Small Things Considered

It Was The Worst of Times, It Was the Best of Times

2009-11-05T09:02:55-08:00

by Elio

At the end of the Permian period, about 250 million years ago and not long before the dinosaurs appeared, life on Earth experienced its greatest catastrophe: a mass extinction that did away with the vast majority of life forms on land and sea. The question arises, who ate the carcasses of the deceased? Surely bacteria and protozoa digested the animal corpses, most likely reaching unusually high population sizes as the result. But who took care of the masses of dead plant material? Fungi are quite good at digesting plants, especially woody plants. So, did the fungi also become prevalent after the catastrophe?

There is evidence for this belief. As the Permian period came to an end, there was a massive spike in the population of Reduviasporonites, a "morphogenus" of microscopic organisms fossilized as single cells or multicellular chains. (The -ites ending is sometimes used to denote organisms known only as fossils. Reduvius, if you care for such things, comes from the Latin reduvia meaning hangnail or remnant. Just why it was called that, I don't know.) Apparently they thrived at that time, as judged by the abundance and the widespread geographic distribution of their fossils. More recently, they have had a checkered taxonomic career. Originally thought of as fungi, they were later relegated to the microscopic algae. It makes a difference. If wood-decaying fungi, they suggest a massive loss of standing biomass on land. If algae, picture widespread swamping. Now, they are back among the fungi. Maybe.

Chains of fossil Reduviasporonites.
Bar = 40 µm. Source.

Conidiophores and conidia of Cladosporium
oxysporum. Source: Dr. Fungus Library.

The evidence? A new paper in Geology is one of the latest to deal with this question. They used "high-sensitivity equipment, partly designed to detect interstellar grains in meteorites." Their pyrolysis GC-MS of fossil Reduviasporonites showed that they do not contain the algal biomarkers, such as breakdown products of chlorophyll—a strike against the algal theory. In addition, these researchers found the ratio of N¹⁵ to N¹⁴ in the material to be characteristic of that seen in fungi, and distinct from that for algae. Another point for the fungal side.

What do these "disaster species" look like? They are usually seen as chains of ovoid cells, each around 25 µm in diameter. Many fungi on today's Earth look quite similar. If Reduviasporonites were indeed fungi, much of the planet must have been covered by a mushy mess of mycelia. For the time-traveler, it would have been the worst of times.

News Flash!

2009-11-03T20:46:24-08:00

by Elio

Jefferson Middle School. Source.

The average age of the readers of this blog has just decreased considerably. Small Things Considered is included in the list of educational sites provided to the youngsters in Ms. Putnam's sixth grade science class at Jefferson Middle School in Columbia, Missouri.

And we had thought that we presented advanced microbiological material!

Five Questions About Microsporidia

2009-11-02T09:19:47-08:00

Preface by Elio

Bacteria, archaea, microfungi, and many protists are "mainstream" in the sense that everyone in our business is aware of them. Other highly consequential microbes such as the microsporidia and the oomycetes commonly elicit reactions such as "I really should look into them sometime." I have been a microbiologist for upward of sixty years but have paid only the scantest attention to these large and important groups of organisms (and therefore hang my head in shame). But it's never too late to correct such oversights. We are starting out by reproducing, with permission of the author, a particularly illuminating article on the microsporidia by Patrick Keeling.

by Patrick Keeling

What Are Microsporidia?

Microsporidia are a diverse group of obligate intracellular eukaryotic parasites. There are approximately 1,300 formally described species in 160 genera [1], but this certainly represents a tiny fraction of the real diversity because most potential host lineages have been poorly surveyed. Nearly all microsporidia are known to infect animals, and some are responsible for a number of human diseases (13 species of microsporidia have been documented to infect humans) predominantly associated with immune suppression [2]. They also infect several commercially important animal species such as bees, silk worms, and salmon, and various domesticated mammals. They are thought to be especially common in insects and fish, although most invertebrates have been so poorly surveyed this may change.

Figure 1. Light micrograph of Antonospora locustae with pressure-
induced polar tube eversion. The scale bar is 10 µm. (A) Many
ungerminated spores (one example labeled U) and a few germinated
spores, showing the residual spore wall (one example labeled G).
(B) A germinated spore where the everted polar tube (PT) has
extended far from the cell and can be seen to be many times the
length of the spore.

Their infective stage is a thick-walled spore, which is also the only stage that can survive outside their host cell [3]. The spore contains a sophisticated infection apparatus, primarily distinguished by a long, coiled filament called the polar filament. When the spore germinates, an inflow of water leads to pressure in the spore that eventually ruptures the wall and forces the polar filament to eject, turning inside out to form a tube (Figure 1) [4]. This process takes place very quickly, so the polar tube is in effect a projectile. At the completion of germination, the parasite cytoplasm is forced through the tube and either delivered to the surface of the host cell, or perhaps injected into the host cytoplasm if the projectile tube has actually penetrated the host cell. It has also been shown that microsporidia can be taken up by phagocytosis, and then use the polar tube to escape from the vacuole [5], so there appear to be more than one mode of infection.

Are They Protists, Fungi, or What?

There has been considerable debate about the origin of microsporidian parasites. Aside from their elaborate infection mechanism, they have few distinguishing features, and have thus been difficult to compare to other eukaryotes. This is illustrated by their tumultuous taxonomic history (Figure 2), and the tendency to lump them with what we now know to be unrelated organisms. When microsporidia were discovered in 1857, they were considered to be schizomycete fungi, but this was an artificial group that included various yeasts and bacteria. They were soon transferred to Sporozoa and ultimately to the subgroup Cnidosporidia. This too was a grab-bag of four unrelated groups (Microsporidia, Myxosporidia, Actinosporidia, and Helicosporidia) that were falsely grouped because of their intracellular parasitic way of life. Remarkably, Microsporidia, Myxosporidia, and Helicosporidia have since been shown to be fungi, animals, and green algae, respectively, underscoring just how distantly related these parasites really are.

Figure 2. Timeline of the changing taxonomic position of microsporidia, from their discovery in 1857 to the present. When first described in 1857, they were classified as schizomycete fungi. Later they were consisdered sporozoan protists (and more specifically members of the subgroup Cnidosporidia), a position favoured for over 100 years. In 1983 a new hypothesis radically departed from this idea, suggesting they were an ancient, primitive lineage that evolved before the origin of mitochondria. Molecular data originally supported this possibility, but as data accumulated, it became clear that they were in reality highly reduced fungi, a conclusion broadly supported by the genomic data now available, although their exact relationship to fungi remains contentious.

Eventually the absence of many “eukaryotic” features in microsporidia, in particular mitochondria, led to the proposal that microsporidia never had these features because they diverged from other eukaryotes prior to the origin of these features [6]. Microsporidia and a few other lineages without obvious mitochondria were collectively called “Archezoa” and were thus proposed to be ancient, primitive lineages of great importance to understanding the origin of eukaryotes. Molecular data originally supported this “ancient origin” hypothesis [7], but as the sampling of genes increased, another hypothesis emerged: that microsporidia are related to fungi [8],[9]. Since the 1990s, most well-supported gene trees have shown this fungal connection [10], and the support for trees that showed the deep-branching position has been undermined by analysis with more sophisticated models that take into account rate heterogeneity [11],[12]. The completion of the Encephalitozoon cuniculi genome underlined this transition in our thinking [13], and also revealed the correlation between substitution rate and “deep-branching” [14]. The conclusion that microsporidia are fungi has one unwanted implication: all taxa that were named based on the view that they are protists (about 1,000 names) are invalid because fungi are subject to botanical rules of nomenclature. Accordingly, it has now been proposed that microsporidia are excluded from the International Code of Botanical Nomenclature, despite their fungal nature [15]. Our view of the origin of microsporidia has thus come full circle, in a way. Their original classification as fungi was actually based on a misguided view of microbial diversity, but we have nonetheless returned to the view that microsporidia are fungi, although where they might branch in relation to the various fungal phyla has remained a source of debate [16].

Are They Really “Amitochondriate”?

The Archezoa hypothesis (that microsporidia were an ancient, primitive lineage) was based on the absence of mitochondria in microscopy studies [6]. However, the phylogenetic evidence that microsporidia are closely related to fungi made it impossible for them to have been ancestrally amitochondriate, thus begging the following question: do they still have mitochondria, or did they lose them? The first evidence for mitochondrial relicts came in the form of nuclear-encoded genes for mitochondrion-targeted proteins. The first of these to be found was HSP70, followed by pyruvate dehydrogenase [17]–[20], and eventually a handful of other genes in the complete genome of E. cuniculi [13]. These confirmed the mitochondriate ancestry of the microsporidia, but left some room for doubt about whether a relict organelle actually persisted because the evidence that these genes encoded organelle-targeting transit peptides was far from clear-cut. Because of this, even the complete genome of E. cuniculi could only provide indirect support for the presence of an organelle in the cell.

Direct evidence for the retention of mitochondria came from the immuno-localisation of HSP70 in Trachipleistophora hominis, which reveled multiple, tiny (50×90 nm) organelles bounded by two membranes, but lacking any other distinguishing structural features [21]. This derived and reduced mitochondrion was named a mitosome. Subsequently, several genes involved in the assembly of iron-sulfur clusters were localized to the mitosomes of E. cuniculi and T. hominis [22],[23]. Interestingly, however, not all mitochondrion-derived proteins still function in the mitosome: the E. cuniculi, glycerol-3-phosphate dehydrogenase, and some components of iron-sulfur cluster assembly in T. hominis localize to the cytosol, despite their mitochondrial ancestry [22],[23]. These proteins have therefore found a new cytosolic function as the organelle degenerated, suggesting the functions of the organelle are even more limited than the genomic data led us to believe.

What Are Microsporidian Genomes Like?

Microsporidian genomes are made up of multiple linear chromosomes much like that of other eukaryotes, but they are otherwise quite reduced and unusual. For a start, many microsporidian genomes are quite small. At the extreme, the Encephalitozoon intestinalis genome is only 2.3 Mbp, smaller than many bacterial genomes. The complete genome of E. cuniculi is only 2.9 Mbp, and only encodes about 2,000 protein-coding genes [13]. The genome is highly compacted, with short intergenic regions, almost no repeats, and little evidence of selfish elements, altogether leading to a gene density approximately twice that of Saccharomyces. Even the genes themselves are shorter than homologues in other fungi, a likely consequence of domain loss due to the reduction of interaction networks. E. cuniculi genes have also massively reduced the number of introns they contain: only 14 have been annotated at present in the entire genome, and in Enterocytozoon bieneusi they appear to have been eliminated altogether [24]. The close proximity of the genes to one another in these compact genomes seems to have an effect on transcription, since a high frequency of overlapping transcripts has been observed in both E. cuniculi and Antonospora locustae [25],[26]. In particular, transcription termination is often not immediately after a gene, but well into or beyond the adjacent gene, so that an mRNA can contain sequence from more than one gene, although only one gene appears to be translated (as often as not, the additional genes are in the opposite strand). This makes it difficult to interpret large scale transcription patterns, since the presence of RNA corresponding to a gene does not necessarily mean that gene is being expressed. The genomes of these highly compacted species have attracted the most attention, but we are now beginning to see that other microsporidian genomes are quite different. There is a nearly a 10-fold range in genome size between different species that have been investigated [27], and genomes on the larger side of the spectrum have been shown to have a low gene density and many transposons [28],[29].

How Do Microsporidia Depend On Their Host?

Microsporidia cannot grow or divide outside their host cell, but exactly how they interact and use resources from their host is only partially known. It has long been known that infection induces changes in the host that appear to be related to metabolic dependency. For example, infection by several species leads the host to surround the parasite with mitochondria, presumably supplying the parasite with energy. In one extreme case, infection leads the host cell to grow to an enormous, mutinucleate cell called a xenoma, which becomes a highly organized spore production assembly line [30]. Prior to the widespread use of genomic methods, the metabolism of microsporidia was difficult to study because they were difficult to separate from their host cells, but it has long been clear they lack many metabolic pathways, such as oxidative phosporylation, electron transport, and tricarboxylic acid cycle [31]. Characterisation of genes involved in metabolism, and in particular genome surveys of microsporidia, have confirmed and refined this view. In all well-sampled species there are no genes for many metabolic pathways (e.g., tricarboxylic acid cycle), and few genes relating to the synthesis of small molecules such as amino acids and nucleotides. Interestingly, several ATP transporters have been found in microsporidia. Some of these localize to the outer membrane of the parasite and seem to import ATP from the host cell, while others import ATP into the relict mitosome [32],[33]. There is also some variability in the metabolic capacity of different species of microsporidia, the most extreme case being the human parasite E. bieneusi, with a genome sequence survey revealing virtually no genes for central carbon metabolic pathways [24]. This includes glycolysis, which is the backbone of energy generation in other microsporidia, suggesting that this species is unable to generate energy from sugar, and is therefore dependent on its host directly for ATP, making this species one of the most host-dependent parasites known.

To access the original article in PloS Pathogens and view the references cited, click here.

Patrick Keeling is a scholar of the Canadian Institute for Advanced Research, Evolutionary Biology Program, and assistant professor, Department of Botany, University of British Columbia.

Ed. Note: Recently, microsporidia were found to be a (and so far the only) natural parasite of the worm, Caenorhabditis elegans. Given the importance of this host as a model for infection, this bodes well for increasing our understanding of microsporidial pathogenesis.

There's Gold in That Thar Periplasm

2009-10-29T10:36:46-07:00

by Elio

John Stone with Gold Mining Pan ca. 1939.
Credit: California Gold: Folk Music from
the Thirties. Collected by Cowell,
Library of Congress.

When thinking of a gold prospector, most of us conjure up a wizened old character leading a mule that carries his pick, gold pan, and rusty coffee pot. Nowadays, think of bacteria instead. Specifically, picture the bacterium Cupriavidus metallidurans (formerly Ralstonia metallidurans), which carries out the biomineralization of gold. It transforms toxic gold compounds into their metallic form via an active mechanism.

The leader of a large international research group working on this subject, Frank Reith of the University of Adelaide, explains: A number of years ago we discovered that the metal-resistant bacterium Cupriavidus metallidurans occurred on gold grains from two sites in Australia. The sites are 3500 km apart, in southern New South Wales and northern Queensland, so when we found the same organism on grains from both sites we thought we were onto something. It made us wonder why these organisms live in this particular environment. The results of this study point to their involvement in the active detoxification of Au complexes leading to formation of gold biominerals.(Science News)

Transmission electron micrograph (TEM)
of a C. metallidurans ultra-thin section
containing a Au nanoparticle in the
periplasmic space. Source.

In a new study, they demonstrated that these organisms defend themselves against the oxidative stress induced by toxic Au(III) complexes by reducing the gold in them to particulate Au⁰. Using such fancy techniques as synchrotron µ-Xray fluorescence (µXRF), they localized a number of metals within the bacterial cells, something they could confirm with regular transmission EM. They also found that the genes involved are located in a new operon, and that they are upregulated when the bacteria are challenged with the toxic minerals. They suggest that this work may lead to the development of Au-specific biosensors. In other words, bacteria may be the gold prospectors of the future.

This is a revisit to this topic. For a previous post, click here.

Microbial Art

2009-10-28T13:27:04-07:00

by Elio

Apple Tree by Dr. Niall Hamilton of New Zealand. Source.

Curious about what all you can paint using bacteria as your pigments? Use your artistry to inoculate agar plates by design, then wait for the bacteria to grow into colonies in esthetic shapes and colors. To whet your appetite and see what can be done, given appropriate talent, visit Microbial Art at http://www.microbialart.com/ Here you'll find original works grown by several microbiologists of artistic bent.

This website, the creation of T. Ryan Gregory at the University of Guelph, has links to lots of other visual goodies. There's almost no end to them.

Mad Dogs and Microbiologists

2009-10-26T09:32:15-07:00

by William C. Summers

Pasteur’s laboratory (around 1885). Source.

Jean-Baptiste watched the powerful dog with unsteady gait approach and then attack a group of six of his friends. He picked up his whip and rushed to meet the animal, only to be savagely bitten on his left hand. In fierce hand-to-hand combat, Jean-Baptiste finally managed to throw the animal to the ground, pinning him to the ground under his knee. With his right hand he forced the dog's jaws apart, sustaining new bites, then used his whip to tie the muzzle of his enemy and finally beat the beast to death with one of his wooden shoes.

Thus it was that Louis Pasteur recounted to his colleagues at the Académie des Sciences in Paris how his heroic patient, Jean-Baptiste Jupille, a 15-year-old shepherd boy from the Jura, came to be exposed to rabies on October 14, 1885. Pasteur was reporting on the success of his treatment of his first rabies patient, Joseph Meister, who was still alive and well more than three months after having been severely bitten by a rabid dog. Meister's survival was considered something of a miracle, because rabies was, and still is, considered a lethal disease in the absence of effective treatment. Young Jupille was treated as Pasteur’s second rabies patient and he, too, survived this heretofore universally fatal disease.

Still, Pasteur had been hesitant to treat Jean-Baptiste because the disease had such a head start on him. From his experimental work on dogs and rabbits, Pasteur and his protégé, Emile Roux, knew that their new treatment was most effective before or very soon after inoculation of the infectious agent. The longer the interval between inoculation and the start of treatment, the less likely the cure. This very early conclusion remained one of the unsolved problems in rabies treatment, at least until very recently.

The Vision of Saint Hubert by Jan Brueghel the Elder and Peter
Paul Rubens. Early 17th century. Source.

Since ancient times rabies (known also as "hydrophobia" or "la rage") has been a dread disease with virtually 100% mortality. The rare survival was usually attributed to supernatural intervention. Most often invoked in miraculous cures was Saint Hubert, the first bishop of Liege in Belgium and the patron saint of hunters, hence associated with dogs. The standard legend relates that during his investiture as bishop, one of the required vestments was missing. At the last moment, it was miraculously supplied when Saint Peter appeared with a stole woven on the looms of heaven and embroidered by the Virgin Mary. Threads from this garment, inserted into a small incision in the forehead of the rabies victim, became a sure cure for rabies—provided some additional rituals were observed. Another legend describes cauterization of the wound site from the bite of a rabid animal with a heated "key of St. Hubert" in the form of a metal cross. Even now, on St. Hubert's feast day, November 3rd, in Ghent local pastries known as mastellen are taken to Mass for blessing, thus becoming remedies for rabies. It is reported that in Flanders there are open air Masses where hunting dogs and other animals are given "The Bread of Saint Hubert" as a sort of spiritual rabies shot.

The standard treatment for rabies is a direct descendant of Pasteur's original concept: injection of attenuated virus before the infection reaches the central nervous system and before it does substantial damage. In this way, one can induce sufficient immunity to destroy the advancing infection. For immunogen, Pasteur employed a preparation of dried spinal cord material from infected rabbits. Drying for varying lengths of time provided virus preparations of varying virulence. He injected the exposed patients with increasingly virulent samples as their induced immunity increased. This procedure was based on Pasteur's theory of the mechanism of immunity. Although his theory has been discredited, he nevertheless devised a regimen that does, indeed, provide effective treatment for rabies victims. While the source of the attenuated virus for immunization has moved on from desiccated rabbit spinal cords to duck eggs, then to cultured cells, the approach has remained the same: induce antiviral immunity before the virus can damage the brain. Thus, to be effective, treatment must begin soon after inoculation.

Both Pasteur's dilemma and the need for miracles, however, may soon be relegated to the dustbin of medical history. A recent report in PNAS by Bernhard Dietzchold and his colleagues at the Thomas Jefferson University in Philadelphia suggests that a new approach to rabies vaccines may make it possible to treat rabies infections effectively even after some time has elapsed. Drawing on new understanding of the pathogenesis of rabies, including its replication and its expression of the relevant immunity-inducing antigens, these workers designed a highly attenuated—but highly effective—vaccine strain of rabies that shows promise as a significantly improved post-exposure treatment for rabies.

Rabies virus, purified from an
infected cell culture. Negatively
stained virions: note their char-
acteristic "bullet shape." Magni-
fication approximately x70,000.
Source.

There have been two major obstacles to rabies vaccines: relatively weak immunogenicity and the problem of attacking infections across the mysterious blood-brain barrier. The new work discussed here seems to overcome both of these obstacles. First, in the vaccine strain, the gene for the major antigen required to induce antiviral immunity, the virion surface glycoprotein (G), has been triplicated. Apparently simply because of the increased gene dosage, the G protein is substantially over-expressed. Because G is also involved in the neuropathogenicity of the rabies virus, two mutations were constructed in the G gene that destroy its pathogenicity, but not its immunogenicity. The authors call this mutant form GAS. Interestingly, this avirulent GAS mutant is dominant over the pathogenic strain, thus ensuring that the "triploid" GAS virus has a very low probability of reversion to the virulent wild type virus. The use of two mutations in G further reduces the probability of reversion to wild type. These predictions were confirmed by demonstrating that the GAS strain is non-virulent even when directly inoculated into the central nervous system of immune-compromised mice.

The second problem, that of immune access to the privileged nervous system, the site of the rabies infection, seems to be overcome by this vaccine strain, as well. Paradoxically, one of the features of the rabies virus that is important in its pathogenicity is its low level of replication. This "stealth strategy" seems to allow it to infect neurons without breaking down the barriers to entry of antibodies. The effective vaccine strains, including the new triple GAS strain, replicate much more rapidly than the wild type virus and, by some mechanism that is still unclear, enable the induced, antiviral antibodies to penetrate the nervous system.

When experimentally testing for post-exposure prophylaxis in a mouse model, the GAS vaccine effectively prevented any signs of infection when administered a short time after inoculation, and reduced deaths and/or paralysis when give at longer times after inoculation. Since it is known that the distance between the site of inoculation into peripheral nerves and the central nervous system is a crucial factor in determining the "window of opportunity" for effective post-exposure prophylaxis, it can be expected that the short "window” in mice will translate into longer time intervals in humans simply based on host size and the distance the virus must travel.

Nearly 125 years after the momentous introduction of the rabies vaccine, we may finally be able to close the circle. The new vaccine, like Pasteur's, also involves an attenuated strain. One would guess that Pasteur would have approved.

Happy World Rabies Day (28 September 2009)

William C. Summers is at the Yale University School of Medicine.

Fiddling with Fungi: And the Winner Is…

2009-10-22T09:58:30-07:00

by Elio

X. longipes. Source.

Late in August of 2008 we promised to update you on the attempts to out-Stradivarius Stradivarius by crafting violins made from wood treated with fungi. Here is the latest news.

Scientists at the Swiss Federal Laboratories for Materials Testing and Research made violins from wood treated with two different fungi: Physisporinus vitreus for the spruce top plate and Xylaria longipes (aka Dead Man's Fingers) for the sycamore bottom plate. The treatment lasted six or nine months, by which time the wood had become covered with a fuzzy growth of mycelium.

P. vitreus. Source.

They made four violins from the same wood: one treated for six months, another for nine months, and two untreated. The British violinist Matthew Trusler played all four for an audience of more than 180 people at a forestry conference. More than 90 people ranked the bioviolin treated for nine months ahead of a real Stradivarius, which came in second, followed by the violin treated for six months. The two untreated violins came in last.

The idea here was that treating the wood with fungi might artificially recreate the structure of the wood found naturally during Stradivarius's lifetime. The Little Ice Age, a period of abnormally cool weather between 1645 and 1715, produced trees with more uniform wood. Treating today's wood with the fungi artificially results in wood with similar properties.

When these modern Stradivarius soundalikes become commercially available, musicians will be able to have the sound of a Stradivarius without the price─for a mere $25,000.

Small Friends of Fungi

2009-10-19T10:15:48-07:00

This article first appeared in the splendid Cornell Mushroom Blog. Reading it, we were jolted into realizing that we had a distorted view of the living world, no less. Like most people, we believed that in natural habitats, some animals eat plants, those animals are eaten by predators, and that's about it. We thank Bob Mesibov for setting us straight. We are delighted to share his insights with you by reproducing his article here, with permission from him and from Kathie Hodge, editor and blogificator of the Cornell Mushroom Blog.

by Bob Mesibov

The millipede Narceus americanus photographed by
Kent Loeffler, copyright Cornell University. You can
find this borescopic image among the many in the
book Beneath Notice: Adventures with a Borescope
by Kent Loeffler, with "Fungal Outbursts" by Kathie
Hodges.

If you studied the traditional sort of biology, you're probably carrying around an unfortunate prejudice.

You see terrestrial habitats as a simplified nutrients-and-energy pyramid. At the bottom are green plants, feeding on sunlight, carbon dioxide and soil water and minerals. Next layer up on the pyramid is the herbivore mob: leaf and stem eaters, sapsuckers, root nibblers, seed and fruit gobblers. Above these green feeders are a couple of layers of predators. And that about sums up the world, right?

Wrong. That's only part of the world, and a small, very specialised part of it, too. To begin with, most animals can't eat green food. Herbivores are dietary specialists among the insects, mollusks, birds and mammals. Your average green leaf or stem doesn't show much herbivore damage, and for good reason. It's mainly water boxed in by cellulose and other structural carbohydrates, which are impossible or extremely hard for animals to digest. Other nutrients are present, but at low concentrations. You need to eat a great mass of indigestible green stuff to get a decent return of elements like nitrogen, phosphorus, potassium and calcium. As for animals eating wood, which makes up most of the biomass in a forest – well, there are termites, and…um…termites…

The truth is that in the real world outside the biology classroom, only a tiny proportion of terrestrial primary production goes through the stomachs of the few evolutionary lineages brave enough to tackle what green plants produce. In any terrestrial habitat, the great bulk of primary production just does not get eaten. It sits, instead, at the bottom of a very different food pyramid. I call it the Dead Plants Society (DPS), as opposed to the Green Feeders Guild (GFG).

In the absence of fire, all that uneaten primary production is first attacked by fungi and bacteria. By 'attacked' I mean 'converted from low-nutrient indigestibles to concentrated yummies', i.e. fungal and bacterial bodies. Stacked on top of this microbial layer in the pyramid are microbivorous layers of nematodes, mites, springtails, earthworms, millipedes and other soil animals. On top of those are predators – but picture 'centipede', not 'eagle'.

The GFG and DPS animal communities differ in many ways. To begin with, in any given habitat the GFG has very high species diversity (think of plant-eating insects) but low higher-taxon diversity, while the DPS has great higher-taxon diversity (lots of strange sorts of animals), but low species diversity. Next, GFG herbivores tend to specialise on particular plants, while DPS microbivores will eat anything that's rotting nicely. There are also a lot of winged GFG members ('gotta find that particular plant I like…'), whereas almost no DPS members have wings, at least in their younger, feeding stages. There's an architectural difference, too. The GFG extends well up in the air, to ca. 100 m in some tall forests, while the DPS is largely confined to the ground.

Then there's the matter of heritage. The earliest DPS fossils are of mites, springtails and millipedes, and they're more than 400 million years old, from a time when terrestrial vegetation was mainly mossy and ground-hugging. The first solid evidence for green feeding (early insects with spores in their guts) appears much later in the fossil record, from coal swamp times. The DPS is vastly older than the GFG, and when you handle richly organic soil you're holding animal communities which are spectacularly ancient and robust. You can almost imagine a springtail thinking: 'Seen the dinosaurs come and go, mammals are nearly done. Wonder what great lumbering dopes we'll see in the next 100 million years? Yum, love these hyphae with yeast sprinkles!'

Dr. Robert Mesibov is a taxonomist from down under who studies seriously fungophilic animals: millipedes. Check out his other many-legged doings via his website. A previous article on the Dead Plants Society is available here.

Talmudic Question #54

2009-10-15T10:12:50-07:00

Some believe that the extent of anaerobic respiration on Earth is usually underestimated. What is your guess for the proportion of biogenic CO₂ that is made by this mechanism?

The Far Side of Microbiology

2009-10-15T10:10:15-07:00

by Elio

How to spot a microbiologist without a name tag.

Source: www.CartoonStock.com. From The Far Side, by Gary Larson. Artist: Randall McIlwaine.

Getting a Handle on Cell Organization

2009-10-12T10:13:50-07:00

by Franklin M. Harold

Mitotic spindle of a human cell: microtubules in green,
chromosomes in blue, centrosomes in red. Source.

Structural organization is one of the most conspicuous features of cells, and possibly the most elusive. No one really doubts that that cell functions commonly require that the right molecules be in the right place at the right time; or that spatial organization is what distinguishes a living cell from a soup of its molecular constituents. But the tradition that has dominated biological research for the past century mandates a focus on the molecules, and so our first step is commonly to grind the exquisite architecture of the living cell into a pulp. Few molecular scientists have asked whether anything irretrievable is lost by this brutal routine. Such questions as how molecules find their proper place in a framework orders of magnitude larger, or how spatial order is transmitted from one generation to the next, have been largely neglected until recently.

Two current and quite excellent short reviews afford an entry into the wilderness. Eric Karsenti takes an historical approach to the role of self-organization in creating order on the cellular scale. The physical principles are arcane, but some aspects are actually quite familiar. We have known for half a century that supra-molecular complexes often arise by self-assembly, without any input of either information or energy; examples include lipid bilayer membranes, ribosomes, microtubules, S-layers and virus particles. But the scope of self-organization has been greatly enlarged in recent years by the discovery that an array of dynamic structures can be generated in the presence of an energy source, usually ATP or GTP. The mitotic spindle of eukaryotes has been identified as a self-organizing machine, the endomembrane system may be another. Like self-assembly, self-construction (my term) requires no external source of information, but it does entail continual energy consumption. In a complementary article, Allen Liu and Daniel Fletcher survey a selection of efforts to reconstitute cellular functions in simplified systems, starting with cell-free extracts or purified proteins. Ingenious experimenters have managed to reconstitute the essentials of actin-based motility, membrane protrusion, the oscillatory system that localizes the midpoint of bacterial cells, and now also the contraction of the Z-ring. Though much remains to be learned, it is safe to conclude that the lower levels of cellular order, at least, are products of pure chemistry: they arise by interactions among the molecular constituents in ways that require the cell as a whole to supply energy and a permissive environment, but no spatial instructions.

This is excellent science, which takes us some way towards bridging the gulf between nanometer-sized molecules and cells in the range from micrometers to millimeters. It also extends the genome’s reach deep into cellular structure. In a self-organizing system, the “instructions” must be wholly inherent in the molecular parts, and ultimately derive from the corresponding genes. It is the genome that specifies the architecture of the mitotic spindle, not explicitly but indirectly: the form and even functions of the spindle are implied in the structure of the spindle proteins, and in their interactions. And if the spindle can be envisaged as a creature of self-organization, why not the entire cell? Yes, indeed─but as we ascend the hierarchy of biological organization, the meaning assigned to self-organization and its underlying mechanisms undergo significant changes. Cells do not construct themselves from pre-fabricated standard parts; instead, they grow. And that mode of self-organization is not purely chemical, for it must produce parts that have biological functions, performed in the service of a larger entity that can compete and thrive in the wide world (discussed here).

We are quite well informed about just how cells grow, and it is clear now that they do so by modeling themselves upon the existing structural framework, which is thereby transmitted to the next generation. To be sure, all the macromolecules involved in this sort of self-organization are gene-specified, but spatial order is not. There appear to be very few individual genes that prescribe dimensions, position or orientation on the cellular scale. Instead, thanks to the ways in which cells grow, spatial cues are sometimes inherited in a manner quite independent of genes (a well-established phenomenon known as “structural inheritance”; discussed here). The form and organization of cells thus stem from two distinct informational roots: the genomic instructions that specify the parts, and the continuity of cellular architecture that guides their placement. Specified proteins and cellular guidelines operate synergistically, reinforcing each other to generate form and organization. As Rudolf Virchow might have said, it takes a cell to make a cell.

Evidence to support such a holistic view of what happens during growth is scattered, but continues to accumulate. Let’s glance at some examples. First, while many sub-cellular structures can be envisaged as products of self-construction from preformed parts, others cannot. A familiar instance is the peptidoglycan wall of bacteria, which consists of a network as large as the cell, made up of covalently-linked subunits. Enlargement during growth calls for extensive cutting, splicing and cross-linking, even while keeping the wall physically continuous from one generation to the next. Second, even self-organizing structures must do so in a manner that ensures their correct placement in cellular space. A particularly neat example comes from recent work on the role of microtubules in cell morphogenesis of fission yeast, Schizosaccharomyces pombe (reviewed by Martin). Microtubules define the poles of elongating cells by depositing there various members of the Tea complex, which in turn recruit additional factors. Cells of a certain mutant, orb6, grow as spheres even though they possess all this machinery. When, however, the mutant cells are grown in microfluidic channels that force them back into the cylindrical shape, the normal longitudinal orientation of the microtubules recovers, and so does deposition of polarity factors at the poles (Terenna et al.). Clearly, the microtubule system and cell form collaborate to organize the cell. Just how this comes about is uncertain, but we can borrow a clue from another admirable study, this one in Bacillus subtilis. Ramamurthi et al. found that the peripheral membrane protein SpoVM localizes to a particular patch of membrane during sporulation by recognizing its curvature; perhaps microtubule ends do likewise.

All this makes good sense, at least to me, but it reopens the question how, and indeed whether, the genome specifies cell morphology and organization. The classical conception, which has been articulated by such luminaries as August Weismann, Francois Jacob and Richard Dawkins, sees the cell as a creature of its genes, and its form and functions as little more than epiphenomena. In the past, this gene-centered view of life has rested on extrapolation more than direct evidence, but confirmation has recently come by way of a remarkable paper from Craig Venter’s laboratory. Lartigue et al. described the transplantation of the entire, naked genome from one species of Mycoplasma to another, tuning the recipient into the donor by both genetic and phenotypic criteria. I hasten to add that the rate of success was vanishingly small, one cell transformed out of 150,000; that no one yet knows just what transpires during genome transplantation; and that it remains to be seen whether it can span genera as well as species. All the same, this is surely a landmark study. If one takes its conclusions literally (which the authors were careful not to do), one must infer that a cell represents the execution of the instructions spelled in its genes, and nothing more.

On the face of it, there seems to be a glaring conflict between the geneticist’s understanding of cell organization, and the physiologist’s. The former insists that form and organization obey the genome’s writ. The latter sees the genome as a key subroutine within the larger program of the cell; and it is the cell, not its genome, that grows, reproduces and organizes itself. They can’t both be true—or can they? Note that reproduction and heredity operate on different timescales. A growing cell relies on self-organization to transmit much of its spatial order, by mechanisms quite independent of the genetic instructions. But the genes specify the parts, and mutations commonly affect the higher levels of order; on the evolutionary timescale, it will be the genes that chiefly shape cells. Having said this, there remains a long stretch between the straightforward specification of an amino acid sequence by its corresponding sequence of nucleotides, and the devious and cryptic manner in which the genome can be said to specify the whole cell. Intellectual subtleties must not obscure the conceptual shift, from a linear chain of command to a branched and braided loop of causes and effects reverberating in a self-organizing web. The only agent capable of interpreting the E. coli genome as "a short rod with hemispherical caps" is the cell itself.

There is a whiff of vitalism about this view of life, even a hint of heresy. Stop now and take a deep breath; for once you begin to wonder where all this organization came from in the first place, you are headed for the blue water.

Franklin M. Harold, Department of Microbiology,
University of Washington, Seattle, WA 98195.
E-Mail: frankharold@earthlink.net

Of Terms in Biology: Neuston

2009-10-08T10:00:40-07:00

by Merry

A water strider. Surely handsome, albeit not microbial.
Source.

When I stumbled across the term bacterioneuston, I discovered a whole new world where the air meets the sea. I found that marine neuston had long been used to refer to the diverse flora and fauna inhabiting the topmost 5 cm of the oceans—a distinctly different assemblage than found in the waters below. This broad grouping had been further subdivided. Floating organisms, such as Physalia, the Portuguese Man o' War, make up the pleuston (the small organisms floating on or near the surface of a body of water, from the Greek pleustikos, fit for sailing). The 40-some species of water striders are part of the epineuston (the organisms living in the air on the surface film of a body of water). Just beneath them, living immediately below the surface film, are the organisms of the hyponeuston. There are no hard and fast lines here. A diving epineustonic water strider finds itself briefly a hyponeuston. The planktohyponeuston gather near the surface at night but spend their days below. And, of course, the plankton float or drift throughout the rest of the water column. (For these neustonic terms and more, click here.)

Notice how those definitions shifted from the topmost 5 cm to the surface microlayer film as our focus of interest shifted. Today, unadorned neuston is often used to mean the organisms in the surface film. Its etymology? From the Greek neustos swimming, from nein to swim.

The presence of a gelatinous surface microlayer film had been proposed in 1983, based partly on observations of the slick associated with blooms in the Sargasso Sea of the filamentous cyanobacterium Trichodesmium. (It struck me that the surface microlayer might be a most desirable location for a filamentous, nitrogen-fixing, photosynthetic cyanobacterium.)

What about the ocean surface film from the microbial neustonian point of view? It might appear to be an immense biofilm stretching from horizon to horizon. Thickness? The authors of one paper wryly note: In most investigations, the surface microlayer is operationally defined based on the depth of the sample layer collected, which is in turn dependent on the sampling method employed. Estimates of its thickness range from 1 to 50 µm, with some of the difference likely due to the variable layers of bacteria present. Surface layer bacterial concentrations are commonly said to be 10³−10⁵ times higher than in the underlying water column. One study of the North Sea found that the surface layer microbes were predominantly from two genera, Vibrio and Pseudoalteromonas, both of which have characteristics suggestive of adaptation to life in biofilms.

Oh, what about the bacterioneuston, where this article began? This is—of course—the bacterial component of the neuston. So far Google finds no mention of an archaeaneuston. The poor Archaea, ignored and neglected yet once again.