Small Things Considered

Kryptonian Vision

by Jennifer Gutierrez

Superman, a Kryptonian, uses his X-Ray
vision. Source.

X-ray vision, once the exclusive domain of Superman and his super hero kin, is now a tool in the biological researchers kit. Granted, not every researcher has access to this superpower; the required synchrotron light sources are found only at large research facilities that happen to have a particle accelerator at hand. Still, it is surely worth the bother. Compared to electron microscopy, samples can be imaged in a more natural state without being either embedded or frozen, and they can be larger. Compared to light microscopy, the smaller wavelength of x-ray radiation has the potential to provide higher resolution. Resolution had been complicated by the difficulty in making efficient lenses, but new techniques in both lens design and manufacture are being developed. The lens problem can be circumvented using lens-less microscopy, as employed in a recent paper by Klause Giewekemeyer et al.

The Swiss Light Source at the Paul Scherrer Institut,
a third-generation synchrotron light source. Source.

The data they reported was generated at the Swiss Light Source (SLS) in Villegen, Switzerland, an international center for x-ray microscopy. This facility utilizes a third generation synchrotron light source (whatever that is. I warn you, this is complicated. This and the next paragraphs are for those who care to know how it works). Here the light is generated by first accelerating electrons using a large series of magnets in a linear accelerator, next increasing the acceleration further with more ‘booster’ magnets, and then storing the accelerated electrons in a series of ‘undulators.’ These undulators were developed in the 1970’s at Berkeley and move the electrons around a circular array of periodically-spaced magnets, causing them to slalom as they circle. This step allows the researchers to select the wavelength of electron radiation to be used in the illumination beam (synchrotron light), ranging from ultraviolet to hard x-rays, depending on their imaging goals.

A Holin One

by Merry

Cryoelectron tomography of a λ holin (S105) lesion. Segmentation
of the envelope densities shows the outer membrane (blue) and
inner membrane (orange). Source.

When time's up, an infecting phage lyses its host cell, thus releasing the assembled virions. (See our previous post for more about lysis.) This process has been intensively studied in the coliphage λ. Here the phage-encoded holin proteins are said to "permeabilize" the E. coli membrane, thus allowing the endolysins accumulated in the cytoplasm to pass through and attack the cell wall. "Permeabilize" has such a polite sound, suggestive of an orderly modification of the membrane to allow the folded endolysins to quietly slip through. But what sort of channels or pores do the holins actually make?

First of all, the holes have to be large—large enough to pass the folded 18 kDa endolysin protein. That means they have to be way larger than the largest channels known for bacterial cell membranes, which are the 5 to 9 nm diameter channels formed by the twin-arginine translocation pathway (Tat). To gauge the size of the holin holes, researchers fused a series of protein domains of varying sizes to the C-terminus of the endolysin and tested the ability of the chimerae to exit through the cell membrane. A fusion protein almost 8 times the size of the endolysin passed right through and so did tetramers of that protein. The holins aren't very exacting gate-keepers, either. When the holes made by one phage were tested using endolysins with different folded structures from different phages that infect unrelated hosts, the endolysins exited as usual.

Time’s Up

Source.

by Merry

Holins are the smallest known biological timers. Timers, not clocks. Timers tick along, then go off after the specified interval. These small, phage-encoded proteins time the length of lytic infections of some phages. When they go off, the game is over and the host cell lyses. This is important work. The phage that gets the timing right is one-up in the evolutionary race.

Since most bacterial hosts have a murein (peptidoglycan) cell wall, the challenge for the phage is to breach that structure. Two different strategies are known, only one of which uses a holin timer. (An excellent 2005 review by Ry Young and Ing-Nang Wang can be found in Ch. 10 of The Bacteriophages, available via Google books.) Phages with small, single-stranded DNA or RNA genomes go the economy route using but a single protein to do the job. These proteins have been called "protein antibiotics" because they effect lysis by inhibiting a specific enzyme in the murein biosynthesis pathway—just like the β-lactam antibiotics. And like the β-lactams, their effectiveness requires continuing cell growth. Unrelated proteins that inhibit different enzymes in that pathway have been found in different phages. (Potential insights for clinically useful antibiotics here?)

In contrast, all double-stranded DNA phages (so far) use at least two proteins: a muralytic (murein-degrading) enzyme and at least one other helper protein, a holin. The holin enables the endolysin to pass through the cell membrane and access the cell wall, thus triggering lysis. Because the endolysins lack a secretory signal sequence, during an infection they accumulate fully-folded in the cytoplasm, waiting for the holin to let them out. Phages that infect Gram-negatives encode a few additional proteins that handle the demise of the outer membrane.

What follows here about lysis describes how coliphage λ does it. What is known about other phages suggests that the λ strategy is representative of many, but surely not all. (For example, the lambdoid phage 21 uses a remarkably different holin-endolysin strategy.)

And The Winner Is…

by Elio

The transposases! The contest, in case you wonder, was for the most abundant set of genes in the known universe, or at least in the genomic data banks available on Earth. Aziz, Breitbart, and Edwards examined some 10 million genes from 2137 sequenced genomes (47 archaeal, 725 bacterial, 29 eukaryotic and 1336 viral) plus 187 metagenomes, in search of the gene set that is both most abundant and most ubiquitous. (Ubiquitous refers to genes that carry essential functions and are thus needed by every genome. A gene that is highly abundant in some ecosystems but not ubiquitous is likely to play an important habitat-specific role.) The winners, by far, were the genes for transposases. One could squawk that the genomes selected for sequencing to date reflect a bias towards “germs” and other microbes of human interest, and that so far the source environments for metagenomes represent only a small sample of what’s out there. But given the off-scale preponderance of the transposase genes, there is little to make one question their dominance.

Abundance of different functional roles in 2137 genomes plotted against the ubiquity of these
functional roles (defined as the number of genomes in which the functional role is represented
at least once). Source.

Not everybody seems to have transposases; only 1/3 of the viruses in this study have them. But those organisms that do tend to possess multiple copies—close to 40 on average. If you take a set of 2000 randomly sampled genes (the number found in a typical bacterial genome), 22 will encode transposases. They are also more common in free-living bacteria than in obligate pathogens or endosymbionts. The champion transposase-carrier? Possibly the marine cyanobacterium Crocosphaera watsonii, which has more than 400 copies.

So, why transposases? The authors point to their general usefulness to both viruses and cellular organisms in promoting the spread of mobile elements, thus leading to diversification. “Selfish genes” indeed! As the authors write, By securing their own replication and dissemination, transposases guarantee to thrive so long as nucleic acid-based life forms exist. This rather implies that they might imagine non-nucleic acid based life forms exist somewhere. Who says that bioinformaticists are just computational nerds?

Aziz RK, Breitbart M, & Edwards RA (2010). Transposases are the most abundant, most ubiquitous genes in nature. Nucleic acids research PMID: 20215432

When Crenarchaeota Divide, They Multiply

by Jenn Tsau and Suzy Szumowski*

Sulfolobus acidocaldarius, an extreme thermophile occurs in
geothermally-heated acid springs, mud pots and surface soils;
it can withstand temperatures from 60 to 95 ºC, and a pH of
1 to 5. Left: Electron micrograph of a thin section (X85,000);
Right: Fluorescent photomicrograph of cells attached to a
sulfur crystal. Source.

When cells divide, it’s really important that they do so equitably so you don’t end up with some having too much and some too little cell material; the results of either could be deleterious. Therefore, over the billennia that life has been around, mechanisms to carefully divvy up intracellular goodies like DNA and cytoplasm have evolved. You can imagine that interest peaked in those who pay attention to such things when it was found that a group of microbes belonging to the Crenarchaeota lack any of the known cell division machinery. Crenarchaeota are a kind of Archaea that includes species known to survive in extremely hot or cold environments. And when we say extreme, we mean deep arctic-cold and volcano-hot—literally. Other known forms of life have some variant of actin (known as MreB in bacteria) or tubulin (aka FtsZ), which are involved in cell division or maintenance of cell shape. Until late 2008, when a pair of papers was published, no such proteins were found in the crenarchaea and their division remained quite mysterious.

Regardless, these hardy little archaea are getting the job done, one way or the other. It has been known for some time that the Crenarchaeota possess homologs of a system called ESCRT (endosomal sorting complex required for transport). This is an attractive candidate for managing division in these organisms. Why? Because the ESCRT system helps detach daughter from mother cell during cell division in metazoans—such as insects, octopuses, and elephants. So it seemed plausible that these proteins might be helping here as well.

Fine Reading: March of the Microbes by John Ingraham

by Elio

A good friend has written a good book. March of the Microbes, just published by Harvard Univ. Press, presents tales from the microbial world, wide and deep. It emphasizes microbial activities that you can see, smell, touch, taste, and, if you include cows belching, hear. John calls these encounters sightings. Some are manifested in our daily doings, such as the teeth we brush, the bread we eat, the beer we drink, or the fish we left overlong in the ‘fridge. Others require us to go farther afield, making trips to the hot springs of Yellowstone Park, fields of legumes, munition factories, or the San Francisco Bay. You may have heard (or seen) many of the sightings described, although some are not commonplace. Did you know that starch becomes the ubiquitous high fructose corn syrup via degradation by bacterial enzymes? Or that the famed Carlsbad Caverns are the result of bacterial mining? The microbiologist will find the stories both illuminating and authoritative. The lay person will be astonished at the marvels of our unseen world.

This is a book written with love. John is passionate about all microbes he has ever encountered, bar none, and shares his fervor in a clear, unpretentious manner. I should know. Along with Fred Neidhardt, we have our names on four (or is it five?) books.

All Is Fair in Love and Warfarin

by Shigeki Miyake-Stoner and Spencer Diamond

Warfarin, up close and personal. Source.

It turns out the drug warfarin has nothing to do with war, but it does involve a recently discovered link between bacteria and humans. Warfarin derives its name from WARF, the Wisconsin Alumni Research Foundation where it was first developed, and -arin from its coumarin-like structure.

Warfarin is used in two roles: to kill pests, and to save human lives. It was first designed for use as a rodent poison, an anticoagulant that would cause mice and rats to gradually hemorrhage and bleed to death internally. Some years later, it was realized that this drug would be useful for humans too! Patients susceptible to dangerous blood-clotting or thrombosis can take appropriate amounts of this drug to "thin" their blood. Warfarin works by inhibiting Vitamin K epOxide Reductase (VKOR), an enzyme that recycles vitamin K after it acts as a cofactor in activating clotting factors. Now you might ask, what does this have to do with bacteria? Well it turns out bacteria have more in common with mammals than we thought!

Talmudic Question #59

Which prokaryotic cells are more abundant on Earth: planktonic (free living) or sessile (adhering to surfaces)? Keep in mind that the ocean waters contain abundant floating surfaces, e.g., marine snow.

On the Continuity of Biological Membranes

by Franklin M. Harold

The complex world of cell membranes. Source.

Thirty years ago, Günter Blobel of the Rockefeller University published a short paper entitled Intracellular Protein Topogenesis, which laid the conceptual foundations for our understanding of how cells build membranes. To serve their functions, peripheral and integral proteins must be inserted into the right membrane with the correct orientation, and most of the article focused on the manner in which this may be achieved. But it also underscored two startling implications of the proposed procedure: first, that every membrane must be derived from a pre-existing membrane; and second, that all extant biological membranes are descendants of the plasma membrane of the first primordial cell.

Blobel’s article became a classic, and spawned a small industry concerned with the molecular mechanisms that target proteins to the recipient membrane and then either translocate or insert them. In a nutshell, the information that specifies a nascent protein’s disposition is contained in its sequence. One segment of that sequence recognizes a receptor protein embedded in the target membrane, commonly part of the translocon; other segments specify whether the amino acid chain is to be taken clear across the membrane or inserted, and with what orientation. Membrane proteins may be processed concurrently with their translation, or after their production is complete. In prokaryotic cells the proteins are produced and handled directly; in eukaryotic cells they are first inserted into the membrane of the endoplasmic reticulum, and then transferred to their target membrane by cargo vesicles. The details can be found in textbooks of molecular cell biology. What concerns us here is the inference that membrane heredity is a fundamental principle of biology. A functional membrane, studded with a particular set of enzymes, transport carriers and receptors, can never be generated de novo; it must arise from a pre-existing membrane, either by modification (for example, the membranes that surround bacterial spores) or else by growth and division or vesiculation. Moreover, since proteins will only be inserted after interaction with a complementary receptor (and that includes the receptor protein itself), a growing “genetic” membrane propagates its own kind.

The idea that membranes are inherited was by no means novel in 1980; cytologists had been musing on it for two decades. But it was quite another matter to assert that it must be so, that “omnis membrana e membrana.”