Small Things Considered

The Limitations of LB Medium

by Hiroshi Nikaido

LB medium, also known incorrectly as Luria-Bertani medium, is widely used to grow bacterial cultures, mainly because it is easy to prepare and provides a broad base of nutrients. LB broth contains, per ml, 10 mg tryptone (a mixture of peptides formed by the digestion of casein with the pancreatic enzyme, trypsin), 5 mg yeast extract (an autolysate of yeast cells), and 5 or 10 mg NaCl. It was formulated by Giuseppe Bertani in 1951 for studying lysogeny in Escherichia coli. He called it “Lysogeny Broth,” or LB. Notably, the original formulation included 1 mg/ml glucose, which has been dropped in more recent times. This medium was designed for work at low bacterial densities, a key point of this article.

While this may appear a tasty dish for many bacteria of research interest, it is an inappropriate choice for physiological studies wherein reproducibility is required. Since only bacterial cultures in balanced growth (achieved by sufficient time in exponential growth) have a reproducible average cell size and chemical composition, none of the components of liquid media should become exhausted during growth of the culture. Is this the case with LB broth? To answer this question, we must know what limits bacterial growth in LB broth.

Growth of E. coli usually stops, even in the presence of the large total concentration of organic nutrients in LB broth, when the OD600 reaches around 2, corresponding to about 0.6 mg of E. coli (dry weight) per ml. The reason is not difficult to find: LB medium provides only a scant amount of carbohydrates, and surprisingly small amounts of other utilizable carbon sources. Tryptone and yeast extract are mostly composed of peptides of varying length. In their definitive 1968 study of Bacto Neopeptone using gel filtration, Payne and Gilvarg found that there was a clear size limit for the usable peptides at about 650 daltons—which corresponds to the exclusion limit of porin channels determined several years later. The smaller, usable peptides were a minority, perhaps a quarter of the entire mixture. Free amino acids were an even smaller minority, approximately 1% or less of the entire preparation. If we assume a similar size distribution for the peptides in tryptone and yeast extract, we can postulate that the yield of E. coli is limited primarily by the available carbon sources.

Growth of E. coli MG1655 in Luria-Bertani broth. An
overnight culture was diluted 5,000-fold in Luria-Bertani
broth and cultivated at 37°C with vigorous aeration. (A)
The OD600 (triangles) and cell concentration (crosses)
were measured periodically. (B) The ratio of the OD600 to
the cell concentration was calculated at each point and
multiplied by 10⁹. Source.

In 2007, D'Ari and associates did a careful study of growth of E. coli in LB broth. They showed that very early in the period usually considered to be well within the exponential phase, when the OD600 reached around 0.3, there is an abrupt change in physiology and the cell size begins to decrease. This observation is not new. As acknowledged by the D'Ari group, Wang and Koch in 1978 had similarly shown that, very early in the "exponential" growth phase, E. coli in LB broth goes through a diauxie-like change in growth behavior. D'Ari's group went further and suggested that what matters is not the general availability of amino acids generated by the intracellular hydrolysis of imported peptides, but the availability of amino acids easily utilized as carbon sources. Although these workers monitored only the free amino acids in the medium (whereas the cells were undoubtedly utilizing peptides for the most part), they concluded that during growth in LB broth E. coli cells undergo alterations in carbon nutrition, using the easier-to-utilize amino acids until they were depleted, then switching to the harder-to-use amino acids.

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

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!

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

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.

There's Gold in That Thar Periplasm

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

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

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.

Fiddling with Fungi: And the Winner Is…

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

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

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

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

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).