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.

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

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.

This Week's Citation Classic

This week's citation classic is A.S Sarabhai, A. O. W. Stretton, S. Brenner and A. Bolle. 1964. Colinearity of the gene with the polypeptide chain. Nature 4914:13-17.

Following Crick and Watson's big breakthrough, the biology world sparkled with new hypotheses regarding the nature of the gene and the genetic code. The DNA molecule's structure implied a number of these hypotheses, but without empirical confirmation, they were nothing but speculation. One of these hypotheses was that the linear sequence of bases in a DNA strand coded for a complementary linear sequence of amino acids constituting the protein product of that gene. While this is the most obvious and parsimonious hypothesis, one need not think hard to imagine other possibilities.

Sidney Brenner was not reluctant to give his students difficult thesis problems. His graduate student, Anand Sarabhai was given the task of demonstrating colinearity of gene and polypeptide chain. For a graduate student to be given such a fundamental, but risky, problem is quite exceptional. Luckily Sarabhai was up to the task. He obtained phage T4 nonsense mutants from Dick Epstein of Geneva. Epstein called his mutants amber mutants, after the mother of Harris Bernstein, a Caltech grad student. German speakers will identify the connection; bernstein is the German word for Amber.

Sarabhai writes: "These mutants (it was believed) did not make a full polypeptide in a normal cell but did so in a suppressor-positive cell. What was not known was whether the amber mutations kept terminating and releasing the synthesized peptide or simply got jammed at the amber site. I told Dick that I could test this in Cambridge quickly. What I found was that the amber mutants kept terminating and releasing the polypeptide, so that you got large amount of fragments of polypeptide of lengths dictated by the position of the amber mutations in the gene. This broke open the co-linearity problem." J. Biosci. 2003, Vol. 28, p. 668.

Analysis of the broken fragments allowed Sarabhai to define 8 segments of the polypeptide chain that are in the same order as the segments on a defined genetic map. Thus Sarabhai, a graduate student, made a fundamental contribution to biology. DNA sequence = amino acid sequence.

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.

This Week's Citation Classics: Host Induced Variation

Today epigenetics is all the rage, but it has its roots in a pair of papers appeared nearly simultaneously in 1952-1953.

Luria SE and Human ML. 1952. A nonhereditary, host-induced variation of bacterial viruses. J. Bact. 64: 557-569.

Bertani G and Weigle JJ. 1953. Host controlled variation in bacterial viruses. J. Bact. 65: 113-121.

Luria & Human and Bertani & Weigle independently discovered that bacterial hosts can affect the growth and phenotypic properties of their bacteriophages.

As Luria and Human put it, "In analyzing the relation between certain phages and certain mutants of their bacterial hosts, we have encountered a novel situation: the genotype of the host in which a virus reproduces affects the phenotype of the new virus. The phenotypic change suppresses the ability of the virus to reproduce in certain hosts but not in others....

Several B/4 mutants of Escherichia coli, strain B, when infected with phages T2 or T6, liberate these phages in a form designated as T*, which does not multiply in young cells of strain B or of its mutants. T* can multiply in a small proportion of old, starved cells of strain B, giving rise to a yield of the corresponding normal T phage."

This finding was quite puzzling at the time, especially since it appeared to subvert traditional Mendelian genetics. Later it was discovered that a number of different mechanisms were responsible for host-induced modification including DNA methylation, restriction modification, and glucosylation. Werner Arber, Daniel Nathans and Ham Smith eventually shared a Nobel prize for their discoveries relating to restriction modification.

When I was working in the Turner Lab at Yale, I noticed that some of my phages grew better on a novel strain when they were previously grown on native strain than when they were previously grown on a novel strain.
I found this phenomenon was kind of interesting and thought I might have been the first to discover this. I called it a "maternal effect". Naturally it was with considerable chagrin when I found out that Luria, Bertani, Weigle and Human had discoved this over 50 years before. On the other hand, I found it neat that I rediscoved something that those giants of microbiology had discovered. And I could console myself with the I was the first to find host-induced modification among RNA phages (maybe).

One more aspect of this discovery that deserves mention is that it highlights the congeniality of the phage group. Not only did each of these authors acknowledge the competing group in their citations, but Bertani and Luria even came up with a media recipe together. Today we call it LB broth.

Figure: Epigenetic Mechanisms Nature 441, 143-145 (11 May 2006)

This Week's Citation Classic: The Fluctuation Test

Luria S. and Delbruck M. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 8: 491.

This week's citation classic comes from Nobelists Salva Luria and Max Delbruck and is one of the most famous experiments in biology. Luria and Delbruck wondered about the nature of mutations. Are mutations spontaneous? Or do they occur in response to environmental conditions? The latter view, common to scientists of the day (e.g. Cyril Hinshelwood), was one of the last vestiges of Lamarckism in evolutionary biology.

Since the time of d'Herelle, it was known that a culture of bacteria exposed to bacteriophage would eventually become clear, as if all the bacteria in the culture were killed. However, eventually the culture would grow cloudy again. It was surmised that the bacteria acquired resistance to the phage, and were able to repopulate the culture. The question was, how can the system be used to demostrate the role of chance in mutations?

Luria struggled with the problem for several months, trying to devise a test to show that mutations were spontaneous. Then at a faculty dance at Indiana University, Luria had his eureka moment.

"During a pause in the music I found myself standing near a slot machine, watching a colleague putting dimes into it. Though losing most of the time he occasionally got a return. Not a gambler myself, I was teasing him about his inevitable losses, when he suddenly hit a jackpot... gave me a dirty look at walked away. Right then I began giving some thought to the actual numerology of slot machines; in doing so, it dawned on me that slot machines and bacterial mutations have something to teach each other." (From Luria's autobiography: A Slot Machine, A Broken Test Tube).

Luria returned to the lab and set up a large number of bacterial cultures containing just a small number of bacteria in each, to which he added some bacteriophage. Luria reasoned that if mutations were spontaneous, then their distribution would resemble jackpots. Here the number of surviving bacteria would be small in most cultures, but large in a handful. On the other hand, if mutations were directed as the Lamarckists supposed, then their payoffs would be evenly distributed. Each culture would contain a small number of mutants, as the figure here shows:

Luria and Delbruck's experiments showed unequivocally that mutations were spontaneous and emphasized the role of chance and historicity in evolutionary biology, thus putting the final nail in the coffin of Lamarckism. See Fig. 2 from L&D's paper where the number of jackpots (>9 resistant bacteria) far exceeds that expected by chance. The reason I selected the Fluctuation Test as this week's citation classic is because of a recent exchange between Rich Lenski and I, of which I reprint portions of here:

"I've always been fascinated by the tension between chance and necessity, between randomness and repeatability. As a kid, for example, I especially liked games with dice and cards that involved both luck and skill.

Then, when I was at Oberlin College, I took a wonderful course in which we used Gunther Stent's "Molecular Genetics: an Introductory Narrative" as a text. Unlike most science textbooks, it emphasized the history of who did what experiments and why. I remember reading about the "fluctuation test" performed by Salvador Luria and Max Delbruck, and trying to make sense of it, and then having that eureka moment when the whole point of the experiment hit me. It's my all-time favorite experiment and to this day, whenever I think about it, I'm struck not only by its elegance, but also by the subtlety of the interpretation and by an appreciation of why the problem had been so difficult until they did their experiment.

As you know, a main focus of the long-term evolution experiment with E. coli has been to better understand the repeatability of evolution that arises from the tension between random mutation, on the one hand, and the systematic process of natural selection, on the other hand, that pushes populations toward greater fitness in the environments in which they live. So in a way, you might think of my long-term evolution experiment as a descendant of the fluctuation test, one that examines the role of random mutation in producing statistically quantifiable variation between replicate lineages, not in overnight cultures but across, now, more than 40,000 generations of evolution."

It is precisely this randomness of evolution that led to Lenski's and colleagues latest discovery that, after 33127 generations, a strain of E. coli evolved the ability to digest citrate. Carl Zimmer does a bang up job of covering that story.

Lenski also had a recent dustup with the IDiots, and his tolerant response is covered here.

Update:P Jonathan Eisen of Tree of Life also wrote about this paper here.

Photo: Max Delbruck, Salvador Luria, and Frank Exner at Cold Spring Harbor Laboratory from the National Library of Medicine.

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 Clyde A. Hutchison, III, Sandra Phillips, Marshall H. Edge Shirley Gillam, Patricia Jahnke, and Michael Smith. Mutagenesis at a Specific Position in a DNA Sequence. The Journal of Biological Chemistry, 253: 6551-6560.

Nobel Laureate Michael Smith doesn't get enough recognition, and I attribute it to his relatively conventional name. His 1993 co-laureate Kary Mullis seems to get much more attention, perhaps because of his more unusual name*, but the technique that Smith developed, in my opinion, ranks with the PCR in terms of utility. This technique is site-directed mutagenesis (SDM).

Back in the old days, if you wanted to change the genotype of your study organism, you needed to bombard it with chemicals or radiation, then screen the mutants, hoping you might chance upon the right mutation. Obviously this technique is kind of inefficient. Joshua Lederberg once commented, "“The ignis futuus of Genetics has been the specific mutagen, the reagent that would penetrate to a given gene, recognize it, and modify it in a specific way. ”

SDM is this ignis futuus. Where radiation works like a sledgehammer, SDM works like a laser. One can target specific nucleotides for mutation into whatever change is desired.

SDM relies on constructing DNA oligionucleotides that are identical for the sequence of interest, but contain the mutation of interest.

Using the bacteriophage X174, the study's lead author Clyde Hutchinson "teamed up with Smith, and the pair realized that an obvious route to a mutagenic method was to use a mutant oligonucleotide primer for E. coli DNA polymerase I on a circular single strand template, which would produce a product that could be converted to a closed circular duplex by enzymatic ligation."

Today SDM is coupled with the PCR, and is used widely to study gene regulatory elements, DNA-protein interaction, and protein structure/function are all typical targets for mutagenesis studies, and in my own lab to study life history variation among phage lambda.

A Journal of Biological Chemistry article describing the discovery is available here.

Any connection between a recent Sandwalk post, the fact that Smith is Canadian and that this article is biochemical in bent is purely coincidental.

*I suppose there are other hypotheses, but let's be kind.

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

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.

Spora and Gaia: How Microbes Fly with Their Clouds

WD Hamilton is my favorite scientist of the 20th century. He may not have had the largest impact, nor may he have had the most awards, but his relentless creativity is an inspiration to us all. This week's citation classic is an example of his wide ranging interests.

WD Hamilton and TM Lenton. 1998. Spora and Gaia: How Microbes Fly with Their Clouds. Ethology, Ecology and Evolution 10:1-16.

In the paper, Hamilton and Lenton hypothesize that some microbes use chemical induction of water condensation (ice nuclei) to enhance their own dispersal between habitats. The bacteria are supposed to create ice nuclei by releasing a gas called dimethyl sulfide (DMS). But this is an example of a cheater-prone trait. Non-producers can reap the benefits of DMS production without paying the energetic costs. So why are microbes producing DMS?
The idea that algae might produce DMS to get themselves into the air occurred to Hamilton first. 'Tim had mentioned that DMSP has a possible function as an antifreeze,' he recalls. 'Now why would a cell in a tropical ocean need antifreeze? Perhaps they sometimes end up high in the air, shot up there by a waterspout. Or maybe there are other ways they could go. Convective energy created by cloud formation would help them.' Flying high, the algae would be exposed to very low temperatures. Idle speculation rapidly led to the formation of a theory that beautifully explains why algae produce DMS. 'Seldom have I had a run of reading where so many papers were relevant or connected and nothing contradicted my ideas," says Hamilton. "I felt certain that there was something interesting here.'

As the Hamilton and Lenton write, "Dispersal is extremely important to life, indeed for self or progeny, it can be considered an organism's third priority after survival and reproduction". Thus microbes may be creating wind, clouds, rain, and snow to carry them around the globe. A great synopsis of the article appeared in the New Scientist, when the paper was first published.

When I first heard about these ideas, I thought them sort of fuzzy, cute and entirely without substance.

But some recent reports are suggesting otherwise. An article by Brent Christner et al. in today's issue of Science shows that ice nucleators are microbial in origin.

"Despite the integral role of ice nucleators (IN) in atmospheric processes leading to precipitation, their sources and distributions have not been well established. We examined IN in snowfall from mid- and high-latitude locations and found that the most active were biological in origin. Of the IN larger than 0.2 micrometer that were active at temperatures warmer than -7C, 69 to 100% were biological, and a substantial fraction were bacteria. Our results indicate that the biosphere is a source of highly active IN and suggest that these biological particles may affect the precipitation cycle and/or their own precipitation during atmospheric transport."

One of the coolest aspects (to me) is that one of the organisms responsible may be Pseudomonas syringae, an organism I use in my own lab. Although it remains unclear which microbes may be most responsible for snowfall or rainstorms, one leading candidate is the plant pathogen Pseudomonas syringae, which infects wheat, corn and other crops. It is a major pest—and the target of genetic modification—because it causes immediate crop damage if the temperatures drop below freezing.

The reason I find this interesting is that I study P. syringae's phage: the cystoviridae. A recent report found that "phages isolated from single clovers were not consistently more similar to each other than to phages isolated from sites across the country [i.e. opposite coasts] or from other previously isolated phages... These data are thus consistent with frequent continent-wide migration in the Cystoviridae."

Olivia Judson writes about Life and Clouds.
A synopsis of Christner et al.'s work is available at Science Daily.
Photo from Nicholas T

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.

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.

This Week's Citation Classic

Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71-94.

Sidney Brenner is one of the founding fathers of molecular biology, having identified mRNA and the nature of the triplet code. Brenner, like other far-sighted scientists, felt that mo-bio was pretty played out by the late '60s and early '70s. Benzer switched to fruit fly behavior. Crick decided to study consciousness. Brenner decided to focus on behavior and development. In his Nobel speech, Brenner stated, "choosing the right organism for one’s research is as important as finding the right problems to work on."

Brenner certainly benefited from bacteriophage, the organism of choice for many molecular biologists, but he was drawn in that direction by Delbruck and the Phage Group. This time, now a mature scientist in his own right, Brenner had a chance to select an organism of his own. He chose wisely: Caenorhbabditis elegans. C. elegans is an ~1mm long bacteriophagous soil nematode. Large populations can be maintained in the laboratory on bacteria cultured on agar, and they have the added advantage of being almost entirely transparent (a huge benefit for neuronal studies).

Brenner's classic 1974 paper asked “How genes might specify the complex structures found in higher organisms?", a major problem in biology today as well as then. Brenner's approach was to link genetics with detailed studies at the cellular level, and introduced C. elegans as the worm of choice for this work.

As Brenner wrote, “Behaviour is the result of a complex ill-understood set of computations performed by nervous systems and it seems essential to decompose the question into two: one concerned with the question of the genetic specification of nervous systems and the other with the way nervous systems work to produce behaviour.”

Thus Brenner launched a two-pronged effort to map the genetics and complete structure of the worm. This effort succeeded marvelously. Today we have unprecedented knowledge of this humble beastie. The developmental fate of each of the worm's 959 cells has been determined. The patterns of connectivity for each of its 302 neurons has been completely mapped. Its entire genome has been sequenced (the first multicellular eukaryote to be sequenced).

Brenner's choice of study organism was enormously influential. I don't think there are many full-fledged biology departments around the country that don't have at least one "worm person". Brenner has produced an amazing legacy.

Some last thoughts...
I am still, at the age of 76, excited by scientific research and the prospect of what can be done in biology. Science is something one is tied to for life and one should never retire from anything until one has secured one's next job. The endless quest for knowledge will continue as long as humans exist.

Brenner won the Nobel Prize in 2002 for his contributions; his speech was wonderful and can be accessed here (video and text). Brenner wrote an autobiography here. Visit the Brenner lab here. The Worm Nation maintains a web presence here.

Image: C. elegans neurons expressing green fluorescent protein.

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

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.

I'm #1 on Google!

From David Ng: I'd like to suggest a meme, where the premise is that you will attempt to find 5 statements, which if you were to type into google (preferably google.com, but we'll take the other country specific ones if need be), you'll find that you are returned with your blog as the number one hit.

Looks like I've cornered the market in all things Evilutionary.
Was I the first to ask "What has phage done for you?"
How is it that I'm not the number one John Dennehy?
Admittedly bacteriophage art is kind of an exotic interest.
Is there anything bacteriophages cannot do? I dunno. You tell me.

Hat tip Larry Moran