Small Things Considered

Talmudic Question #64

by Ramy Aziz

Are any human-associated biofilms "useful" or "beneficial" to human health?

A Giant Among Giants

A colorized SEM showing numerous particles
of the phycodnavirus PBCV-1 attached to a
chlorella NC64A host. Bar = 500 nm. Source.

by Merry

Without a doubt, Mimivirus is remarkable. For a virus, it is extraordinarily large and complex. But it is hardly one of a kind. The more that researchers look for large viruses, the more they find.

Although phages generally tend to have small genomes, some managing with but a handful of genes, a glance at the current NCBI list reveals that there are now eight with sequenced genomes that amount to more than 200 kb. A Pseudomonas phage tops the list with 317 kb, but the not-yet-sequenced genome of Bacteriophage G of Bacillus megaterium is reported to be ~670 kb.

EM of novel Bacillus thuringiensis phage from
soil—the first representative of a new group
of large myoviruses. Bar = 0.1 μm. Source.

Serwer and colleagues have pointed out that the procedures used to isolate phages are biased against the giants. Typical plaque assay protocols call for at least 0.3% agarose in the overlay. However large phages, such as Bacteriophage G, can't make plaques of significant size when the agarose concentration is 0.2% or higher. Using 0.15% agarose, these researchers isolated a novel Bacillus thuringiensis phage from soil—the first representative of a new group of large myoviruses. It too has a genome with more than 200 kb packaged inside a 95 nm capsid that sports a tail measuring half a micrometer!

Phycodnavirus OtV5, with a 122 nm capsid, infects Ostreococcus
tauri, the smallest known free-living photosynthetic organism, ~1
µm in diameter. TEM. (A) At high multiplicity of infection (moi),
many viruses can adsorb to a single cell. (B) Virus replication
results in the accumulation of viral particles in the cytoplasm
before cell lysis occurs. Bar = 500nm. Arrows = virus particles;
Chl = chloroplast; Cyt = cytoplasm, n = nucleus, m = mitochon-
drion, sg = starch grain. Source.

Most of the known giant viruses infect eukaryotes and are members of a monophyletic group known as the nucleocytoplasmic large DNA viruses or NCLDVs. They earned the "nucleocytoplasmic" label because they either replicate entirely in the cytoplasm or initiate the process in the nucleus and then complete it in the cytoplasm—thus independently of the host's transcriptional apparatus. Here you find the pox viruses of vertebrates and invertebrates, the phycodnaviruses (phyco- meaning algae) of marine and freshwater algae, and the amoeba-infecting Mimivirus. Phycodnaviruses of note include the coccolithovirus that plays a role in the termination of blooms of an abundant marine alga, the coccolithophore E. huxleyi, as well as a large virus that infects Ostreococcus tauri, the smallest known free-living photosynthetic eukaryote.

The phycodnaviruses are quite remarkable themselves. Their genome lengths are mostly in the 300 kb range, but one is 560 kb. The archetypal phycodnavirus that infects chlorella-like algae has ~373 protein coding genes—more than the number often touted as the "minimum" required to support life. However, gene numbers don’t tell the whole story as these viral genomes lack many of those listed in the "essential" gene set. In an unvirus-like manner, this genome also encodes 11 tRNAs and three kinds of introns plus genes for multiple DNA methyltransferases and DNA site-specific endonucleases—the enzymes that make up the restriction modification systems found in many Bacteria. These genes are functional; all chlorella viruses have methylated bases in their genomes, each virus with its own characteristic site-specificity. And most intriguing of all, this chlorella virus has the gene needed to synthesize hyaluron, and synthesize it it does, eventually covering its chlorella host with a dense fibrous network. Hyaluron synthesis had been thought to be an art exclusive to vertebrates (and a few pathogenic bacteria that include it in their capsules to fool our immune system). Even more bizarre, some chlorella viruses make chitin instead, and yet others make both and accumulate both on the surface of their host.

Infection of Chlorella NC64A by PBCV-1. (B) Attachment of PBCV-1 to the algal wall and initial digestion of the wall. (D) Complete digestion of the algal wall. (F) An empty viral capsid remaining on the surface of the host. Bar = 100 nm. Source.

Mimivirus has the phycodnaviruses beat by just about any yardstick you choose, and it even crosses that illusory line intended to separate viruses from cellular life. Its ~500 nm capsid is larger than the smaller bacterial cells such as Mycoplasma. Its 1.2 Mb genome contains 981 predicted protein-coding genes—double the number found in the smallest known Bacteria (Mycoplasma genitalium) and Archaea (Nanoarchaeon equitans). But a virus it is, firmly placed phylogenetically within the NCDLV group, albeit on its own branch.

Close-up view. Credit: Didier Raoult.
Source.

Mimivirus infecting an amoeba. The vrion has
been phagocytosed and resides within a vacuole.
The inner membrane of the virion (light circle)
will later fuse with the vacuole membrane to
discharge the virion contents into the cytoplasm.

We don't know what most of those 981 genes do as they lack identifiable homologs in the sequence databases, but at least 95% of them are transcribed during infection. Where did they all come from? Many appear to be paralogs produced by gene duplication events in Mimivirus. Of those with clear homologs, most are related to bacterial genes, a few to genes in Acanthamoeba and other protists. These likely came via horizontal gene transfer from a host, from other parasites present in the host, or from Bacteria phagocytized by the host for food.

Having a 1.2 Mb genome presents some challenges. One is simply synthesizing enough DNA for >300 progeny viruses in about 12 hours. In one experiment, researchers measured a 7-fold increase in total DNA within the host in the first 8 hours, so recycling of host DNA by viral endonucleases simply won’t suffice. Not surprisingly, Mimivirus (and other NCLDVs) encodes numerous enzymes for nucleotide metabolism and synthesis. Next comes the task of packaging those genomes into the preassembled capsids, a process that takes place in a cytoplasmic "virus factory." The unique "stargate" that opens upon infection to rapidly deliver the genome is a story in itself (click here and here).

A model of the complex virion of Mimivirus (cross
section viewed perpendicular to the unique five-fold
axis). From the outside in: head proteins (black) and
shafts (green) of the surface fibers that are attached
to the anchor proteins (blue spheres) that cover the
lattice forming the icosahedral capsid (red spheres).
Next, an additional protein/lipid layer (gold), uniden-
tified fibers (orange), and the bilayer lipid membrane
(green). Inside the membrane is the genomic DNA
(black) with associated proteins (green) and other
proteins (pink). Thick blue lines on the surface
represent the stargate. Source.

The protein capsid measures ~0.5 µm; the dense layer of reticulated polysaccharide fibers covering it surface increases the diameter to ~0.75 µm. It was the faint Gram-positive staining of those fibers combined with the virion size that earned Mimivirus its name: Microbe mimicking virus. Such large size may be necessary to efficiently infect amoebae and other protists via their feeding phagocytosis pathway. Studies using precisely-sized beads found that individual beads greater than about 0.6 µm are taken up immediately, whereas smaller ones accumulate on the cell surface until combined they reach sufficient volume to trigger uptake.

With such fascinating stories being told by Mimivirus and the other giants, people are now looking for them in more environments. Modified techniques are called for, as those used previously to spot viruses may have excluded many of them. For example, when collecting marine samples for viral metagenomes, researchers often use filters with 0.16-0.2 µm pores to catch the "microbial" fraction and allow the "viral" fraction to pass through. Realizing that many NCLDVs are apt to be caught with the microbes, Monier and colleagues searched the "microbial" sequences from the Sorcerer II Global Ocean Sampling (GOS) Expedition for NCLDVs using their conserved DNA polymerase sequences as a handle. They found Mimivirus sequences in 86% of the samples and chlorella viruses in a third.

Claverie and colleagues see no limitations that would preclude the existence of even larger viruses. Unlike cellular organisms, there are no metabolism-based constraints on particle volume. Of course, a virus must be smaller than its host, and Mimivirus is <1/30 of the size of its host amoeba. Bacteriophage G may be approaching the limits here: a 200 nm diameter phage infecting a 2 µm Bacillus. Given that Mimivirus can fit 1.2 Mb of DNA into its 0.5 µm diameter capsid, they surmise that a virus with a 10 Mb genome would be possible. It would require only a 1 µm capsid, a size easily accommodated by large amoeboid protists.

What do you think is the likelihood that Mimivirus will still be #1 giant five years from now? We'd bet not, as much of the virosphere is yet to be explored, and likely there is more than one researcher with dreams of discovering the next viral leviathan. Large protists that feed on bacteria would be the place to look.

Van Etten JL (2003). Unusual life style of giant chlorella viruses. Annual review of genetics, 37, 153-95 PMID: 14616059

Claverie JM, Ogata H, Audic S, Abergel C, Suhre K, & Fournier PE (2006). Mimivirus and the emerging concept of "giant" virus. Virus research, 117 (1), 133-44 PMID: 16469402

Hello Again, Metabolism!

by Amy Cheng Vollmer

Source.

Years ago, pathways of intermediary metabolism made up a significant portion of biochemistry and microbiology courses. Therein, students learned about interconversions and connections between pathways, and they could follow the carbons as they moved from acetate into the cholesterol molecule and many others. But the advent of exciting new methodologies—structural biology, recombinant DNA, molecular genetics, immunochemistry, probes, blots, microarrays, metagenomics, and more—crowded much of metabolism right off the syllabus. Given that teaching pathways could be dry and boring, faculty often elected to substitute more trendy and exciting topics instead. To be sure, they thought metabolism was important, but assumed ‘someone else must be teaching it.’

The result is a generation of well-trained scientists who can clone or crystallize just about anything and can harvest bushels of data from vast microarrays. But once those gene names are converted into enzymes, they are not so adept at mapping their enzymatic steps into a coherent and integrated system of pathways. In fact, a survey I instigated at an IMAGE (Integrating Metabolism and Genomics) meeting in 2004 showed that the vast majority of individuals trained after the 1970s knew little about the pathways of photosynthesis or of amino acid, purine, and pyrimidine biosynthesis, nor did they think that needed to be taught at the undergraduate level.

So we now have faculty (in the assistant and associate ranks) who admit to me that they would have a tough time teaching pathways effectively because they don’t know much beyond glycolysis and the Krebs cycle. Yet, in the past 5 years, I have found time and again that some of the most revealing presentations at the ASM meetings (and others) have shown that important signals in microbial processes are, in fact, small metabolites, and that the key enzymes are not specific to pathogenesis, development (e.g., sporulation) or differentiation (morphotypes), but rather they are the enzymes of the Krebs cycle or for key steps in nitrogen or phosphate metabolism. Imagine that!

In this era of metagenomics, metabolism has resurfaced dressed fashionably as metabolomics: the study of the universe of small molecule metabolites that characterize a biological sample, usually one containing many different species, e.g., the metabolome of the human gut or the cow’s rumen. Somehow the profiles of small molecules reveal important aspects of the health of such ecosystems. So now we find faculty and students scrambling to teach and learn about primary and secondary metabolic pathways so that they can find their way through the vast databases that are being assembled from structures, pathways, regulatory networks, etc.

Metabolism is enjoying a renaissance in our curricula and it is about time! There are vast secrets about biology to be revealed by the small molecules. Understanding how their levels rise and fall will take a careful study of the enzymatic pathways leading to and from them. So hello again, Metabolism! It’s so nice to have you back where you belong.

Amy is Professor of Biology, Swarthmore College, and President of the Waksman Foundation for Microbiology.

Microbiology in the Andes: Ancient and Unexpected

by Elio

The Ancient Gate of the Universidad de San Gregorio
Magno.

Thanks to the investigations by the Ecuadorian physician and scientist, Dr. Byron Núñez Freile, I learned of a surprisingly high level of scientific development that took place long ago in a remote region of the world. Quito, the present-day capital of Ecuador, is nestled amidst the high Andes and was the northern capital of the Inca empire. It was conquered by the Spaniards in 1534. In this exceedingly distant land, Jesuits established a college within a year of their coming in the late 16th century. By 1622, they founded one of the oldest universities in the Americas, the Universidad de San Gregorio Magno. This was earlier than the founding of Harvard, which happened in 1642. With the passing years, the two universities may not have enjoyed a parallel development, but early on they were likely of comparable quality. Soon, San Gregorio became a major institution, with a most impressive library of 16,000 volumes, the largest in South America at the time. In its first thirty years of existence, the university granted 160 masters degrees and 120 doctorates, mostly in philosophy and theology. Nevertheless, the library holdings also included numerous scientific and medical treatises.

Panorama of Quito. Source.

Quito can be reached by an easy flight these days, but in olden days getting to its lofty location (an altitude of 9,000 feet) required a week-long mule trek from the Pacific coast. Remote indeed! A major event of scientific relevance took place in 1736, when a geodetic French mission led by Charles-Marie La Condamine arrived, intent on measuring the circumference of the Earth at the equator. The French delegation interacted closely with members of the university, which resulted in a strong scientific legacy.

The scientific concerns of the times included the world we now call microbiology. No wonder. In 1589 a smallpox epidemic killed 37.5% of Quito’s inhabitants. A description of the disease in a letter by one of the priests makes clear allusion to its contagiousness. Later on, several of the Jesuits made insightful observations about the etiology of infectious diseases. Among them was Juan Magnin (1701-1753), a Swiss missionary who became a member of the French Academy of Sciences, who stated: There are microbes that can only be seen with a microscope that are 27 million times smaller than the smallest that can be seen with the naked eye. These facts and others seem incredible.

A microscope such as was available in Quito
in the 18th century, this one made ca. 1745
by John Cuff in London. Source.

And …(the microscope) allows to establish that the dirt on the teeth is due to the accumulation of innumerable microbes; furthermore, it is likely that many of the diseases of the human body, especially leprosy and venereal diseases, are due to the accumulation of microbes.

The other major microbiological issue of those times, the theory of spontaneous generation, became the concern of a native-born member of the faculty, Juan Bautista Aguirre (1725-1786). He wrote: I affirm…that the forms of animals, even insects, are not engendered by putrefactions but they arise from eggs or germs. He also stated: …with the aid of the microscope one discovers innumerable germs incredibly small in size, in the air, water, vinegar, blood, milk, etc. The most ingenious Leuvoiseck (sic) bore witness to having seen such small germs in a drop of water that 90,000 of them did not reach the size of a grain of wheat. What he lacked in spelling skills, he made up for by a good understanding of the literature!

Eugenio Espejo (1747-1795). Source.

There is more to this history. Soon after the Jesuits were expelled in 1767, the major Ecuadorian intellectual of that age, Eugenio Espejo (1747-1795), made important contributions to hygiene and the containment of smallpox. His words: Within the infinite variety of these living particles (“atomillos”) we have an admirable resource to explain the prodigious multitude of diseases and symptoms… Born of an Indian father and a mestizo mother, Espejo was a notable polymath, a true product of the Enlightenment. Not only was he the most notable physician of his time in Quito, he was also a lawyer, a philosopher, and the founder of Quito’s first newspaper. As a public figure, he laid the groundwork for the independence movement that eventually led to the liberation from Spain.

In the 17th and 18th centuries, Quito was a notable center of learning and discovery. Here, in splendid isolation, far from other universities and libraries, arose a sophisticated understanding of the world of microbes, both regarding their medical importance and their biological essence. This is nothing short of remarkable.

Quito at night. Source.

I confess to personal glee in this story. I spent my teen years in Quito and eventually became a student at the Universidad Central, the public institution that was built on the one founded by the Jesuits so long ago.

I am grateful to Dr. Núñez Freile for having brought this remarkable story to my attention. Dr. Núñez Freile is in charge of two highly informative blogs (in Spanish), one on infectious diseases here, the other on hand washing here.

Power of Ten

Tenfold Power. A gorilla can lift 10 times its own
weight, it is said. It can also sit wherever it wants.
Source.

by Elio & Stanley

How often have you heard it said, or seen it stated in writing, that we carry ten times more microbial cells than cells of our own? We don't dispute this figure, at least not as a ballpark estimate. But we were curious to find out where it came from. The paper that seems to be quoted most often in this regard is Microbial Ecology of the Gastrointestinal Tract by Dwayne Savage in the Annual Review of Microbiology, 1977, 31:107-33. Savage was an eminent intestinal microbiologist and those of us who knew him believe that he was a stickler for details. So he should have known. He stated: The various body surfaces and the gastrointestinal canals of humans may be colonized by as many as 10¹⁴ indigenous prokaryotic and eukaryotic microbial cells (70).

So now let’s go to reference # 70. It is to a paper by T.D. Luckey in the American Journal of Clinical Nutrition. 1970, (11) 1430-1432. Now we’re getting somewhere. Luckey writes: Assume one viable microbial mutation each 10⁸ cells, a microbial count of 10¹¹/g intestinal contents and 10³ g intestinal contents, then at a given time each of us harbors about 1 million newly mutated microbes (10¹¹ x 10³/10⁸ = 10⁶).

Just how authoritative is that? References to experimental work? None are given. Surely, the number of bacteria in feces must have been determined countless times through the years. A search of older literature is called for (but who’s got the time!). Moreover, is the number of human cells based on more solid ground? We have no idea.

Nowadays, a quantitative PCR using universal bacterial primers should give a reasonable estimate of the total intestinal bacterial DNA, a figure that can be converted with some assurance to the number of bacteria present. For good measure, add a few percent to account for eukaryotic microbes. Note, however, that we take leave from a good portion of our intestinal microbiome every day, the lucky among us with satisfying regularity. So, the ratio fluctuates daily. No big deal, but let’s qualify the ten-to-one mantra by saying “estimated.” To say the least.

Stanley Maloy is Dean of Sciences and Associate Director of the Center for Microbial Sciences at San Diego State University.

The Uncultured Bacteria

by Kim Lewis

Fig. 1. "The Great Plate Count Anomaly."

The majority of bacteria will not grow on nutrient medium in the lab. The basic experiment is simple: take a sample from the environment, such as marine sediment or soil, mix with water, vortex, allow it to settle, dilute supernatant and take two droplets. Plate one on a Petri dish with LB medium, and place the other on a microscope slide (adding a dye such as DAPI helps). Count the number of cells under the microscope and the number of colonies on the Petri dish – the result will be “The Great Plate Count Anomaly.” About 100 times more cells will be observed microscopically than colonies counted on the Petri dish (Fig. 1). This simple result is one of the most profound puzzles in microbiology, and one of the most significant unsolved questions in biology. Indeed, microorganisms probably make up the bulk of the total biodiversity of species on the planet, but we do not have access to the vast majority of them. Importantly, most Divisions—the largest taxonomic units—do not have a single cultivable representative, and we know of their existence only from 16S rDNA isolated directly from the environment.

There are two basic approaches to solving a problem, and one of them is to decide that it does not exist. It has been suggested on the pages of Nature and Microbe that the anomaly only exists in our minds and results from the insufficient number of microbiologists with a “green thumb” willing to tinker with growth conditions. However, the anomaly has existed for over 100 years, and tinkering by generations of scientists produced only minor improvements. A serious effort was mounted by several groups to culture representatives from the ubiquitous TM7 division, for example, but produced no results.

Fig. 2. Growing the uncultured in agar, between semi-permeable
membranes, in their original environment.

What I find most fascinating in the problem of uncultured bacteria, however, is not the existence of exotic strangers such as TM7, but the fact that many—if not most—“unculturable” organisms are close relatives of common garden-variety bacteria such as Bacillus subtilis or Pseudomonas aeruginosa. Judging by the genomes of their relatives, the uncultured organisms should grow on almost anything, yet they grow on nothing in the lab, including yeast extract which contains the entire metabolic map. They do grow in soil or marine sediment, so should be able to consume degradation products of plants and animals, which would be sugars and amino acids. The puzzle is truly perplexing.

Given the unsuccessful 100 year-old quest for a good medium, we gave up on the hope of recreating it in a Petri dish. Instead, we decided to grow the uncultured in their natural environment, where one cannot possibly fail! Enclosing a marine sediment sample mixed with agar between semi-permeable membranes and placing it back in its original environment then allows for free diffusion of compounds through the chamber (Fig. 2). The bacteria are tricked into perceiving this diffusion chamber as their natural habitat, and form colonies. Similar approaches of culturing in their natural environment resulted in cultivation of dominant pelagic Pelagibacter ubique by Steve Giovannoni’s group, and Prochlorococcus by Zinser and co-authors.

Fig. 3. A sample from marine sediment biofilm is inoculated on
a Petri dish with rich medium (left panel). Inoculating colonies
from this plate pairwise (right panel) allows to identify uncul-
tured organisms and their helpers. Material from one colony,
KLE1104 (close relative of cultivable M. polysiphoniae) was
spread evenly over the plate, while cells from another colony,
KLE1011 (related to M. luteus) were patched in a single spot.
KLE1104 colonies only form around KLE1011.

One useful clue to the nature of uncultivability came from our observation that some uncultured organisms will only grow in the presence of cultivable species from the same environment (Fig. 3). Using this as an assay, in collaboration with Jon Clardy’s group we discovered that growth factors for many uncultured species from biofilms enveloping marine sand grains are siderophores, chelators of insoluble Fe(III) (Fig. 4). Some species are fairly promiscuous in taking up siderophores, while others, such as a distant relative of Verrucomicrobia, will only grow in the presence of a siderophore from a particular neighbor.

Fig. 4. A culturable helper releases a siderophore that captures insoluble
Fe(III) and brings it into the cell. The same siderophore/Fe(III) complex
is captured by an uncultured bacterium which is unable to make its own
siderophores.

But why would bacteria lose the ability to make a factor necessary for capturing an essential nutrient, thus becoming dependent on their neighbors? This dependency, of course, leads to loss of liberty; uncultured species can no longer colonize new territory. Simple advantages of thievery are unlikely to provide a credible explanation. Indeed, siderophore piracy is well-known among bacteria, but the cultivable pirates retain their own siderophore operons and turn expression on when iron gets low. I think that the reason for the loss of siderophores is to prevent adaptive evolution in new environments. Any time a cell finds itself in new surroundings, it will evolve to increase its fitness. This newly evolved organism is unlikely to outcompete resident species that spent millions of years adapting to the same environment, and will die out. Going back to their original environment is not an option either; they are not the same now and will be less fit than their parents (Fig. 5).

Fig. 5. (Upper panel) Death by an evolutionary dead-end. A culturable
bacterium propagates in its familiar nich (yellow) and sheds off a cell
that travels to a new environment (grey) where it attempts to adapt
by evolving new features and losing some old ones. It is unable
however to compete with a resident species (purple), and is no longer
competitive with the parent strain. (Lower panel) An uncultured
organism depends on a growth factor from a neighboring species. If
a cell finds itself in an unfamiliar environment, it stops dividing and
waits for a ride back, where it resumes productive propagation.

This scenario has been described for our pathogens such as P. aeruginosa which looses a large number of genes, including virulence factors and proteases, while adapting to the environment of the lung of cystic fibrosis patients. The result of such runaway evolution is a dead end; the pathogen can neither infect new hosts nor return to soil, its other major habitat. This is probably the main fate of weeds like P. aeruginosa or E. coli—death by adaptive evolution. The other 99% of species choose where they live and grow. If they find themselves in an unfamiliar environment, then the best strategy is to go into dormancy and wait for a ride back home. This strategy is very similar to what we know about spores of cultivable species such as B. subtilis. Spores will germinate on any nutrient environment, but the probability of germination is greatly increased in the “correct” environment. Alanine (for reasons we do not understand) is a good germinator, and so are products of peptidoglycan hydrolysis from neighboring species. B. subtilis seems to be an intermediate between a true uncultured species and a weed like E. coli.

What is next? The siderophore story suggests that there are other growth factors to be discovered that indicate a familiar environment to the unadventurous uncultured. (Next year: the uncultured from the Human Microbiome.)

All figures courtesy of Kim Lewis.

Kim Lewis is Professor of Biology and Director of the Antimicrobial Discovery Center at Northeastern University.

D'Onofrio A, Crawford JM, Stewart EJ, Witt K, Gavrish E, Epstein S, Clardy J, & Lewis K (2010). Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chemistry & biology, 17 (3), 254-64 PMID: 20338517

Comments on the “Synthetic Cell”

Creation of Light, by Gustave Doré (1832-1883).
Source.

by Elio & Merry

The now famous announcement by the Venter group is based on their paper in Science entitled Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. We applaud this work for its impressive technical achievement and we acknowledge its future potential. However, we find the term “creation” to be misleading because the "new" cell was assembled using mainly preexisting constituents. We also find the term worrisome because, somewhat perversely, it may bring up creationism. We reprint a comment submitted by Bernie Strauss in response to the timely article published in Nature: Life After the Synthetic Cell. His words accurately express our sentiments.

Bernard S. Strauss wrote:

The first time I recall reading that biochemists had succeeded in synthesizing life was in 1967 when Arthur Kornberg and his colleagues succeeded in replicating a øX174 DNA template with E. coli polymerase I and ligase. According to the National Library of Medicine, U.S. President Lyndon Johnson looked at the Stanford press release and to Kornberg's dismay announced: "Some geniuses at Stanford University have created life in the test tube!" There was much discussion at the time including arguments similar to those published in the May 27th issue of Nature. The accomplishments are reasonably similar making allowance for the increase in technology over the past 40 years. A sequence of DNA has been replicated and then introduced into a cell where it takes over the functional apparatus to produce materials specified by the introduced DNA. This is certainly an important technical achievement and one with potential practical application. Does it tell us much about the origin or nature of life?

I would argue that the result is of little theoretical importance. The critical part of Kornberg's work 40 years ago (and one which he acknowledged) and of Venter's work this year is the necessity of a preformed cell. It is only this preformed cell that can convert the coded information in the DNA into functional products. In addition, that cell is not only a passive translator of genetic information but seems to include inherited information of its own, apart from the nucleic acid. As an example, membrane structures require preexisting structures as a template. It may be that at some date it will be possible to mix component membrane parts to form a structure into which all of the other cytoplasmic components can be added along with a synthetic DNA. So far however, all that has been done is to insert programming instructions into a completed machine. The basic unit of biology, and of life, remains the cell. Recent work emphasizes just how malleable the cell is but still requires the preexistence of this elementary life unit.

Anyone who has done science knows just how much work it takes to make any new finding. It is perhaps inevitable that having done all that work, one wants to make the most of it.

This brings to mind two earlier contributions to this blog by Franklin Harold (click here and here) where he discusses the essentialness of pre-existing membranes. It also prompts us to muse that every virus does something similar, with less expense and fanfare.

As always, we welcome your comments.

Bernard Strauss is Professor Emeritus, Department of Molecular Genetics and Cell Biology, The University of Chicago.

It's a Book!

by Merry

Well over two years ago, through this blog I had the opportunity to edit a research paper for Forest Rohwer. One thing led to another, and we then spent the better part of two years writing a book together—the first book for either of us. Coral Reefs in the Microbial Seas is now published! It is a relatively small book written for a broad audience. Minimal science background is required, even though we weave together concepts from many scientific disciplines as we discuss how coral reefs work and what might be ailing them today. And, of course, the microbes are front and center. There is a bit of metagenomics, some epidemiology, an ode to the microbial members of the coral holobiont, the effects of rising atmospheric CO₂ on the oceans, fish farming in the Gulf of Aqaba, and on and on, all spiced with playful stories from Forest's research expeditions and illustrations by Derek Vosten, sculptor and tattoo artist. Forest has been studying coral reefs for nearly a decade, and has ~25 coral-related research papers and book chapters to his credit.

To sample the book, visit our book page at Amazon and use "look inside this book." Or click here to download a book excerpt (1.5 Megs) that includes the Table of Contents, Preface, Introduction, two of the anecdotal stories, and more. You are welcome to distribute the excerpt to others, make it available on your blog, etc. And then please do let me know what you think.

The Sea Slug's Guide to Plastid Adoption

by Merry

A handsome E. chlorotica. Its rich green
pigmentation is courtesy of plastids captured
from its food source, the siphonaceous marine
alga Vaucheria litorea (seen in the background).
Source.

Having an intimate relationship with photosynthetic microbes is a widespread strategy adopted by numerous unicellular and multicellular organisms. Some eschew a committed relationship, and simply nab the plastids, sequestering them inside vacuoles where they continue to photosynthesize for a while. Previously we reported on a ciliate that captures the algal nuclei, as well, to support the plastids, and a flagellate that seems to in the process of converting their plastid into a well-mannered organelle. What about us metazoans? So far there is only one group known to practice this kleptoplasty—the sap-sucking sacoglossan sea slugs—and, so far, only one genus among them is known to have made this a long-term relationship.

The best-studied species here is Elysia chlorotica. These naked molluscs pass their quiet lives in salt marshes from Chesapeake Bay to Nova Scotia. Eggs laid each spring hatch into planktonic veliger larvae that home-in on the one particular species of algae that they use for food, Vaucheria litorea. The larvae attach to the algae where, within 24 hours, they metamorphose into hungry juvenile slugs that begin feeding. During winter months the slugs are inactive; with the arrival of spring and warmer water, the hermaphroditic adults lay their eggs and die soon thereafter.

The large cells of Vaucheria are coenocytic, each containing many nuclei and plastids. The slug slits open the cells with its radula (a rasp-like tongue), then slurps out the algal contents, plastids and all. (The movie below includes a stunning closeup view of a young slug's first meal.) The plastids are selectively taken up by specialized cells lining a portion of the gut and sequestered in vacuoles (by a mechanism as yet unknown). Here, the plastids keep on working. When given ¹⁴CO₂, the ¹⁴C is later found in various slug metabolites. Young slugs that have acquired their first plastids can complete their entire life cycle (~10 months) in aquaria without algae so long as they have light.

In these algae, more than 90% of the plastid proteins are encoded by nuclear genes. How do these algal plastids survive without support from the algal genome? This is an urgent matter as some of the proteins that make up the light-harvesting apparatus suffer ongoing damage and need to be replaced frequently. Researchers incubated algae and plastid-containing slugs with [³⁵S] methionine, then isolated their plastids. The plastids showed similar arrays of labeled proteins. Moreover, some of those plastid proteins synthesized in the slugs are synthesized on cytoplasmic ribosomes, not in the chloroplasts.

Subsequently, these researchers showed that the slug’s nuclear genome contains genes for at least three plastid proteins that are encoded in the algae’s nuclei. The slug also expresses several genes for the chlorophyll synthesis pathway and synthesizes chlorophyll a. These genes are present even in pre-hatched larvae that have never fed and do not contain plastids—strongly suggesting that the genes have been incorporated into the slug germline DNA and are vertically inherited. Another lab zeroed in on a gene from photosystem II (psbO) and found it in the DNA from E. chlorotica eggs—more evidence for germline transmission. Looks like this is an instance of horizontal gene transfer (HGT) between two eukaryotes, from the nuclear genome of the algae to that of the slug.

Portraits of E. chlorotica. (A) Free-swimming veliger larvae. The green coloring in the digestive gut is due to planktonic feeding, not plastid acquisition, at this stage. (B) The first meal of a metamorphosed juvenile. Plastid acquisition by feeding is required for continued development. (C) Young adult 5 days after first feeding. (D) Adult. (Bar = 100 µm in A, 500 µm in B-D). Source.

E. chlorotica is not the only "solar-powered" sea slug. E. clarki, found in the Florida Keys, maintains its plastids for up to 4 months. More accomodating than E. chlorotica, it acquires plastids from four different species of algae—sometimes housing plastids from two or more species in the same cell.

Mature icosahedral viruses with double envelopes budding
into cytoplasmic vacuoles in a hemocyte from a dying slug.
Bar = 0.5 µm. Source.

These stories leave us wondering just how this particular HGT might have occurred. It's a long way from the nucleus of an alga being digested in the gut of a sea slug to the nuclear DNA in the slug's germ cells. No definite answers yet, but some suspect that an endogenous retrovirus may have played a role. Evidence? First, a brief digression back to the slug life cycle. The entire adult population dies synchronously each spring. Slugs in lab aquaria die at the same time as those in the field, regardless of the time of year when they had been collected.(How’s that for timing?) TEM of dying slugs, but not of younger slugs, revealed viruses in their cells. The enveloped icosahedral virions resemble those of known retroviruses; most tellingly, their bloom is associated with a hundred-fold increase in reverse transcriptase activity. Smaller capsids are also seen in the plastids.

Are these 'retroviruses" killing the slugs? Some of the researchers don't think so. Many cells in the dying slugs show characteristics of apoptosis, suggesting programmed cell death. The viruses might maintain a latent infection and then reactivate when the slug's defenses have been compromised. Did these viruses have something to do with the algal→slug gene transfer? Maybe. Retroviruses are a feasible vector to ferry the algal genes to the slug genome. Would be nice to have the complete genome sequence for this virus so we could see if algal genes are on board. Meanwhile, it is early summer in the marshes and another generation of juvenile slugs have gone green.

Pierce, S., Curtis, N., & Schwartz, J. (2010). Chlorophyll a synthesis by an animal using transferred algal nuclear genes Symbiosis, 49 (3), 121-131 DOI: 10.1007/s13199-009-0044-8

Rumpho ME, Worful JM, Lee J, Kannan K, Tyler MS, Bhattacharya D, Moustafa A, & Manhart JR (2008). Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica. Proceedings of the National Academy of Sciences of the United States of America, 105 (46), 17867-71 PMID: 19004808

Of Ancient Curses, Microbes, and the ASM

It is our pleasure to begin an annual tradition of hosting a few reflections from the incoming president of the ASM.

by Bonnie L. Bassler

On July 1, as I start my term as ASM President, I am reminded of three ominous curses of dubious ancient origin:

1. May you live in interesting times.
2. May you come to the attention of those in authority.
3. May you find what you are seeking.

May you live in interesting times: Clearly, these times qualify. Microbes will be at the heart of solutions to our most pressing problems: the environment, food, energy, and health. The BP oil spill began on day -79 of my term. Microbes are coming to the rescue and ASM expertise is on the scene (see the earlier post in this blog). Let us hope that lessons have been learned. In his inauguration address, President Obama promised to “restore science to its rightful place.” It may be happening. The National Academy of Science’s Board on Life Sciences recently released their report: A New Biology for the Twenty First Century. The US Cabinet Secretaries of Energy and Agriculture requested a series of workshops to discuss how to implement the new biology. A first workshop, focused on food and fuel, was held in DC last month and I was invited to participate (day -27 of my term). Workshop members were asked to develop scientific challenges for the decade to be proposed to Congress for funding. Together with several other ASM members in attendance, I strongly advocated the understanding and appropriate use of microbes to synthesize new fuels, clean up the environment, optimize crop production, etc. Our rallying sound bite: Microbes: the world’s only unlimited renewable resource!

May you come to the attention of those in authority: On May 20, with quite some fanfare, Science published a manuscript: Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. On that same day (day -42 of my term) the ASM Public and Scientific Advisory Board (PSAB) released a position statement offering a balanced perspective on the manuscript, the status of the field of synthetic biology, and its regulation. A number of ASM members, including myself, gave expert opinions in newspapers, on the radio, and on TV. President Obama requested his Presidential Commission for the Study of Bioethical Issues to undertake a study of the implications of this scientific research and other advances that may lie ahead in this field. I will testify at the first Commission hearing on July 8 (day +8 of my term—at last we’re into positive numbers!). I feel well equipped to represent us. Last year I was the organizer and chair of the National Academies of Sciences Keck Futures Conference on Synthetic Biology. I continue to learn about new developments in the field, and I am expertly advised by our PSAB staff, our new PSAB Chair Roberto Kolter, and other knowledgeable ASM members. My specific role is to compare and contrast the engineering perspective with that of the biological and genetic sciences, and to explain how approaches represented by synthetic biology differ from other approaches to biological manipulation. I will also address what has been accomplished and what is likely to be accomplished in this field and what I think are important obstacles to the advancement of synthetic biology.

May you find what you are seeking: Over this past year (day -365 to day 0) as President Elect, I got to know the Society inside and out. I learned what remarkable accomplishments 40,000 volunteers can achieve. I saw our members donate huge blocks of time for the good of our discipline and the health of our planet. I became fully convinced that collectively we have the potential and the expertise to make the world healthier, to enable a sustainable relationship with our environment, and to ensure the promise and prominence of science and technology in our culture. It has been a magical and eye-opening year. I am beginning to understand the vast breadth and depth of this organization and the position of its members in leading the nation and the world in all in matters touched by microbes.

There’s nothing like a few ominous curses to get the blood flowing. I am eagerly looking forward to this coming year with the hope that I can make significant contributions to the ASM and to our community at large. I will do my best to further enhance our reputation in public policy, education, outreach, and scientific advancement. Now at day +1 and counting, I look forward to meeting you!

Bonnie Bassler is the Squibb Professor of Molecular Biology at Princeton University and a Howard Hughes Medical Institute Investigator. She has been a member of the ASM for over 25 years. She is also a fellow of the American Academy of Microbiology and a member of both the National Academy of Sciences and the American Academy of Arts and Sciences. Her lab studies quorum sensing as a mechanism of chemical communication among bacteria.

A Fastidious Bacteriophage

by Michael Yarmolinsky

Patrons of upscale seafood restaurants are given the opportunity to see that the unfortunate creatures destined for the lobster pot are waving their antennae about. Savvy customers at downscale seafood markets evaluate questionable claims of freshness by smell. A fastidious bacteriophage would welcome the opportunity to gauge the quality of a potential meal, if only it could make that assessment. I was recently reminded, in the course of disposing of old reprints, that a bacteriophage named Chi can do so. It attacks only motile strains of bacteria, and then only if the flagella are active. How is this circumspect appraisal accomplished?

Phage Chi X 220,000. Source.

It has been several years since the publication of convincing support for a mechanical model that can account for the discrimination exhibited by Chi. Why revisit it now? First, for the benefit of those who have overlooked the remarkable story of Chi’s fastidious behavior and second because I suspect that we have learned only the half of it.

What sort of phage is Chi?

Chi is a virulent, double-stranded DNA phage with a long, uneven history and a long, tapered tail ending in a single, long, kinky tail fiber. It was first characterized in 1936 in the laboratory of Félix d’Herelle, the adventurer-scientist who is jointly credited with the discovery of the viruses he designated “bacteriophages.” Chi’s genome (60 kb) has recently been sequenced (Andrew Kropinski, Kelly Hughes and Roger Hendrix, in preparation).

The initial report showed that Chi attacks only flagellated bacteria. Growth of sensitive strains on agar containing phenol, at a concentration known to prevent the development of flagella, rendered the bacteria Chi-resistant. Chi-resistance could be used to select mutants defective in flagellation. Mutants altered in several of the 44 known flagellar genes of Salmonella were subsequently selected in this way.

The flagellar organelle. Source.

What sort of organelle is a flagellum?

Flagella are long, thin, helical filaments commonly much longer than the bacterial body from which they emerge at various sites. Their somewhat flexible, sinusoidal appearance was interpreted, until the early 70s, as evidence that they act like whips, but their helicity is intrinsic and their action that of a propeller rotated by a motor embedded in the cell body. Normal rotation speeds for Escherichia coli flagella are around 6000 rpm, but a record speed, set by a vigorous Vibrio, is 100,000 rpm. Each flagellum is both a reversible motor organelle and a protein export and assembly apparatus that fabricates the external filament by extruding flagellin monomers through a central channel and adding them to the growing flagellum at its very tip.

Talmudic Question #63

by Stan Zahler

When you streak out Your Favorite Bacterium on an appropriate agar plate, the colonies reach an expected diameter and stop growing. Why?

Corollary: If you inoculate YFB in the center of an agar plate and incubate it, it would reach its expected diameter. What do you predict would happen if you touch the tip of a sterile hair to the edge of the colony, draw it out on the agar radially, and re-incubate the plate?