Small Things Considered

Recent Blog Items We've Enjoyed—Spring Harvest

by Elio

Alan Cann’s MicrobiologyBytes has a great video showing magnetotactic bacteria darting back and forth in response to a moving magnetic field.

Besides a lot of other interesting news, Tara Smith’s Aetiology has an update on the case of Robert Farrell, the geneticist who got into trouble for supplying generally harmless bacteria to an artist who used them in an exhibition. The good news is that the case was dismissed (although the Feds can still appeal it).

For the aficionados of Epulopiscium, that Gargantua among bacteria, a new article reports that cells of this fish-gut-dwelling organism are polypoid in the extreme. This gives them some of the advantages of a multicellular organism, a point that Cesar Sanchez discusses (along with some highly pertinent links) in his blog Twisted Bacteria.

In Spanish, Miguel Vicente’s Esos Pequeños Bichitos (Those Small Little Bugs) has a revealing and informative article on Robert Koch and aspects of his private life. Worth polishing up the Spanish you learned in school.

And while you’re brushing up on languages, a blog in French, Le blog des bactéries et de l'évolution deals with the 150th anniversary of the birth of Theodor Escherich, the German physician after whom our best known cellular organism is named. This occasion was also celebrated in 2007 in Nature Reviews Microbiology.

In Evilutionary Biologist, John Dennehy deals with the perennial question: Are viruses alive? We maintain that this should only be asked after a few beers. As a good geneticist, John answered: Living entities are those that evolve via natural selection, and surely viruses should be included in that category. The argument continues, no?

Carl Zimmer’s The Loom talks about his soon-to-appear book: Microcosm: E. coli and the New Science of Life. Having read some of it in draft form, I can heartily applaud this birth.

Fine Reading: Fungal Alchemy Revisited

by Elio

The Chernobyl reactor encased in
cement. It is said that it will remain
radioactive for some 100,000 years.

One of the most breathtaking reports in this blog has been the finding that ionizing radiation stimulates the growth of some fungi, such as those found in the encased Chernobyl reactor. A recent review in FEMS Microbiology Letters expands on this phenomenon and surveys considerably more data. Here you can read about how fungi accumulate some radionuclides and resist powerful jolts of ionizing radiation. You can also learn about their amazing ability to grow their hyphae towards the radiation source and flourish better as the result. In toto, this is an example of the stunning ability of living things to hack it in even the most atrocious of environments. But the point is not just academic. The authors say: Using this recently acquired knowledge, we may be in a better position to suggest the use of fungi in bioremediation of radioactively contaminated sites and cleanup of industrial effluent.

Where Art Thou, O Ribosome?

by Elio

In a recent post (The Age of Imaging), I mused about advances in microscopy that are revolutionizing our concept of bacterial cell structure. High on the list of amazing developments is cryoelectron tomography (CET), a technique that allows one to look inside bacterial cells. Flash-freezing cells at very low temperatures embeds them in vitreous ice, and this preserves their structures when subsequently subjected to the high vacuum of the electron microscope. This process is minimally invasive and requires no staining. Bacteria frozen in this manner can swim away after thawing! A great tutorial about this technique and its uses is provided on Grant Jensen’s web page.

Cryoelectron tomography has its limitations, some of which are discussed by Jensen. Most pertinent to bacterial microscopy is the thickness of the object viewed. It turns out that bacteria like E. coli are close to the upper limit of what can be conveniently looked at, at least for now. Most of the articles written so far concern smallish bacteria, such as Bdellovibrio, Caulobacter, Flavobacterium, Spiroplasma, and Prochlorococcus, among others. However, studies on subcellular structures near the cell surface or protruding from it have been reported in larger cells as well. An overview of such studies can be found in a recent review. One can reasonably expect an avalanche of reports of this sort. It’s already happening.

Cryoelectron tomography of a vitrified
cell showing ribosomes and the membrane
(grey) in an optical slice. Ribosomes
determined with high fidelity are green,
those with lesser fidelity are yellow. Source

How to choose one example from such a rich offering? I opted for one that deals with a question that I worried about long ago: where are ribosomes located in bacterial cells? Are they mostly attached to nascent messenger RNA strands, making proteins even as the mRNA is being transcribed? This question was recently approached by CET using the slender mycoplasma, Spiroplasma melliferum. Although the full answer to my question is not yet available by this technique, the authors showed that most ribosomes seem to be more or less randomly distributed in the cell. About 40% may well be in polyribsomes—those strings-of-pearls made of ribosomes attached to mRNA. (This figure is lower than that seen in fast growing bacteria, where it is about 80%.) About 15% of the ribosomes appear to be lying close to the membrane, with their 50S subunits facing it. This is the orientation one would expect if the proteins being made are in the process of of being inserted in the membrane. (For a report imaging this process, click here.) This process, which links transcription, translation, and insertion of proteins into the membrane, was termed transertion by Woldringh.

One more thing. When it comes to ribosomes, their concentration is highly dependent on the growth rate as imposed by the nutritional conditions. Therefore, parameters such as the proportion engaged in translation or transertion should be studied in cells growing at different rates. Some of us have been saying this for a long time.

The View From Hereby CharlesYanofsky

We have invited a number of distinguished microbiologists to share some of their thoughts with us.

The Evolution of the Genetic Code

Although the genetic code is well established, a very exciting unsolved problem is discovering how codons were related to amino acids in the evolution of protein synthesis. How did a tRNA and a tRNA synthetase first evolve, and what was their ancestral source? How were the genes for the first tRNA and tRNA synthetase duplicated and how was their specificity varied? Can we offer any explanation for why there are two classes of tRNA synthetases? Can one predict which tRNAs evolved from one another? Similarly, can we predict which tRNA synthetases evolved from an existing tRNA synthetase?

A related series of exciting experiments would be to attempt to reproduce some evolutionary events and determine how many mutational changes it would take─and where─to evolve a tRNA synthetase with new specificity from an existing synthetase. Suppression studies many years ago showed that tRNAs may acquire new decoding specificity following single mutational changes, but synthetase suppressors were not recovered, as I recall. Also, we now know that synthetases recognize both the anticodon and acceptor end sequence of each tRNA─is this consistent with what is known about suppressing tRNAs?

Reference: Carter, C. W., Jr. 2008. Whence the genetic code?: Thawing the 'Frozen Accident'. Heredity 100: 339–340.

Yanofsky is Professor Emeritus of Biological Sciences at Stanford University and a 2003 recipient of the National Medal of Science.

Talmudic Question #32

Which group of bacteria and archaea is more abundant on Earth (by mass): obligate aerobes, obligate anaerobes, facultatives, or obligate anaerobic respirers?

The Loss of a Good Word

by Elio

Photomicrograph of nanobacteria from ice
cores. Courtesy of V. I. Miteva and J. E.
Brenchley

A recent report goes a long way towards dispelling the notion that “nanobacteria” found in human blood are living entities. They seem to be inorganic precipitates, instead. Fine with me. What is less fine is that those making the original claim have mangled a perfectly good term and perhaps rendered it useless. Many, if not most, bacteria in the environment are very small, some passing through filters that retain “regular” bacteria. See our previous post entitled Old, small, cold… or a most informative review. These tiny organisms deserve to be called nanobacteria. This may be a lost cause. When entering “nanobacteria” in Google, the first 100 hits (out of 186,000) are about the apparently spurious entities, none about real bacteria. Abuse of words happens.

Staph And Cholesterol: An Odd Couple

by Elio

Cholesterol and MRSAs vie for headlines. Both are formidable topics of enormous interest to readers of mass media and to scientists (many of whom read mass media, probably in the bathroom). It turns out that Staphylococcus aureus and arteriosclerosis have something in common, although not in the way you might think. It happens that the yellowish-golden pigment of S. aureus is made by a pathway that, in its early steps, resembles the one for making steroids.

Let’s back up a moment. The S. aureus pigment is a carotenoid called staphyloxanthin. Although the discoverer of the organism, the Scottish surgeon Alexander Ogston, coined its species name in 1880, the role of the pigment has only been recently uncovered. It is essential for the virulence of the organism, acting as an antioxidant that detoxifies host-produced reactive oxygen species, such as O₂^–, ·OH, and HOCl. Mutants lacking the pigment are much less virulent. Sounds like blocking the synthesis of staphyloxanthin would be a good way to combat staph infections, no?

The pathways for making staphyloxanthin and steroids. Source

The early steps in the biosynthesis of staphyloxanthin and cholesterol are the same, up to the synthesis of an aliphatic precursor, presqualene diphosphate. Thereafter, the two pathways diverge. The next step is catalyzed by two different enzymes (the dehydrosqualene synthase CrtM for staph, the squalene synthase SQS for humans) that were shown in a recent paper to be structurally very similar. The authors asked if known SQS inhibitors might also inhibit the staph's CrtM and thus block staphyloxanthin synthesis. Sure enough, some did. Not only that, treating the staph with some of these compounds rendered them not only colorless but also less able to defend themselves from oxidant-based attacks by human leukocytes. Peritoneally infected mice survived better if given one of these new drugs. This is a neat new strategy. These inhibitor's are not antibiotics per se but anti-virulence factors. The organisms can grow perfectly well when cultured in their presence. But by blocking staphyloxanthin synthesis, these inhibitors render the staph defenseless against the reactive oxygen species produced by our immune system.

These compounds differ from statins, which work further downstream in cholesterol biosynthesis (and which would not be expected to affect the biosynthesis of staphyloxanthin). However, the inhibitors used here may also be medically useful for lowering cholesterol levels, a point that is under investigation. It is conceivable that some day one and the same drug could lower one’s cholesterol and prevent infections by S. aureus. But even without going that way, this work bodes well for the discovery of potent anti-staph drugs. Look out, MRSA!

A Red Letter Day (on Merry's calendar)

by Merry

Not every one makes it to the age of 80. And of those who do, only a precious few are still doing what they love. Elio's 80th birthday is this Saturday, April 26. He told me to restrain my eloquence here, but to see him at 80, still being a learner as well as a teacher, publicly writing and speaking about The Small Things, and enjoying life—well, that's an inspiration for me. Happy birthday, Elio! Mazel tov!

On being 80

by Elio

Long ago, I thought that reaching 72 years of age would be a reasonable accomplishment, as this would bring me into the new millennium. Done, some time ago. I now have no mileposts in mind, just a still horizon. If anything, it’s this blog that counts. So, let me tell you why this has been and continues to be such an exhilarating, if demanding, experience. I will quote from the very last part of my book, In the Company of Mushrooms. I still like the sound of it and hope that it will help explain my interest in mushrooms, as well as microbes. Before doing that, I want to thank my collaborator, Merry, without whom the task would be far more arduous.

I am a microbiologist by profession, now near the completion of my career. When I collect and study mushrooms, however, I do not act as a professional biologist. Most of what I love about mushrooms and how they fit in people’s lives is far remote from my research and teaching. My life in science has been spent at the laboratory bench: I studied how bacteria grow and make their DNA. I am of the generation that witnessed the beginning of molecular biology and its offspring, genetic engineering. It was only after my career was established that I stepped into the world of forests, pastures, and mushrooms.

Still Life with Mushrooms, Insects and Amphibians. Otto Marseus Van Schrieck,
Dutch (1662). Herzog Anton Ulrich-Museum. Braunschweig, Germany

The distance between these two interests—microbiology and mushrooms—may not appear to be very fundamental to a non-scientist, but it is in fact quite considerable. It is true that biology is biology, in the sense that the basic question is always the same—What is life?—but at the level at which we participate in the profession of biology there are marked differences in attitude between those who study living things in the field and those who work in laboratories.

This gap is of recent origin—it was unknown until the nineteenth century—and, happily, it gives signs of closing. On the one hand, biologists who study the evolution of living things and their place in the environment are coming into the laboratory to take advantage of modern molecular tools. On the other hand, those who study the functions of living cells have found great opportunities in probing the wondrous diversity of the natural world. The rift between the field worker and the lab worker, in the questions they ask and the attitudes they convey, is narrowing, and we can welcome the fact that biology is reemerging as a unified science. It is worth noting that the “history” in “natural history” is derived from the Greek word for “learning by inquiry,” which today we would name “science.”

For most of my professional life, however, the distinction between “field biology” and “laboratory biology” was quite substantial. My colleagues seemed content to study one or two kinds of bacteria under laboratory conditions and only rarely seemed concerned with the “real world.” The view has been put forth that different personalities are attracted to the two approaches to biology. To overstate the point, the “naturalists” are seen as more caring, more accepting of their role as stewards of living things, whereas the “experimentalists” are thought to be more analytical, interested only in how things work.

That’s the theory, at least. I have always had a hard time with this notion because it seemed, at best, to describe people at the extremes. I feel that I straddled these two worlds. Strange as it may sound, I have developed, if not a love, at least a personal closeness to the bacteria I study. The strains I have worked with are, by and large, harmless to people. To me, they are living things, not just bags of enzymes and DNA. They are, in other words, as alive to me as mushrooms are—and as trees and animals are. So, is the jump from the lab bench to the woodland glade as big as all that? In both places one can find, or make, the opportunity to study nature, to experience life, and it is my hope that this book (and blog) will lead you at least part of the way toward that end.

Play It Again, Cyan

by Merry

The evidence is quite compelling. All plastids (photosynthetic organelles and non-photosynthetic kin) are thought to be descended from one cyanobacterium that was engulfed by one unicellular heterotrophic eukaryote approximately one billion years ago. The descendents of that host are today's green algae (and plants), red algae, and glaucophytes (a group of microscopic freshwater algae). Several other eukaryote lineages acquired plastids second-hand by engulfing a plastid-containing red or green alga (a process called secondary symbiosis to distinguish it from the original primary symbiosis). For an interesting review of the convoluted history of plastid acquisition, click here.

Granted, it is not a simple matter to transform one’s lunch into a well-behaved photosynthetic organelle, but still one wonders why this appears to have happened only once, especially since plastid acquisition by secondary symbiosis—also not a simple thing to do—has occurred several times. Still, for some reason(s), only one eukaryote has managed to transform a cyanobacterium into a photosynthetic cellular entity.

Differential interference contrast
image of P. chromatophora.
Source: Micro*scope

Or so it was thought, until recently when an obscure freshwater amoeba, Paulinella chromatophora, attracted attention for possibly hosting an unrelated plastid. In other words, it may have acquired its cyanobacterium all on its own, which would be big news. It is a phototroph, but its closest relatives are heterotrophs, some of which feed on cyanobacteria.

When P. chromatophora was first described over a hundred years ago, its prominent sausage-shaped photosynthetic entities were named chromatophores—an unfortunate choice because the term has other meanings as well—but it has stuck. Other names are used as well, adding to the confusion. Some call them cyanelles because they have retained their peptidoglycan cell walls and thus look more like the cyanobacteria they once were. Some call them photosynthetic endosymbionts, while yet others argue that they are organelles deserving of the name plastid.

Names aside, what do we know about them? The complete genome sequence of the Paulinella chromatophore was recently published. It has a single chromosome containing only 1,021,616 bp—a reduction of more than a third compared to the smallest known genome of a cyanobacterium (Prochlorococcus marinus). Of the 39 sequenced cyanobacterial genomes, the one most closely related to the chromotophore genome is that of a Synechococcus. Interestingly, Prochlorococcus and Synechococcus form a monophyletic lineage apart from all other cyanobacteria and all known plastids. Thus it is clear that this chromatophore is no wayward plastid. The genomes of the chromatophore and Synechococcus WH5701 are remarkably similar. The Synechococcus genome contains orthologs for 855 of the chromatophore's 867 protein-encoding genes, in virtually the identical gene order. Can't get much closer that that.

Are the chromatophores true plastids or mere endosymbionts? This is being hotly debated. Although there is no clear line between the two, there are some generally accepted criteria for organelle status: organelle replication is controlled by the host; neither party can survive independently; organelles haves undergone massive genome reduction including horizontal transfer of some genes to the host nuclear genome. And most tellingly, host-translated proteins required by the organelle are directed to the organelle by a dedicated transport system.

So, how do the chromatophores measure up? So far so good (for the organelle fans). Chromatophore replication is tied to host cell replication. The host-chromatophore relationship appears to be stable (more than 15 years in laboratory culture) and obligatory. Genome reduction has occurred, and the presence of pseudogenes indicates that it may be ongoing. Intriguingly, a few genes thought to be needed for intracellular life appear to be missing, too. Perhaps they now reside in the host nuclear genome, or perhaps their role is filled by metabolites provided by the host. As to the key criteria—transfer of cyanobacterial genes to the host genome and transport of proteins back to the chromatophore—answers must await further experimental results. It is difficult to be patient, as this is exciting news. But given that this symbiosis is estimated to have occurred roughly 60 Mya, I guess we can wait a little longer.

Talmudic Question #31

Many unicellular protists have a very complex body plan. One can find “legs” (see our posting on Euplotidium), “mouths,” food vacuoles, etc. Some of these structures reflect the feeding habits of the organisms. The question arises, why haven’t they have become multicellular?

Some Like It Linear

by Merry

The classic 1962 autoradiograph by John
Cairns of a replicating E. coli chromosome.
(The insert is an interpretation of the auto-
radiograph.) This work confirmed the notion
of a circular chromosome, an idea which
had arisen from genetic studies showing the
E. coli chromosome was circularly permuted
(i.e., its genes were circularly linked).

Last year, a team of Japanese researchers deliberately converted the circular chromosome of E. coli into a linear one. To do this, they borrowed some tricks from the lambdoid coliphage N15, a temperate dsDNA phage whose prophage exists and replicates as a 46 kb linear plasmid. When tested in L broth at 37°C, the modified E. coli were indistinguishable from wild-type in terms of growth rate, nucleoid appearance, and overall pattern of transcription—provided that the linearization occurred at or near the chromosome replication terminus.

Should we be surprised?

At one time, chromosome structure was one of those cherished quirks that separated the biological world into two camps: eukaryotic cells with separate linear chromosomes and prokaryotic cells with a haploid, circular chromosome and often one or more circular plasmids. This turned out to be wrong because the reality for prokaryotes is more varied than that. (For a review article, click here.) The first linear bacterial chromosome was discovered in 1989 in the spirochaete Borrelia burgdorferi, the agent of Lyme disease. Since then, linear chromosomes have been found to be sporadically distributed among very different bacteria, ranging from the Borrelia with their small (950-1000 kb) genomes to free-living, filamentous, gram-positive Streptomyces with 6-9 MB genomes. Then there is the α-Protobacterium Agrobacterium tumefaciens with one circular and one linear chromosome. It appears likely that linearization occurred independently in different lineages.

Having a linear chromosome requires solving the end replication ("telomere") problem—how to replicate both DNA strands all the way to the end. At least two solutions are in use among the bacteria with linear chromosomes. Some cap the ends of their chromosome with covalently bound terminal proteins which prime DNA replication. Some use covalently-closed hairpin ends formed by inverted terminal repeats that fold back on themselves. Some use a little bit of both. These are the same tricks used by linear plasmids and by viruses with linear chromosomes. Generally, linear bacterial chromosomes have a central origin of replication, oriC, and replication proceeds bidirectionally, much as in circular chromosomes.

Linear thinking. Source: shizzknits

Switching from circular to linear needn't be complicated. A single crossover between a circular chromosome and a linear plasmid could produce a linear chromosome ending with the plasmid termini. Linear plasmids have now been found in numerous Streptomycetes and in all members of the genus Borrelia. In all cases the plasmid and host use the same type of telomere. At times the distinction between circular and linear replicons seems to disappear as some plasmids can replicate in either form, some organelles (e.g., plant chloroplasts) appear to make use of both. Various circular replicons, including the conjugative plasmids, routinely go linear at some point.

The linear chromosome of S. lividans can be circularized in the lab by deleting the terminal repeats. These cells grow as well, or almost as well, as wild-type. Nevertheless, as of 2000, all natural Streptomyces isolates tested had a linear chromosome. And the species with circular chromosomes appear to stubbornly stick to their way of doing business, as do those that prefer linear thinking.

So, should we be surprised?