Small Things Considered

Plasmalogens Have Evolved Twice

by Howard Goldfine

Some biologists go blissfully through life without paying much attention to lipids. They do this at their own risk, because there are innumerable things to be learned from their study, including, as we will see here, many relevant to the understanding of evolution. Lipids come in unexpected and exciting varieties, a point that has been acknowledged in this blog (for examples, see here and here)

The lipids that make up the membranes of prokaryotes are polar, that is, they have a moiety such as phosphate, linked to one of the carbons of their glycerol backbone (non-polar triglycerides are generally not known to be made by prokaryotes). The lipids of aerobic and facultative bacteria are mainly of the phospholipid or glycolipid type, in which the first two carbons of the glycerol backbone are linked to long-chain fatty acid esters (Figure). The situation, however, is different in many anaerobes, including both Gram-positive and Gram-negative species, in which the membranes contain both diacyl lipids and compounds known as plasmalogens, in which the chain linked to the first carbon of the glycerol is attached through an O-alk-1’-enyl ether bond (Figure) rather than an ester bond. Plasmalogens were accidentally discovered by Robert Feulgen in 1924. He observed that the cytosol of animal cells turned red when stained with a colorless fuchsin-sulfurous acid reagent, aka known as the Schiff reagent. Such staining reveals compounds that contain aldehyde groups. The red color did not appear if Feulgen’s preparations were first treated with alcohol. He called these compounds plasmalogens meaning “aldehyde-forming substances found in the plasma.” It wasn’t until the 1950s that the correct structure of these ether lipids was worked out. Feulgen and others found plasmalogens in many animal tissues; they are present in especially high concentrations in the brain, CNS, and muscles. Indeed, >60% of the phosphatidylethanolamine (PtdEtn) of our brain is in the plasmalogen form.

Robert Feulgen. Source.

In the late 60s and early 70s the biosynthetic pathway for plasmalogen synthesis in animal tissues was elucidated. It was found to start with the acylation of dihydroxyacetone phosphate (DHAP), rather than with glycerol-3-P as is the case for diacyl lipids. A saturated ether bond is formed by replacement of the acyl chain on DHAP with a long chain alcohol. A number of remodeling steps lead to a form of PtdEtn containing the saturated ether bond. The last step is an aerobic desaturation of the saturated ether between the first and second carbons yielding plasmenylethanolamine, the plasmalogen equivalent of PtdEtn (figure).

But among bacteria today only anaerobic species contain plasmalogens. In these organisms, the final oxygen-requiring desaturation reaction is not possible. Furthermore, we know through studies with tritium-labeled glycerol, that bacteria do not use DHAP for plasmalogen formation. Clearly they have another pathway, one that most likely evolved long before these lipids were made by animal and plant cells. The early earth had an anaerobic atmosphere; hence the first living things were anaerobes. Indeed, reactions essential for making bacterial cells—including amino acids, purine and pyrimidine bases, lipids, and essential cofactors—are still anaerobic, in line with their anaerobic ancestry. Present evidence suggests that plasmalogen synthesis in bacteria follows the same pathway used for the formation of diacyl phospholipids, a pathway starting with glycerol-P, but in this case the conversion of diacylphospholipids to the corresponding plasmalogens is carried out via an unknown mechanism.

So, plasmalogens appeared early, but did not survive in aerotolerant and aerobic bacteria. Why not? A clue comes from the finding that the alk-1-enyl ether bond in plasmalogens is broken by reactive oxygen species (ROS) such as superoxide, hydroxyl free radicals, and singlet oxygen. As oxygen increased in the earth’s atmosphere, respiration, with its ability to generate lots more ATP than fermentation, evolved in bacteria. ROS are formed in cells during respiration, typically at the last step in the electron transport chain. This created a problem that aerobes had to solve. The simplest solution was to get rid of plasmalogens and replace them with lipids containing only acyl esters. This process can even be replicated in the laboratory today (see here and here). The separate evolution of Archaea reveals another solution; they make lipids with chemically stable, saturated ether bonds.

Structure and conformation of a phospholipid and a plasmalogen.
A. Phosphatidylcholine (a phospholipd). B. Plasmenylcholine
(a plasmalogen). Source.

As plants and fungi evolved, they appear to have done well without plasmalogens, but in the simplest animal cells plasmalogens reappear, here made by an aerobic mechanism. Oxygen became an essential reactant for protozoa and metazoa. It is needed for making the sterols and steroid hormones, polyunsaturated fatty acids and eicosanoids formed from them, for the synthesis of certain amino acids, and as noted here, for the synthesis of plasmalogens as well.

But why are plasmalogens needed by animals and why have they been retained in anaerobes? Plasmalogens have a different three-dimensional structure from diacyl lipids, one that allows them to pack more closely, thus rendering membranes less permeable. This may account for their presence in conductive tissues such as the CNS. Further, they can be cleaved by specific enzymes, thus regulating the production of free fatty acids such as arachidonate, the precursor of eicosanoids. It seems that nature rediscovered a useful molecule and learned to make it by a new mechanism. This poses another puzzle: how do animal cells which make ROS avoid the potential damage they can do to plasmalogens? The answer appears to be that, unlike bacteria, they can repair the damage by acylation of the resulting hydroxyl group at the first carbon of the glycerol backbone. Interestingly, this has been proposed as a mechanism to detoxify ROS. It seems that nature has turned the tables!

Howard Goldfine is Professor of Microbiology at the University of Pennsylvania School of Medicine.

Goldfine, H. (2010). The appearance, disappearance and reappearance of plasmalogens in evolution Progress in Lipid Research DOI: 10.1016/j.plipres.2010.07.003

An Inactive Mine Provides Active Opportunities

Acid mine drainage in Spring Creek down-
stream from the Richmond Mine, part of
the Iron Mountain Mine Superfund Site
nine miles northwest of Redding, Calif.
Microbes inside the mine eat pyrite─fool's
gold─to produce sulfuric acid, creating the
most acidic groundwater ever measured.
Spring Creek flows eventually into the
Sacramento River. (UC─Berkeley) Source.

by Elio

“...a riddle, wrapped in a mystery,
inside an enigma...”
                                  Winston Churchill

Metagenomics is a fine tool indeed for surveying a microbial community in concert, treating both the cultured and uncultured equally. When the sample studied is rich in microbial variety, as often is the case, the pieces of genomes can be reluctant to reveal the genetic heritage of whole microbes. But there are a few particular environments that are dominated by a handful of species at most, and here this approach allows the reconstruction of complete genomes. That is the case with the acid mine drainage from mineral or coal mines. When mining ceases, all hell can break loose (anthropocentrically speaking). Microbes oxidize sulfides such as pyrite (iron sulfide) into sulfuric acid, which in turn solubilizes iron, copper, arsenic, silver, gold, and other heavy metals. Water no longer being pumped from the mine, this gemish of minerals emerges from seeps and other openings to become a highly toxic, low pH stream that eventually pollutes larger bodies of water. But for some bacteria and archaea, this is a juicy place to live and thrive.

A researcher collects samples of the pink
biofilm floating atop hot, green, acidic pools
in the Richmond Mine at Iron Mountain,
California. (Brett Baker/UC Berkeley) Source.

The most acidic water known anywhere emerges from the Richmond Mine, near Redding, in Northern California. This mine, inactive since 1963, has been studied by members of Jill Banfield’s lab at Berkeley, who have amassed an impressive amount of microbiological information. Favored for study are thick, slimy, pink microbial mats sitting on green water. Here, the dominant organism tends to a bacterium, Leptospirillum sp., sufficiently abundant that its genome could be reconstructed from metagenomic data. In addition, some unusual archaea found here turned out to be, as the authors say, enigmatic. They can be identified with fluorescent probes within biofilms that they term “blanket strips.” We asked Dr. Banfield for further enlightenment: Blanket strips is a term to describe the appearance of the biofilm. Imagine a pink fluffy blanket cut into strips. These are floating on hot water at a pH between 0.5 and 1.5 (low enough to dissolve the metal eyelets of the researchers’ boots). As an aside, she also shared with us that working underground at this site is like an extreme sport (it is extremely hot and humid). These organisms have been called ARMAN (for Archaeal Richmond Mine Acidophilic Nanoorganisms) and have been found in other environments as well, such as boreal wetlands and a New Zealand hot pool.

When it comes to the unique ARMAN attributes, one hardly knows where to begin. First, ARMANs are very small, even for environmental organisms, which tend to be on the slim side. ARMANs are 200-400 nm in diameter and able to pass through 450 nm filters. Their volume is a puny 0.009 µm3 to 0.04 µm3.

The taxonomic affiliations of the top blast hit for all proteins from each
genome to the KEGG genome database. Source.

Next, their genomes. For some of the analyses, the cells in question were concentrated by filtration. Genomes of the three strains under study have about 1 million bp and therefore are among the smallest known for free-living organisms. As expected from such parsimony, most of the DNA codes for proteins. Many genes are about 10% shorter than usual, which is also true for some obligate parasites such as the archaeon Nanoarchaeum equitans and the bacterium Neorickettsia sennetsu. So far, so good, but what is surprising is that their genomes straddle major evolutionary divides. Phylogenetically, they fit within the Euryarchaeota, yet the majority of their genes that can be assigned to COG’s (clusters of orthologous groups) are of the Crenarchaeota type. Just as surprising, as many as 21% of their genes look like they are bacterial! And some 25-38% of the genes, depending on the strain, have no homologies with anybody else. Where then do these waifs belong? Analysis of their 16S RNAs and several protein genes suggest that they fit near the root of the Euryarchaeota. But is this shoehorning them? We expect that the parentage of these odd organisms will be scrutinized further.

(A) An ARMAN cell on the right is being penetrated by a Thermoplasmatales lineage archaeon
on the left. Also shown is a virus attached to an ARMAN cell. Bar = 300 nm. (B) Details of the
penetration of the ARMAN wall by the Thermoplasmatales cell. Bar = 50 nm. Ar = ARMAN;
Tp = Thermoplasmatales lineage cell; Va = vacuole; VI = virus. Source.

Even now, mining the ARMAN genomes is a rewarding undertaking. One finds a number of characteristics shared with symbiotic or parasitic microbes, e.g., genes that are short, some that are split, and some that overlap with others. All this, plus the small genome size, suggests that ARMANs may not be able to make a living alone, relying instead on other members of their community for some resources. Indeed, looking at these organisms by cryo-EM reveals that, at least in some 3D reconstructions, ARMAN cell walls look like they are penetrated by other archaea belonging to the cell wall-less Thermoplasmatales. If so, who is parasitizing whom? Is this a way for the ARMANs to get nutrients? And yet more puzzles. To quote the authors: almost all cells contain a mysterious tubular organelle that is roughly 200 nm long and 60 nm wide. What’s this all about? Also visible are viruses attached to their surface. Lacking CRISPR defensive sequences, ARMANs may be ready prey to viral infections.

Slice of an ARMAN cell with the inner
membrane in orange and outer membrane
in yellow. Ribosomes are represented
with light blue spheres drawn to scale.
A tubular structure is shown in orange.
Such tubes range in diameter from 58 to
62 nm and the length is ~200 nm. Source.

One last point, perhaps not as surprising as the rest, but noteworthy nonetheless. These cells have only a handful of visible ribosomes, as few as 92 per cell, which suggests that they are not growing fast. For perspective, consider that a fairly slow-growing E. coli has some 10,000 of them! But in this special environment, perhaps one need not hurry to outcompete one's neighbors.

The ARMAN names may not last long, so fancier names have been proposed. Being uncultured, they have to bear the “Candidatus” designation, hence Ca. Micrarchaeum acidiphilum for one, Ca. Parvarchaeum acidiphilum for another. The Garden of Archaeal Wonders continues to surrender some of its secrets. And well it might, as more and more “archaeologists” are presently at work. Surely many more surprises are in stock. But it’s worth noting that now, given only a short few years, dedicated researchers can go from the discovery of novel and exciting organisms to the elucidation of many of their characteristics.

Baker BJ, Comolli LR, Dick GJ, Hauser LJ, Hyatt D, Dill BD, Land ML, Verberkmoes NC, Hettich RL, & Banfield JF (2010). Enigmatic, ultrasmall, uncultivated Archaea. Proceedings of the National Academy of Sciences of the United States of America, 107 (19), 8806-11 PMID: 20421484

A Fastidious Bacteriophage

by Michael Yarmolinsky

Patrons of upscale seafood restaurants are given the opportunity to see that the unfortunate creatures destined for the lobster pot are waving their antennae about. Savvy customers at downscale seafood markets evaluate questionable claims of freshness by smell. A fastidious bacteriophage would welcome the opportunity to gauge the quality of a potential meal, if only it could make that assessment. I was recently reminded, in the course of disposing of old reprints, that a bacteriophage named Chi can do so. It attacks only motile strains of bacteria, and then only if the flagella are active. How is this circumspect appraisal accomplished?

Phage Chi X 220,000. Source.

It has been several years since the publication of convincing support for a mechanical model that can account for the discrimination exhibited by Chi. Why revisit it now? First, for the benefit of those who have overlooked the remarkable story of Chi’s fastidious behavior and second because I suspect that we have learned only the half of it.

What sort of phage is Chi?

Chi is a virulent, double-stranded DNA phage with a long, uneven history and a long, tapered tail ending in a single, long, kinky tail fiber. It was first characterized in 1936 in the laboratory of Félix d’Herelle, the adventurer-scientist who is jointly credited with the discovery of the viruses he designated “bacteriophages.” Chi’s genome (60 kb) has recently been sequenced (Andrew Kropinski, Kelly Hughes and Roger Hendrix, in preparation).

The initial report showed that Chi attacks only flagellated bacteria. Growth of sensitive strains on agar containing phenol, at a concentration known to prevent the development of flagella, rendered the bacteria Chi-resistant. Chi-resistance could be used to select mutants defective in flagellation. Mutants altered in several of the 44 known flagellar genes of Salmonella were subsequently selected in this way.

The flagellar organelle. Source.

What sort of organelle is a flagellum?

Flagella are long, thin, helical filaments commonly much longer than the bacterial body from which they emerge at various sites. Their somewhat flexible, sinusoidal appearance was interpreted, until the early 70s, as evidence that they act like whips, but their helicity is intrinsic and their action that of a propeller rotated by a motor embedded in the cell body. Normal rotation speeds for Escherichia coli flagella are around 6000 rpm, but a record speed, set by a vigorous Vibrio, is 100,000 rpm. Each flagellum is both a reversible motor organelle and a protein export and assembly apparatus that fabricates the external filament by extruding flagellin monomers through a central channel and adding them to the growing flagellum at its very tip.

Mysteries of the Bacterial L-Form: Can Some of Them Be Unveiled?

by Hans H. Martin

Figure 1. Peptidoglycan sacculi isolated from L-form
spheroplasts of P. mirabilis. Electron micrograph of
Pt-Ir-shadowed sample. 20,000X.

L-forms are bacterial variants with defective cell walls and irregular growth and multiplication. They arise after peptidoglycan, the exoskeleton of the bacterial cell wall, has been either degraded by bacteriolytic enzymes, or its biosynthesis has been disturbed by antibiotics and other inhibitors, or by defect mutations in essential genes for cell wall synthesis. L-forms with different degrees of wall defects can arise. International experts, headed by nobelist Sidney Brenner, recognized the need to distinguish between entirely cell wall-less protoplasts, surrounded only by a cytoplasmic membrane, and spheroplasts with residual, fragile cell walls. L-forms were discovered in 1935 by Emmy Klieneberger and subsequently described by many authors (examples here and here). Much interest in L-forms arose from their assumed but still unconfirmed roles as concealed pathogens and as survivors of antibiotic action. They are also useful tools for the study of basic mechanisms of cell biology, such as cell division. Yet, as justly deplored in a recent review, L-forms are still "unfamiliar to many microbiologists" and are often regarded "with scepticism." One hears complaints about the unusually labor-intensive and time-consuming process of L-form isolation and cultivation, and the uncertain outcome. However, in my experience, this can be overcome by patient determination.

A special type of resistance to antibiotics, which we studied extensively in my lab, is reflected in the ability of the Gram-negative bacterium Proteus mirabilis to evade the inhibitory action of penicillin and other β-lactam antibiotics by growing as spheroplast- L-forms (references here and here). It is important to note that the most common resistance mechanism, inactivation of β-lactam antibiotics by β-lactamase, is not involved in this phenomenon.

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.

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!

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.”

Of Archaeal Periplasm & Iconoclasm

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

A Close Encounter of the Enological Kind

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.

Measuring the Strength and Speed of the Microbial Grappling Hook

by Amber Pollack-Berti

Source.

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

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