Small Things Considered

Microtubules in the Verrucomicrobial Closet

by Daniel P. Haeusser

Figure 1. Global phylogeny of the tubulin superfamily. Source.

General biology textbooks, like the one I use for teaching, often depict the prokaryotic cell as an oval of homogenous-appearing cytoplasm surrounded by two membranes. This simplified image of a Gram-negative bacterial cell contrasts with the depiction of a well-organized, feature-laden eukaryotic cell with its membrane-bound organelles. While many would agree that the absence of a membrane-bound nucleus defines the prokaryotic–eukaryotic boundary, the recent decade of research has given us a glimpse of the actual organizational complexity of bacteria, which includes encoding cytoskeletal elements. Just as eukaryotes have their tubulin, actin, and intermediate filaments, the prokaryotes have FtsZ, MreB, and crescentin (although the occurrence of each varies greatly).

The first of these prokaryotic cytoskeletal proteins to be discovered, FtsZ, is a distantly-related member of the tubulin family (Figure 1). FtsZ is a highly conserved protein involved in cytokinesis in most bacteria, certain archaea, chloroplasts, and in the mitochondria of some protists. Although FtsZ shows only ~17% amino acid identity with tubulin, their crystal structures are remarkably similar (a fine example of structure trumping sequence).  Notably, FtsZ, unlike tubulins, is unable to form microtubule-like structures; instead it just self-associates into loosely bundled protofilaments. Although a recent paper shows that FtsZ forms tubule-like structures in vitro in the presence of a binding partner/assembly regulator, these tubules are quite distinct from eukaryotic microtubules in both size and structure, and their in vivo relevance is uncertain.

For many years FtsZ remained the only member of the tubulin family to be identified in prokaryotes. However, reports of microtubule-like structures observed in specific bacterial species have continued to appear (reviewed here). As further evidence of their tubulin nature, many of them have been found to be sensitive to tubulin depolymerization agents and to react with anti-tubulin antibodies. Unfortunately, none of these observed structures have been matched to particular genes, so their genetic identity remains a mystery.

Figure 2. TEM of P. dejongeii showing a prosthecae (PT) with
inset highlighting the intracytoplasmic membrane (ICM): an
example of prokaryotic compartmentalization. Source.

The study of bacterial tubulin received a tremendous boon in 2002 with the discovery of true α- and β-tubulin homologues (named BtubA and BtubB) in bacteria. These proteins share 37% sequence identity with tubulin although they cluster phylogenetically in their own group. They were found in the Prosthecobacter, a free-living genus in the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum that has been debated as being an evolutionary link to eukaryotes. Prosthecobacter are morphologically defined (and named) by an elongated polar appendage (the prosthecae) (Figure 2).

The discovery of BtubA/B naturally led researchers to investigate whether the proteins formed microtubule-like structures in a Prosthecobacter cell. Disappointingly, no such structures were seen either in vivo or with in vitro-assembled BtubA/B. Thus, despite the homology between BtubA/B and tubulin, it appeared as if they were as incapable of forming true microtubules as FtsZ.

This has now changed. As is often the case, new techniques and imaging capabilities have revealed structures that previous studies failed to visualize, specifically BtubA/B structures that may have been destroyed by conventional electron microscopy sample preparation. Last month, researchers using electron cryotomography (ECT) published a report of BtubA/B microtubule-like structures in several Prosthecobacter species (Figure 3). These microtubules run parallel to the membrane, individually or in small bundles, predominantly in the cell stalk or in the region where the stalk transitions into the cell body. The authors would have liked to demonstrate that the microtubules consisted of BtubA/B by knocking out the btub locus and observing the loss of the tubules, but the required genetic system of manipulation is not yet available. Fortunately, another species could substitute for the nonexistent mutants. This species, P. fluviatilis, lacks the btub genes, makes no BtubA/B, and possesses no discernible microtubules. More evidence: microtubules formed when BtubA/B were expressed in E. coli, and they could also be assembled in vitro.

Figure 3a. BtubA/B assemble into five-protofilament
microtubules. Shown here is an 11 nm thick cryo-
tomogram slice of P. vanneervenii with microtubule
structure bundles (arrows), and in 3D reconstruction.
Bar = 100 nm. Source.

In ECT tomograms, the Prosthecobacter microtubules are ~200 – 1200 nm long, with an average diameter of 7.6 nm and a repeat distance of 4.4 nm (Figure 3a). Notably, these dimensions are similar whether the microtubules are produced by Prosthecobacter species, expressed in engineered E. coli, or assembled in vitro. How does their fine structure compare to canonical eukaryotic microtubules? The published crystal structure of the BtubA/B dimer fits best with a five-protofilament tubule model with 4.6 nm spacing (Figure 3b). In contrast, the number of protofilaments in eukaryotic microtubules is usually 13. Spacing, however, is conserved (~5 nm in eukaryotes), which suggests that in both groups the filaments form one-start helical tubules.

Figure 3b. Pseudo-atomic model of five-
protofilament bacterial microtubule (blue;
built from Protein Data Bank ) superimposed
on the image of a cryo-sectioned BtubA/B
tube (black). Bar = 10 nm. Source.

One might expect that BtubA/B or related genes could be found in other bacteria as well, particularly considering that microtubule-like structures have been observed in several other species. For example, consider the uncultured ectosymbionts of Euplotidium ciliates called epixenosomes (meaning external alien body), an amazing defense system featured in this blog several years ago. The epixenosomes are also members of the phylum Verrucomicrobia, and they contain observable microtubules. However, the lack of a genetic system again makes genetic verification impractical, thus their actual relation to BtubA/B, or the tubulin family in general, is still unclear.

So, where do BtubA/B fit evolutionarily within the tubulin family (Figure 1)? On the one hand, they share traits with FtsZ that tubulin lacks, namely chaperone-independent folding and weak dimerization. However, BtubA/B can form heterodimers, something that is generally not possible for FtsZ, except in rare species or in chloroplasts that encode more than one type of FtsZ. The authors of the ECT report agree with the suggestion that BtubA/B represent an ancient form of tubulin that preceded the larger, 13-protofilament microtubules seen in eukaryotes. The unique presence of BtubA/B in Prosthecobacter and related species suggests that they originated via horizontal gene transfer, but from whence they came remains enigmatic.

Figure 4. Immunofluorescent staining of
BtubA/B coexpressed in E. coli. Bar = 2 μm.
Source.

The big question remaining for BtubA/B and the microtubules they make is what function they serve. An early hypothesis, given the reduced genomes of many PVC superphylum members, was that they functioned in cell division in place of FtsZ. Subsequent work showed, however, that Prosthecobacter do encode ftsZ, making a role for BtubA/B in cell division less likely. However, Prosthecobacter FtsZ diverges in key residues important for assembly and GTP hydrolysis. Similarly, the Thaumarchaeota are known to encode ftsZ with a highly divergent tubulin/FtsZ signature sequences, and they utilize the Cdv system for cytokinesis instead. Alternatively, given the localization of BtubA/B observed in Prosthecobacter, one might expect they contribute to the structure or formation of the prosthecae, but similarly-shaped species exist without the btub genes or observable microtubular structures (e.g. P. fluviatilis). When co-expressed in E. coli, BtubA/B form microtubules that run parallel to the membrane the entire length of the cell (Figure 4), but I know of no specific phenotype that they confer outside of those that could occur from general foreign protein overexpression.

The development of a genetic system in a species that encodes BtubA/B would be ideal, but until then the study of existing model systems may be the only route for further exploration of BtubA/B assembly and function. Regardless, the unique properties of BtubA/B in sharing characteristics of both tubulin and FtsZ make even their in vitro study highly relevant to the understanding of this cytoskeletal family. Such studies may answer pending questions about the regulation of bacterial microtubule assembly, as well as their directionality, dynamic instability, and accessory protein requirements—all properties that the more divergent tubulin-family member FtsZ appears to lack. Another distant tubulin-family member, TubZ (Figure 1) from a virulence plasmid of B. thuringiensis, does display treadmilling without any apparent tubule formation, and will aid such comparative studies within the tubulin family.

Finally, it leads one to wonder: What other eukaryotic garments are residing unidentified and uncharacterized in the prokaryotic closet?

Daniel is a postdoctoral fellow in the Margolin Lab in the Department of Microbiology & Molecular Genetics at the University of Texas, Houston Medical School. He also teaches as an adjunct professor at the University of Houston-Downtown in the Department of Natural Sciences. In addition to science, he enjoys reading, writing, and film. He can be found on Google+.

Pilhofer M, Ladinsky MS, McDowall AW, Petroni G, & Jensen GJ (2011). Microtubules in bacteria: ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton. PLoS Biology, 9 (12) PMID: 22162949

Green Flypaper

by Merry

Source.

Troubled by pesky flies in your home or office? Tempted to reach for the toxic flypaper? Here’s a green alternative long known by the Indians of the Central Mexico highlands. Step outside and look around for a tree with entire branches covered by a large spiderweb. Clip out a section of the web and hang it indoors, complete with resident spiders. A natural fly trap. Common house flies will be attracted to the web, lured to their death by a sweet smelling bait with a microbial source.

Make certain, though, that you’ve got the right kind of web. Only those made by the social spider Mallos gregalis, will work. Social spiders? Granted, most spiders are cannibalistic loners, but approximately 23 of the 39,000 spider species have opted for varying degrees of permanent, non-territorial sociality (quasisociality). The spiders in these casteless communities cooperate in hunting, feeding, nest construction, and, in some cases, caring for the spiderlings, as well.

The web of a different social spider from the Amazon River
region of Brazil. Source.

Solitary M. gregalis spiders kept in the lab can construct webs and hunt pretty successfully, but the webs are simple structures. In nature, as many as 20,000 live together in a single, intricate, communal web. The outer surface is the primary site for prey capture. The interior labyrinth of silk-lined tunnels and chambers is where the spiders hang out except when vibrations from struggling prey send them to the surface to feed. By cooperating, this spiders can capture more and larger prey, with less silk production required per individual.

Muscoid (housefly-like) flies like to swarm around M. gregalis webs in particular, drawn by  their  noticeable “sweet, yeast-like” odor. It is the not silk that draws flies, but the odor from “web decorations.” These spiders, unlike most others, leave the carcasses of partially consumed flies in their webs. Remove the carcasses from the web and the scent disappears.

What’s going on here? Researchers in the 1980s using simple culturing assays determined that fed-upon fly carcasses are host to abundant yeasts of several types, whereas unfed-upon carcasses or those fed-upon by other spider species host predominantly bacteria. Cultures of yeasts derived from the carcasses fed-upon by M. gregalis, emanated the same sweet scent and attracted flies. They concluded that the feeding by the spiders alters the conditions in the carcasses so as to either give the yeasts a growth advantage or inhibit the growth of bacteria or both. To my knowledge, this is the first example of a predator using microflora to attract prey by means of odor. The tactic, though good, has a flaw. If more prey are attracted than can be  consumed by the spiders, their bodies will be decomposed by the bacteria, thus releasing a dominant ammonia odor that repels the flies. The spider population will now crash or emigrate en masse.

Theridion nigroannulatum, another social spider, with egg sacs. Credit:
Ingi Agnarsson. Source.

The researchers referred to this as a spider-yeast symbiosis, but I suspect there is another spider-associated microbial player that may be the source of an antimicrobial that keeps the bacteria in check. Just my guess. But as the story stands now, it provides postscripts to two of our earlier blog posts by providing one more reason why spiders decorate their webs and another example of an arthropod ringing a microbial dinner bell. The time seems ripe for someone to pick up the thread and pursue this story further using current ‘omics techniques.

Tietjen, W., Ayyagari, L., & Uetz, G. (1987). Symbiosis Between Social Spiders and Yeast: The Role in Prey Attraction Psyche: A Journal of Entomology, 94 (1-2), 151-158 DOI: 10.1155/1987/67258

The Paenibacillus Moving Company

by Elio

Enlightenment, a bacterial colony. Displayed in the
Esher Ben-Jacob online art gallery. Source.

Microbes get around. They can be carried by the wind, by insects, or by water currents, sometimes across large distances, sometimes from one grain of soil to the next. Some bacteria contribute to their own dispersal by their motility or, rarely, one may think, by the motility of other microbes. It’s hard to generalize here because there are just too many variables, too many conditions where microbes are on the move. But this business can be pure fun. So it’s with pleasure that I read about a delightful account of how bacteria can transport fungal spores to the benefit of both.

But first a little background, both microbial and personal. Some bacteria, when placed on an agar surface, do not stay put but spread out from the point of inoculation. Certain ones, such as Bacillus mycoides, do it simply by extending their long filaments, but others do it by moving on the agar surface. There are several ways to move along, either with flagella (a process called swarming) or without them (e.g., gliding or twitching). Swarming, whether in Gram-positives like certain Bacillus species or in Gram-negatives such as Proteus, is a communal activity. Single cells don't swarm, aggregates of several cells do. What you see under the microscope are rafts of bacteria arranged side by side, moving along a path that is sometimes regular, sometimes not. To download a movie of a ‘raft’ of ceils swarming together, click here (or to go to the web page where you can select additional file 4, click here). The speed of movement across the agar surface is pretty amazing, about 180 µm/min or 10 mm/h. This means that if you place such a bacterium in the center of a 14 cm Petri dish, it will reach the edge in about seven hours. When such a swarm has covered a certain distance, the cells either stop moving, as in the case of Proteus, or make round disc-like colonies that have the odd ability to rotate. For a movie of a ‘colony’ of cells swarming together, click here (or to go to the web page where you can select additional file 2, click here). The speed at which this rotation happens is also quite fast, one revolution in about 10 seconds. Towards the end of my career, but while I still had a lab, my collaborator Dana Boyd and I played around with these organisms. This was the last time that I worked at the bench, so I quite cherish this topic.

A typical colonial pattern generated by P. vortex when grown on 2.25%
(w/v) agar with 2% (w/v) peptone in a 9 cm Petri dish. (A) Whole colony
view. Each vortex (the condensed group of bacteria, the bright dots) is
composed of many cells that swarm collectively around their common
center at about 10 μm/s. (B) An individual vortex. The diameter of the
vortex is 75 μm. Source.

Peering at bacteria that are swarming is a game most anyone can play. If you have access to a Petri dish with agar in it, take a few grains of almost any soil and place them in the center. Within six hours or so, you will see that among the many small colonies on the surface there will be some that moved away from the inoculum site. What you will see are tracks with ‘colonies’ at the end. Most likely, you are now looking at Paenibacillus, the organism I’m featuring here.

Paenibacillus, once merely known as Bacillus (the current name means almost Bacillus), of which there are many species, is one of the champion swarmers that has puzzled microbiologists for over 100 years. So, you ask, why do they swarm? Hmm, it would seem intuitively obvious that they must be scavenging for food. Not so fast. The swarms do not exhibit chemotaxis or any other preference for needed nutrients, as some other bacteria do. We found that our strains could swarm on agar containing just phosphate buffer, period. For good measure, we added chloramphenicol, to which these bugs were sensitive, and that didn't impair their travels. At no time did we see the swarms move preferentially towards a place on the plate where we had put any of a variety of nutrients.

In our efforts to figure out what this is all about, we confirmed something that had been known, namely that the bacteria could carry around other bacteria, such as cocci. To take this to the limit, we used 1 µm diameter latex particles and, guess what, they were spread around just like the bacteria themselves. These particles seemed to be pushed along, either at the head of a raft or trapped within it. So at that point we started to think that swarming could be used for interactions with other organisms, possibly to carry along species that could furnish the swarmers with food in certain fallow environments. We never demonstrated this, although it seems as plausible today as then.

However, to my satisfaction, others are having a longer and more productive look. In particular, microbiologist Colin Ingham teamed up with physicist Esher Ben-Jacob and colleagues Oren Kalisman, and Alin Finkelstein to ask probing questions of these bacteria. Not only that, but they are having fun with the patterns that bacteria can form on agar as witnessed by a gallery of sensational pictures. In a recent paper, these investigators showed that these bacteria are able to move the spores of a fungus, Aspergillus fumigatus. This fungus is a common soil organism known to cause disease in immunocompromised patients. The fungal spores are not motile, thus dependent on water or air currents for dispersion. It turns out that if you mix these spores with cells of a species of Paenibacillus called P. vortex, the spores are moved along for quite a distance, at the same rate as the bacteria. After suitable incubation, fungal colonies appear all over the plate. It’s up to you to imagine the practical implications of these findings.

Volga Boatmen, by Ilya Repin. Source.

These microscopic ferrymen can haul quite a cargo. It can consist not just of individual spores but also spore aggregates 25 µm across (each spore is about 3 µm in length). This reminds one of ants that can carry up to one hundred times their body weight. It gets better. These bacteria can even impel the fungal spores across an area on the agar impregnated with an antifungal growth-inhibitory compound, a veritable minefield.

In searching for the mechanism involved, the authors found that not all fungal spores can be so translocated. Spores of even some close relatives of A. fumigatus do not play the game but stay put on the agar. Also, germinating spores are not transported efficiently. Under the electron microscope, spores being carried are seen surrounded by bacterial flagella. And, sure enough, adding purified flagella inhibited their transport. This suggests the presence of receptors for flagella on the surface of certain spores and not others. So, are there two different mechanisms involved, one for non-specific particles such as latex beads, another specific for certain cargoes? Does this have anything to do with the size of the object being moved, the larger ones requiring chemical recognition by the bacteria?

Bacteria crossing a canyon in the Petri dish along
natural bridge of fungal mycelia (top, courtesy of Colin
Ingham), much like animals crossing a canyon in Nepal
on a man-made bridge (bottom, courtesy of Aya Ben-
Yakov). Source.

It is easy to see what's in it for the fungus. It gets translocated, and thus is dispersed to new sites. But what's in it for the bacteria? The investigators did a simple but clever experiment: they cut a slit in the agar a half millimeter wide. The bacteria could not cross it alone, but the fungi had no problem spanning the gap with their hyphal threads. And when both bacteria and fungi were present, guess what? On the other side they now found bacteria that had done a high wire act on the fungal tightrope. In other systems, it has been shown that bacteria make biofilms on the surface of hyphae so, in a way, this was to be expected.

Not only do these bacteria move on agar at a fine clip, but they do it even under conditions of starvation. As I mentioned already, in my lab we saw swarming taking place at normal speeds on agar that contained only phosphate buffer. No added food. They seem to want to move at all costs, consuming precious metabolic reserves in the process. Swarming is their top physiological priority. So one would guess that this is a property that has been selected for in a big way during evolution. Whatever the purpose, we may have been premature in ruling out some kind of chemotaxis. In the supplemental material to one Ben-Jacob paper (To see the movie, click here, or to go to the web page where you can select additional file 5, click here), there is an indication that swarms of bacteria move towards extracellular material left in the trails, although this may not be for nutritional reasons

Distribution of the relative Social IQ scores of 502 bacteria. Green indicates bacterial strains
that belong to the Paenibacillus genus. Source.

Now a final word about Paenibacillus and its genome. It turns out that it’s one smart bug, in fact, brilliant by one criterion. Ben-Jacob scored the genomes of many bacterial species for the number of genes potentially involved in social behaviors. He proposed a bacterial Social IQ scale, which he describes as: a quantitative score.… to assess the genome potential of bacteria to conduct successful cooperative and adaptable behaviors (or social behaviors) in complex adverse environments…. The score is based on the number of genes which afford bacteria abilities to communicate and process environmental information (Two-component system and transcription factor genes), to make decisions and to synthesize offensive (toxic) and defensive (neutralizing) agents as needed during chemical warfare with other microorganisms. Now for the punch line: Three bacteria species stand out with significantly high Social-IQ score among all sequenced bacteria,—over 3 standard deviations higher than average, indicating a capacity for exceptionally brilliant social skills). Notably, all these species belong to the same bacteria genus—the Paenibacillus…^.

Ingham CJ, Kalisman O, Finkelshtein A, & Ben-Jacob E (2011). Mutually facilitated dispersal between the nonmotile fungus Aspergillus fumigatus and the swarming bacterium Paenibacillus vortex. Proceedings of the National Academy of Sciences USA, 108(49): 19731-6. PMID: 22106274.

Talmudic Question #83

Assume the Earth is hit by a large asteroid that lowers the average temperature of the planet to the extent that would eliminate most multicellular life. What would be the short term consequences for the microbial world? And later on?

Pushing the Thermodynamic Envelope into the Proteomic Edge

by Tracey McDole

A schematic potential energy funnel for the folding of
proteins without sufficient water present. It highlights the
many barriers to the preferred minimum energy structure on
the folding pathway. There are numerous local minima that
might trap the protein in an inactive three-dimensional
molecular conformation. The top rim represents the high
energy of the unfolded protein, with folding lowering the
energy towards a minimum energy structure that is at the
bottom of the funnel. It should be noted that this funnel
represents a three-dimensional landscape, whereas the
actual energy landscapes are multidimensional. Source.

The word marginal means to be at the outer or lower limits; minimal for requirements; almost insufficient. Certainly being marginal sounds bad, but is it always? In a recent issue of PNAS, Dill and colleagues show that the proteome—a cell’s collection of thousands of different types of proteins—is only marginally stable to denaturation under normal physiological conditions. Little did you know, we’re all just a few kcal mol^-1 away from being a pile of unfolded proteins.

Just exactly how close are we to becoming a marinade of melted matter at 37.5 °C? The answer lies in the distribution of lengths of our proteins. This is because the dynamics of protein folding largely depends on just one factor, the number of amino acids in the protein (N). In other words, at a given temperature and pressure, a number of fundamental thermodynamic properties scale linearly as a function of amino acid chain length. So, if you want to visit Uncle Thermophile in Yellowstone, you better pack plenty of short proteins. This is unexpected, considering that most important characteristics of proteins (e.g., native structure and biochemical mechanism), depend much more on the details of design—things like the number of hydrophobic amino acids or hydrogen bonds, counts of salt-bridging ion pairs, or structure, both secondary and tertiary.

Energy landscape diagram. The blue line represents every
possible structure of a pretend protein laid out in a line,
with similar structures near each other and higher-energy
(worse) structures placed higher on the Y axis. Source.

One thermodynamic quantity that can be used to predict the conformational stability of a protein at a given temperature is the amount of energy that is stored in an orderly way and is therefore free to do useful work. This measure is called the Gibbs free energy difference of folding (ΔG = G_unfolded – G_folded). Reactions tend to proceed spontaneously in the direction corresponding to decreasing Gibbs energy. This means that the conformational state (folded vs. unfolded) with the lowest Gibbs free energy will be favored.

Distribution of free energies of folding of the
proteins in the proteome of E. coli at 37 °C.
The free-energy bin size is 1RT. Area under the
curve equals the total number of proteins in the
proteome (4,300 for E. coli). Source.

When the authors looked at the distribution for Gibbs free energy differences of folding across entire proteomes (predicted from amino acid chain lengths), they discovered that the bulk of the proteins in the proteome have relatively small ΔG values; implying that for the most part, the transition from folded to unfolded occurs en masse. And just like solid ice turning into liquid water, cells undergo a sharp heat-death transition because the bulk of proteins in their proteome denature near the same temperature.

Mush. Source.

Roughly 50% of the dry component of a cell’s mass is protein, and for E. coli, the shift from model organism to a denatured mush occurs at around 50 °C. Jack up the incubator even a few degrees (from 37 °C to 41 °C) and the fraction of denatured proteins in the proteome goes up by nearly 20%. This is proportionally true even for hyperthermophiles the champions of thermostability, whose protein melting transitions are also quite sharp. Life is indeed at the edge for everyone.

Two other thermal properties of proteome stability (that go hand in hand) are enthalpy and entropy (ΔH, ΔS). You can think of the enthalpy of unfolding as a measure of the amount of heat energy released when you unfold a protein due to the breaking of bonds. To see for yourself how enthalpy of unfolding affects the fraction of a two-state protein that has melted at temperature (T), click here. By comparison, entropy is not a measure of heat, but rather a (logarithmic) measure of the volume in the space of all possible configurations (as it is for a gas). The entropy of unfolding represents the increase in the number of possible configurations that results from unfolding. When mesophiles (25-40 °C) were compared with thermophiles (40 °C), the thermophilic proteomes had, on average, less positive enthalpies and entropies of unfolding than mesophilic proteomes. The reasons for these differences are not known; however, this suggests that the denatured states of thermophilic proteins may be more compact than the denatured states of mesophilic proteins. Looking for somewhere to relax your proteins, and reduce the entropy in your life? Take a cue from the thermophiles and visit the hot springs…just don’t stay in too long.

So, once again, why is marginal stability a good thing? Well, it turns out that livin’ on the edge isn’t as reckless and imprudent as it sounds. That’s because evolution has the power of a yogi master, in this case, the skill to balance between meltdown and maximal growth. Luckily, life is very flexible by design, and performs many balancing acts to avoid thermodynamic equilibrium. Here’s an important one: besides temperature, the rate at which proteins can diffuse across a cell can also limit biochemical reaction rates. So, another way to maximize the speed of biochemical processes could be to vary cell size, and thus protein density, so that proteins don’t need to travel so far. If a cell’s protein density were too low (cell size too large), reaction rates would be limited by the time required for the proteins to diffuse and collide with reactants.

On the other hand, if the cell’s protein density were too high, (cell size too small), diffusion would be slow because of the crowded cellular milieu through which the proteins must move. It turns out that, just like proteome stability, the rate at which proteins can diffuse through the cell can be predicted from the amino acid chain length distribution of proteins in the proteome. This has to do with the fact that the hydrodynamic radius of the native protein is also related to the number of amino acids in the protein chain (N). Go figure! Happily, when Dill and colleagues used their model to predict the optimal density for maximal biochemical reaction rates, the value was extremely close to the protein densities observed in cells of various sizes (~20% volume fraction).

Diagram representing the unfolded and folded structures of a
protein. Source.

Insights into the Zen life of a cell obtainable from simple scaling relationships between cell properties and the number of amino acids in a protein don’t stop here. Since chain length (N) also correlates with protein folding rate (the longer the protein, the slower the folding), Dill and colleagues used chain length distributions in whole proteomes to predict the folding rates of all the proteins. So, how slow can you go? The slowest proteins fold at rates similar to their rates of synthesis (assuming a translation rate of 15 amino acids per second). This is a nice example of how a cell’s physical limits can contribute to its evolutionary limits.  

Thankfully, evolution is the peace-bird perched precariously close to the threshold between life and death. Cells have evolved the machinery to run as ‘hot’ as possible in order to maximize the speeds of their biochemical reactions. Since small changes in temperature can lead to large changes in the fraction of the proteome that is denatured, stress-responsive signaling pathways are essential. These pathways can help stabilize the proteome by transcriptionally upregulating chaperones (to speed up protein folding), folding enzymes, and coupled disaggregation and degradation activities for dealing with misfolded proteins.

So, to return to the macroorganisimal perspective with the microscopic in mind, the way I see it, the next time you’re feeling feverish you have two options. If you would like to test your proteome’s denaturation phase boundary try dancing to Hot Blooded by Foreigner. If, on the other hand, a threshold shift is something you prefer not to experience, try reducing your enthalpy by meditating on evolution’s many paths to equilibrium.

Tracey is a PhD student in the lab of Dr. Forest Rohwer, San Diego State University.

Dill KA, Ghosh K, & Schmit JD (2011). Physical limits of cells and proteomes. PNAS USA, 108 (44), 17876-82 PMID: 22006304

That Scary Restroom Microbiota

by Elio Schaechter and Joshua Fierer

Source.

Newspapers and other media are reporting with regular frequency that restrooms, ATM machine pads, money bills, and other sites carry many different microbes upon their surfaces including potentially pathogenic bacteria and viruses. Headlines call attention to such scary-sounding news and alarm the general public. We can expect that this practice will engender a widespread concern of the public for their safety. The outcry will encourage the implementation of unreliable, unnecessary, and potentially counterproductive “protective” measures. People may, for example, resort to using soaps with bactericidal compounds that have the potential to alter the generally protective normal flora. Inappropriate or non-essential use of bactericides may well accelerate the development and spread of resistance to microbicides and antibiotics within the microbial community. Resistance capabilities, including the upregulation of efflux pumps, can evolve particularly rapidly, and these same mechanisms may also increase resistance to antibiotics. For a reference to this topic, click here.

Let us suppose, as implied by these reports, that we do often encounter potential pathogens on ‘fomites,’ that is, inanimate objects. This is nothing new. We have known for over one hundred years that we live in a buggy world, surrounded by actual and potential pathogens, and that we often carry them within us. How often do we get sick from such encounters? Only the most immunosuppressed people must live in an aseptic “bubble” to avoid contact with all environmental microbes. With the exception of some epidemic-type diseases such as the common cold and influenza, most of us living in hygiene-blessed environments lead a life relatively free of externally-acquired infectious disease. In micro classes for medical students, we used to quip that the only people who get sexually transmitted diseases from toilet seats are members of the clergy! The exceptions to such relatively peaceful coexistence are important, viz. the spread of MRSA or Clostridium difficile in hospitals and, of recent, in the community, but these are specific concerns requiring their own focus when epidemiological studies indicate there is a problem.

The studies underpinning these scary headlines commonly involve analysis and/or amplification of the DNA that can be swabbed from surfaces. This methodology does not assess the viability of the organisms nor do these studies necessarily estimate the amount of specific DNA present. It is obvious that not knowing if the agents are alive or dead or if they’re present in amounts that could initiate an infection impacts on conclusions about the danger they may pose. In other words, there is no basis for linking these findings to the realities of public health.

Of particular concern to us, the authors of these scientific articles do not, in general, acknowledge their limitations. Instead, we read such statements as: More generally, this work is relevant to the public health field as we show that human-associated microbes are commonly found on restroom surfaces suggesting that bacterial pathogens could readily be transmitted between individuals by the touching of surfaces. This research does provide potentially useful information by listing the vast number of bacterial species that are found in the human environment, many of human origin. However, this merely opens possibilities about their relevance to human health, and, broadly speaking, these facts are far from novel. Do the justifiable conclusions from these studies warrant alarming the public? We think not. We wish that the authors clearly pointed out the limitations of their studies.

Joshua Fierer is Chief of Infectious Diseases at the VA San Diego Health Care System and Professor of Medicine and Pathology, University of California at San Diego.

It’s Raining Viruses!

by Merry

Caterpillar cadavers containing viral particles (OBs) are
eaten by predatory invertebrates such as this spider.
Since the alkaline pH of the insect midgut is required to
release the virions from the OBs, the OBs pass through
the predator’s digestive tract intact and are deposited
in their feces. In this way the virus gets a free ride across
relatively large distances, there to await ingestion
by a hapless caterpillar. Source.

It’s true! Each year it rains viruses, more than a trillion of them per acre over thousands of forested acres in the USA. This is the work of the airborne arm of the USDA Forest Service, part of their efforts to reduce the devastation to hardwood forests caused by the imported gypsy moth, Lymantria dispar. Each year they spray with a registered “general use pesticide” called GYPCHEK, the active ingredient of which is Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV), an ideal ‘green’ pesticide. The virus is host-specific, self-replicating, biodegradable, and deadly. And I would add, it makes a most interesting story, provided you’re not a caterpillar.

LdMNPV is a member of the genus Alphabaculovirus, family Baculoviridae. These viruses infect arthropods, most commonly lepidopteran larvae—what you and I would call caterpillars. Because one virus can efficently convert one caterpillar into more than 10⁹ viruses, with each of those progeny poised to repeat the process, they have received attention as potential biological control agents to help us in our perpetual war against the insects.

SEM of baculovirus OBs (polyhedra). Arrows
indicate three polyhedra of different sizes.
Bar = 2 μm. Source.

Viruses are supposed to be small, something you look at with an electron microscope. But these viruses form unusually large polyhedral particles that range in size from 0.15 to 15 μm, thus are readily visible at 400× or 100× magnification. The particles, called occlusion bodies or OBs, are how the virus journeys from one caterpillar to the next. An OB is made up of many virions embedded within a stable, paracrystalline protein matrix. Nested inside each virion are several rod-shaped nucleocapsids, a nucleocapsid being a protein shell enclosing a copy of the viral genome. The whole conglomeration is assembled in the nucleus of the host cell. Thus the multinucleocapsid and the nuclear in the virus’s name. What about the polyhedrosis? This is a general term for any disease of caterpillars that is caused by polyhedral virus particles and that leads to the gruesome liquefaction of the host and the accumulation of OBs in the fluid.

These OBs are good at their job. They can persist in the environment and remain infectious for months or years. Infection requires that the caterpillar inadvertently ingest one. It takes the alkaline pH of an insect midgut (pH 10.5–11) to dissolve the matrix and liberate the virions within. The virions fuse with midgut epithelial cells and the nucleocapsids migrate to the nucleus. There, the DNA is uncoated and viral transcription gets quickly underway. As this initial infection proceeds, progeny virions bud from the cell, enveloped in a bit of the cell membrane. These budded viruses spread the infection to other susceptible host tissues.

Baculovirus matryoshka dolls. (A) Baculovirus particles, called
occlusion bodies (OBs) or polyhedra; (B) Cross-section of an OB;
(C) Diagram of a polyhedron or OB cross-section showing multiple
nucleocapsids inside each virion, the virions embedded in the pro-
tein matrix. Credit: (A&B) Jean Adams; (C) © V. D'Amico. Source.

Most virion production takes place during the very late phase of infection. Virions form in the nucleus when several nucleocapsids are enclosed together within a membrane. At the same time large quantities of the OB matrix protein accumulate in the cell nucleus. The virions become embedded in the matrix to form OBs that accumulate until released by cell lysis. The caterpillar, by this time a cadaver, disintegrates, sowing the OBs.

The whole process, from OB ingestion to cadaver dissolution, is efficient and inexorable. For various steps along the way the virus makes use of metabolic genes that it carries in its genome. For example, viral proteases and a chitinase included in the OB matrix help the virions to digest their way through a chitinous peritrophic matrix to reach the epithelial cells lining the midgut. These enzymes are also synthesized in a tightly regulated fashion in the very late phase of infection to dissolve the host tissues, facilitating release of the OBs.

Baculoviruses also manipulate the caterpillar’s immune defense. The host’s midgut cells are primed to trigger apoptosis in response to viral infection. This would prevent the virus from gaining a toehold as the infected cells would die before the virus had time to replicate. However, the virus encodes an effective anti-apoptotic gene (p35) that counters the host defense. This gene is particularly intriguing because no cellular homologs had been found as of 2001. Yet, it is the only gene known to block apoptosis in all three of the primary model systems used to study cell death—C. elegans, drosophila, and the mouse. In addition, these viruses encode members of the large IAP gene family, so named because Inhibition of APoptosis was the first function identified for these proteins. Members of this family are found among eukaryotes, from yeast to humans, where they play a variety of cellular roles. Some baculoviruses use one IAP gene to inhibit apoptosis early in the infection to allow viral replication to proceed, and a different IAP gene late in infection to induce apoptosis to facilitate release of the intracellular OBs—thus showing that you can have it both ways.

Infection of a caterpillar by a nucleopolyhedrovirus. (NPVs, or nucleopolyhedrosisviruses, are baculoviruses in which each virion contains only one nucleocapsid. The infection steps shown here for an NPV also apply to LdMNPV.) Source.

Overall, the goal of the virus is to convert as much caterpillar biomass into OBs as possible and distribute those OBs where they are most likely to be eaten by potential hosts. In a fiendishly clever way, LdMNPV encodes another gene acquired from its gypsy moth host that furthers both of those aims. This gene encodes an enzyme, EGT (ecdysteroid UDP-glucosyl transferase), that inactivates the molting hormone. The caterpillar uses this enzyme to regulate hormone levels appropriately during molting and pupation. By expressing egt immediately after infection, the virus quickly blocks molting. Why is this good for the virus? Because larvae don’t eat while they molt. Prevent molting, and your host keeps eating and growing and making more biomass for you to convert into viruses.

A recent paper by Hoover et al. in Science created a stir by linking the egt gene to the modified behavior characteristic of virus-infected caterpillars. Just what effect does this virus have? In online media, we typically read that the virus makes the gypsy moth caterpillar climb to the top of a tree to die. Even in the cited paper, we are told: Just before death, infected larvae climb to the top of their host trees to die… ‘Tis true that these virus-infected caterpillars usually die in the tree tops, which is why the disease was named Wipfelkrankheit (tree top disease) in a German paper published in 1891. A more precise, albeit less dramatic, picture of what is going on can be found in the supplemental online material accompanying the Hoover paper:

Once EGT titers become high enough in virus-infected larvae to inactivate 20E [the molting hormone], the molting cycle is disrupted, inducing larvae to remain up in the tree in a feeding state rather than crawling down to hide from predators during the day. As they become more moribund, larvae remain at elevated positions and die…

This fits better with what information I could find online about gypsy moth caterpillars. During the first three instars, uninfected caterpillars stay in the middle or crown of their tree feeding. When the caterpillar population is high, fourth instar larvae do the same, feeding continuously day and night until the tree is stripped bare, at which point they set out to find another tree. But when there are few caterpillars on the tree, fourth instar larvae feed in the top branches only at night, descending at dawn to spend the day hiding from predators in crevices in the bark. In the evening they return to the top branches to feed. So there already is a programmed climbing behavior that is normally manifested at a specific developmental stage. Since the virus disrupts normal development by altering hormone levels, is it so surprising that the caterpillars, unable to molt, stay in the tree tops to feed both day and night? Nevertheless, that the larvae die high up in the trees does provide a second benefit to these viruses. As the larval cadavers disintegrate, OBs rain onto the foliage below, where they are more apt to be ingested by other caterpillars.

This is scary stuff, a virus pulling its host’s strings. It reminded me of two previous STC posts about pathogen-modified insect behaviors, one a story of ants infected by entomopathogenic fungi, the other about nematomorph worms that prompt grasshoppers and crickets to go for a fatal swim. It seems that if your successful evolutionary path led to hard-wired behavioral patterns, some pathogen—maybe a fungus or a worm or a virus—will come along and make you misbehave to their advantage.

Hoover K, Grove M, Gardner M, Hughes DP, McNeil J, & Slavicek J (2011). A gene for an extended phenotype. Science (New York, N.Y.), 333 (6048) PMID: 21903803

What’s the Score on the Microbiome?

Source.

by Elio

Just for kicks, let’s count up recent papers that say the microbiome is a good thing and those that say otherwise.

Just count titles.

For instance:

Socially transmitted gut microbiota protect bumble bees against an intestinal parasite

Drosophila Microbiome Modulates Host Developmental and Metabolic Homeostasis via Insulin Signaling

Successful Transmission of a Retrovirus Depends on the Commensal Microbiota

Intestinal Microbiota Promote Enteric Virus Replication and Systemic Pathogenesis

So, right now, the score is 2 to 2. It will change soon.

Especially if you send us some to add to the list.

Retrospective, December 2011

We’ve prepared this quick tour for you, with brief visits to all the major articles we posted since our May, 2011, retrospective.

Source.

Microbial Behavior

Are You Me or Am I You?  Proteus mirabilis is one sentient bug: it can distinguish itself from its parents and relatives.

Ringing a Microbial Dinner Bell.  Bacteria make smelly stuff, some of which provides appetizing cues for insects. Associate blogger Mark Martin explains.

Source.

Structure and Function

Hard Biology.  How do diatoms make their glass-hard shells? Well, they assemble subunits within the cells, then export them and insert them into preformed structures.

Some Like It Curved.  How do bacteria make curves in their cell wall? It turns out that cardiolipin is a curved and curve-inducing lipid molecule found in the curvilinear cell poles of rod-shaped bacteria.

Rafting Through Time.  Do bacteria have lipid rafts in their membranes, a la eukaryotic cells? Possibly.

Vital or Not Vital: That Is the Question.  Guest Gemma Reguera clarifies the confusion about what is viable and what is vital, including what ‘vital dyes’ tell us about such states.

Source.

Viruses

Virus Hacks Intercellular Communications Network.  Cells of our immune system talk to each other when mobilizing a defense, but vaccinia has many clever ways to tamper with those messages for its own benefit.

Is a Good Offense the Best Defense? Yeasts have to choose between having RNAi to defend against incoming viruses and such, or hosting killer viruses that eliminate the competition.

Phage Lambda’s Polar Expedition.  Are all regions on the surface of an E. coli equally inviting to an adsorbing lambda phage, or does lambda have a preference?

Phage DNA: Going with the Flow.  To launch an infection, a phage needs to transfer its DNA into the host cell, but a phage is not a hypodermic needle.

And We Thought We Knew What CRISPRs Do!  Here’s a new twist. A prophage in Pseudomonas aeruginosa inhibits biofilm formation and the host’s CRISPRs are required.  

Now That's Using Your Head!  We thought all tailed phages use their tails to recognize and attach to their host cells, but here’s one that uses its head instead.

Viruses that Infect Parasites that Infect Us: The Matryoshka Dolls of Human Pathogens.  Graduate student Jamie Schafer takes us, layer by layer, through two stories of viruses that infect parasites that infect us, both with possible clinical implications.

Source.

Pathogenesis

A Pestis from the Past.  Associate blogger Marvin Friedman recounts the history of the Great Plague, as illuminated by recent genomic studies of old bones.

The Lyme Disease Spirochete Feasts on Tick Antifreeze.  A fellow blogger, Microbe Fan, allowed us to use his article on how borrelias use of antifreeze in ticks as a nutrient. Who would have thought?

The Janus Bug.  And you thought that Helicobacter pylori  was a bad bug, causing ulcers and cancer! Well, it may also stimulate the immune response to protect people from asthma.

Going Next Door Without Getting Your Feet Wet.  By using novel techniques, Daniel Portnoy’s lab can introduce Burkholderia into a cell, thus bypassing early stages of infection and isolating later ones. New pathogenesis mechanisms are now revealed.

A Clever Bug That Is Difficile to Control.  Marvin Friedman discusses how Clostridium difficile adenylates its teichoic acids to ward off the action of host defensive peptides.

How We Tell The Good Bacteria From The Bad.  MD/PhD student Micah Manary tells us how the inflammasome (which some of us need to look up) is involved in selecting members of the intestinal microbiome.

Source.

Genomics and Evolution

The Façade of E. coli K-12.  Nothing was thought to be less dangerous than the K-12 strain of E. coli. Guess again. It’s full of genes encoding factors that can be turned on in HU protein mutants.

Fine Reading:  Microbial Evolution.  On the 150^th anniversary of publication of On the Origin of Species, 34 microbiologists met in the Galapagos to discuss the role of microbes in evolution.

Bacteria Activate Fungal Gene Clusters.  Cross-talk among very different organisms are the rage. Marvin Friedman presents another exciting example.

What’s Old is New: Genome Wide Manipulation of the Bacterial Chromosome in Vivo.  Guest Michael Schmidt discusses a novel and clever way of manipulating the bacterial chromosome on genome-wide basis.

Source.

Ecology

Shipwreck Microbiology.  One shipwreck can devastate a coral reef for decades. The culprit? Iron leached from the wreckage.

One Size Sometimes Can Fit All.  And this is of great concern when it is one antibiotic resistance factor that can counter every antibiotic in our arsenal.

Some Like it Cold.  Marvin Friedman explains that the trigger factor protein (a chaperone involved in keeping nascent peptides from folding prematurely) is selectively upregulated in the cold.

Taking Bugs Out For a Spin.  Graduate students Linh Truong and Shabana Din describe the unexpected resistance of bacteria to very high g forces.

A Microbe By Any Other Name Would Smell As Sweet…  Mark Martin waxes eloquent on the subject of smells emanating form bacteria and their uses.

Symbiosis

Source.

A Bug in a Bug in a Bug.  Bacteria living inside bacteria? Astounding as this sounds, you find them as nested endosymbionts of mealy bugs.

Wolbachia: The Difference Between a Nuisance and a Threat?  Graduate student Jamie Shafer tells a great story about a phage altering how the endosymbiont Wolbachia affects its insect host.

A Wormful of Bugs.  Here’s another example of an animal that considers a gut obsolete. Instead, it employs a sack of densely packed bacteria that feed it by oxidizing sulfur.

Source.

Fungi

The King of the Fungi.  Imagine a fungus half the size of a bowling alley growing from a fallen tree. A big tree.

Fungi That Spit Like Baseball Players.  Guest Gemma Reguera reveals new insights on how mushrooms shed their spores. Fast! Very fast!

How to Escape a Deadly Embrace.  There lurk fungi in soils that can trap nematodes. Aha! The nematodes have evolved an escape tactic.

Source.

Miscellanea

How to Improve Vaccines (or Not), 1912 Style.  Antibodies, it turns out, make a toxic vaccine less toxic. So it was thought 100 years ago, and now we find out that this holds up.

Fine Reading: Microbial Genomics and Infectious Diseases.  We call attention to a seminal paper by David Relman on this topic.

Preaching to a Prokaryotic Choir.  Mark Martin directed some probing questions about the microbial world to students in his undergraduate micro class and shares with us the results.

Of Terms in Biology: Riboswitch.  Twenty classes of these talented RNAs are known, and they do interesting and diverse jobs.

The Microbial Weltanschauung. Elio muses on the effect of knowing that there are so many prokaryotic cells on this planet.

Talmudic Question #82

Are there any surface structures or components on bacteria that are NOT used as receptors by some phage?

A Wormful of Bugs

by Elio

Paracatenula cf. polyhymnia. Light microscope
micrograph. The light portion is the ‘head’ or
rostrum, the dark portion the trophosome.
Source.

 

Let’s start out with a little quiz. What examples can you name of endosymbiotic bacteria so tightly packed that they’re nearly wall-to-wall within cells of their host? If you said legume root nodules, you can claim a prize. (Ask someone else for it, as we don’t have any.) You would get bonus points if you also mentioned the bacteria-filled bacteriocytes of certain insects or the celebrated giant tube worms found near the deep sea black smokers. By the way, the root nodule symbioses, though not mandatory, are highly beneficial to the host, but the other two are obligatory.

Here is another recently described example to add to this thought-provoking repertoire. In the sandy bottoms beneath shallow ocean waters one finds worms that have neither a mouth nor a gut. Although such areas seem sparse in life, in fact they’re home to a rather rich biota. Microbes, both pro– and eukaryotic, are present in abundance and so are animals, such as the worms we’re talking about here. So, how can animals be mouthless and gutless? Taking a page from the giant tube worms, a group of investigators found that indeed these worms are filled with a sack of bacteria. As in the tube worms, these bacteria provide food for the host by oxidizing H₂S using oxygen as the electron acceptor. This kind of biochemical endowment is widespread in nature and is found in groups as diverse as ciliates, arthropods, mollusks, and other kinds of worms. There is even a giant archaeon that is covered with sulfur oxidizing bacteria. Despite this huge variety of hosts and the many other sites where sulfur metabolism takes place in nature, the great majority of the bacterial sulfide-reducers are either Gamma- or Epsilonproteobacteria. Here, the symbionts are Alphas. The Alphaproteobacteria include others prone to intracellular life such as the nitrogen-fixing rhizobia and the rickettsiae (from which the mitochondria appear to be derived). Should we expect more examples of symbionts in this class?

EM micrograph of a complete cross-section of Paracatenula cf.
polyhymnia. Source.

The worms in question, Paracatenula galateia, belong to the platyhelminths or flatworms, but inside they don't look like your usual fluke or tapeworm. Their bacteria-filled sac, called the trophosome, accounts for over 90% of the worm’s body and has cells called bacteriocytes that are filled with bacteria. In some species, the bacteria make up close to 50% of the total volume of the worm. By the way, the bacteria are quite big—5 to 8 μm in diameter. Incidentally, such large sizes are also seen in some insect bacterial endosymbionts.

So, how did these researchers find out what the bacteria do for their host? The first hint that they oxidize sulfur came from just looking at the worms. They are full of white granules. By physical analysis, these granules indeed consist of elemental sulfur and make up 5-19% of the tissue mass. More hints: the symbiont’s genome contains the gene for a subunit of DsrAB, the dissimilatory sulfite reductase, a key enzyme that works in reverse to participate in sulfur oxidation. The bacteria also carry the gene for AprA, a subunit of adenosine-5’-phosphosulfate reductase, another enzyme in sulfur energy metabolism. And they encode RubisCO, the main player in autotrophic carbon fixation. The sulfur granules and the presence of these enzymes strongly suggest that the bacteria can carry out a sulfide oxidizing, energy-producing metabolism. And, just like in the tube worms, this energy-generating mechanism ultimately allows the bacteria to synthesize the metabolic building blocks that provide the host with needed organic nutrients. Little is known about how H₂S and oxygen enter the trophosome. The worms not having a respiratory or circulatory system, this presumably occurs by diffusion through the worm’s body wall.

Metabolism that depends on the oxidation of sulfides using oxygen in marine environments poses a problem. H₂S is found in the sediments, oxygen in the water column. Neither is commonly abundant in the other’s realm. The worms in the rifts do not have this problem because they live near the black smokers that spew large amounts of sulfides. How about these worms? The authors have this to offer: By migrating through the redox potential gradient in the uppermost 5- to 15-cm sediment layer, millimeter-sized worms can supply chemoautotrophic symbiotic bacteria alternately with spatially separated electron donors and acceptors such as sulfide and oxygen, as has been described for Nematoda and Oligochaeta. They may even move up and down, in the style of some sheathed bacteria we have discussed before.

Well worth mentioning is the evolution of host and symbiont. If you place the dendrograms for the bacteria (based on 16 S rRNA ) and that of the various worms species studied (based on 18S and 28S rRNA) side by side, they line up most satisfactorily. This is known as cocladogenesis, and it suggests that the worms had a common ancestor who acquired a bacterial progenitor, and that the two further evolved together, each one changing as the other did. The authors posit that the original pairing took place 500 to 620 million years ago. If so, this would be the oldest known bacterial-metazoan symbiosis.

Cocladogenesis between Paracatenula and Candidatus Riegeria. Tanglegram of strict consensus cladograms of four reconstruction methods for both symbiont 16S rRNA and host concatenated 18S and 28S rRNA. No conflicting nodes are statistically supported in the results of the four phylogenetic reconstruction algorithms, indicating close coevolution between the partners. Source.

The symbionts have ben given a name, Candidatus Riegeria galateiae. The genus name is in honor of the late zoologist Reinhard Rieger, who described the host genus. (I’ll remind you of the practice of bacterial taxonomists not to allow bacteria into the guild unless they have been cultivated. Candidatus means just that. I will have more to say about this sometime later.)

The more examples we have of symbioses involving microbes, the more our juices start running. They are vivid examples of ingenious and intricate choreographies between different organisms, all leading to the conclusion that no opportunity for evolution is left unexplored.

Gruber-Vodicka HR, Dirks U, Leisch N, Baranyi C, Stoecker K, Bulgheresi S, Heindl NR, Horn M, Lott C, Loy A, Wagner M, & Ott J (2011). Paracatenula, an ancient symbiosis between thiotrophic Alphaproteobacteria and catenulid flatworms. Proceedings of the National Academy of Sciences of the United States of America, 108 (29), 12078-83 PMID: 21709249

Leisch N, Dirks U, Gruber-Vodicka HR, Schmid M, Sterrer W, & Ott JA (2011). Microanatomy of the trophosome region of Paracatenula cf. polyhymnia (Catenulida, Platyhelminthes) and its intracellular symbionts. Zoomorphology, 130 (4), 261-271 PMID: 22131640

The Astonishingly Rich Smorgasbord of New Methods for Light Microscopy

by Elio

A replica of a Leuwenhoek microscope. Source.

The two age-old problems of the light microscope are resolution and contrast. Resolution is a function of the wavelength of light, so that in the visible it is, at best, about 200 nm. (It also depends on the lenses, but they have been pretty good for a long time.) This means that if you were looking at, say, a virus 100 nm across, you could possibly see it, but you couldn’t tell if you were looking at a single particle or several stuck together. An irreducible problem, it seemed. Besides, that is if you could see it at all. Most biological objects have little contrast, thus are practically invisible. This was countered early on by staining the object with deep-hued dyes, but most of the time the cells were killed in the process.

One tactic for enhancing the contrast in living cells was the advent in the 1930’s of the phase contrast microscope. By taking advantage of small differences in refractive index between object and background, it could make a nearly transparent object visible. That was about it, until recently. Regarding resolution, an important breakthrough in the early 1950s was the introduction of fluorescence in microscopy (although other uses of this technique have made this a secondary point). Think about it. You are now looking at emitted light, which means that you can see the object as long as it gives off enough light. Size does not matter. Think of a very distant star, which can be seen even though, to the eye, it’s insignificant in size.

A more modern microscope. Source.

That was it for a while. Starting sometime in the 1990’s, a number of advances were made and new light microscopes were fabricated. Such microscopes now allow one to do things that only a few years ago were thought impossible. Using a variety of tricks, one can now obtain 20-30 nm resolution, about ten times greater than with a conventional microscope set-up. They are known by a variety of acronyms, e.g., SIM, STED, STORM, and many others.

The editors of Cell have done us a great service, publishing a table that lists a large number of techniques. (Click here to download the table as a pdf file.) We provide a link because the table is too large to display here, though we have included a snippet below. Some of these methods do not deal with resolution or contrast alone, but permit you to measure chemical properties, such as the protein or lipid content of a structure. A fine review covering some of this ground but with emphasis towards visualizing biofilms was written in 2010 by Neu and colleagues.

Many are the breakthroughs that have been made thanks to these technological developments. To take one example, the visualization of single molecules in living cells, once a distant dream, is now a routine event. The Age of Imaging is indeed upon us.