Small Things Considered

Naegleria’s Split Morphology Disorder

2010-02-08T08:30:00-08:00

by Psi Wavefunction

The complex structure of flagellates. EM section through
a mature flagellate Tetramitus. K1 = kinetosome. Cm =
cytostomal canal. FV = food vacuole. Rz = rhizoplast.
Source.

By default, a membrane-bound entity like a cell should be a spherical, formless blob. However, most cells are not such formless blobs, but rather have adopted one or more forms from a vast repertory of stunningly complex morphologies. To wit, see (and admire) the radiolarians, metazoan neurons, giant parabasalians or the endlessly weird and sophisticated ciliates. Even prokaryotes have a highly complex cellular structure, and are not, as some biochemists are prone to think, mere bags of enzymes. The deeper you venture into the realm of cellular diversity, the more awe-inspiring becomes the cornucopia of cellular structural and morphological variety. Luckily, there is some order to it as there are two fundamental 'genres' of cellular morphology, at least in the protists: flagellates and amoebae. Of course, there are also cysts, but since those are mostly resting stages (being a round ball isn't particularly helpful while feeding or fleeing from predators...) they can be ignored for now.

Cell shape depends on the cytoskeleton. As you know, its two main component systems are actin and tubulin, ignoring the plethora of miscellaneous proteins that are used for various structural jobs. Tubulin makes microtubules, the spindle fibers of mitosis, but is also important for the flagellar apparatus (we've yet to find one composed of actin and probably for good reasons). You also know that actin is a key player in cell motility and morphology in animal systems. It is also heavily involved in endomembrane trafficking within a cell, as well as endo- and exocytosis. If interested, a recent issue of Science has a nice overview of actin in morphogenesis and cell movement.

The role of the cytoskeleton in morphogenesis is much less clearly defined. It depends largely on the species. Plants, for example, rely very heavily on tubulin for morphology, with actin being a minor player. Amoeboid cells are primarily actin-based. In fact, amoeboid cells resort to tubulin largely for spindle formation during mitosis. They hate tubulin about as much as plants hate actin. Actin-based cells don't have to be amorphous; they are still able to achieve complex morphologies. But there is a positive correlation between amoeboid-ness and actin-ness ('actinity'?).

In contrast, flagellates are primarily tubulin-based. Of course, they still use actin for some intracellular work, but the shape depends largely on the whims of their microtubules. Perhaps not relying much on flagella allows the amoeboids to dispense with the microtubule organization pathways, thereby switching to actin. Flagellates, relying heavily on intact tubulin systems, may be less prone to losing their structure. Also, if you're a flagellate, you need shape for a modicum of streamlining. Try swimming around as a formless or floppy blob of some sort! Keep in mind that life at that scale is very different. Viscosity calls the shots when considering unicellular motility. Perhaps being hydrodynamic isn't even as important as simply retaining shape. Otherwise you'd be like a blob of molasses trying to swim through a sea of maple syrup. Not gonna get very far.

Whatever the reason, amoeboid cells tend to have a predominantly actin-based cytoskeleton, flagellates have a penchant for tubulin. Of course, not all organisms are decisive enough to make this commitment, so we've got amoeboflagellates in the middle.

Plenty of other organisms fancy transitioning between being more amoeboid or more flagellate. But few cells actually dispense with flagella and basal bodies altogether, to form them anew when special conditions arise. It's time to introduce one that does.

Naegleria and Tetramitus: Heteroloboseans with 'split morphology disorder'

Meet Naegleria, a Heterolobosean, famous to medical biologists as a brain-eating opportunist, and to actual biologists as the master of de novo flagellar creation.

Naegleria gruberi. Source.

And before someone accuses me of focusing on biomedically relevant organisms, Tetramitus, another Heterolobosean, does it too, and has more flagella to oogle at.

Dramatic transformation of Tetramitus between amoeboid and flagellate forms. Source.

The nice picture of the Tetramitus flagellate form at the beginning of this post reveals its marvelous complexity. Keep in mind this thing was a 'formless' amoeba a few hours before. Quite a complex structure to be formed within a 4 hour period! Note that while the organelles seem to position themselves freely in amoeboid cells, they become substantially more fixed in the flagellate form. This one even has subpellicular microtubule bundles, a cytostome, and the oral groove characteristic of its 'kingdom,' the Excavata.

Of Naegleria's culinary indulgences

Because of Naegleria's pesky habit to occasionally get into human brains and eat them (oops!), it gets a lot more research attention (which, in the world of Heterolobosean biology, doesn't really mean very much). It should be noted that while there are some waves of Naegleraphobia out there, incidences of human infection are very very VERY rare (small handful of cases a year; some years without any cases whatsoever). Unfortunately, it is almost universally fatal, but so are more ubiquitous diseases. But that's enough excuse for the media to put up a scary article on Naegleria every once in a blue moon, often accompanied by an image of Amoeba proteus or Chaos spp. Hey, at least they're still eukaryotes!

Before we continue on with our hard core cell biology, let's clarify that Naegleria is not a parasite, but rather an opportunist that seems to not mind the 37 °C heat of the human body. It gets into the human brain through the nose, generally from swimming in warm water. Once inside, it devours the brain, inadvertently killing its ‘host, but it does not require transmission to another host to live on. So Naegleria's gastronomic inclinations may well just be an accident, albeit a rather costly one to the few humans it infects.

Amoeboid to flagellate transformation

Summary of Naegleria's life cycle based on C. Fulton in
Methods in Cell Physiology, Vol. 4. 1970. (Don't pay any
attention to the portrayal of the flagellar root apparatus.)

Our exploration of Naegleria's life cycle begins with its dominant life stage, the eating and mitotically-able amoeboid form. Interestingly, mitosis happens without basal bodies (like it does in land plants and diatoms). The addition of water induces the flagellate form, which is capable of feeding via its elaborate oral groove and cytostome, but not dividing. While an amoeboid shape is optimised for a benthic environment (crawling on surfaces), excessively fluid environments favour the swimming flagellate. The third life stage is a resting cyst, which it forms and hibernates in when times are bad (e.g., drought). There seems to be no mention of a sexual life cycle, most likely due to this group being so understudied. (Each year brings us more and more evidence of sex in organisms previously thought to be asexual, with accumulating evidence suggesting sex is a shared eukaryotic feature.) Naegleria's life cycle conveniently involves all three fundamental cell types, which is why it's such a wonderful system for studying the transformations between them (and cellular differentiation.)

The transitions between the principal cell morphologies involve the regulation of actin and tubulin monomer levels: that is, the amount and concentration of the 'building blocks' for each cytoskeletal system. In fact, the reason many organisms encyst for mitosis (or at least reduce their flagellar length) is to redirect tubulin from the cytoskeleton and flagellum to form the mitotic spindle. Otherwise, you'd have to synthesise a bunch of new tubulin that would soon become useless. If you look at some images of Naegleria amoebae undergoing mitosis, you'll see that the cells are much rounder than normal.

There's probably also a regulation of the actin- and microtubule-associated proteins involved in cytoskeletal organization for each system (and the interactions between them), but that doesn't seem to have been studied at all in Naegleria. Simply synthesising tubulin is insufficient for any semblance of a well-organised cytoskeleton; auxiliary proteins are required to regulate the distinctly non-random morphology of a flagellate.

Changes in the actin and microtubule cytoskeleton as amoebae
differentiate into flagellates. Immunostaining of the cytoskeleton:
red = anti-actin; green = anti-α–tubulin; yellow = co-localisation
of anti-actin and anti-α-tubulin. Time: 0 to 120 minutes. Bar: 10 µm.
Note that these are different cells, as they had to be fixed (killed)
for immunostaining. I would love to see a live cell timelapse of this
process! Note how the flagellate morphology is 'molded' by the
longitudinal microtubule bundles. Source.

The Naegleria system could be fruitful for isolating microtubule-associated proteins and other microtubule regulators (e.g., via microarray chips). Considering the cytoskeleton is the most exciting cell structure (fact), cell biologists should stop neglecting Heteroloboseans. We do know that the commitment to flagellation involves a rapid increase of tubulin production, since the amoeboid form has barely any of it.

Naegleria takes ~120 min at 25 °C to transition from an amoeba to a flagellate. The earliest event in this process is the initiation of tubulin synthesis, followed by basal body assembly 30 min later. Cells round up as actin filaments depolymerise, and basal bodies begin to sprout flagella at about the one hour mark. In another 20 min, the tubulin cytoskeleton is molded into the proper flagellar body shape, with flagella reaching full length at ~110-120 min.

Doesn't look like Naegleria has had its genome sequenced yet, although ESTs and a draft for N. gruberi are underway. Of course, the other heteroloboseans are being thoroughly neglected. And yet we may be about to sequence ten thousand vertebrate genomes.

Hopefully I've convinced you at least that Naegleria would make an interesting model for studying cell differentiation and transitions in morphology, as well as cytoskeletal dynamics. While the field is still miniscule—at least its non-clinical component—some work is being done right now on the regulation of cytoskeletal development in Naegleria (such as this paper on the localisation of microtubule-related mRNAs), but not as much as you'd expect. I'm not aware of any other system that involves such a complete transition between three fundamental cell types. For studies of morphology, what more can you ask for?

Note: For a list of research papers drawn upon, click here.

Yana, aka Psi Wavefunction, is an undergraduate in the Department of Botany, University of British Columbia, and the host of the blog Skeptic Wonder: Protists, Memes and Random Musings. Her original post on Naegleria presented on her own blog was adapted, with her permission, for publication here.

Talmudic Question #58

2010-02-04T09:10:31-08:00

What if all phages on this planet went on strike and refused to have their genes expressed?

A Close Encounter of the Enological Kind

2010-02-01T08:29:00-08:00

This article is adapted from one published in the January issue of the magazine Wines and Vines, with permission of the publisher and the author. Microbiologists with epicurean interests in microbiological end-products would do well to become acquainted with this magazine.

by John Ingraham

Early in my career, by good fortune, I encountered the malolactic fermentation. Investigating it by standard microbiological methods led to results that changed the way California red wines are made, for the better most agree. How satisfying it is to think that I was following in the footsteps of Pasteur, no less, and his early career encounter with wine making.

The story started when a highly skilled wine maker, Brad Webb, was freshly hired by James D. Zellerbach of paper and ambassadorial fame shortly after building the fabulously beautiful, Hanzell Winery in Sonoma County, California. Brad was given his complete instructions in a bottle. Zellerbach presented a bottle of Romanée Conti Pinot Noir telling him, “This is what I want.”

The winery, nestled among new hillside plantings of Pinot Noir and Chardonnay, was meticulously built in the Burgundy style. Made of native stone, with resident moss kept intact during construction, its walls were maintained lush and verdant with distilled water (tap water would have left an unsightly white crust). The lampshades on interior lights were French, double-lobed, grape-picking baskets. But much more relevant to his ambitious goals were the custom stainless steel equipment (such as temperature-controllable fermenters fabricated to Brad’s specifications), French oak barrels, and nitrogen-protecting bottling capabilities. This impressive combination of quality facilities along with Brad’s skills and dedication paid off handsomely. He made superb Pinot Noirs and Chardonnays, recently making Wines and Vines’ list of “Wines that Changed the Industry.”

Some of the stainless steel equipment at Hanzell Winery.
Source.

But not at first. Brad’s initial Pinot Noir wines, made from purchased grapes, refused to undergo what is known as the malolactic fermentation, an alarming and perplexing disappointment to him. Alarming, because Brad well knew that the malolactic fermentation, by converting malic acid to the less-acidic lactic acid and CO₂, not only adds flavor components, but is the sine qua non for conferring the softness and subtle flavors of a quality Pinot Noir. And it was perplexing because grapes from the same vineyards regularly underwent the fermentation in other cellars. He tried a number of maneuvers, none of which worked. Even wines that had undergone the malolactic fermentation elsewhere failed at Hanzell. Using such wines to inoculate batches made at Hanzell didn't work either. Brad was exasperated, but he realized that malolactic-free Hanzell offered the opportunity to research the fermentation and solve his dilemma.

Malolactic fermentation in California had a long, solid reputation for capriciousness and independence. Most winemakers of the era became aware that the fermentation was underway only when tanks of wine began to rumble softly, usually in midwinter. Winemakers didn’t start it; they couldn’t stop it; it just happened. But why only then? Malolactic fermentation seemed to have a mind of it own.

Knowing that I, then an Assistant Professor of Enology at UC Davis, was venturing into the microbiology of the malolactic fermentation, Brad dropped by to see me. He offered Hanzell as a negative control for experiments on spontaneous malolactic fermentations. This began a brief but richly rewarding collaboration as well as a warm friendship. I began my studies by searching for bacteria capable of causing the malolactic fermentation. From samples of dry wines and lees collected at seven California wineries, I had isolated about 50 strains of lactic acid bacteria (the parent class to which malolactic acid bacteria belong). The isolates proved to be a diverse lot, representing all four major types of lactic acid bacteria: rods and cocci that did (heterofermentative) and did not (homofermentative) produce gas from glucose. But strikingly, all were able to utilize malic acid in laboratory culture, converting it readily to lactic acid and CO₂. That is, all were metabolically able to mediate a malolactic fermentation, at least in the benign environment of laboratory culture. But individual samples almost always contained only a single type, suggesting that these bacteria weren’t randomly spread throughout wineries. Only one established itself and became dominant.

Brad and I evaluated all 50 of these “ML” strains for their ability to grow and remove malic acid from fresh and partially fermented must. We selected number 34, the most vigorous, for further studies. ML-34 had been isolated from a sample of Barbera (as I recall) that I had taken from a redwood tank at Louis Martini’s winery in Saint Helena. Louis was welcoming and fully cooperative, but preferred that his winery not be identified as the source of this bacterium. Bacteria in wineries didn’t project a positive image. After all, they are the bête-noir, causative agents of Pasteur’s “maladies des vins.”

In 1959, Brad and I took ML-34 to Hanzell to study it under winery conditions. There, we added 100 ml of a culture to 4 gallons of freshly pressed grape juice (must). Two days later all its malic acid was gone. It worked. In various assays and with different grapes, malolactic fermentation was complete in 21 days.

We were delighted. We were, we believed, the first to induce a malolactic fermentation from a pure culture of a known strain of a bacterium. It turned out that others in Europe had achieved the same thing somewhat earlier. And to our satisfaction, we had learned how to control malolactic fermentation.

What was the key to this control? Why hadn’t the malolactic fermentation occurred spontaneously at Hanzell as it did so readily elsewhere? That seemed obvious enough. There were no resident malolactic bacteria in these new sanitary facilities. But why had an inoculum from a malolactic-fermented wine failed to induce a fermentation at Hanzell? Certainly the wine must have contained malolactic bacteria. We found several reasons, the main one being that ML-34 needed many growth factors. Now, it turns out that when yeasts stop growing, they glean growth factors from their surroundings and store them inside their vacuoles until they have nutritionally impoverished their environment. The trick, then, was to add ML-34 before, not after, the yeast fermentation took place, and thus before the yeast had sequestered the needed growth factors.

Oenococcus oeni. Source.

Nothing is absolute when dealing with the rich complexity of wines as well as the numbers and condition of bacterial cells used as inocula for malolactic fermentations. Subsequent investigators have experienced little difficulty starting malolactic fermentations in finished wines. But the ability of yeast to deplete must of nutrients essential for malolactic bacteria has also been established. Over the years, ML-34’s name changed, becoming increasingly more elegant and wine specific. It eventually graduated to Oenococcus oeni (from the Greek, oinos, for wine).

In spite of their important contributions to wine quality, malolactic bacteria have not escaped suspicion and criticism. As they convert malic acid to softer lactic acid and CO₂, they marginally increase volatile acidity, and they decarboxylate some amino acids forming corresponding amines. For example, they convert histidine to histamine and tyrosine to tyramine. These amines induce allergy-like symptoms. Although their concentrations in wine are minute, some speculate that accumulation of histamine and tyramine could present a hazard for persons taking antidepressants that inhibit monoamine oxidase, the enzyme that metabolizes these amines. On the plus side, we should remind ourselves that the delightful Portuguese vinho verde wines rely on the CO₂ from a malolactic fermentation for their slightly sparkling spritz of “pétillance”. It’s a mystery why California vintners have never accepted this other gift that malolactic bacteria proffer.

Outside the wine world malolactic bacteria have attracted attention for their remarkable ability to scrape by on very little energy. A number of bacteria are well known energetic misers, but malolactic bacteria might well be the world-record holders among heterotrophs. The conversion of malic acid to lactic acid and CO₂ releases only about a third of the energy needed to generate a single molecule of ATP. By pushing one proton (hydrogen ion) out of their cell each time they utilize a molecule of malic acid, malolactic bacteria incrementally accumulate a reserve of potential energy until they have enough to make a molecule of ATP.

Important as it is to the winery, that’s not malolactic fermentation’s only home. It occurs wherever appropriate lactic acid bacteria (and most are) encounter malic acid or malate. One such venue is our mouths. Streptococcus mutans, the causative agent of dental caries, which most of us host, is an example of a lactic acid bacterium that mediates that conversion. So each time we ingest malic acid-containing foods, principally fruits and vegetables, we carry out our own malolactic fermentation. Thus, ingested malate exerts an alkalizing effect right at the site of incipient caries formation. Some dental researchers reason that the resulting neutralization helps to control dental caries, yet another of the fermentation’s gifts.

If bacteria capable of the malolactic fermentation can be found almost everywhere, even on our teeth, what’s special about Oenococcus oeni? It’s special because it can grow in wine. It tolerates wine’s low pH and considerable alcohol content. O. oeni probably achieved these abilities rather quickly in evolutionary terms because we now know that it’s an exceptionally rapid evolver. But that’s another intriguing aspect of its complex and still expanding story. We’re just pleased that it did.

Upon retirement from UC Davis as Professor of Microbiology, John took up writing and consultation in biotechnology. His latest book, March of the Microbes: Sighting the Unseen, which takes a bird-watcher’s approach to microbiology, will be released by Harvard University Press in February.

Fine Reading: Yet Another Reason to Appreciate Fungi

2010-01-28T08:34:00-08:00

by Elio

This blog, known as it for taking up the cause of the underdog, was fortified by reading How Fungi Have Shaped Our Understanding of Mammalian Immunology in a recent issue of Cell Host and Microbe. Not only that, but this is an exceptional piece of reviewing. It’s short (a mere three pages), fun to read, and to the point.

So, what’s the case for fungi shaping our understanding of immunology? (Note the inclusion of “mammalian” in the title. A nice departure from the customary anthropocentricity). The author, Gordon Brown of Aberdeen, guides us through both early and late developments, starting with Elie Metchnikoff, the discoverer of phagocytosis who saw yeast cells being engulfed by the water flea Daphnia, through C-type lectins, Toll and Toll-like receptors, and intracellular signal pathways.

Selected seminal discoveries from 1884 to 2006 are shown along with representative images,
including Metchnikoff (1884); zymosan engulfment by PMN (1941); properdin (1954, trimeric
structure); Aspergillus infection in TLR-deficient Drosophila (1996); fungal recognition by
Dectin-1-expressing fibroblasts (2001); collaborative signaling between TLR and CLRs (2003);
ITAM-like motifs and Syk kinase recruitment (2005); the requirement of multiple PRRs for
optimal anti-Candida responses (2006); and enhanced Candida infection in the kidneys of
CARD9^−/− mice (2006). Source.

In the author’s words: …fungi and their components have long been known to influence immune function, and the contributions made from the study of fungal infections are often underappreciated. Well, here’s for trying.

Measuring the Strength and Speed of the Microbial Grappling Hook

2010-01-25T09:43:36-08:00

by Amber Pollack-Berti

Source.

I’ll admit, I’ve been in love with the type IV pili (T4P) for a long time. After memorizing all those complex pathways for regulation and metabolism, there was something so refreshing and accessible about pili. These bacterial surface appendages are, by their nature, mechanical structures. They are easy to visualize. Their composition is simple: a Type IV pilus is a flexible filament made up of thousands of repeating pilin subunits. Bacteria employ this one structure for a variety of purposes: T4P play structural roles in motility and biofilm formation, and have also been implicated in DNA binding and uptake, in host cell signaling, and even in communication of electrical signals as ‘nanowires’.

T4P are long and thin and have great tensile strength. They are involved in bacterial motility using a special mechanism: the pili are retracted into the cell when the pilin subunits are removed at the pilus’ cell end by an ATPase, the protein PilT. This results in the ratcheting of the pilus back into the cell. When a pilus is attached by its distal end to a surface of agar, the retraction is so strong that the cell is pulled forward in a kind of movement called twitching motility. When a bacterium attaches via the pilus to the surface of a host cell, it retracts until the two come in contact. In these cases, the pilus acts as a grappling hook, pulling the cell along in short bursts of pili elongation, attachment, and retraction.

Experimental setup and force generation during T4P
retraction. (a) A cell is immobilized on a polystyrene-
coated cover slide. When a T4P bound to a bead in
the laser trap is retracted, it displaces the bead by
a distance d from the center of the trap. The force
feedback is triggered by moving the slide by a dis-
tance x to maintain d at a constant value. (b) Typical
deflection, d, of the bead during pilus retraction as a
function of time. F, force; t, time.
Source.

The force exerted in pilus retraction as well as its speed have been recently measured. Just how much force can be exerted during the retraction of one pilus? How fast can it retract? If you apply force to the pilus, does it change the speed of retraction? These are physics experiments, albeit at a microscopic scale. The authors used laser tweezers to measure the force and velocity of pilus retraction. The experimental setup consists of immobilizing a cell on a microscope slide and linking a bead to the end of a pilus. The bead can be localized via a laser trap, and the distance it travels recorded as a function of force and time.

Using this approach, they pitted two different microbes—Myxococcus xanthus and Neisseria gonorrhoeae—against one another in the equivalent of a bacterial tractor pull. They compared M. xanthus T4P to previously reported stats for N. gonorrhoeae. Could two distinct bacteria, living very different lifestyles and gazing at each other from far across the phylogenetic tree, pull their pili with similar force? Similar velocity?

Model of type IV pili assembly and retraction. Prepilin
leader sequences are cleaved by PilD. Processed PilA is
assembled on a base of minor pilins (PilE, V, W, X, and
FimU) by the action of the cytoplasmic membrane protein
PilC and the NTP-binding protein PilB. The pilus is extrud-
ed through the outer membrane via a pore of PilQ multi-
mers. Pilus retraction is by the action of PilT. Source.

When it comes to stalling force (the force at which retraction drops by an order of magnitude), M. xanthus may be the winner, with a stalling force of ~150 pN. But N. gonorrhoeae is no slouch, with a previously reported force of ~110 pN. Whatever the details, the authors state: to our knowledge, the pilus motor is the strongest linear motor reported in the literature to date. By comparison, the stalling force of an actin filament is 7.7 pN per filament, that of myosin 2.5 pN. But while both N. gonorrhoeae and M. xanthus may retract their pili with the same general amount of force and at similar velocities, there was a striking difference: when force is applied to N. gonorrhoeae there is an increased likelihood of direction switching: the pilus stops retracting and begins to elongate. In contrast, applying force to a M. xanthus pilus does not increase the likelihood of elongation.

Similar values for retraction strength and velocity suggest common machinery at work in two very different bacteria. Does the difference in direction switching reflect the different lifestyles of a soil-dwelling microbe and a human pathogen? T4P systems are employed by a wide variety of bacterial species. Certainly studies that investigate this mechanism in other twitching bacteria will further expand our knowledge of T4P retraction across diverse bacterial lifestyles.

Amber is a Graduate Research Assistant in the Ruby Lab at the University of Wisconsin, Madison, and the host of a blog that also features The Small Things: Tiny Topics.

Fine Reading: Classics from the Archives of the Royal Society

2010-01-21T08:45:00-08:00

by Elio

The Royal Society announced that it has put 60 of its most memorable papers online. One of them concerns the discovery of bacteria by Antonie van Leeuwenhoek. The original paper as published in the Society’s Philosophical Transactions of 1677-1678 was entitled:

Observations, communicated to the Publisher by Mr. Antony van Leewoenhoeck, in a Dutch Letter of the 9th of Octob. 1676, here English’d: Concerning little Animals by him observed in Rain-Well-Sea- and Snow water; and also in water wherein Pepper had lain infused.

Here are two excerpts that can be expected to tickle the present day microbiologist’s mind. (Note that in the style of the time, the letter “s” looks like an “f.”)

The eye of a body louse is approximately 100 µm in diameter or 10⁴ µm³. The organisms van Leeuwenhoek saw were 1/10 that length, or 10 µm, and 1/1000 that volume, around 10 µm³. Many bacteria reach such dimensions. The movement described matches the runs and tumbles of chemotactic bacteria.

Let’s assume he was talking about lengths. A honey bee is about 1.2 cm in length, a horse, about 250 cm. Thus, the ratio of their lengths is approximately 200x. Cheese mites are around 0.5 mm in length, thus making the organisms he observed approximately 2.5 µm long. I haven’t found out how thick the hair of a cheese-mite is.

Cryptic Life in the Antarctic Dry Valleys

2010-01-18T08:30:00-08:00

by Merry

A ventifact (a stone sculpted by wind-driven sand) in
the "Martian" landscape of the McMurdo Dry Valleys.
Source.

On first inspection the habitat seems as sterile as the surface of autoclaved glassware... but the trained eye, aided by a microscope, sees otherwise.
E.O. Wilson, The Future of Life

For the microbiologist, the next best thing to a trip to Mars might well be an expedition to the McMurdo Dry Valleys of Antarctica. Here, in the Earth's coldest and driest deserts, the conditions approach those on our neighboring planet and are thought to also approach the cold-arid limit for life. Not all of the Dry Valleys are equally dry. Some have perennially frozen, glacier-fed lakes and ephemeral streams, and thus have some soil moisture. Here one finds more of life, even three taxa of multicellular animals—tardigrades, rotifers, and, most numerous, bacterial-feeding nematodes. (Click here for our earlier post about the microbes found in a subglacial lake in this region.)

Lacking such a source of water, thus being one of the truly dry Dry Valleys, is McKelvey Valley. What little snow falls here or blows in sublimates in the cold, hyper-arid conditions. How cold? The average air temperature is around –20 °C. Winter brings prolonged periods of −55 °C cold; in summer, air temperature can rise to a balmy 0°C. Humidity is low (<10% RH in winter). And then there are the ceaseless katabatic winds (from the Greek word katabatikos meaning "going downhill"). Cold, dense air falls downhill off the East Antarctic Ice Sheet and races through the valley at speeds commonly exceeding 50 km/h, sometimes reaching 320 km/h (200 mph). These winds evaporate all moisture and carry sand grains that scour the barren landscape. All in all, these conditions bear some resemblance to those used to freeze-dry biological samples.

Endolithic community layer in fractured sandstone.
Source.

The valley floor is an unstable, gravelly, desiccated mineral soil with high salinity, little organic material, and the lowest nitrate concentrations known for any terrestrial soil. Surface temperatures fluctuate wildly under the intense summer sun, seesawing between –15° and +27.5°C in a few hours. UV is intense. And, of course, the winds scour the land and carry off any hint of moisture. Nevertheless, life carries on. Some intrepid researchers left the comforts of Hong Kong or New Zealand or even relatively balmy Minnesota to look for organisms living under such extreme conditions. As reported in their recent paper, there are some bacteria living in these dry surface soils, mostly Acidobacteria and Actinobacteria. As you might expect, at the top of the list are desiccation tolerant taxa such as Deinococcus and Rubrobacter. There are also numerous nitrogen fixers. With neither photoautotrophs or chemoautotrophs present, organic carbon is at a premium, and its lack is thought to preclude further community development.

Chasmoliths: a lichenized microbial community protrud-
ing from a granite rock fracture. Source.

Both qualitatively and quantitatively, most of the life in this valley is associated not with the soil, but with the rocks. Here and there the flat-lying sedimentary bedrock is exposed. The rock surfaces, and even the shallow cracks, are likely sterile due to temperature fluctuations and abrasion from windblown sand. But any life that burrows in even just a few millimeters finds more stable temperatures and shelter from the incessant wind, even in the intense cold. Given the estimate that -6 ºC or -8 ºC is the lower limit for metabolic processes, some activity would be possible in these rock niches for 1000 hours per year at the most. However, that is enough to sustain microbial communities, both the endoliths, organisms that live within the porous structure of the rock itself and the chasmoliths that live within the cracks and crevices.

Due to their structure and mineralogy, sedimentary rocks such as the local sandstone are particularly well-suited for housing endoliths a few millimeters beneath their surface. Enough light penetrates for photosynthesis (at least during the months when there is sun), while damaging UV is reduced. In contrast to the soil community, these rock dwellers are mostly photoautotrophs. Two distinct communities of bacteria and eukaryotes are detectable, both visibly and experimentally. The upper 2 mm, called the lichen zone, is dominated by a lichenized fungus, Texosporium sancti-jacobi, associated with the green alga Trebouxia jamesii. Below 2 mm, the photosynthetic cyanobacteria rule, mostly Chroococcidiopsis, a genus noted for having radioresistance comparable to that of Deinococcus radiodurans. Low light levels not withstanding, light may not be the factor limiting these endolithic communities, but rather lack of available CO₂. The chasmolith communities in the crevices are made up primarily of various lichens and cyanobacteria, combined with a distinctive sprinkling of other bacterial groups.

A typical quartz hypolith showing no external evidence
of the underlying microbial community. Source.

There is yet a fourth cryptic community here, the hypoliths that live underneath light-colored, translucent stones. These are almost exclusively cyanobacteria, making do in an environment that receives less than 0.1% of the incident light. There are no fungi here, and only a very few algae.

There is a tendency to think that the more extreme the environment, the less the biological diversity. The communities in the Dry Valleys don't conform to that pattern. The endoliths and chasmoliths combined comprise more than 50 bacterial species (based on 98% identity of their 16S rDNA sequences). Although eukaryotes represent only 5% of these communities, they include four genera of fungi (both Ascomycota and Basidiomycota) as well as three groups of algae. Add in the hypoliths, and these three lithic communities span 16 phyla in two domains.

What about the missing archaea, those masters of extreme environments? Not a one to be found so far, not even one lurking under a rock. Surely there are yet even more surprises to be found by the cryophilic researcher.

Leaf-Cutters Get Their Fix (nitrogen fix, that is)

2010-01-14T20:38:34-08:00

by Elio

A single leaf-cutter ant fungus garden chamber with the
queen (black arrow). Scale bar: 1 cm. Source.

Ants span a great distance within the human psyche, from being insignificant little nuisances to occupying a prominent place in the biological scheme of things. They surely deserve the latter. Not only do they make up a large proportion of the animal biomass (up to 25% of the total in the tropics), but their social qualities make for substantial mind food. It was not always so. Their very name is tendentious: it is derived from the West Germanic amaitjo, meaning "the biter," from Proto-Germanic ai-, "off, away" + mait- "cut." (If you see little connection between these roots and the word ant, keep in mind the Rules of Etymology, as expressed by a linguist friend: The vowels don't count and the consonants have arbitrary values.)

Few things have the psyche-tickling value of the leaf-cutter ants, those master agronomists that some 50 million years ago learned to cultivate their fungal foodstuff. The leaves they so assiduously bring into their nest become the substrate on which the fungi grow. To deter unwanted fungal species that would otherwise move into their “fungus gardens," the ants harbor bacteria (Pseudonocardia) that make selective antifungal products. But their agricultural endeavors still leave us with a question: Where do the ants get their nitrogen? The plant tissues they use are poor in nitrogenous compounds and, of course, neither they nor their fungi can fix nitrogen. We’ll tell you in a minute, but first a startling digression.

A partly excavated nest of a mature colony. Source.

The nests these leafcutter ants make are astounding in size, containing as many as eight million worker ants and occupying an underground volume of more than 20 m³! That’s about the size of a 100 square foot room, about the size of my small den. Researchers excavated such a nest, with a stunning visual effect (although we don't expect the ants approved of it).

Now, how about the source of nitrogen? The recent paper by the pioneer myco-myrmecologist (that’s a term we just made up for fungus-ant expert) Cameron Currie and colleagues comes up with the answer: the fungus gardens contain nitrogen-fixing bacteria that provide a substantial proportion of the needed nitrogen. The evidence? The nitrogen content is low in the plant food, greater in the fungus gardens, and greater still in the spent plant material that the ants carry to their midden-like refuse dumps. In addition, the authors measured acetylene reduction, a tried and true method for measuring the activity of nitrogenase, the enzyme that carries out nitrogen fixation. Nitrogenase activity was highest in the fungus gardens, although the level varied with the species of ants. Little enzyme activity was associated with the ants or their Pseudonocardia symbionts, but activity was found in bacteria isolated from the fungus gardens using nitrogen-free media. Prevalent were nitrogen-fixing species of Klebsiella and Pantoea.

What does it take for these symbiotic consortia to be successful? As the authors point out: The ecological success of leaf-cutter ants is derived, in large part, from the combined ability of the ants to break down plant antifungal barriers and of the fungus garden to neutralize plant antiinsect toxins. As a result, leaf-cutters can subsist on a large variety of plants, many more than other herbivores. But they must rely on nitrogen-fixing symbionts for their nitrogen. In this, they resemble termites, which are known to have such bacterial symbionts. Not only does nitrogen fixation by the symbionts help the ants, it may also add fixed nitrogen to the environment, via the detritus from the spent fungus gardens.

Leaf cutting ants left their hunting-gathering mode of life and became farmers long before humans did. In the process, they have acquired some pretty amazing uses for symbiotic relationships. They sure know how to farm sustainably!

Through the Looking Glass: Silicate in Bacterial Spores

2010-01-11T08:30:00-08:00

by Peter Setlow

Silicon location in spores. Spores of a high silicate-
adsorbing Bacillus isolate of the B. cereus group
were prepared in silicate-containing medium. TEM
Panel: an ultrathin section of a spore by transmission
electron microscopy. The granules on the outer sur-
face of the coat were absent from spores prepared
in silicate-free medium. (CX = peptidoglycan cortex;
UC = undercoat; CT = coat; SX = silicon-containing
granules; EX = outermost exosporium layer.) SI-K
Panel: STEM-EDX image with the silicon pseudo-
colored green. Merge Panel: TEM and Si-K images
combined. Bar = 100 nm. Source.

Amid the furor surrounding the anthrax attacks in the USA in 2001, significant attention was focused on whether the Bacillus anthracis spores used had been “weaponized” by the adding of a coating of fumed silica to aid in their dispersal. This information might have helped pinpoint where the spores had originated. However, a confusing factor was that for more than 20 years, significant levels of silicon had been reported in spores of at least some Bacillus species, including those of Bacillus cereus, a close relative of B. anthracis. Clearly, the natural presence of silicon in B. anthracis spores makes discrimination between weaponized and non-weaponized spores more difficult. However, in the older reports the silicon was not localized at the outermost surface of the spores, as would be expected if they had been artificially spiked with such compounds.

Anyhow, what is silicon doing on a bacterial spore? A recent paper provides some clues. Starting with soil from a paddy field, these researchers identified a number of novel bacterial isolates that efficiently take up silicate present in media at concentrations comparable to levels found in the water in soils. Strikingly, all of these isolates were Bacillus species and most were members of the B. cereus group that includes the pathogens B. anthracis, B. cereus, and Bacillus thuringiensis. Silicate uptake by the novel isolates was associated only with spore formation (sporulation), and was a late event in this process. However, while sporulation was required for efficient silicate uptake, silicate uptake itself was not essential for sporulation.

Visualizing silicate on the spores of these new isolates required some fancy techniques, namely scanning electron microscopy coupled with energy dispersive X-ray spectrometry (STEM-EDX). The silicate taken up was present, at least in part, as granules on the outer surface of the spore coat, with some perhaps in the coat itself. Note that the outermost layer in B. anthracis and B. cereus spores is not the coat but rather the exosporium, a loosely-fitting outermost structure found on some, but not all, bacterial endospores. Analysis of silicate uptake showed that laboratory B. cereus and Bacillus thuringiensis strains, as well as a more distantly related Bacillus megaterium strain, also accumulated silicate on spores, although at lower levels than the novel Bacillus isolates. Interestingly, the well-characterized Bacillus subtilis 168 laboratory strain took up no detectable silicate. These new results make it clear that spores of at least some Bacillus species incorporate significant amounts of silicate and deposit it on the spore coat. It is notable that this silicate is not on the spore’s outer layer, and thus it should be possible to discriminate between weaponized and non-weaponized B. anthracis spores by STEM-EDX analysis.

Bioterrorism aside, this work raises several interesting questions. The first concerns the mechanism of silicate accumulation on spores. It appears to be mediated by the mother cell in which the spore matures, but how the mother cell does this is not known. Finding this out may lead to an understanding of why some Bacillus strains/species accumulate silicate and some do not. The second obvious question is why would a spore accumulate silicate at levels up to 6% of the spore’s dry weight. One possibility is that this might facilitate dispersion of single spores, much as does an outer coating of fumed silica. However, according to this paper, high levels of silicate on the spore coat did not enhance the dispersal of dry spores. Thus, silicate accumulation must play another role.

Indeed, whereas the spores’ silicate plays no role in spore resistance to heat, hydrogen peroxide, UV radiation or NaOH, it significantly increases spore resistance to killing by 0.1-0.4 N mineral acids. This increased acid resistance might be particularly important in spores of pathogens such as B. cereus and B. anthracis that may pass through an acidic mammalian digestive tract. On the other hand, this would not be important in the alkaline digestive tract of the insect forms for which B. thuringiensis is pathogenic. Therefore, it seems likely that the spores’ silicate layer may serve an additional function. Since silicate accumulation in other organisms can impart structural rigidity, perhaps silicate plays such a role for spores as well. This leaves us with yet more interesting questions to address to these spores.

Peter Setlow is Professor of Molecular, Microbial and Structural Biology at the University of Connecticut Health Center in Farmington, CT.

An Open Invitation to Argue With Me

2010-01-08T08:30:00-08:00

by Elio

I have written a “think piece” published in the December 2009 issue of ASM’s journal, Microbe, in which I list my choices of what constitute paradigm shifts in modern microbiology. The choice was entirely idiosyncratic. I am sure that others would include different important developments on their roster. I also make the point that some apparent P.S.’s are really “paradigm drifts. Chacun à son goût, if you pardon my French. So, here’s your chance to expound you views on this matter. What breakthroughs in microbiology do you consider worthy of being called paradigm shifts?

Talmudic Question #57

2010-01-07T10:35:30-08:00

by Frank Harold

Does anyone know of a solid example of a biological membrane that arises de novo, rather than by the extension of a pre-existing membrane?

How Proteomics Got Started

2009-12-21T08:30:00-08:00

by Fred Neidhardt

Van Gogh's Starry Night. (1889)

The following is a personal account of the beginning years of what is now called proteomics. Odd that I, almost a Luddite, should be writing about the origin of a field initiated by a dramatic technical advance; I tend to avoid complex new scientific instruments and techniques. As a graduate student under Boris Magasanik at Harvard Medical School during the early 1950s, I was glad that my project (induced enzyme synthesis in bacteria) could readily be approached with simple technology. Bacterial growth could be monitored turbidimetrically with a Klett colorimeter; the same instrument could provide colorimetric assays of enzyme activities. Only the phage geneticists of that era, using sterile toothpicks to pick viral recombinants or mutants from plaques on Petri dishes, had it technologically easier.

Around me at that time in Harvard University’s Department of Bacteriology and Immunology (now Microbiology and Molecular Genetics) were gifted individuals who on occasion were forced to purify proteins using laborious and personally onerous techniques. Not a life for me, I decided, even though H. Edwin Umbarger assured me that purifying an enzyme “developed character.”

Beside laziness, there was a second, more fundamental, reason I never purified a protein. Cell growth was the biological event that had hooked me as a graduate student, and work that began by smashing cells into little bits seemed inappropriate.

Nevertheless, within the next six years I would find myself absorbed in two major aspects of cell growth physiology that involved proteins, and these subjects would prove more intractable than the purification of proteins. Catabolite repression (or, more generally, how bacterial cells choose to utilize multiple carbon sources), and growth rate modulation (how bacterial cell size and composition are interrelated with growth rate) were two processes directly related to cell growth rate.

The comprehensive work of Schaechter, Maaløe and Kjeldgaard on Salmonella cell growth rate and composition appeared shortly before my studies with Klebsiella aerogenes. Fundamental laws of bacterial growth were established by these studies in the early 1960s. Nevertheless, these laws were supported only by observation and by the easy rationale of their selective value to the cell; they were bereft of biochemical explanation. Both catabolite repression and growth rate modulation proved to be fascinating, but vexing; only now, fifty years later, are these processes approaching mechanistic solution.

A major reason for their intractability lay in limitations in our ability to approach the living bacterial cell. For most of the 20th century the study of the physiology of bacteria (and, indeed, all other organisms) was largely reductionistic. The living cell was taken apart and studied biochemically, or was dissected by the increasingly powerful marriage of biochemistry and genetics. The triumphs of this approach were notable.

Still, catabolite repression and growth rate modulation joined a list of questions that could not be answered by the reductionistic approaches of biochemistry, even when augmented by the power of genetic analysis. Questions of the following sort had to be postponed (or were never asked) because the tools to approach them were not available:

Why don’t bacteria of a given species grow at the same rate on all carbon and energy sources?

Is there a growth rate-limiting step during steady state growth of a bacterial culture?
How many changes take place in a bacterium transitioning from growth to non-growth?
What causes the size and macromolecular composition of a bacterial cell to be much more dependent on its rate of growth than on the chemical nature of its food?
How do bacterial cells prioritize their choice of food when given options?

By the mid-1970’s my mind, filled with unanswered questions about growth physiology, was searching for a new way to approach the bacterial cell. That way was revealed, not by anyone in my laboratory, but by a graduate student named Patrick O’Farrell at the University of Colorado at Boulder. A postdoctoral fellow in my laboratory at the University of Michigan, Steen Pedersen, one of the keenest of disciples of Ole Maaløe in Copenhagen (and one of his most honest critics) returned from a visit to Colorado in 1974 and reported to our laboratory that a graduate student there had produced a two-dimensional polyacrylamide gel system that could resolve the proteins of a bacterial cell on an array that looked as cool as “the sky on a starry night.”

Source: EcoSal.

Steen’s information electrified us, for we realized that a fundamentally new approach to bacterial growth physiology had become possible. Instead of asking the cell for information about a protein of interest to us, we could finally interrogate the cell about the proteins that were important to IT in any given situation. The cell could now reveal to us what lay behind the biological Green Door (in reference to an infamous American pornographic film of that era). For the first time the road to a global analysis of cell physiology could be imagined. And, in retrospect, it is clear that the era of proteomics began in 1975, the date of publication of Patrick O’Farrell’s thesis research in the Journal of Biological Chemistry. His paper was quickly recognized by a variety of molecular biologists as a true technological breakthrough. Citations in the next 30 years numbered over 16,000 (in spite of the fact that the manuscript was initially rejected with two disparaging reviews which had to be overruled eventually by members of the journal’s editorial board).

For the first time we could now learn what the cell had to teach us about its complement of proteins and about adjustments to different environmental conditions. This new ability to listen to the cell led soon to new insights into growth rate physiology. But before this could happen it was necessary to add several features to the O’Farrell technique.

First, we recognized that we had to standardize the two-dimensional gel system of O’Farrell in order to compare the protein arrays from different samples. This required extreme attention to details of procedures and quality of reagents. The genius of O’Farrell’s system was that it employed two independent properties of proteins to separate them: their molecular weight and their isoelectric point. Isoelectric focusing in a gel tube containing ampholines to establish a pH gradient produced the first dimension—proteins lined up by their charge. Placing the resulting tubular gel on an electrophoretic gel slab containing sodium dodecyl sulfate, allowed the polypeptides previously resolved by charge now to be segregated by their size. The resulting two-dimensional polyacrylamide gel (2-D gel) was then stained and dried for subsequent inspection. A beautiful picture—but to be useful, 2-D gels had to be reproducible, and this was not an easy task for a number of reasons. In the end it took years of perfecting sample preparation and gel casting (not to mention improvements in ampholines) to get to the stage where computer-driven pattern matching could align a whole series of “starry patterns” from the multiple samples of an experiment.

Second, once the pattern-matching problem was in hand (no small feat), the issue became one of accurate measurement of the quantity of protein in the individual spots across the gel set. Clever uses, first of isotopes, then of differentially colored samples, were devised to obtain reasonable quantification. As a result, it became possible for the cell to display much of the array of changes made in its proteome (the totality of its several thousand proteins) as the cell adapted to its environment.

Fortunately, these tasks of standardizing and quantifying O’Farrell gels were approached by many individuals skilled in scientific technology. James Garrels at Cold Spring Harbor Laboratory, Norman G. and N. Leigh Anderson at Argonne National Laboratory, and Julio Celis at the University of Aarhus, Denmark, were some of the people who early on used their considerable skills to expand the usefulness of 2-D gel technology.

But still a third attribute had to be added to 2-D gels for maximum usefulness: the identities of the “starry” spots on the gels had to be determined. For the bacterium Escherichia coli and its close cousins, my laboratory in Ann Arbor mounted a full-scale effort to correlate spots on the 2-D gels with known proteins. Hundreds of protein spots were identified through the use of purified proteins (donated, naturally, by others) and mutants in known genes. Everyone in my laboratory contributed to this effort; unfair as this is, I’ll single out only two because of their germinal work in identifying spots and because of their tireless energy in teaching the 2-D gel process to all the others: Ruth A. VanBogelen and Teresa Phillips.

Needless to say, the identification of spots might be regarded as tedious drudgery—and it was—save for the thrill that we were simultaneously making discovery after discovery using the 2-D gels: heat-shock and cold-shock proteins, proteins under stringent control, proteins that vary monotonically with growth temperature, proteins that vary with growth rate—and we were not simply learning which proteins exhibit a certain behavior, but what fraction of the cell’s proteome was involved in different physiological responses to stress or starvation. These discoveries led Ruth VanBogelen and her colleagues to the concept of protein signatures. A protein signature is the set of proteins that, by their amplification or suppression, signal a particular physiological stress state of the cells. One learned how to recognize when a cell was in a state of energy starvation, or oxidative stress, or membrane damage, or… the list goes on. One can imagine the gigantic usefulness of this approach when a pharmaceutical company is exploring how a potential therapeutic agent acts.

But we should bring this story to a close quickly, because from the mid 1990s onward the explosion of cell protein technology transformed the field from what Pat O’Farrell had created to one with a formidable arsenal of techniques for protein resolution and measurement. The term proteome was introduced in 1996 to refer to the totality of proteins in a cell, and this quickly gave rise to the noun, proteomics, to designate studies of the proteome. The 2-D gel technique introduced by Pat O’Farrell has inspired others to develop improved techniques for monitoring the global pattern of a cell’s total protein complement. The availability of DNA sequences with reasonably accurate annotations, for the genomes of hundreds of species has made it possible to develop separation techniques that enable tandem mass spectrometry to provide the “second dimension” to primary fractionation procedures, and as a result, enable protein identifications an order of magnitude beyond that which was achieved in the first two decades of the 2-D era.

To be sure, the current armamentarium of proteomics is being used in highly targeted ways to explore previously identified sets of “proteins of interest” (as our law enforcement agencies might call them), but I want to emphasize that Pat O’Farrell’s development of the first method of spreading out the proteins of a cell was at the start, and particularly for me, the initiation of an exciting new grammar of scientific questioning.

Frederick C. Neidhardt is F.G. Novy Distinguished University Professor, Emeritus, Department of Microbiology and Immunology, University of Michigan Medical School at Ann Arbor.