Small Things Considered

Fine Reading: March of the Microbes by John Ingraham

2010-03-11T08:45:00-08:00

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

2010-03-08T09:08:50-08:00

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!

Source.

A VKOR homolog was identified in Mycobacterium tuberculosis (Mtb), the bacterium responsible for human tuberculosis. Why might this bacterium have a VKOR? Surely it doesn't need to regulate its blood clotting. Mycobacterial VKOR (MtbVKOR) is involved with making disulfide bonds in proteins, and is quite important for the bug's life processes. A recent paper by Dutton and colleagues investigated the role of MtbVKOR in bacteria.

Knocking out the gene encoding MtbVKOR severely affected growth of Mtb. So did growing Mtb in the presence of warfarin. The authors had previously noted that adding this gene to E. coli restored flagellar function in a mutant lacking the cell-envelope protein DsbB. Since they knew that DsbB is an enzyme involved in forming disulfide bonds, they performed experiments to see if MtbVKOR had the same activity. Indeed, both enzymes mediated the same electron transfer step involved in disulfide bond formation in the E. coli flagellar protein FlgI. Notably, these same transgenic E. coli grown in the presence of warfarin lost both MtbVKOR's disulfide activity and their motility.

SEM of Mycobacterium tuberculosis. Source.

To further compare VKOR and MtbVKOR, warfarin resistant mutants of MtbVKOR were sequenced and found to have point-mutations similar to those that appear in humans resistant to the drug and who require higher dosage.

Although found in vastly different organisms, VKOR and MtbVKOR have the same basic function. The downstream effects of their activity are definitely divergent, but both can be inhibited by the same small molecule, warfarin. It is now clear that this enzyme can be studied for the development of better anticoagulants and better tuberculosis treatments.

The story of warfarin should be of interest to drug companies. In an age of blockbuster drugs and a pill for just about anything, one would think drug companies are doing rather well. Over 12 million Americans take cholesterol-lowering statins, and practically everybody you know has taken at least one prescribed pill in the past year. However, drug companies are begining to feel a pinch as many of them have put all their eggs in one basket, relying on one best-selling drug or another. Many of them are now seeking to expand the list of conditions that can be treated with each one of their drugs. By doing so, the companies can sell more of their drug while it is still patent protected. The evolutionary conservation of enzymatic function and structure will likely lead to more warfarin-like examples, cases where we can find new tricks for an old dog.

The bottom line is that all of this crosstalk is rooted in evolution. When change proceeds without design, as in evolution, it is not easy to create something complex from scratch. We often see that the useful products of evolution get re-used and modified throughout time.

Shigeki (left) and Spencer (right) are students in the University of California at San Diego/San Diego State University Integrative Microbiology graduate course during the 2010 winter quarter.

Talmudic Question #59

2010-03-04T08:45:00-08:00

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

2010-03-01T08:45:00-08:00

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.”

Biology is notoriously so riddled with exceptions that such a sweeping generalization is bound to raise eyebrows: never? Indeed, possible exceptions do crop up from time to time. If this prospect piques your curiosity, take a look at the work of G.H. Kim and his colleagues (here and here), which describes the astonishing capacity of naked blobs of algal cytoplasm to reconstitute a membrane and resume growth. Blobs endowed with nuclei and a sample of organelles survive transiently in the absence of a plasma membrane (sic!), construct a temporary one made of polysaccharides, and finally produce a proper membrane made of lipids; how they do this is quite unknown and would provide a nice test of Blobel’s dictum. In years of reading I have never come across an authentic example of a membrane made afresh, and a query to readers of this blog elicited no response. Like the second law of thermodynamics, the verity that membranes must be grown rather than made rests not on proof positive, but on the absence of any known exceptions.

Even though membrane heredity enjoys general acceptance, it seldom comes up in the literature. The reason, I believe, is that it holds the answer (more correctly, part of the answer) to a question that few scientists are asking, but an important question all the same. As cells grow and divide, the form and arrangement of their internal organelles (many of them membrane-bound, especially in eukaryotes) is quite faithfully transmitted to the next generation; just how does that come about? Time was when transmission of the cognate genes was deemed to be a sufficient reason; though as far back as the sixties scholars such as Boris Ephrussi and Tracy Sonneborn insisted that the inheritance of genes cannot by itself account for the persistence of structural organization. The principle that membranes must be inherited unambiguously sides with those “cytoplasmic heretics” and their followers. Thomas Cavalier-Smith, one of the few prominent scientists to fully embrace Blobel’s thesis, puts it clearly and forcefully:

Two universal constituents of cells never form de novo: chromosomes and membranes…… Just as DNA replication requires information from a pre-existing DNA template, membrane growth requires information from pre-existing membranes—their polarity and topological location relative to other membranes… Genetic membranes are as much a part of an organism’s germ line as DNA genomes; they could not be replaced if accidentally lost, even if all the genes remained.

Structural order is transmitted jointly by copies of the genes and by architectural continuity. One of the reasons that every cell comes from a pre-existing cell is that there is no other way to make a membrane.

Not only are membranes passed from one generation to the next, they are remarkably persistent on the evolutionary timescale. This is most vividly illustrated by the membranes of mitochondria and chloroplasts, both of which descend from endosymbiotic eubacteria. No one knows for sure how ancient these partnerships are, but since all extant eukaryotes apparently derive from a common ancestor endowed with mitochondria, this one must go back one or two billion years, and possibly more. Chloroplasts were probably acquired later, but even that event dates back to at least 600 million years ago, and probably longer. In the course of their “enslavement” and reduction to the status of organelles, most of the endosymbionts’ genes were either transferred to the host’s nucleus or lost altogether. Nevertheless, the membranes of both organelles clearly proclaim their bacterial ancestry, both in their chemical composition and in their morphology. In the case of chloroplasts, the number of membranes that surround the organelle tracks the history of successive episodes of symbiosis. The chloroplasts of green plants and algae, red algae and glaucophytes, offspring of the primary endosymbiosis, are encased within two bilayer membranes, derived respectively from the inner and outer membrane of the cyanobacterial endosymbiont. But the chloroplasts of many other photosynthetic protists are enveloped in three or even four bilayer membranes, which are believed to report a history of secondary or tertiary endosymbiosis: cases in which a non-photosynthetic protist engulfed and assimilated a photosynthetic algal cell in its entirety. (For a review of this complicated story, click here.) Membranes are not immune to evolutionary change; they are subject to radical alteration and reduction of function, and may also be lost altogether. A striking example of membrane transformation is supplied by hydrogenosomes, metabolic organelles of anaerobic protists, which are thought to derive from mitochondria with the loss of the respiratory chain; even more extreme reduction produces residual membranous bodies known as mitosomes. It seems to be the membrane-bound compartment, not its functional proteins, that has the propensity to endure; sometimes what matters is the bag, not its contents.

Organelles make an impressive example of the persistence of membranes, but one could wish for more of them. A likely one comes from the Archaea, whose membranes all display a distinctive complement of lipids and ion-translocating ATPases, even though their environments range from volcanic hot springs to the open ocean and the stomach of cows; it cannot be natural selection alone that maintained the archaeal signature! As for eukaryotic cell membranes, they are evidently of dual origin. Those of mitochondria and chloroplasts were inherited from the endosymbionts; the provenance of the others is in dispute, but the most plausible hypothesis at present is that the membranes of the nucleus and endoplasmic reticulum represent infoldings of the host’s plasma membrane. Let me reserve this minefield for a future comment; in the meantime, should you know of other examples of membrane heredity, do please let me know!

The doctrine that it takes a membrane to make a membrane has profound implications for the origin and evolution of cells. First, if the molecular machinery of protein translocation is required to put in place integral membrane proteins, how could functional membranes have existed before there were translocons, let alone proteins? If every membrane must grow from a pre-existing membrane and reproduces (or modifies) its topology, how could this lineage have begun when there were no membranes to copy? This is one of the many chicken-versus- egg paradoxes that bedevil the mystery of cellular origins, and one that is not at all laid to rest by postulating that an RNA World preceded the DNA/RNA/Protein World that we inhabit today. Second, persistence of membranes carries a strong hint that the conventional view, which derives the first cells from aggregation of biological molecules produced by abiotic chemistry, is fundamentally mistaken. Instead, life must have been in some degree cellular from the very beginning: the product of co-evolution of genes, catalysts and membranes in a structured setting, as Cavalier-Smith has argued for many years.

Liposomes: Lipids like to organize themselves into
membranes, but this does not appear to be the way
cells make their membranes. Source.

The genesis of membranes, and of cells, quite passes understanding; nevertheless, the field presently displays a ferment of experiments and ideas that must owe something to the relentless challenge from advocates of intelligent design. In his original paper, Blobel suggested that the first precursors of cellular life were lipid vesicles that had formed spontaneously in the primordial broth. Their outer surfaces provided capturing devices for the coalescence of ancestral molecules involved in replication, transcription, and translation, as well as metabolic enzymes, all assumed to be present in the surrounding medium. Translocation of molecules or segments thereof across the lipid bilayer into the interior phase would have evolved at this stage. Note that the polarity of these protocells would have been inside-out relative to cells as we know them (enzymes and ribosomes on the outer surface, not in the lumen). So Blobel sketched a scheme to make them invaginate, close up into a “gastruloid” enveloped by a double membrane, and thus assume the familiar polarity of contemporary cells, with all the machinery on the inside. These ideas have been adopted by Cavalier-Smith, who explains in much detail how “obcells” would have formed, functioned and at last turned outside-in. An important element of Cavalier-Smith’s thinking is that the first true cells were enveloped in two bilayer membranes; cell evolution must, therefore, have begun with “negibacteria,” i.e., Gram-negatives. It all makes sense, if you can believe that at least the rudiments of life’s molecular machinery took form out there in the soup, with inorganic polyphosphate tossed in as an energy source. But the obcell hypothesis has never caught on, presumably because readers judge it to be just too implausible, and so do I. A recent re-formulation by Griffiths addresses some of the difficulties, but falls short of eliciting a “Eureka!” reaction.

But what are the alternatives? Lipid membranes can form abiotically (ingredients are even found in carbonaceous meteorites), and they can encapsulate macromolecules such as RNA and a polymerase. But the spate of recent publications in this vein (here and here) never touches on the issues raised by membrane protein topology, nor on membrane inheritance. So let me instead draw attention to a very different idea that has languished on the fringes of serious science ever since the geochemist Michael Russell first articulated it two decades ago, but is now gaining traction. Believers find the cradle of life (here and here) in the nooks and crannies of porous mineral deposits formed at the edges of submarine hydrothermal vents, specifically warm and alkaline ones such as the Lost City field. Alkaline hydrothermal vents make an attractive venue for the early stages of chemical evolution: sequestered spaces, reasonable conditions, and an ample supply of precursor molecules including hydrogen gas, methane, and small organic compounds. Geochemistry even supplies a potential energy source: the large difference in pH between the alkaline vent fluids and the acidic bulk water (as much as four units). It does not strain credulity to suggest that among the products of vent chemistry may have been amphipathic molecules that aggregated upon surfaces and occasionally generated primitive membranes. If (and what a big If that is!) chemical complexity burgeoned in the honeycomb to the point of simple metabolism and heredity, some of those membranes may have grown, propagated their kind and come to enclose cell-like bubbles with the correct polarity. In the fullness of time, could some of those bubbles have escaped from their inorganic hatchery, setting forth to seek their fortune and inherit the earth? Might the fundamental differences in lipid chemistry between Eubacteria and Archaea report separate origins from different hydrothermal mounds? Well, let’s not get carried away. The notion that cells were born of hydrothermal vents also has multiple pitfalls, notably the lack of any obvious driving force to channel chemical evolution in the direction of biological functions; but it is a fantasy well worth pondering.

This is all good, clean fun—as long as we prize the doubt, keep a sense of humor, and do not pretend to the authority that comes only with hard, experimental science. Karl Popper taught us that science advances best by the interplay of conjecture and refutation; unfortunately, students of cell evolution do the former rather better than the latter. Even in this Age of Omics, when it comes to making sense of the incomprehensible we can only place our trust in tales of the imagination.

Franklin M. Harold, Department of Microbiology,
University of Washington, Seattle, WA 98195.
E-Mail: frankharold@earthlink.net

The Next Generation (Or Two)

2010-02-25T08:45:00-08:00

by Elio

Source.

Student blogs there are that gladden an old man’s heart. Here’s a sampling.

In Catalogue of Organisms, Christopher Taylor, a student of arachnids in Perth, Australia, posted a new interpretation of the mysterious Prototaxites—giant, 8 meter tall fossils some 400 million years old that predate any plants of that size. It was thought that these megastructures were fungi (see our post on this). It has now been proposed that they are sheets of liverworts that rolled up as they cascaded down slopes. Christopher points out things that may be wrong with this scheme.

In Skeptic Wonder, Psi Wavefunction, an undergraduate in British Columbia, takes on the term “Oncogene” and explains why it should disappear forever. Her writing is so lively that we published a guest post by her recently.

In Micro Writers (“written by students to students”) that comes to us from Cairo University, Mariam points to the wisdom of escaping from anthropocentric to biocentric microbiology. This post is based on a commentary by Ramy Aziz published in Gut Pathogens that was highlighted on our blog not long ago.

In Extreme Biology, students post about "anything biology-related." These students have not yet graduated from high school! To our delight and awe, Amy Ciardiello, a 9th grade violinist, writes about "violin-making and fungi"—a topic we had previously posted (here and here) on STC. She accompanies her post with a superb performance of the 1st movement of Haydn's Concerto No. 2 in G Major. Go there and feast both your mind and your ears.

We welcome notices of other microbiological research blogs presented by students.

Mother’s Love

2010-02-22T08:45:00-08:00

by Elio

Binary fission is a most impressive invention. In one fell swoop, it ensures that progeny cells are born alike and endowed with the same potential for growth and survival. Simple as it sounds, it must have taken considerable evolutionary contortions to make it function so well throughout the living world. But there are cells that have adopted an alternative mechanism, where cell division is asymmetrical, where one progeny cell is made from a “mother cell” that keeps generating “babies.” The best known example is, of course, budding in yeast. But other cells also arise in this fashion, including some bacteria, the sexual spores of mushrooms, and even some plant cells.

The polarisome in a forming yeast bud. Actin cables
growing out from the polarisome transport protein
aggregates to the mother cell. Source.

So, is there an advantage to bypassing binary fission and budding instead? It would seem that way. Recent work from Tom Nyström’s lab has shown that proteins that become damaged in the course of cell growth flow back into the mother cell and leave the young bud free of such impediments. The damage to proteins is often due to oxidation by reactive oxygen species. Damaged proteins tend to form aggregates. This can be bad, getting rid of them is good. How do these proteins accumulate in the mother cell? Interestingly, protein aggregates hitch a ride on actin filaments that grow from the growing bud to the mother cell. Such filaments are assembled at the tip of the bud in a structure the authors call a “polarisome,” which is made up of core proteins plus some involved in actin polymerization (formins). Also required is the age-retardant deacetylase, a protein called Sir2 and aka sirtuin. Previously known as a life span modulator—not just in yeast but in worms, fish, and mammals as well—Sir2 has now been found to also be involved in actin-related processes, hence in polarisome formation. It gets more complicated. For an overview, we suggest a commentary by Guarente.

(A) Exponentially growing young yeast cells to compare their size and
morphology to that of old cells. (B) A typical lifespan determination.
The number of budding cycles that each of a set of 50 ‘virgin’ cells
undergoes before it stops dividing was determined by micromanipu-
lation and counting of budding cycles. (C) M is the terminal mother cell
after 15 cell cycles. Note the enormous size as compared to young cells
and the surface changes. D14 is the second but last daughter that did
not completely separate from the mother and did not give rise to new
living cells. Source.

Now let’s look at this story in a broader context. It’s not just about shipping dirty laundry off to mother. One consequence of the asymmetry of budding is that the mother cell retains its bodily integrity bud after bud, whereas this is lost when a cell divides by binary fission. In yeast, a mother cell can bud some 15-30 times, after which it conks out. How do we know? Counting the number of times a cell gives off buds is done patiently under the microscope, using a micromanipulator to remove each daughter cell once it separates from the mother cell. Keep this up until the mother cell gives off no more buds. Imagine teasing the new cells away for the mother cell 30 times or so! (This seems to have been done first in 1950 by A. A. Barton, working for a British brewing company.) This phenomenon is called senescence, and is visually illustrated by wrinkles of old age and the unusually large size of the Grande Dame. Newly formed cells start the process anew, each daughter cell becoming a mother cell of its own. However, in time, the newly made cells are less competent for further budding. Not surprisingly, yeast is a favorite for studies of cell polarization and its possible role in senescence. Many papers have been written on this subject.

A favorite connection between yeast budding and aging relies on a 120-year-old theory by August Weismann. He postulated that aging evolved from the need to separate germ cells from somatic cells. Germ cells need to be protected from damage; somatic cells can ”take it.” One of the reasons given is that additional resources must be bestowed on germ cells to ensure their genetic stability. Somatic cells, in contrast, do not have such mechanisms and thus accumulate damage.

This is one way to think about asymmetric cell division. Formally speaking, the mother cell acts as a somatic cell that produces multiple germ cells, the buds. Each, when grown, becomes a mother cell with full reproductive potential, able to produce a full complement of buds of her own. During budding, the young bud escapes from the cell damage represented by the aggregated proteins, thus foiling at least that aspect of cell aging.

If asymmetric cell division affords such protection of the germ line, why don't all cells do this? The question is not terribly relevant to multicellular somatic cells, given that they are not involved in germ line propagation (unless some investigator teases their nuclei out and introduces them into eggs). But how come more unicellular microbes haven't adopted the budding strategy? That is a question for another time.

Given that yeast is the best known of all eukaryotic organisms, allowing endless kinds of genetic manipulations, it’s no wonder that it has become a model for the study of aging. And here I thought that I would be a good subject for researching what happens in old age!

Addendum: As Qetzal noted (see his comment below), I could have mentioned that a possibly analogous phenomenon has been reported for bacteria. When E. coli divides, the "old" cell pole accumulates chaperones involved in the aggregation of (presumably) damaged proteins. Eventually the "old" cells lose their reproductive capability. Something analogous takes place in Caulobacter crescentus. Thus, bacteria may also use the strategy of segregating damaged proteins in aging cells, to the advantage of the population as a whole.

Liu, B., Larsson, L., Caballero, A., Hao, X., Öling, D., Grantham, J., & Nyström, T. (2010). The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates Cell, 140 (2), 257-267 DOI: 10.1016/j.cell.2009.12.031

Prophage Masquerade

2010-02-18T08:45:00-08:00

by Merry

Source.

Roseovarius nubinhibens recently joined the exclusive club of about a thousand bacteria whose genomes have been sequenced. Why this honor? It’s a member of one of the most ubiquitous and most intensely studied clades of α-Proteobacteria, the marine roseobacters. This populous group participates in important jobs, including the global cycling of sulfur, climate regulation, and even modulation of our day-to-day weather. (For the latter, see our earlier post.) To appreciate the sulfuric importance of R. nubinhibens in particular, we need to begin with the major quantities of dimethylsulfoniopropionate (DMSP) made by marine algae for whom it serves as an osmolyte. Roseobacters then enter the picture, their work being to break down the DMSP by either of two pathways. One route converts DMSP into volatile DMS that can give rise to sulfate aerosols that act as cloud seeds. The other pathway leads to methanethiol, a primary sulfur source for marine bacteria. The relative amount of sulfur that flows each direction matters. R. nubinhibens was one of the first bacteria found to be able to carry out both conversions, thus is a potentially important switch point. The researchers reporting its isolation (José González and colleagues in Mary Ann Moran's lab) dubbed their new species Roseovarius nubinhibens (from the Latin nubes, for clouds, and inhibens, for inhibiting).

Virus-like particles generated by mitomycin C induction of R. nubinhibens
ISM. Siphovirus particles are commonly seen in the induced lysate, and
many of them appear to be “empty.” Bars = 50 nm. Source.

The R. nubinhibens genome has been sequenced. Any prophages on board? A routine search using Prophage Finder (software that looks for clusters of phage-related genes) turned up several candidates. Let's refine the question and ask whether there are any inducible prophages on board. Add mitomycin C (a potent DNA cross-linker) to a growing culture and wait 24 hours. Voilà! You get ~10¹⁰ VLPs per ml compared to ~10⁵ for control cultures. (What is a VLP? It's a Virus-Like Particle, i.e., something that looks like a virus but hasn't been shown to infect like a virus.) The induced VLPs have the long, flexible tails and polyhedral heads characteristic of siphoviruses, but most of these VLPs are empty-headed and/or have broken tails, suggesting that many are defective.

Was one of the predicted prophages the source of the VLPs? The DNA strands inside the VLPs were shown by pulsed-field gel electrophoresis (PFGE) to be about 30 kb, suggesting that the prophage might be about the same. None of the predicted prophages were near this size. So the researchers then searched the genome for any phage-related genes. They were lucky. They found an integrase gene along with a few other recognizable phage genes in a 27 kb region that Prophage Finder had overlooked. They concluded that they had found the prophage associated with the VLPs because they identified a gene in this stretch of DNA that encodes a major capsid protein and showed it to be identical to the capsid gene in the VLP DNA.

So what else does that prophage DNA encode? A few genes required for the prophage life cycle could be tentatively identified. Of the 28 genes that had significant hits in the GenBank database, 25 are most similar to genes of unknown function previously found in Rhodobacterales bacteria. Might those 25 "bacterial" genes actually be phage genes that are part of the unsequenced majority and that are residing in prophages? No wonder Prophage Finder—looking as it does for known phage-related genes—didn’t find the real prophage! Since more than 99.9% of phage diversity has not been captured in any database, hunting for prophages with Prophage Finder is like going birding with a field guide that includes less than one species out of a thousand.

Other sequenced members of the Rhodobacterales harbor related prophages, suggesting that this type of prophage is common and may be active in horizontal gene transfer (HGT) among these bacteria. Tantalizingly, there may be a subplot to this story, with even more HGT going on. The PFGE analysis of the VLP DNA also found a few short segments of 3, 4, and 12 kb. The authors suspect that these may be random bits of host DNA packaged inside capsids, i.e. gene transfer agents (GTAs). (For our earlier post about GTAs, click here.) GTAs transfer genes between members of the same species. Their production is controlled by the bacteria, and neither viral infection or cell lysis is involved. GTAs have been found only in α-Proteobacteria, with a particularly high incidence among the roseobacters. Previously, 21 of the 22 sequenced members of the marine Roseobacter clade had been found to carry the genes for GTA production; now R. nubinhibens makes it 22 out of 23.

What do GTAs have to do with prophages? Did one evolve from the other? If so, in which direction? In the words of two researchers in this field: The overall pattern of relationships between these various GTA, phage and prophage elements supports the notion of a continuum of sequence relationships resulting from transfers throughout a global phage gene pool. And we have barely dipped our toes into that global pool.

Zhao Y, Wang K, Ackermann HW, Halden RU, Jiao N, & Chen F (2010). Searching for a "hidden" prophage in a marine bacterium. Applied and environmental microbiology, 76 (2), 589-95 PMID: 19948862

Five Questions About Lysogeny

2010-02-15T08:44:00-08:00

by Merry

Lysogeny—a nasty time bomb or a mutually beneficial symbiosis? A prophage gone lytic will murder its host, but a symbiotic picture can well be argued. Here are some thoughts about the ongoing give-and-take. More details are still emerging.

1. If you are a phage, why be temperate?

A phage at the crossroads. Lambda (λ), the intensely-
studied temperate phage of E. coli, chooses between
the lytic pathway and lysogeny. Source.

For a phage, temperance offers the obvious advantage of providing a safe haven when host cells are few and far between or when conditions are not good for their rapid growth. Indeed, one does find more lysogens in nutrient-poor environments or during winter months. While nestled within a host chromosome, the prophage is faithfully replicated pari passu with host DNA. If the host clone prospers, so does the prophage. Here the prophage is protected from the heat-labile factors (proteases secreted by bacteria?) that can chew up a virion. Even while inside an intact virion, the phage DNA can be damaged by UV light. In sunlit waters, the number of infective virions is typically less than the number of intact virions, the difference being accounted for by virions that contain UV-damaged DNA. Prophages are also subject to UV damage, but being a segment of the host chromosome the damage is often mended by the host's DNA repair machinery. Of course, there are trade-offs here. Lysogeny eliminates the risky business of extra-cellular survival and locating a host, but one hungry protist can end the game for all concerned.

2. If you are a bacterium, why tolerate a prophage?

Source.

Having a prophage on board burdens you with more DNA to be replicated and with a passenger that just might kill you should conditions change. But said prophage protects you from infection by related phages and often supplies genes of immediate usefulness. Good examples of the latter are prophage-encoded toxins and other virulence factors, such as those essential for the pathogenesis of V. cholerae, E. coli O157, and C. diphtheriae. Even the temperate lambdoid phages of E. coli provide genes that make their hosts resistant to killing by serum complement. Some adaptations of nonpathogenic bacteria to a specific ecological niche almost surely also rely on useful prophage genes. It is thought that most of these genes had been acquired from previous hosts. Their conveyance by phage is central to horizontal gene transfer among prokaryotes.

This role of prophage as a source of new genes is of colossal importance. Bacterial genomes are composed of a core genome shared by virtually all members of a species, plus a halo of strain-specific genes. Prophage (along with related genomic islands) appear to be a primary source of these defining genes.

3. How do prophage genomes evolve?

Looking at this evolutionarily, one would not expect an entire prophage to become fixed in the bacterial genome since it is only the rare prophage gene that increases host fitness. If any of the prophage genes necessary for induction are inactivated, all the better for the host. The time bomb has been defused without the loss of potentially useful genes. (Click here for a paper about an archaeal prophage that has lost the ability to excise but still protects its host from viral infection and, under conditions of starvation, makes a lytic enzyme that lyses the host.) Once the prophage can no longer go lytic, its genes are released from selection pressure and gradually decay. Thus, in hindsight, it is not surprising to find many bacteria carrying noninducible prophages that have suffered varying degrees of gene loss. The rare useful genes, typically ones picked up by the phage from a former host, may become part of the host genome—testaments to horizontal gene transfer.

4. Are prophage genes active?

Coliphage lambda. Source.

Prophages account for a significant fraction of the host genome, especially in pathogens. E. coli O157:H7 strain Sakai contains 18 prophage elements that total 16% of its DNA; the 4-6 prophages found in Streptococcus pyogenes make up 12% of its genome. The percentages in non-pathogens are lower, but still prophages contribute many genes to the lysogen. Early studies on a few model systems (the temperate lambdoid phages of E. coli in particular) suggested that most prophage genes are repressed, that only the few regulators needed to maintain lysogeny are transcribed. When more lysogens were scrutinized, more prophage genes were found to be active during lysogeny. In some cases transcription is up- or down-regulated in response to changes in host state or environmental conditions. Expression of some prophage genes is under host control, such as the diphtheria toxin gene that is governed by the same mechanism that regulates the expression of some bacterial genes in the presence of iron. In free-living bacteria, transcription of some prophage genes of unknown role (morons) is constitutive.

5. How common is lysogeny?

Many numbers are bandied about. Of all terrestrial bacteria cultured as of 1987, 47% were reported to be lysogens. A 2008 paper estimated that 60–70% of all sequenced bacterial genomes contain prophages. Those with 6 or more are mostly pathogens. Prophages are less common in Archaea and in bacterial endosymbionts that have undergone pronounced genome reduction. All of these numbers are best viewed as approximations. Since formerly active prophages in varying stages of decay can linger in the host genome, when does a decaying prophage cease to qualify as a prophage? This is like asking when that burrito you had for breakfast ceases to be a burrito. Should one count only those prophages that can be induced to enter the lytic cycle by mitomycin C or UV irradiation? By that criterion, roughly half of the marine bacterial isolates contain prophages. This gives you some idea of how many bacteria likely harbor active prophages that might be induced under various environmental conditions.

Suppose that you want to know how many bacteria carry prophage genes, not just those with inducible prophages. For bacteria whose genome has been sequenced, the first step would be to scan their genome using ab initio gene prediction software to locate every potential gene—an imperfect business in itself. Next, use BLASTx to compare the ORFs, one by one, to all known phage genes. When you find an ORF that has significant similarity to a phage gene, closely examine its neighborhood for more phage-related genes. Find a cluster of phage-related genes and you might have located a prophage genome. One major flaw here: all of the currently "known" phage genes are estimated to represent less than 0.0002% of the total number in existence. So a lot of prophages and prophage relics can be missed.

You could instead zero in on one particular prophage gene. For example, all active prophages encode an integrase (the enzyme that catalyzes the insertion of the prophage into the host genome). Integrases can be efficiently detected by in silico analysis, but spotting an integrase does not necessarily mean you've found a prophage since integrases are used by other types of mobile elements, as well.

So to answer the original question, we don’t know exactly how many, but there are a lot of lysogens out there, and prophage relics abound.

Meanwhile, a sixth and clearly unanswerable question has come to mind. If lysogeny is so great, why aren't all bacteria lysogens? John Paul offered some thoughts on this. Phages typically have very narrow host ranges. Thus, when a prophage protects its host from infection by related phages, this may protect it from virtually all infection. It follows that as the percentage of lysogens increases, it becomes more and more unlikely that a phage virion will meet up with a susceptible host. At some point, the phage population would decline and lytic infection would come to a halt. That might sound like a boon for the bacteria, but not really. In aquatic environments lytic infection plays an important evolutionary role by fostering bacterial diversity through "kill-the-winner" dynamics. Paul posited that it may be that one could not have more than half lysogens and still keep lytic processes and kill-the-winner ongoing. And about half is what we have.

Canchaya, C., Fournous, G., & Brussow, H. (2004). The impact of prophages on bacterial chromosomes Molecular Microbiology, 53 (1), 9-18 DOI: 10.1111/j.1365-2958.2004.04113.x

Paul, J. (2008). Prophages in marine bacteria: dangerous molecular time bombs or the key to survival in the seas? The ISME Journal, 2 (6), 579-589 DOI: 10.1038/ismej.2008.35

Of Archaeal Periplasm & Iconoclasm

2010-02-11T08:46:00-08:00

by Elio

Rough work, iconoclasm, but the only way to get at truth.
Oliver Wendell Holmes

A thin section of Ignicoccus hospitalis under
the EM. C = cytoplasm. CM = "cytoplasmic"
membrane. P = periplasm. OS = outer sheath
(membrane). Bar = 0.5 µm. Source.

Biology is the iconoclast’s paradise. Over and over, cherished beliefs, some dating back for centuries, fall to the ground as exceptions to the rule are discovered. To the long list of such exceptions, we now add the finding by groups in Regensburg and Frankfurt that the outer membrane of an archaeon, Ignicoccus hospitalis, is energized and capable of generating ATP. Granted, this is a hyperthermophile who helped shatter the ancient belief that life at high temperatures is not possible, thus hardly a conformist. But this discovery is, to say the least, unexpected.

The old tenet is that the energetic business-end of prokaryotes is the cell membrane, whether surrounded by an outer membrane, as in Gram negatives, or not, as in Gram positives. I should know, I taught this for umpteen years. True, in Gram-negatives, energy can be transmitted to the outer membrane via the Ton system (a system that provides energy for the transport of iron siderophores, vitamin B12, and some colicins), but the reverse, making energy on the outer membrane and sending it to the cytoplasm, is not part of the old belief. Yet it’s been known for some time that a goodly number of bacteria can energize their outer membrane. They do this by having cytochromes inserted in the outer membrane where they carry out a process known as extracellular electron transfer. This ability stands the organisms in good stead, allowing them to utilize metals in rocks as electron acceptors.

Localization of A₁A_O ATP synthase on I. hospitalis cells in ultrathin sections. (A) Labeling
with antibodies against the purified ATPase complex, (B) Labeling with antibodies against the
membrane-bound subunit a. For both images the secondary antibody with ultrasmall gold
particles is visualized. C = cytoplasm. IM = inner membrane. V = vesicles in the periplasm.
OM = outer membrane. Bar = 1 µm. Source.

First of all, dual membrane systems have not be found in archaea other than Ignicoccus. What are the new conclusions about power generation in its outer membrane based on? Mainly on immunoelectron microscopy of sections using gold-labeled antibodies and immunofluorescence, which revealed that ATP synthase and H2:sulfur oxidoreductase are located entirely in the outer membrane. These two enzymes are required for energizing membranes and for ATP production. Thus, ATP can be expected be made in the outer membrane and released into the periplasm (which in this organism is huge—larger than the cytoplasm). You may ask, are these two enzymes also found in the inner membrane? The answer is no. Since the periplasm is so large and the two membranes so far apart, enzyme localization to one membrane or the other can be readily discerned. This introduces the question: which is the cytoplasmic membrane in this organism? The figure tells you something about the rare structural complexity of this organism. Note that the two membrane system is different from that of ordinary Gram-negatives, as here the outer membrane is not known to contain LPS or porins.

Co-culture of Ignicoccus spec. (green) and
Nanoarchaeum equitans (red). ss rRNA sequence-
specific fluorescence staining. Bar = 1 µm. Source.

Is the Ignicoccus story relevant to other prokaryotes? Who’s to say at this point. Ignicoccus is mightily idiosyncratic, e.g., it’s unique among archaea in having a two-membrane system. Not only does it grow at very high temperature and use reduction of elemental sulfur as its main energy source, but it also lives in intimate association with another archaeon, the smaller Nanoarchaeum equitans, which has a reduced genome and apparently gets its energy from its larger partner. The unusual ignicoccal ability to make ATP within its periplasm may help it to supply ATP to its associates across the outer membrane.

The authors propose a tantalizing notion: if the eukaryotic cell arose by an archaeon having swallowed a bacterium (hold on, we’re not getting into that discussion right now), then Ignicoccus or something like it would have been the ideal ancestor, able as it appears to be to donate ATP to anyone residing within its boundaries. True or not, one should further respect the outliers in the biological scheme of things as potential sources of novel and deeper relationships.

Research Blogging Citation
Kuper, U., Meyer, C., Muller, V., Rachel, R., & Huber, H. (2010). Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0911711107

Naegleria’s Split Morphology Disorder

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

by Psi Wavefunction

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

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

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

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

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

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

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

Naegleria and Tetramitus: Heteroloboseans with 'split morphology disorder'

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

Naegleria gruberi. Source.

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

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

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

Of Naegleria's culinary indulgences

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

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

Amoeboid to flagellate transformation

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

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

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

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

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

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

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

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

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

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

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

Talmudic Question #58

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

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

A Close Encounter of the Enological Kind

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

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

by John Ingraham

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

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

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

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

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

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

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

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

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

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

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

Oenococcus oeni. Source.

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

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

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

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

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

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