Small Things Considered

Dr. Rous’s Prize-Winning Chicken

2010-09-01T16:36:55-07:00

by Welkin Johnson

If one were to apply contemporary principles of evaluation to the work of Peyton Rous in his time and without the benefit of hindsight, this work would certainly be assigned a low priority, not fundable, and probably also not publishable in the trendsetting journals that today alone can confer recognition and prestige. Even in 1966, the year of the [Nobel] prize, it was impossible to guess at the full significance of the Rous sarcoma virus for human medicine and biology…The urgent lesson from the Rous experience, then, should be that it is the quality of the science that counts, not its compliance with a fashionable trend and not its perceived future value, which cannot be predicted.
Peter K. Vogt (1996. The FASEB Journal 10:1559-1562.)

Peyton Rous. Source.

One hundred years ago today, Peyton Rous published the first in a series of papers describing experiments that began with work on a sarcoma from a single Plymouth Rock hen. In that initial paper, Rous showed that bits of the tumor could establish new tumors when injected into healthy chickens. A year later, in 1911, Rous published what has become a classic in the annals of virology—A Sarcoma of the Fowl Transmissible by an Agent Separable from the Tumor Cells—reporting that the tumors were transmissible from one chicken to another by injection of a cell-free, filtered homogenate of the tumor tissue. The conclusion, since confirmed a thousand times over, was that a virus caused the tumors. The virus, now known as Rous sarcoma virus (RSV), went on to play a starring role in 20^th century biomedical research.

Historical accounts tell us that Rous’s findings were initially dismissed by many as a peculiarity of chicken sarcoma, with little or no relevance to human cancer. The view one hundred years later is far more spectacular. In the 1930’s, reports of oncogenic viral agents began to emerge from laboratories studying cancer in inbred mice. Some of these cancer-causing viruses, like Rous sarcoma virus, had RNA genomes, and as a group they came to be known as the RNA Tumor Viruses. In 1958, Howard Temin and Harry Rubin published a description of a quantitative assay for studying Rous sarcoma virus in tissue culture. The assay took advantage of the rapid and reproducible way in which RSV transformed cells. Specifically, diluted solutions of virus could be applied to monolayers of cells, which were then overlaid with agar. Localized “foci” of transformed cells would then form. Virologists could count foci in a monolayer of cells the way bacteriologists count plaques on a lawn of bacteria, back-calculating to determine multiplicity of infection. Thus, by the 1960s, the study of RNA tumor viruses had become a mature field encompassing both in vivo animal studies and tissue culture-based, bench-top experiments. In 1966, Peyton Rous’s contributions were belatedly recognized with a Nobel prize.

Barred Plymouth Rock hen (like the one that
started it all). Source.

Howard Temin’s studies of RSV transformation in tissue culture, and in particular the observation that the transformed phenotype remained stable during passage of transformed cells, led him to the startling hypothesis that the RNA tumor viruses replicated through a DNA intermediate, or provirus. Although Temin published several papers in support of his provirus hypothesis, most of his contemporaries did not see the light until 1970, when he and David Baltimore independently proved that virions of RNA tumor viruses contained RNA-dependent DNA polymerase activity. This enzyme came to be known as reverse-transcriptase (RT). An opinion piece summing up the ensuing flood of follow-up studies was appropriately titled Apres Temin, le Déluge. In the wake of this flood, Francis Crick even felt obliged to defend the so-called Central Dogma of molecular biology. In addition to revolutionizing the study of RNA tumor viruses (subsequently called retroviruses), the discovery of RT was a watershed event in molecular biology, providing the means for generating cDNA and the key to reverse transcription–PCR (RT-PCR). In recognition of their work, Temin and Baltimore received the Nobel Prize in 1975.

By the 1970’s several groups had begun zeroing in on the molecular nature of acute transformation of cells by RSV. Peter Duesberg and Peter Vogt reported that the RSV genome contained an extra bit of sequence not found in the genomes of very closely related, non-transforming viruses. In 1975, Dominique Stehelin, J. Michael Bishop, Harold Varmus, and Peter Vogt reported that this extra sequence was homologous to a host gene. The host gene, called src (from sarcoma) was the first proto-oncogene, and v-src (the version found in RSV), its oncogenic counterpart. Like RT before it, the discovery of viral oncogenes incited a dramatic shift in experimental emphasis, this time by giving scientists a molecular beachhead in the war on cancer. For discovering the cellular origins of viral oncogenes, Bishop and Varmus received the Nobel Prize in 1989.

It is noteworthy that during the sixty-six years separating Rous’s chicken experiments and the discovery of src (1910-1976), no retroviral pathogens of humans were known (it wasn't until 1980 that Robert Gallo’s group reported the isolation of HTLV-I, the first human retrovirus). Thus, seven decades of work on retroviruses had already had a profound impact on biomedical research when, in 1983, the RT-assay described by Temin and Baltimore helped Francoise Barré-Sinoussi and Luc Montagnier detect a new retrovirus in the cells of an AIDS patient. In 2008, Barré-Sinoussi and Montagnier were awarded the Nobel Prize for discovering the human immunodeficiency virus (HIV-1).

All thanks to Dr. Rous and his prize-winning hen.

Welkin is Assistant Professor of Microbiology and Molecular Genetics at Harvard Medical School, and an Associate Blogger for Small Things Considered.

Plasmalogens Have Evolved Twice

2010-08-30T10:00:00-07:00

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

Biofilms Over Troubled Waters

2010-08-12T10:00:00-07:00

by Mark O. Martin

Source.

The old saying “pouring oil on troubled waters” is a metaphor for bringing peace to a turbulent situation. Recent events in the Gulf of Mexico have proved the contrary, that oil poured (or spilled) upon seawater can produce the very antithesis of calm. After many weeks of concern, and with the long term threat of possible subsurface oil still strong, recent reports note that the oil slicks at the surface have become more difficult to find. What is happening? To be sure, dispersal over time is inevitable, but there may be more to the “vanishing” oil slicks.

What we perceive as the leakage of a toxic fuel into the ocean might be seen as an aliphatic feast by marine microbes. Quite a bit of literature describes the relationship that microbes have with oil (for a review, click here) and the possible role for such microbes in bioremediation of oil spills.

Burning off slicks. Source.

Researchers who investigated the aftermath of prior oil spills found that microbes can be involved in the amelioration of such accidents, acting as natural bioremediation agents, as we'd expect on Planet Microbe. Alkane-degrading microbes can in fact increase in population in response to petroleum contamination, as observed in studies ranging from Antarctica to Spain. However, such studies tend to focus on the impact of oil on shorelines instead of on the ocean surface itself. As I watched televised coverage of the spreading oil slicks in the Gulf, I recalled a short paper I had assigned to my latest microbiology class, which suggested that the very surface of the ocean itself could be thought of as an enormous, but very thin, biofilm. This short review by Cunliffe and Murrell credits the biological oceanographer John McN Sieburth with visualizing the air-sea interface as a gelatinous microlayer—a huge biofilm that may cover 70% of the Earth’s surface! (Click here to listen to an interview with Michael Cunliffe.)

A diagrammatic representation of the neuston including the bacterio-
neuston and TEP. Source.

The upper millimeter of the ocean with its inhabitants is referred to as the neuston, its clearly distinctive microbial communities as the bacterioneuston. These communities reflect the specific environmental and nutritional conditions to be found in that microlayer in marine, estuarine, or fresh water environments. Transparent exopolymer particles (TEPs) synthesized by primary producers form the “lattice” that appears to hold the bacterioneuston in place (see figure). TEPs can act as a food source as well as surfaces for microbial colonization, as they float upward and merge with the presumed surface biofilm.

One study found that the bacterioneuston, in contrast to the microbiota found half a meter below the surface, is dominated by two genera: Vibrio and Pseudoalteromonas. Both appear to be well adapted to life in a biofilm at a liquid-solid interface. It would be interesting to learn if mutants of such bacteria that are defective in biofilm formation on solid substrates are also impaired in their ability to colonize the neuston. The recent oil spill in the Gulf of Mexico obviously is relevant to this topic. Just as “fertilization” of areas where oil reaches the shore can accelerate bioremediation by supporting luxuriant microbial growth (including alkane degraders), it would be no surprise to learn that the bacterioneuston in the area of the oil spill has undergone a change in population structure and function while taking advantage of the oil as an unexpected source of energy and carbon. I am quite certain that microbiologists are investigating this, and I look forward to learning how the Gulf bacterioneuston responds over time. While an oil spill can cause great damage, it may also yield insights into how marine microbial ecology responds to abrupt change, particularly change precipitated by human actions.

Philosophically, it is awe inspiring to think of the entire surface of the oceans as a nearly two-dimensional biofilm spanning the globe, a world apart from the water column beneath. This floating world beckons to us to explore the population structure of the marine bacterioneuston living therein. With the knowledge gained, we may be able to assist Nature in what we perceive as the natural “clean up” processes that are the microbial world’s response to nutritional and environmental change.

In his novel, Solaris, the late science fiction writer Stanislaus Lem described a world-spanning ocean that was itself sentient. I am not prepared to go quite that far with this concept of a thin, ocean-spanning biofilm, even though microbial communication surely takes place therein. Nevertheless, this concept certainly has made me look at the expression of pouring oil on troubled waters in a new light. Perhaps we need a new aphorism for peace making, one that encourages the formation of robust surface biofilms to minimize disruptions!

Mark O. Martin is an Associate Professor of Biology at the University of Puget Sound in Tacoma, Washington, and an Associate Blogger for Small Things Considered. He remains an unrepentant microbial supremacist.

Cunliffe M, & Murrell JC (2009). The sea-surface microlayer is a gelatinous biofilm. The ISME journal, 3 (9), 1001-3 PMID: 19554040

When the End Is the Story

2010-08-09T10:00:00-07:00

by Welkin Johnson

It looks like a herpesvirus, but does it replicate like one?
Electronmicrographs showing mature HHV-6 particles
emerging from an infected cell. Source.

Sometimes, discovery in biology is about discerning rules and sometimes it is about pursuing exceptions. In this spirit, Human Herpesvirus six (HHV-6), the etiologic agent of the common childhood illness roseola infantum, is shaping up to be an intriguing exception. As every virologist knows, members of the Herpesviridae maintain their large double-stranded DNA genomes (typically 100-250kb) as autonomous, covalently closed circles (episomes) during latent infection of host tissues. Nevertheless, there is now convincing evidence that the HHV-6 genome can, at least on occasion, become integrated into host-cell chromosomes. Interestingly, the first hints that this could happen did not come from hypothesis-driven laboratory experiments, but from a handful of clinical case reports of individuals with exceptionally high levels of HHV-6 DNA in peripheral blood.

HHV-6 was discovered in 1986, and its sibling, HHV-7, in 1989. In 1988, a link between HHV-6 and roseola infantum was established. Together HHV-6 and HHV-7 are now categorized as Roseoloviruses, constituting their own genus within the family Herpesviridae. As with the other human herpesviruses, HHV-6 and HHV-7 infections are widespread among humans, but are generally not associated with severe pathogenesis except under conditions such as acquired or induced immunodeficiency. Of the eight known human herpesviruses (officially referred to as HHV-1 through HHV-8), most of us have heard of the Herpes simplex viruses (HHV-1 and HHV-2), the agents behind cold sores and genital sores, respectively, and we are familiar with Varicella Zoster virus (HHV-3), the cause of chickenpox, and Epstein-Barr virus (HHV-4), the cause of “kissing-disease” (infectious mononucleosis).

In contrast to the voluminous research accorded to their more notorious relatives, the scientific literature on the Roseoloviruses is scant. Nonetheless, a quick search of this literature turns up something peculiar—a smattering of case reports describing families in which HHV-6 DNA appears to be inherited vertically. Importantly, the viremic individuals within such families include one parent and at least one child. In some studies, fluorescence in situ hybridization (FISH) using HHV-6 sequences as probes revealed a close physical association between HHV-6 DNA and human chromosomes in the cells of the afflicted individuals. Combined, this gives rise to the rather fantastic notion that HHV-6 can, from time to time, find its way into an individual’s germline DNA and be passed on to the next generation. When this happens, afflicted individuals have HHV-6 viral DNA in every nucleated cell in the body.

The phenomenon of chromosomally inherited HHV-6 is unprecedented in two respects. First, no other human herpesvirus is known to integrate its DNA into the host cell chromosome. (In fact, stable maintenance of episomal DNA is widely viewed as central to the legendary ability of herpesviruses to maintain lifelong, persistent infections.) Second, aside from these studies, there are no reports in which a virus has been documented to enter the germline of a modern human and to subsequently be passed on to a child. Animal genomes, including our own, are full of ancient retroviral sequences (see our post), and the smart money would have been on a retrovirus, rather than a herpesvirus, to be the first to achieve this feat in modern times.

Fluorescent in situ hybridization (FISH) high-
lighting telomeric regions at the ends of cellular
chromosomes (bright spots correspond to
fluorescent, telomere-specific DNA probes).
Like cellular chromosomes, human herpes-
viruses HHV-6 and HHV-7 also have telomere-
like repeats at the ends of their genomes. The
function of telomeres in the viral genomes
remains a mystery, but recent data suggest
that they may facilitate insertion of the
viral genome into host cell DNA. Source.

At first glance, the genomes of HHV-6 and HHV-7 resemble the typical herpesvirus genome. They are >150 kb long, double-stranded DNA molecules bracketed by long, direct repeat regions; they contain specific cis-acting signals required for DNA replication, and, like the typical herpesvirus genome, they are estimated to contain more than one hundred open reading frames. However, a notable peculiarity of the Roseoloviruses is the presence of hexanucleotide repeats composed of the sequence TTAGGG at both the 5’ and 3’ ends of their genomes. Why is this peculiar? Because TTAGGG also happens to be the sequence of the mammalian telomeric repeat, strings of which are found at the ends of every cellular chromosome. Telomeric repeats are added to the 3’ ends of chromosomes by the cellular DNA polymerase known as telomerase, and are essential for the cell to distinguish the ends of linear chromosomes from 3’ ends generated by DNA breaks. A functional role for the TTAGGG repeats in the viral replication cycle has not yet been established, although they most certainly play a role, viral genomes not being known for their tolerance of extraneous, unnecessary sequence. Telomeric repeats have also been seen in two other herpesviruses: Marek’s disease virus (MDV) of chickens and Equine Herpesvirus 2 (EHV-2). Given the phylogenetic distance between these viruses, it is likely that the presence of telomeric sequences has evolved more than once in the history of the Herpesviridae.

FISH analysis of integrated HHV-6 DNA by E.P.Nacheva and
colleagues. One probe (green) binds to the telomeric region of a
specific chromosome, and the other (red) to HHV-6. The merged
image (yellow) shows co-localization of the HHV-6 DNA with the
telomeric region at one end of the chromosome. Source.

Arbuckle and colleagues at the University of Southern Florida undertook a detailed molecular study of several families with inherited HHV-6 and published their findings earlier this year. These investigators first used FISH to confirm the presence of HHV-6 DNA associated with chromosomes in multiple individuals from each of four families with high levels of HHV-6 DNA in blood. Although the particular chromosome involved differed from one family to the next, HHV-6 DNA was invariably found close to the end of one chromosome. Within each family, HHV-6 DNA was associated with the same chromosome, supporting the notion that the HHV-6 sequences in each family were being vertically inherited just like any other genomic locus. The investigators also used PCR amplification to capture and sequence the junctions between HHV-6 DNA and cellular chromosomal DNA by employing primer pairs, one specific for the ends of the chromosome, the other for a sequence within the viral genome. Sequencing of the PCR products revealed virus-host DNA junctions within chromosomal telomeric repeats, confirming that the HHV-6 DNA was indeed covalently integrated into the host genome.

The maintenance of an integrated copy of a herpesviral genome through at least two host generations is nothing short of amazing. The HHV-6 genome is >150 kb long, and contains a wealth of genes, many of which are likely to be involved in viral immune evasion. What are the consequences of carrying viral sequences in the germline, having them present and possibly expressed in cells throughout the body? Are some or all of these sequences seen as “self” by the person’s immune system? Does successful inheritance of chromosomally integrated HHV-6 possibly require additional molecular events, such as a mutation or the inactivation of part or all of the viral genome?

Over 90% of humans are seropositive for HHV-6, yet reports of families with inherited, chromosomally integrated HHV-6 are few. This suggests that integration of the HHV-6 genome into human germline DNA is not a typical outcome of HHV-6 infection. There are several possible reasons for this, e.g., HHV-6 infection of germline tissues may occur with very low frequency, or the majority of HHV-6 integrations into germline DNA may be deleterious and consequently never passed on to a new generation. Nevertheless, the mere existence of integrated HHV-6 DNA in these families raises a somewhat heretical idea: could this particular herpesvirus have evolved to use integration as an essential step in its infectious cycle? In other words, does integration happen as a matter of course in the somatic tissues where HHV-6 normally replicates?

The study by Arbuckle and colleagues provides some clues. Using peripheral blood lymphocytes from families with integrated HHV-6 DNA, the investigators were able to induce lytic viral replication in culture. While not directly proving that integration is essential, this experiment is important because it demonstrates that integrated HHV-6 DNA is functionally capable of expressing progeny virus. In a separate experiment, standard cell lines (JJHAN and HEK293T) were experimentally infected with a laboratory strain of HHV-6 in culture. After allowing the virus to replicate and spread in these cells, the investigators were able to detect newly integrated HHV-6 DNA, proving that integration can also occur here as a consequence of viral replication. Does this mean that integration is the rule for HHV-6? Herpesviruses replicate their DNA in the nucleus, so integration by homologous recombination may happen from time to time simply as a matter of chance, without having any bearing on the biology of the virus. In the case of HHV-6, homology provided by the telomeric repeats may simply increase the probability of this taking place. However, the same investigators were unable to detect episomal forms of HHV-6 DNA in experimentally infected cells. Although this constitutes a negative result, the observation is consistent with the possibility that HHV-6’s strategy for replication and latency is distinct from that of other herpesviruses.

The precise molecular events that give rise to inherited HHV-6 remain to be deciphered. Based on the presence of telomeric repeats at the ends of the HHV-6 genome, Arbuckle and colleagues favor the view that homologous recombination is involved. While plausible, one can also imagine alternative mechanisms, perhaps ones employing viral gene products that target the viral genome specifically to the telomeres. The telomeric repeats associate with a complex of cellular proteins involved in protection and replication of chromosome ends. Is it possible that the viral repeats also interact with the cellular telomeric machinery, possibly hijacking cellular complexes to meet the virus’s own ends (pun intended)? If integration turns out to lie at the core of the HHV-6 replication cycle, how then does an integrated genome embedded in a large chromosome serve as the template for producing unit-length progeny viral genomes? Could viral genome replication have deleterious effects on the physical integrity of the host cell chromosome, or conversely, might the virus regulate integration and replication in a manner consistent with host-cell viability?

The list of questions raised by these observations is long indeed, and should prove intriguing to virologists. Such questions also mean that the Roseoloviruses are primed for a share of the limelight heretofore accorded to their more famous HHV cousins.

Welkin is Assistant Professor of Microbiology and Molecular Genetics at Harvard Medical School, and an Associate Blogger for Small Things Considered.

Arbuckle JH, Medveczky MM, Luka J, Hadley SH, Luegmayr A, Ablashi D, Lund TC, Tolar J, De Meirleir K, Montoya JG, Komaroff AL, Ambros PF, & Medveczky PG (2010). The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro. Proceedings of the National Academy of Sciences of the United States of America, 107 (12), 5563-8 PMID: 20212114

Fine Reading: The Sex Habits of Fungi

2010-08-05T10:00:00-07:00

by Elio

Schizophyllum commune. Source.

As luck would have it, two pieces of writing on the sex habits of fungi appeared within days of each other. One is light reading, a post in the admirable Cornell Mushroom Blog entitled A Fungus Walks Into a Singles Bar. This is a précis into the complex story of fungal sexuality. It takes you friendly fashion through the maze of mating types and multiple “genders.” One mushroom, Schizophyllum commune, has more than ten thousand of them! (I once proposed sitting around and making up names for at least a few dozen of them. Any takers?) While you are there, have a look at some spectacular movies of mushroom development, especially stinkhorns (yes, again).

The life cycle of a basidiomycete, the mushroom Coprinus
cinereus. Source.

More technical is a paper by a duo of Finnish and German researchers who studied the sequenced genomes of 8 basidiomycetes, along with that of yeast (an ascomycete), looking for genes that speak to the structure and organization of mating types. First of all, these genes are highly conserved; fungi don't bother to reinvent the wheel. Mating type genes encode sex pheromones, some of which have been well studied. The authors conclude: In silico analyses now also permit the identification of putative components of the pheromone-dependent signaling pathways. Induction of these signaling cascades leads to development of dikaryotic mycelia, fruiting body formation, and meiotic spore production. In pheromone-dependent signaling, the role of heterotrimeric G proteins, components of a mitogen-activated protein kinase (MAPK) cascade, and cyclic AMP-dependent pathways can now be defined. The story is complex, but far from lacking in fascination.

Raudaskoski, M., & Kothe, E. (2010). Basidiomycete Mating Type Genes and Pheromone Signaling Eukaryotic Cell, 9 (6), 847-859 DOI: 10.1128/EC.00319-09

An Inactive Mine Provides Active Opportunities

2010-08-02T10:00:00-07:00

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

Talmudic Question #64

2010-07-29T10:00:00-07:00

by Ramy Aziz

Are any human-associated biofilms "useful" or "beneficial" to human health?

A Giant Among Giants

2010-07-26T10:00:00-07:00

A colorized SEM showing numerous particles
of the phycodnavirus PBCV-1 attached to a
chlorella NC64A host. Bar = 500 nm. Source.

by Merry

Without a doubt, Mimivirus is remarkable. For a virus, it is extraordinarily large and complex. But it is hardly one of a kind. The more that researchers look for large viruses, the more they find.

Although phages generally tend to have small genomes, some managing with but a handful of genes, a glance at the current NCBI list reveals that there are now eight with sequenced genomes that amount to more than 200 kb. A Pseudomonas phage tops the list with 317 kb, but the not-yet-sequenced genome of Bacteriophage G of Bacillus megaterium is reported to be ~670 kb.

EM of novel Bacillus thuringiensis phage from
soil—the first representative of a new group
of large myoviruses. Bar = 0.1 μm. Source.

Serwer and colleagues have pointed out that the procedures used to isolate phages are biased against the giants. Typical plaque assay protocols call for at least 0.3% agarose in the overlay. However large phages, such as Bacteriophage G, can't make plaques of significant size when the agarose concentration is 0.2% or higher. Using 0.15% agarose, these researchers isolated a novel Bacillus thuringiensis phage from soil—the first representative of a new group of large myoviruses. It too has a genome with more than 200 kb packaged inside a 95 nm capsid that sports a tail measuring half a micrometer!

Phycodnavirus OtV5, with a 122 nm capsid, infects Ostreococcus
tauri, the smallest known free-living photosynthetic organism, ~1
µm in diameter. TEM. (A) At high multiplicity of infection (moi),
many viruses can adsorb to a single cell. (B) Virus replication
results in the accumulation of viral particles in the cytoplasm
before cell lysis occurs. Bar = 500nm. Arrows = virus particles;
Chl = chloroplast; Cyt = cytoplasm, n = nucleus, m = mitochon-
drion, sg = starch grain. Source.

Most of the known giant viruses infect eukaryotes and are members of a monophyletic group known as the nucleocytoplasmic large DNA viruses or NCLDVs. They earned the "nucleocytoplasmic" label because they either replicate entirely in the cytoplasm or initiate the process in the nucleus and then complete it in the cytoplasm—thus independently of the host's transcriptional apparatus. Here you find the pox viruses of vertebrates and invertebrates, the phycodnaviruses (phyco- meaning algae) of marine and freshwater algae, and the amoeba-infecting Mimivirus. Phycodnaviruses of note include the coccolithovirus that plays a role in the termination of blooms of an abundant marine alga, the coccolithophore E. huxleyi, as well as a large virus that infects Ostreococcus tauri, the smallest known free-living photosynthetic eukaryote.

The phycodnaviruses are quite remarkable themselves. Their genome lengths are mostly in the 300 kb range, but one is 560 kb. The archetypal phycodnavirus that infects chlorella-like algae has ~373 protein coding genes—more than the number often touted as the "minimum" required to support life. However, gene numbers don’t tell the whole story as these viral genomes lack many of those listed in the "essential" gene set. In an unvirus-like manner, this genome also encodes 11 tRNAs and three kinds of introns plus genes for multiple DNA methyltransferases and DNA site-specific endonucleases—the enzymes that make up the restriction modification systems found in many Bacteria. These genes are functional; all chlorella viruses have methylated bases in their genomes, each virus with its own characteristic site-specificity. And most intriguing of all, this chlorella virus has the gene needed to synthesize hyaluron, and synthesize it it does, eventually covering its chlorella host with a dense fibrous network. Hyaluron synthesis had been thought to be an art exclusive to vertebrates (and a few pathogenic bacteria that include it in their capsules to fool our immune system). Even more bizarre, some chlorella viruses make chitin instead, and yet others make both and accumulate both on the surface of their host.

Infection of Chlorella NC64A by PBCV-1. (B) Attachment of PBCV-1 to the algal wall and initial digestion of the wall. (D) Complete digestion of the algal wall. (F) An empty viral capsid remaining on the surface of the host. Bar = 100 nm. Source.

Mimivirus has the phycodnaviruses beat by just about any yardstick you choose, and it even crosses that illusory line intended to separate viruses from cellular life. Its ~500 nm capsid is larger than the smaller bacterial cells such as Mycoplasma. Its 1.2 Mb genome contains 981 predicted protein-coding genes—double the number found in the smallest known Bacteria (Mycoplasma genitalium) and Archaea (Nanoarchaeon equitans). But a virus it is, firmly placed phylogenetically within the NCDLV group, albeit on its own branch.

Close-up view. Credit: Didier Raoult.
Source.

Mimivirus infecting an amoeba. The vrion has
been phagocytosed and resides within a vacuole.
The inner membrane of the virion (light circle)
will later fuse with the vacuole membrane to
discharge the virion contents into the cytoplasm.

We don't know what most of those 981 genes do as they lack identifiable homologs in the sequence databases, but at least 95% of them are transcribed during infection. Where did they all come from? Many appear to be paralogs produced by gene duplication events in Mimivirus. Of those with clear homologs, most are related to bacterial genes, a few to genes in Acanthamoeba and other protists. These likely came via horizontal gene transfer from a host, from other parasites present in the host, or from Bacteria phagocytized by the host for food.

Having a 1.2 Mb genome presents some challenges. One is simply synthesizing enough DNA for >300 progeny viruses in about 12 hours. In one experiment, researchers measured a 7-fold increase in total DNA within the host in the first 8 hours, so recycling of host DNA by viral endonucleases simply won’t suffice. Not surprisingly, Mimivirus (and other NCLDVs) encodes numerous enzymes for nucleotide metabolism and synthesis. Next comes the task of packaging those genomes into the preassembled capsids, a process that takes place in a cytoplasmic "virus factory." The unique "stargate" that opens upon infection to rapidly deliver the genome is a story in itself (click here and here).

A model of the complex virion of Mimivirus (cross
section viewed perpendicular to the unique five-fold
axis). From the outside in: head proteins (black) and
shafts (green) of the surface fibers that are attached
to the anchor proteins (blue spheres) that cover the
lattice forming the icosahedral capsid (red spheres).
Next, an additional protein/lipid layer (gold), uniden-
tified fibers (orange), and the bilayer lipid membrane
(green). Inside the membrane is the genomic DNA
(black) with associated proteins (green) and other
proteins (pink). Thick blue lines on the surface
represent the stargate. Source.

The protein capsid measures ~0.5 µm; the dense layer of reticulated polysaccharide fibers covering it surface increases the diameter to ~0.75 µm. It was the faint Gram-positive staining of those fibers combined with the virion size that earned Mimivirus its name: Microbe mimicking virus. Such large size may be necessary to efficiently infect amoebae and other protists via their feeding phagocytosis pathway. Studies using precisely-sized beads found that individual beads greater than about 0.6 µm are taken up immediately, whereas smaller ones accumulate on the cell surface until combined they reach sufficient volume to trigger uptake.

With such fascinating stories being told by Mimivirus and the other giants, people are now looking for them in more environments. Modified techniques are called for, as those used previously to spot viruses may have excluded many of them. For example, when collecting marine samples for viral metagenomes, researchers often use filters with 0.16-0.2 µm pores to catch the "microbial" fraction and allow the "viral" fraction to pass through. Realizing that many NCLDVs are apt to be caught with the microbes, Monier and colleagues searched the "microbial" sequences from the Sorcerer II Global Ocean Sampling (GOS) Expedition for NCLDVs using their conserved DNA polymerase sequences as a handle. They found Mimivirus sequences in 86% of the samples and chlorella viruses in a third.

Claverie and colleagues see no limitations that would preclude the existence of even larger viruses. Unlike cellular organisms, there are no metabolism-based constraints on particle volume. Of course, a virus must be smaller than its host, and Mimivirus is <1/30 of the size of its host amoeba. Bacteriophage G may be approaching the limits here: a 200 nm diameter phage infecting a 2 µm Bacillus. Given that Mimivirus can fit 1.2 Mb of DNA into its 0.5 µm diameter capsid, they surmise that a virus with a 10 Mb genome would be possible. It would require only a 1 µm capsid, a size easily accommodated by large amoeboid protists.

What do you think is the likelihood that Mimivirus will still be #1 giant five years from now? We'd bet not, as much of the virosphere is yet to be explored, and likely there is more than one researcher with dreams of discovering the next viral leviathan. Large protists that feed on bacteria would be the place to look.

Van Etten JL (2003). Unusual life style of giant chlorella viruses. Annual review of genetics, 37, 153-95 PMID: 14616059

Claverie JM, Ogata H, Audic S, Abergel C, Suhre K, & Fournier PE (2006). Mimivirus and the emerging concept of "giant" virus. Virus research, 117 (1), 133-44 PMID: 16469402

Hello Again, Metabolism!

2010-07-22T10:00:00-07:00

by Amy Cheng Vollmer

Source.

Years ago, pathways of intermediary metabolism made up a significant portion of biochemistry and microbiology courses. Therein, students learned about interconversions and connections between pathways, and they could follow the carbons as they moved from acetate into the cholesterol molecule and many others. But the advent of exciting new methodologies—structural biology, recombinant DNA, molecular genetics, immunochemistry, probes, blots, microarrays, metagenomics, and more—crowded much of metabolism right off the syllabus. Given that teaching pathways could be dry and boring, faculty often elected to substitute more trendy and exciting topics instead. To be sure, they thought metabolism was important, but assumed ‘someone else must be teaching it.’

The result is a generation of well-trained scientists who can clone or crystallize just about anything and can harvest bushels of data from vast microarrays. But once those gene names are converted into enzymes, they are not so adept at mapping their enzymatic steps into a coherent and integrated system of pathways. In fact, a survey I instigated at an IMAGE (Integrating Metabolism and Genomics) meeting in 2004 showed that the vast majority of individuals trained after the 1970s knew little about the pathways of photosynthesis or of amino acid, purine, and pyrimidine biosynthesis, nor did they think that needed to be taught at the undergraduate level.

So we now have faculty (in the assistant and associate ranks) who admit to me that they would have a tough time teaching pathways effectively because they don’t know much beyond glycolysis and the Krebs cycle. Yet, in the past 5 years, I have found time and again that some of the most revealing presentations at the ASM meetings (and others) have shown that important signals in microbial processes are, in fact, small metabolites, and that the key enzymes are not specific to pathogenesis, development (e.g., sporulation) or differentiation (morphotypes), but rather they are the enzymes of the Krebs cycle or for key steps in nitrogen or phosphate metabolism. Imagine that!

In this era of metagenomics, metabolism has resurfaced dressed fashionably as metabolomics: the study of the universe of small molecule metabolites that characterize a biological sample, usually one containing many different species, e.g., the metabolome of the human gut or the cow’s rumen. Somehow the profiles of small molecules reveal important aspects of the health of such ecosystems. So now we find faculty and students scrambling to teach and learn about primary and secondary metabolic pathways so that they can find their way through the vast databases that are being assembled from structures, pathways, regulatory networks, etc.

Metabolism is enjoying a renaissance in our curricula and it is about time! There are vast secrets about biology to be revealed by the small molecules. Understanding how their levels rise and fall will take a careful study of the enzymatic pathways leading to and from them. So hello again, Metabolism! It’s so nice to have you back where you belong.

Amy is Professor of Biology, Swarthmore College, and President of the Waksman Foundation for Microbiology.

Microbiology in the Andes: Ancient and Unexpected

2010-07-19T10:00:00-07:00

by Elio

The Ancient Gate of the Universidad de San Gregorio
Magno.

Thanks to the investigations by the Ecuadorian physician and scientist, Dr. Byron Núñez Freile, I learned of a surprisingly high level of scientific development that took place long ago in a remote region of the world. Quito, the present-day capital of Ecuador, is nestled amidst the high Andes and was the northern capital of the Inca empire. It was conquered by the Spaniards in 1534. In this exceedingly distant land, Jesuits established a college within a year of their coming in the late 16th century. By 1622, they founded one of the oldest universities in the Americas, the Universidad de San Gregorio Magno. This was earlier than the founding of Harvard, which happened in 1642. With the passing years, the two universities may not have enjoyed a parallel development, but early on they were likely of comparable quality. Soon, San Gregorio became a major institution, with a most impressive library of 16,000 volumes, the largest in South America at the time. In its first thirty years of existence, the university granted 160 masters degrees and 120 doctorates, mostly in philosophy and theology. Nevertheless, the library holdings also included numerous scientific and medical treatises.

Panorama of Quito. Source.

Quito can be reached by an easy flight these days, but in olden days getting to its lofty location (an altitude of 9,000 feet) required a week-long mule trek from the Pacific coast. Remote indeed! A major event of scientific relevance took place in 1736, when a geodetic French mission led by Charles-Marie La Condamine arrived, intent on measuring the circumference of the Earth at the equator. The French delegation interacted closely with members of the university, which resulted in a strong scientific legacy.

The scientific concerns of the times included the world we now call microbiology. No wonder. In 1589 a smallpox epidemic killed 37.5% of Quito’s inhabitants. A description of the disease in a letter by one of the priests makes clear allusion to its contagiousness. Later on, several of the Jesuits made insightful observations about the etiology of infectious diseases. Among them was Juan Magnin (1701-1753), a Swiss missionary who became a member of the French Academy of Sciences, who stated: There are microbes that can only be seen with a microscope that are 27 million times smaller than the smallest that can be seen with the naked eye. These facts and others seem incredible.

A microscope such as was available in Quito
in the 18th century, this one made ca. 1745
by John Cuff in London. Source.

And …(the microscope) allows to establish that the dirt on the teeth is due to the accumulation of innumerable microbes; furthermore, it is likely that many of the diseases of the human body, especially leprosy and venereal diseases, are due to the accumulation of microbes.

The other major microbiological issue of those times, the theory of spontaneous generation, became the concern of a native-born member of the faculty, Juan Bautista Aguirre (1725-1786). He wrote: I affirm…that the forms of animals, even insects, are not engendered by putrefactions but they arise from eggs or germs. He also stated: …with the aid of the microscope one discovers innumerable germs incredibly small in size, in the air, water, vinegar, blood, milk, etc. The most ingenious Leuvoiseck (sic) bore witness to having seen such small germs in a drop of water that 90,000 of them did not reach the size of a grain of wheat. What he lacked in spelling skills, he made up for by a good understanding of the literature!

Eugenio Espejo (1747-1795). Source.

There is more to this history. Soon after the Jesuits were expelled in 1767, the major Ecuadorian intellectual of that age, Eugenio Espejo (1747-1795), made important contributions to hygiene and the containment of smallpox. His words: Within the infinite variety of these living particles (“atomillos”) we have an admirable resource to explain the prodigious multitude of diseases and symptoms… Born of an Indian father and a mestizo mother, Espejo was a notable polymath, a true product of the Enlightenment. Not only was he the most notable physician of his time in Quito, he was also a lawyer, a philosopher, and the founder of Quito’s first newspaper. As a public figure, he laid the groundwork for the independence movement that eventually led to the liberation from Spain.

In the 17th and 18th centuries, Quito was a notable center of learning and discovery. Here, in splendid isolation, far from other universities and libraries, arose a sophisticated understanding of the world of microbes, both regarding their medical importance and their biological essence. This is nothing short of remarkable.

Quito at night. Source.

I confess to personal glee in this story. I spent my teen years in Quito and eventually became a student at the Universidad Central, the public institution that was built on the one founded by the Jesuits so long ago.

I am grateful to Dr. Núñez Freile for having brought this remarkable story to my attention. Dr. Núñez Freile is in charge of two highly informative blogs (in Spanish), one on infectious diseases here, the other on hand washing here.

Power of Ten

2010-07-15T10:00:00-07:00

Tenfold Power. A gorilla can lift 10 times its own
weight, it is said. It can also sit wherever it wants.
Source.

by Elio & Stanley

How often have you heard it said, or seen it stated in writing, that we carry ten times more microbial cells than cells of our own? We don't dispute this figure, at least not as a ballpark estimate. But we were curious to find out where it came from. The paper that seems to be quoted most often in this regard is Microbial Ecology of the Gastrointestinal Tract by Dwayne Savage in the Annual Review of Microbiology, 1977, 31:107-33. Savage was an eminent intestinal microbiologist and those of us who knew him believe that he was a stickler for details. So he should have known. He stated: The various body surfaces and the gastrointestinal canals of humans may be colonized by as many as 10¹⁴ indigenous prokaryotic and eukaryotic microbial cells (70).

So now let’s go to reference # 70. It is to a paper by T.D. Luckey in the American Journal of Clinical Nutrition. 1970, (11) 1430-1432. Now we’re getting somewhere. Luckey writes: Assume one viable microbial mutation each 10⁸ cells, a microbial count of 10¹¹/g intestinal contents and 10³ g intestinal contents, then at a given time each of us harbors about 1 million newly mutated microbes (10¹¹ x 10³/10⁸ = 10⁶).

Just how authoritative is that? References to experimental work? None are given. Surely, the number of bacteria in feces must have been determined countless times through the years. A search of older literature is called for (but who’s got the time!). Moreover, is the number of human cells based on more solid ground? We have no idea.

Nowadays, a quantitative PCR using universal bacterial primers should give a reasonable estimate of the total intestinal bacterial DNA, a figure that can be converted with some assurance to the number of bacteria present. For good measure, add a few percent to account for eukaryotic microbes. Note, however, that we take leave from a good portion of our intestinal microbiome every day, the lucky among us with satisfying regularity. So, the ratio fluctuates daily. No big deal, but let’s qualify the ten-to-one mantra by saying “estimated.” To say the least.

Stanley Maloy is Dean of Sciences and Associate Director of the Center for Microbial Sciences at San Diego State University.

The Uncultured Bacteria

2010-07-12T10:00:00-07:00

by Kim Lewis

Fig. 1. "The Great Plate Count Anomaly."

The majority of bacteria will not grow on nutrient medium in the lab. The basic experiment is simple: take a sample from the environment, such as marine sediment or soil, mix with water, vortex, allow it to settle, dilute supernatant and take two droplets. Plate one on a Petri dish with LB medium, and place the other on a microscope slide (adding a dye such as DAPI helps). Count the number of cells under the microscope and the number of colonies on the Petri dish – the result will be “The Great Plate Count Anomaly.” About 100 times more cells will be observed microscopically than colonies counted on the Petri dish (Fig. 1). This simple result is one of the most profound puzzles in microbiology, and one of the most significant unsolved questions in biology. Indeed, microorganisms probably make up the bulk of the total biodiversity of species on the planet, but we do not have access to the vast majority of them. Importantly, most Divisions—the largest taxonomic units—do not have a single cultivable representative, and we know of their existence only from 16S rDNA isolated directly from the environment.

There are two basic approaches to solving a problem, and one of them is to decide that it does not exist. It has been suggested on the pages of Nature and Microbe that the anomaly only exists in our minds and results from the insufficient number of microbiologists with a “green thumb” willing to tinker with growth conditions. However, the anomaly has existed for over 100 years, and tinkering by generations of scientists produced only minor improvements. A serious effort was mounted by several groups to culture representatives from the ubiquitous TM7 division, for example, but produced no results.

Fig. 2. Growing the uncultured in agar, between semi-permeable
membranes, in their original environment.

What I find most fascinating in the problem of uncultured bacteria, however, is not the existence of exotic strangers such as TM7, but the fact that many—if not most—“unculturable” organisms are close relatives of common garden-variety bacteria such as Bacillus subtilis or Pseudomonas aeruginosa. Judging by the genomes of their relatives, the uncultured organisms should grow on almost anything, yet they grow on nothing in the lab, including yeast extract which contains the entire metabolic map. They do grow in soil or marine sediment, so should be able to consume degradation products of plants and animals, which would be sugars and amino acids. The puzzle is truly perplexing.

Given the unsuccessful 100 year-old quest for a good medium, we gave up on the hope of recreating it in a Petri dish. Instead, we decided to grow the uncultured in their natural environment, where one cannot possibly fail! Enclosing a marine sediment sample mixed with agar between semi-permeable membranes and placing it back in its original environment then allows for free diffusion of compounds through the chamber (Fig. 2). The bacteria are tricked into perceiving this diffusion chamber as their natural habitat, and form colonies. Similar approaches of culturing in their natural environment resulted in cultivation of dominant pelagic Pelagibacter ubique by Steve Giovannoni’s group, and Prochlorococcus by Zinser and co-authors.

Fig. 3. A sample from marine sediment biofilm is inoculated on
a Petri dish with rich medium (left panel). Inoculating colonies
from this plate pairwise (right panel) allows to identify uncul-
tured organisms and their helpers. Material from one colony,
KLE1104 (close relative of cultivable M. polysiphoniae) was
spread evenly over the plate, while cells from another colony,
KLE1011 (related to M. luteus) were patched in a single spot.
KLE1104 colonies only form around KLE1011.

One useful clue to the nature of uncultivability came from our observation that some uncultured organisms will only grow in the presence of cultivable species from the same environment (Fig. 3). Using this as an assay, in collaboration with Jon Clardy’s group we discovered that growth factors for many uncultured species from biofilms enveloping marine sand grains are siderophores, chelators of insoluble Fe(III) (Fig. 4). Some species are fairly promiscuous in taking up siderophores, while others, such as a distant relative of Verrucomicrobia, will only grow in the presence of a siderophore from a particular neighbor.

Fig. 4. A culturable helper releases a siderophore that captures insoluble
Fe(III) and brings it into the cell. The same siderophore/Fe(III) complex
is captured by an uncultured bacterium which is unable to make its own
siderophores.

But why would bacteria lose the ability to make a factor necessary for capturing an essential nutrient, thus becoming dependent on their neighbors? This dependency, of course, leads to loss of liberty; uncultured species can no longer colonize new territory. Simple advantages of thievery are unlikely to provide a credible explanation. Indeed, siderophore piracy is well-known among bacteria, but the cultivable pirates retain their own siderophore operons and turn expression on when iron gets low. I think that the reason for the loss of siderophores is to prevent adaptive evolution in new environments. Any time a cell finds itself in new surroundings, it will evolve to increase its fitness. This newly evolved organism is unlikely to outcompete resident species that spent millions of years adapting to the same environment, and will die out. Going back to their original environment is not an option either; they are not the same now and will be less fit than their parents (Fig. 5).

Fig. 5. (Upper panel) Death by an evolutionary dead-end. A culturable
bacterium propagates in its familiar nich (yellow) and sheds off a cell
that travels to a new environment (grey) where it attempts to adapt
by evolving new features and losing some old ones. It is unable
however to compete with a resident species (purple), and is no longer
competitive with the parent strain. (Lower panel) An uncultured
organism depends on a growth factor from a neighboring species. If
a cell finds itself in an unfamiliar environment, it stops dividing and
waits for a ride back, where it resumes productive propagation.

This scenario has been described for our pathogens such as P. aeruginosa which looses a large number of genes, including virulence factors and proteases, while adapting to the environment of the lung of cystic fibrosis patients. The result of such runaway evolution is a dead end; the pathogen can neither infect new hosts nor return to soil, its other major habitat. This is probably the main fate of weeds like P. aeruginosa or E. coli—death by adaptive evolution. The other 99% of species choose where they live and grow. If they find themselves in an unfamiliar environment, then the best strategy is to go into dormancy and wait for a ride back home. This strategy is very similar to what we know about spores of cultivable species such as B. subtilis. Spores will germinate on any nutrient environment, but the probability of germination is greatly increased in the “correct” environment. Alanine (for reasons we do not understand) is a good germinator, and so are products of peptidoglycan hydrolysis from neighboring species. B. subtilis seems to be an intermediate between a true uncultured species and a weed like E. coli.

What is next? The siderophore story suggests that there are other growth factors to be discovered that indicate a familiar environment to the unadventurous uncultured. (Next year: the uncultured from the Human Microbiome.)

All figures courtesy of Kim Lewis.

Kim Lewis is Professor of Biology and Director of the Antimicrobial Discovery Center at Northeastern University.

D'Onofrio A, Crawford JM, Stewart EJ, Witt K, Gavrish E, Epstein S, Clardy J, & Lewis K (2010). Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chemistry & biology, 17 (3), 254-64 PMID: 20338517