriophage lysogeny.

Usually bacteriophages lyse their hosts following infection, however a few so-called "temperate" phage undergo lysogeny. In lysogeny, the bacteriophage integrates its genome into that of its host. The phage, then, is replicated each time the bacterial cell divides. In the lysogenic state, the bacteriophage can have considerable influence over host physiology.

Check out this page for the full details!

Photo from Viruses (Scientific American Library) by Arnold J. Levine

This Week's Citation Classic: Joshua Lederberg

Joshua Lederberg passed away last Saturday. He was truly a giant in microbiology. When I first joined the Department of Ecology and Evolutionary Biology at Yale, I visited the building on a weekend, and having no key, couldn't enter. As I walked around the building looking for an open entrance, I noticed a plaque near the front portal. I forget the exact wording, but in effect it says, "Here, in this building, Joshua Lederberg made his most important discovery, genetic exchange in bacteria, for which he received the Nobel Prize in Physiology and Medicine in 1958." (He was only 33!). With that, I felt great pride and amazement that I would work in the same building (possibly even the same lab!) as one of the greats in my field.

One of Lederberg's most cited papers is: Lederberg, Joshua, E. L. Tatum, "Gene recombination in E. coli", Nature 158 p. 558, October 19, 1946. It was his first published paper. It is very short and only takes up a quarter of a page in Nature.

Its findings, however, were revolutionary. Previously it was unequivocally assumed that bacteria reproduced asexually by binary fission and no genetic exchange or recombination occured. Lederberg and Tatum were able to find evidence that a wild-type bacterium E. coli K-12, lacking the ability to grow on certain media was able to acquire genes that permitted it to grow on that media, when reared with strains that possessed those abilities.

The most important effect of this discovery was that it established bacteria, K-12 in particular, as appropriate organisms to study genetics.

The experiment is described in greater detail on the National Library of Medicine website and I post the relevant section:

[Lederberg] took two of Tatum's double mutants of the K12 strain, each unable to synthesize two different nutrients (the vitamin biotin and the amino acid methionine in the first mutant, the amino acids threonine and proline in the second), to minimize the possibility of reversion to prototrophy, which in double mutants is extremely rare. When he crossed the two double mutants, Lederberg discovered that some of their progeny regained the ability to synthesize the two respective nutrients which previously had to be supplied in the broth for them to grow, and that this ability was inherited by succeeding generations. Such prototrophs were obtained only when the two mutants were mixed, not when single strains were incubated separately. Moreover, all individual bacteria within a colony of recombinants had the same genotype, or genetic constitution. Furthermore, from among the double mutants Lederberg isolated several that in addition to having the nutritional requirements described above were resistant to a bacteriophage, a bacterial virus, that infected E. coli. Resistance to bacteriophage T1 provided a second, so-called unselected genetic marker in this sub-group of nutritional mutants. When Lederberg tested prototrophs for T1 resistance, he found that some were resistant while others remained sensitive to the virus. Most importantly, he was able to demonstrate that the ratio of resistance to sensitivity depended on which parent carried the resistance marker, and that this ratio was reversed in reciprocal crosses (i.e. when instead of parent strain A, parent strain B carried this marker)."

I still use K-12 today, as well as a phage discovered by Lederberg's wife, Esther, phage Lambda.

Photo: Lederberg receiving the Nobel Prize.

Astronomy Picture of the Day?

This science nerd gets alerts from google when bacteriophages appear in the news. That's how I found out about the 21 April 2008 NASA Astronomy Picture of the Day. The short article is, for the most part, on the money. It's estimated that there are 10^31 phages on earth. That's more numerous than stars in the universe (~10^21). However, phage therapy is a bit more advanced than they let on. You can read more about phage therapy here.

Credit: Wikipedia