Small Things Considered

Retrospective, June 2013

2013-06-17T04:00:00-07:00

We continue our semi-annual ritual and post this quick tour of our blog posts published since December, 2012.

Pretty picture. Source.

Pictures Considered

Our new section dealing with “pictures that made a difference but may be nearly forgotten by now” seems to be off to a good start. Please send us suggestions of pictures you think should be considered.

#1. Visualizing Coupled Transcription and Translation in E. coli. A single EM demonstrates that transcription and translation are coupled in prokaryotic cells.

#2. The E. coli Chromosome Caught in the Act of Replicating. One radioautograph suffices to show that the E. coli chromosome is circular.

#3. How Do You Know There Is a Nucleoid? Nucleoids are visualized in living E. coli cells by using the simple optical trick of increasing the refractive index of the medium.

#4. Koch’s Development of Early InstaGram Positive Photography. Associate blogger Daniel unearths one of the earliest photomicrographs of bacteria, taken by none other than Robert Koch.

#5. The Birth of the FtsZ Ring. Using gold-labeled antibodies, FtsZ, the mother of bacterial cytoskeletal proteins, was found to localize at the septum of E. coli during cell division.

Source.

Ecology and Evolution

Terraforming Mars With Microbes. Graduate student Ben Auch thinks beyond the confines of this planet about what it would take to introduce life to Mars.

Little Known Glomalin, a Key Protein in Soils. A third of the nitrogen in soils is contained in a tough protein made by mycorrhizal fungi that helps bind soil particles to make soil more productive.

Whose Planet Is It Anyway? Elio comments on the epic manifesto by McFall-Ngai and collaborators on the importance of the relationship of animals and microbes—truly an imperative.

A Day in the Life: Eavesdropping on Marine Picoplankton. There is nothing like studying bacteria in their natural habitat, is there? Freelance science writer Heather Maugham tells us about a talented robot called ESP that measures cycles of gene expression in microbes in the oceans, autotrophs and heterotrophs alike.

Who Would Have Thought? One Hundred Million Year Old Polyamines. Tough is tough as molecules go, but sticking around through vast geological time is still pretty amazing.

Domestic Just for the Sake of it – The Evolution of a Fungus with Good Taste. Fungal domestication can lead to fine-tasting foods. Associate blogger Daniel relates how applying positive selection resulted in better sake-making fungus strains.

Wolbachia Make Fruit Flies Lay More Eggs to Make More Wolbachia. The endosymbiotic bacterium Wolbachia is known for altering the sex ratio of its insect hosts. Here, associate blogger Marvin discusses novel ways that the bacterium manipulates reproduction of its host.

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Viruses and Prions

Stuck in Phage Heaven: Heaven, for a phage, is an environment with abundant potential hosts, such as bacteria-rich mucus. Merry relates that some phages have evolved to adhere to mucus, and that while hunting therein they protect the underlying mucus-producing epithelium from bacterial infection.

A Good Defense Is Worth Stealing: Phages are a prime target for the bacterial CRISPR defense system. Merry cheers as a phage co-opts the CRISPR mechanism and uses it to defend itself against another mobile genetic element.

When a Good Peptide Deformylase Gets Better: Merry adds yet another chapter to the ongoing story of how cyanophages maintain and exploit the photosynthetic machinery of their cyanobacterial hosts.

Biting the Hand That Clothes You: Merry shares a new word in her vocabulary, kleptoparasitism, as it applies to the theft of capsid proteins. This is one of the dirty tricks used in the war between competing S. aureus mobile pathogenicity islands.

Book Review: Viruses: Essential Agents of Life. Associate blogger Welkin reviews a fine book that awards viruses the sort of attention they deserve. Reading this may convince you that, indeed, "studying any organism in the absence of its viruses is misleading."

Oddly Microbial: Prions. Marcia Stone, a prominent science writer and contributor to this blog, discusses the possible roles of these challenging entities in Alzheimer’s, cancers, and other human tsuris.

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Pathogenesis, Host/Microbe Ecology, and Taxonomy

Tit-for-Tat: A Bacterial Counterattack System. Graduate students Spencer Scott and John De Friel explain the intricacies of that marvelous mechanism that bacteria use for defense and communication, the Type VI Secretion System.

Fecal Transplants in the “Good Old Days.” Stanley Falkow, the originator of molecular pathogenesis, shares with us his early experience using feces-filled gelatin capsules to improve the lives of patients. Simple and effective. Just wear gloves!

No Bacterium Is An Island. Associate blogger Marvin explains how Pseudomonas aeruginosa senses the number its neighbors (a quorum) and uses the information to decide when to make virulence factors or inhibitors of Gram-positive bacteria.

Microbes or Not, Parasites All. Worms may not be microbes—far from it—but because they share with bacteria and protozoa the ability to cause infection, all three are often discussed together when teaching. And well they should be, as they share common aspects of pathogenesis. Just a reminder…

Putting Redundancy to Work. Graduate student Katrina Nguyen discusses IMAD, a clever way to reveal host-parasite interaction by partly depleting both a function of the host and of the microbe. This uncovers, among other things, redundancies in virulence factors.

A Passing Thought. Elio has fun with a legendary though fictional turd-sectioning character in the Robertson Davis novel, Fallen Angels.

A Whiff of Taxonomy – The Phylum Elusimicrobia. In a continuing attempt to bring taxonomy to the table, Elio tackles an elusive group of nearly ubiquitous “ultramicrobes.”

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Structure and Function

The Art of Microbial Alchemy. Associate blogger Gemma discusses how to make gold nuggets using Cupriavidus metallidurans, a bacterium that reduces gold to an insoluble form and doesn’t find this metal all that toxic. This may beat digging for it!

E. coli Cells Face FACS and Get Back into Shape. Graduate student Kimberley Busiek discusses a clever way that Kevin Young’s lab employs flow cytometry to collect cell division mutants. A most useful technique, it turns out.

Holey Biofilm! Bacterial biofilms are not homogenous structures (not that you thought so). Associate blogger Gemma explores their landscape of tunnels and holes, identifying the motile bacteria responsible for their construction. While this tactic may help their own community obtain nutrients, it can also destroy biofilms of other bacteria by spreading noxious chemicals.

Bdellovibrio’s Appetite for Metabolites. Expert bdellovibriologist Henry Williams discusses how these predators benefit from choosing prey that make specific compounds, in this case polyhydroxyalkanoates. Not only are the strategies of predators not always simple but they embody unexpected complexities.

A PomZ Scheme For Turning One Cell Into Two. Bacterial cell division maven Bill Margolin discusses the central question in the field: how does the ring-making FtsZ protein find the cell center? A positive spatial regulator, PomZ, is part of the protein cabal involved, although just how all this works remains elusive.

Bacterial Antidepressants: Avoiding Stationary Phase Stress. As some bacteria grow to high densities, their metabolism raises the pH, which causes their acute discomfort (death). They counteract the rise in pH by making oxalate, a response under the control of quorum sensing. This is neatly explained by associate blogger Marvin.

The Gram Stain: Its Persistence and Its Quirks. Ah, if it were all simple: Gram-positives have a thick peptidoglycan cell wall, Gram-negatives have two membranes. The exceptions test the rule, or, rather, undo it, especially for environmental species.

Living Wires of the Ocean Floor. Associate blogger Gemma turns our attention to the bottom of the sea where bacteria act as long electric cables. They shuttle electrons from the depths, where they are released by hydrogen sulfide oxidation, to upper layers where oxygen can gobble them up.

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Miscellanea

Short Courses for Long-Term Learning. Experienced microbiology teacher Phoebe Lostroh shares her experience of teaching science courses one at a time, each one studied intensively over a 3.5 week period. What the students lose in variety is more than balanced by what they gain from greater focus on a subject matter.

A Tale of Centenarians. Claudio Schazzocchio relates the tale of the oldest Nobel Prize winner ever and Italian senator-for-life, Rita Levi-Montalcini, and her fellow centenarians’ path to the discovery of the nerve growth factor.

The Gender Bias of Science Faculty. A milestone paper on this subject by Jo Handelsman and colleagues is discussed by Victor Racaniello of TWiV, TWiM, and TWiP fame.

Feynman Said “Just Look At The Thing!” Physicist Richard Feynman told us how to solve biological questions: “Just look at the thing!” In Talmudic Question #67, Elio wondered what one would choose to look at first given such a “Feynman supermicroscope” and what one might see. Chemist and biologist Jan Spitzer responds with his own insights.

Teaching Students To See Through “Microbial Eyes." Famed teacher and passionate microbial supremacist Mark Martin shares vignettes of what he heard when asking his microbiology students to comment on their previous microbiological illiteracy. A challenge to include microbes where they truly belong in the rest of the biology curriculum.

Fine Reading: The gut microbiota of insects – diversity in structure and function

2013-06-13T04:00:00-07:00

by Elio

Now that the mammalian intestinal microbiome has been promoted to organ status, might not such stately respectability be granted to the gut microbiota of other metazoans? If looking for a worthy candidate for such recognition, one could not do better than to consider the varied communities dwelling in the guts of insects. A recent review by Engel and Moran points out that beneficial intestinal microbes here: upgrade nutrient-poor diets, aid digestion of recalcitrant food components, protect from predators, parasites and pathogens, contribute to inter- and intraspecific communication, affect efficiency as disease vectors, and govern mating and reproductive systems. Microbes, it is clear, have contributed mightily to insect success.

A. Generalized gut structure of insects. The foregut and hindgut are lined by a cuticle layer (black line), the midgut secretes a peritrophic matrix (dashed line). B-M: Gut structures of insects from different orders. If not specifically indicated, the gut of an adult insect is shown. Stipples depict the predominant localization of the gut bacteria. For plataspid stinkbug, the magnification shows bacteria localized in the midgut crypts. Source.

The microbial landscape of the insect gut microbiome is much different from that of mammals. The great diversity of insects is reflected in their digestive tracts that also differ greatly in anatomy and physiology, and thus accommodate microbiotas that vary in composition and number. The total number of bacteria present range from 10⁹ per honey bee, to 10⁵ in a fruit fly, to negligible numbers in the sap-feeders, the latter often carrying instead an abundance of bacterial endosymbionts. Pick any individual insect and likely it will have fewer species in its gut than you do, although there are exceptions. Perhaps the most diverse communities among the insects are those in the termites, which are teeming with bacteria and protists. The 19th century American paleontologist and biologist Joseph Leidy observed that when one ruptures the intestine of a termite, myriads of the living occupants escape, reminding one of the turning out of a multitude of persons from the door of a crowded meeting-house.

The authors of this review article serve as expert guides to the anatomic complexity of the insect digestive system—with it distinguishable fore-, mid-, and hindgut. Each molt brings marked changes that disrupt or discard the microbiota, thus calling for extensive re-seeding. The routes of acquisition of these varied partners are likewise diverse. Some insects tend their eggs, thus facilitating vertical transmission, while the social insects have opportunities for transmission within the group. Both of these routes favor the development of gut communities’ characteristic of that species. The interests of the Moran lab being centered on bacterial endosymbionts, those associated with the gut receive considerable attention and serve to contrast with the free-living members of the microbiome.

Bacteria arriving by any route encounter powerful host defensive mechanisms. These barriers vary among the different groups of insects, and so do the bacterial strategies for surmounting them. As expected, the mighty subject of insect innate immunity, which is so conducive to understanding its equivalent in mammals, is treated here in detail. So are nutritional symbioses, the role of the microbes in insect development and physiology, and, especially thought provoking, their participation in intraspecific and interspecific communication. But be prepared for a specialized language: you will encounter terms such as proctodeal trophallaxis (anus-to anus feeding), peritrophic matrix (a layer of chitin fibrils that protects the intestinal epithelium), paratransgenesis (the elimination of a pathogen via the introduction of a symbiont) and others that may prompt a Google search.

This review will acquaint you with what we currently know about the great variety of bacteria found in the guts of insects, and at the same time will convince you that much remains to be explored before we have the grand picture of the inner life in insects. This is to be expected, insects being the most varied and abundant animal clade of all.

Engel P, & Moran NA (2013). The gut microbiota of insects - diversity in structure and function. FEMS microbiology reviews PMID: 23692388

O'Hara AM, & Shanahan F (2006). The gut flora as a forgotten organ. EMBO reports, 7 (7), 688-93 PMID: 16819463

Finally, Farewell to “Stamp Collecting”...

2013-06-10T04:00:00-07:00

by Christoph Weigel

The perspective paper by Margaret McFall-Ngai and colleagues was recently featured by Elio in this blog, strongly emphasizing its Chicxulub-like impact on microbiology. Here I offer a postscript, a few loosely connected thoughts from a historical perspective about its impact on biology and life sciences in general.

Until the 50s of the last century, advancement in biology was largely the product of three overlapping generations—students, active scientists, and emeriti—laboring over methods, paradigms, concepts, and theories. With few exceptions, these were European and North American men. Theories put forward by the emeriti during their active time tended to be overthrown by their former students who now become active scientists themselves: a spiral of slow progress. Since experiments were tedious and methodological progress slow, scientists were inclined to heated debates regarding concepts and theories. Few theories held for more than one generation, notable exceptions being Darwin's insight of evolution, Mendel's concept of inheritance, and the cell theory by Schleiden and Schwann. Collecting thousands of different mosses or pinning thousands of insects for a museum collection was considered at least equally important as experiments, the lattermost often designed to prove an existing theory rather than to generate a new testable hypothesis. Nevertheless, Louis Pasteur's experiments disproving the spontaneous generation of life and Robert Koch's postulates for proving disease causation can be considered to have ushered in the dawn of experimental biology.

By the late 20th century, biologists were diligently striving to disprove Ernest Rutherford's famous dictum that “...science is either physics or stamp collecting.” As molecular biology emerged, it was hoped—and rigorously asserted by its practitioners—that living systems could be completely understood in terms of the properties of their constituent parts (fundamentalist reductionism), and biology ultimately would be reduced to physics, as according to James Watson: “... there are only atoms. Everything else is merely social work” (cited here). Evolution research was dismissed because, being in essence historical, it could not be reduced to physics. Instead, the fitting approach was to solve the structures of biomolecules, which would thereby reveal their function. Likewise, the road to increased understanding of biology was the falsifiable working hypothesis, itself derived from previous experimental results (empirical reductionism).

Influenced to some extent by the New Age idea that our planet is most fittingly perceived in toto as a single living organism, a growing number of biologists in the 80s began to argue for a holistic approach. The reductionist approach then in vogue could not explain the emergent properties of complex biological systems, or, as Steven Rose phrased it: “...watch a flock of birds, startled by a noise, take off from the field on which they have settled—see them wheel and turn in formation, and try to explain or predict the behaviour of the group merely from a knowledge of the wing-musculature of each individual and aerodynamic theory.” However, there was a flaw in this argument: no holistic concept at that time was able to propose meaningful experiments.

At about the same time, a second criticism was put forward by biologists concerned that reducing biology to physics could in the end strangle scientific creativity. They favored curiosity-driven research over technology-driven research. Or, as Elio put it during a meeting in '87 in memory of Luigi Gorini: “On the planet Krypton every experiment works. As a consequence people quickly run out of ideas and so they spend their time sequencing the human genome. With Luigi, experiments did not always work, but he never ran out of ideas.” (quoted here).

Fig. 1. The sequence of the E. coli rrnB gene determined in 1979 by the Maxam-Gilbert technique. Source.

Now, early in the 21st century, the situation is dramatically different. Never before were the life sciences explored by so many researchers from diverse cultural backgrounds, both men and women. Due to the ever increasing speed of technological development, research now spans about five rather than three contemporary generations. Cutting-edge technology of the 80s is at best of historical interest today—who remembers Maxam-Gilbert sequencing? Experiments have become less tedious but they now produce so much data for analysis that hardly any time is left for any of these multiple generations to debate what it all means. When the human genome sequence was published, biology hit a wall of biological complexity. Many biologists saw that fundamentalist reductionism was failing and the spiral of progress arrested as biology was drawn in various directions. Its central narrative seemed lost—almost.

At this point, enter the paper by McFall-Ngai et al., just in time, adding umami flavor to Carl Woese's call for 'New Biology for a New Century'. That call was paraphrased elegantly by Freeman Dyson: “... postulating a golden age of pre-Darwinian life, when horizontal gene transfer was universal and separate species did not yet exist. Life was then a community of cells of various kinds, sharing their genetic information so that clever chemical tricks and catalytic processes invented by one creature could be inherited by all of them. Evolution was a communal affair, the whole community advancing in metabolic and reproductive efficiency as the genes of the most efficient cells were shared. ... But then, one evil day, a cell resembling a primitive bacterium happened to find itself one jump ahead of its neighbors in efficiency. That cell separated itself from the community and refused to share. Its offspring became the first species of bacteria—and the first species of any kind—reserving their intellectual property for their own private use. With their superior efficiency, the bacteria continued to prosper and to evolve separately, while the rest of the community continued its communal life.” Although Margaret McFall-Ngai and her co-workers refrain from expressing it explicitly, I can easily imagine them adding to this narrative: ...In separating itself from the community, refusing to share everything, this first species did not end communication with its siblings and the rest of the bunch, but rather increased its specificity, as witnessed by the ubiquitous communication among and direct interactions—even gene swapping—between the extant prokaryotes and eukaryotes, viruses, and a plethora of mobile genetic elements.

To come full circle—or more precisely to reenter the spiral—Karl Popper had suggested already in 1986 that by adopting 'active Darwinism' biology would avoid the teleological trap and eventually come into accordance with his scientific method of reductionism. Instead of the prevailing view in which selection was the imposed driving force of evolution, Popper’s 'active Darwinism' proposed that: “...the organism itself is not passive and neutral, waiting to be selected, but instead actively participates in its own selection, by choosing appropriate environments and modifying inappropriate ones; organism and environment interpenetrate and modify one another in ways which are determined in part by their own mutual history.” (cited here). I assume Margaret McFall-Ngai and her colleagues would prefer the plural here: organisms and environment interpenetrate and modify one another... This added complexity can then be tackled by the approved methodologies of empirical reductionism without the danger of reverting to "stamp collecting,," as pointed out by Carl Woese.

This is where we stand today. Biology has its 21st century narrative, which is just another word for an extended to-do list for biologists. The good news (for Elio, in particular): acute observation and curiosity have regained their pivotal role in finding out what life is all about. As we move forward, we eventually can teach computers one of the most precious, though enigmatic, of human traits—pattern recognition—so they can help us to cope with the approaching tsunami of data, help us visualize biological complexity.

Christoph is a lecturer in Life Science Engineering at the Hochschule für Technik und Wirtschaft, Berlin, Germany.

Woese, C. (2004). A New Biology for a New Century Microbiology and Molecular Biology Reviews, 68 (2), 173-186 DOI: 10.1128/MMBR.68.2.173-186.2004

Rose, S. (1988). Reflections on reductionism Trends in Biochemical Sciences, 13 (5), 160-162 DOI: 10.1016/0968-0004(88)90138-7

Pictures Considered #5. The Birth of the FtsZ Ring

2013-06-06T04:00:00-07:00

by Elio

Thin section of an E. coli cell in the process of dividing showing gold-labeled anti-FtsZ-antibody particles located at the septum.

The first cytoskeletal protein discovered in bacteria was FtsZ, the tubulin-like maker of the contractile ring involved in cell division of most bacteria. It was found by investigating one of a series of Fts (for “Filamenting temperature sensitive”) conditional mutants, first constructed by Y. Hirota, A. Ryter and F. Jacob in the 1960’s. These mutants do not divide but grew as filaments at a non-permissive temperature. Joe Lutkenhaus became acquainted with such mutants in the late 1970’s while a postdoc in Willie Donachie’s lab in Edinburgh. On his own, he continued studying such mutants and ended up purifying the FtsZ protein. Once he had it on hand, he made antibodies to it and, in an inspired moment, decided to look at the cellular localization of FtsZ using gold-labeled antibodies. What he found was of historic importance: although the antibody–carrying particles were distributed randomly in sections of most of the cells, in those cells that had initiated their septum and were engaged in cell division, the gold particles were located in a ring-shaped structure at the division septum. What this means was clearly stated in the paper: “….. we propose that FtsZ self-assembles into a ring structure on the cytoplasmic surface of the inner membrane where septation will occur and that is the first step in the division process. We also suggest that the initiation of FtsZ into a ring structure is the rate-limiting step for septation and occurs when the amount of FtsZ is sufficient.” One picture is all it took.

Terraforming Mars With Microbes

2013-06-03T04:00:00-07:00

by Ben Auch

Using new advances in synthetic biology and our updated understanding of Martian geochemical conditions, we should be able to inoculate the planet Mars with specially designed extremophilic microbes in an attempt to start (or re-start) life on its surface. This could be the largest and most audacious scientific experiment ever undertaken, aimed at one of the greatest puzzles in biology: how does life evolve on a planet? In so doing, the microbial pioneers we launch could pave the way for future human colonization of Mars and beyond.

A Biosphere is Born

Fig. 1. Source: Jackson Moore.

Four billion years ago the Earth was entirely hostile to the life we find on it today. Our home-to-be was harsh and unrecognizable, with high volcanic activity, asteroid impacts, 1000-foot tides, and a toxic, anaerobic atmosphere. But then something amazing and maybe unique happened: through some mechanism life appeared. No, it did more than that: it exploded, and it changed the planet forever. How this happened is far from clear. The first microbes may well have been chemoautotrophs, using widely available sulfur and iron as a substrate for growth. These early anaerobes were largely supplanted by the photosynthetic cyanobacteria, which released large amounts of toxic oxygen into the atmosphere, killing their predecessors and causing Earth’s longest ice age. When the dust settled, a new Earth was born, rich in oxygen and a protective ozone layer, and eventually permissive to the aptly named Cambrian explosion, the root of most eukaryotic domain of life. Said another way, the initial genesis of life was the spark that unlocked a vast array of biochemical reactions on a global scale, creating an environment and atmosphere capable of supporting the vast diversity of life we see, study, and even represent today. This was the evolution of a biosphere.

Life Out There

Fig. 2. A self-portrait of Curiosity on Mars. Source: NASA

Exactly how this extraordinary transformation proceeded, and whether it has occurred elsewhere in our universe, continues to puzzle us all, scientists and non-scientists alike. While the Earth’s biosphere remains a dynamic and fascinating system, we cannot go back to see it evolve. We rely instead on samples in deep rock layers and fossilized remains. To add more to our understanding of how a biosphere evolves, we might try moving beyond n=1. We could then ask: what do diverse biospheres have in common, and in what ways do they differ? NASA’s Kepler spacecraft has been searching the skies for exoplanets that might be capable of supporting life, and it has found over 100 of them. As many as 1 in 6 stars may harbor Earth-like planets. So perhaps the Earth isn’t so special. While many of these planets lie light-years (and many, many life-times) away, another potentially Earth-like planet is situated a mere 225 million km from us.

Was there, and is there now, life on Mars? Most of our scientific efforts on that planet have focused on these questions. Water is required for all life as we know it, and 2008’s Phoenix lander mission confirmed that water-ice is present on Mars, including in the Martian soil. The Mars Science Laboratory (Curiosity) has recently found evidence of liquid water flowing in very large quantities on an ancient Mars. In terms of direct evidence of life, however, we’ve come up short - the high UV irradiance and presence of oxidizing compounds may have destroyed all traces of surface life. Further robotic lander missions may bring us closer to an answer, but we may never know.

A Grand Experiment

Instead of mere observation, perhaps we should shift our focus to experiments that use Mars as our “petri dish.” The idea of terraforming (making planets Earth-like) has long been on the minds of science as well as science fiction writers, the aim being to industrially induce planets to support human colonies. There may come a time when humanity desires (or is required) to expand beyond the boundaries of its own home.

What would it take to make Mars into a planet capable of supporting human life? Humanity hasn’t even sent a manned mission to Mars, or any planet beyond our gravity well. What can we do to move human colonization of Mars forward? What I discuss here is to approach terraforming by stimulating the evolution of a new biosphere: planetary ecopoiesis.

Fig.3. A candidate for terraforming? Growth of permafrost isolate WN1359 on TSBYS at 0 °C and Earth atmosphere and pressure (circles); simulated Mars atmosphere and Earth pressure (triangles); and simulated Mars atmosphere and pressure (squares). Source.

To bring life to a “dead” planet, we need a suitable seed and suitable terrain. What the lander missions have found is not encouraging to life. Although the life we humans know is unsuited to the Martian surface, I think we can address this challenge with the same tool that changed our own planet so dramatically billions of years ago: microbes. On Mars, an “inoculation” by a hardy collection of microbial species could begin the process of terraforming, improving the chances of growth for other types of life, much like a pioneer species in ecological succession. These microbial pioneers would face some hefty tasks: they must increase Mars’ atmospheric pressure and mean temperature, melt ice to create pools of liquid water, increase atmospheric greenhouse gases, and provide an atmospheric shield to UV radiation. And they must do this with what is available in situ on Mars. What would be the traits of this microbial pioneer? They would have to be highly cold tolerant, anaerobic, photoautotrophic, and UV-resistant, and able to use highly limited available substrates for growth. They would have to be able to grow on solid ice. We have examples of several candidates on Earth today, including cyanobacteria.

Chroococcidiopsis is a rock-dwelling cyanobacterium highly resistant to desiccation, hypersalinity, and temperature swings found in extremely arid environments. Carnobacterium spp. has recently been shown to grow in permafrost at very low atmospheric pressures and without oxygen. Methanogenic archaea combining carbon dioxide and hydrogen could be critical in promoting rapid greenhouse warming. Many of these organisms function best as members of trophic and bioengineered consortia, so they should not be seeded in isolation.

A Five Year Mission?

Key advances in our understanding of Martian geochemistry, Earth-based bioprospecting, genomic sequencing, and synthetic biology make planetary ecopoesis on Mars thinkable at long last. In the next five years, we could make significant progress towards developing microbial pioneers for Mars. First, we must continue to gather data on the physicochemical conditions on Mars. Curiosity has enough power for at least 14 years of operation and has the most sophisticated suite of analytical tools ever sent to another planet. A companion mission is planned for 2020.

Second, we must further bioprospect our own planet for appropriate template organisms and identify candidates that may grow in the Martian environment. Sequencing and metagenomics are paving the way for cataloging the unique adaptations of extremophiles, and a search for microbial pioneers would add to a growing body of knowledge about microbial communities in extreme environments.

Third, we must develop the tools to genetically manipulate natural isolate organisms in order to modify them to fulfill the requirements of the parameters of the mission. Say what you will about the meaning of the term synthetic biology, but it’s clear that on the shoulders of micro, molecular, and systems biology, we are rapidly advancing our ability to modify microbes. Key to these advances has been the plummeting cost of DNA sequencing and, more recently, DNA synthesis. From parts, to modules, to systems, things are moving quickly. An important next step will be the construction of synthetic microbial consortia, key to this experiment. We can extend some of the natural abilities of our most extremophilic organisms by leveraging our new knowledge. In a sense, we can give evolution a head start. We can create rationally designed microbial consortia that are suited to living in the harsh Martian landscape, using Earth-based extremophiles as models and whose growth on a large scale could begin to tip Mars towards being supportive of less extremophilic life, such as our own. In addition to genetic changes for survival on Mars, other changes could enhance the ability of our consortium to adapt and evolve. Viruses could play a role in encouraging rearrangements of genetic information both within and between microbes, and enhanced conjugation could improve the ability of helpful mutations to propagate across the consortium. Additionally, we could send along equipment for DNA synthesis, allowing new adaptations discovered on Earth to be uploaded, synthesized, and transformed into the metagenome: genetic teleportation.

In drawing this to a close, I must leave many potential problems and pitfalls untouched, to say nothing of the ethics of seeding an entire planet with a synthetic microbial consortium. Who knows—maybe our own planet was seeded by a microbe hitching a ride on an asteroid! I can think of few experiments in the past or future as daring, and perhaps hubristic, as the creation of a new biosphere. This aside, I ask you: would it be so bad to have on hand a potential extension of our Earth-bound biological uniqueness? A handful of bugs could be our sojourners to the stars, microbial partners offering a new home and a peek back at our own origins. Maybe now isn’t the right time. We have so many problems on our own planet to take care of. But maybe, in a perhaps not unthinkable scenario, our terrestrial microbes would be our last message to the universe: amidst the collapse of our world, we load on a rocket our little bugs, bound for Mars or elsewhere to preserve for a while longer an extension of our existence. Alas, ad astra per aspera: a rough road leads to the stars. But maybe we can make it a little easier by relying on the unique talents of the microbes in our midst with whom we share this tiny dot.

Ben is a Biology graduate student at UCSD participating in the UCSD/SDSU Joint Doctoral Integrative Microbiology graduate course.

Graham, J. (2004). The Biological Terraforming of Mars: Planetary Ecosynthesis as Ecological Succession on a Global Scale Astrobiology, 4 (2), 168-195 DOI: 10.1089/153110704323175133

Friedmann EI, & Ocampo-Friedmann R (1995). A primitive cyanobacterium as pioneer microorganism for terraforming Mars. Advances in space research : the official journal of the Committee on Space Research (COSPAR), 15 (3), 243-6 PMID: 11539232

Nicholson WL, Krivushin K, Gilichinsky D, & Schuerger AC (2013). Growth of Carnobacterium spp. from permafrost under low pressure, temperature, and anoxic atmosphere has implications for Earth microbes on Mars. Proceedings of the National Academy of Sciences of the United States of America, 110 (2), 666-71 PMID: 23267097

Talmudic Question #99

2013-05-30T04:00:00-07:00

This is our first illustrated Talmudic Question and our last Talmudic Question (at least for a while). We think it may be time to move to new pastures. We hope that our new section, Pictures Considered, will partly fill this space.

TWiM #56: Live at ASM in Denver

2013-05-30T04:00:00-07:00

Hosts: Vincent Racaniello, Michael Schmidt, and Elio Schaechter.

Vincent, Elio and Michael recorded this episode before an audience at the 2013 General Meeting of the American Society for Microbiology in Denver, Colorado, where they spoke with Andrew, Ferric, Suzanne, and Michelle about their research on a phage system for evading innate immunity, retractions of research papers, bacterial infections of the eye, and cytoplasmic defenses against intracellular bacteria.

Right click to download TWiM #56 (73 MB .mp3, 102 minutes).

Subscribe to TWiM (free) on iTunes, Zune Marketplace, via RSS feed, by email or listen on your mobile device with the Microbeworld app.

Send your microbiology questions and comments (email or mp3 file) to twim@twiv.tv, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twim.

Stuck in Phage Heaven

2013-05-27T05:51:32-07:00

by Merry Youle

Figure 1. This image, intended to highlight the mechanism of the DNA packaging motor of phage T4, also provides a vivid representation of the 155 Hoc proteins (yellow) assembled into the capsid with their Ig-like domains extending outward. Green = major capsid protein; purple = vertex protein; blue = outer capsid proteins Soc; yellow = outer capsid protein Hoc. Source.

Ceteris paribus, the more prey, the better the hunting. The successful fisherman fishes where the fish are, the skillful pride of lions hunts where the wildebeest gather, and savvy phage predators hang out where the bacteria are. Such bacteria-rich locations include the protective mucus layer formed by animals to protect their vulnerable exposed surfaces. Bacteria—symbionts and pathogens alike—find mucus hospitable; it offers them food and a structured environment. The congregation of bacteria makes mucus a good place for phages to hunt their prey, and some have evolved to exploit this resource. Thus, while looking after their own interests, phages here also serve as an antimicrobial defense for their metazoan host.

The rule of thumb is that phage virions outnumber bacteria in most environments by roughly ten-to-one. Barr et al. recently reported that this ratio is increased more that four-fold in mucus sampled from a wide range of metazoans, from corals to humans. Unlike chemotactic bacteria, phages can’t actively move towards mucus. That there are more phages in mucus than in the adjacent milieu implies that when good fortune and diffusion brings them to the mucus layer, they adhere and stick around. The obvious question is how.

Figure 2. Samples of mucus were collected from diverse metazoans, along with samples from the adjacent milieu. Both bacteria and presumed phages (virus-like particles) were counted by epifluorescence microscopy and the phage-to-bacteria ratios (PBRs) calculated. On average, PBRs for mucosal surfaces were 4.4-fold greater than for the adjacent environment. Source.

An Introduction to Mucus

What exactly is this mucus”? A complex viscous gemisch. Its main macromolecular components are mucins, giant glycoproteins up to 103–106 kDa. In structure, these proteins are often likened to a “bottle brush.” The central rod is formed by a long “worm-like” polypeptide chain composed of hydrophobic regions that alternate with domains rich in serine and threonine residues. Those serine and threonine residues bear O-linked glycan chains that extend outward 0.5–5 nm forming the “brush” bristles. Mucus is sticky, so sticky that numerous organisms use it to adhere to surfaces or entrap food. Nevertheless, virions diffuse through it unimpeded. Not only are they small enough to slip through the mucin meshwork, but their surface is so densely coated with charged amino acid groups that they escape the hydrophobic binding that traps other particles their size. Something else must be responsible for the increased number of phages in the sampled mucus. Could it be some mechanism by which phages adhere to mucus?

A New Function for the Hoc Protein

These researchers started to track down this mechanism. Given the structure of mucin proteins, they postulated that the exposed glycan chains were likely to be key factors. In many different organisms, the binding of immunoglobulin-like (Ig-like) proteins to specific carbohydrates underlies cell adhesion processes. Surprisingly, about 25% of the tailed phages whose genomes have been sequenced contain Ig-like domains, all within structural proteins where they can be exposed on the surface of the virion. This led to a testable hypothesis: phage Ig-like domains bind to the mucin glycans, thus retarding phage diffusion and increasing phage residence time within the bacteria-rich mucus layer.

T4 phage was the obvious choice for investigating this in vitro. This phage assembles 155 copies of the Hoc (Highly antigenic outer capsid) protein in its capsid, each copy of which contains four immunoglobulin-like (Ig-like) domains that are exposed to the environment (see figure 1). Hoc’s function is unknown; removing the Hoc gene does not affect infectivity or replication under lab conditions. It was thought that perhaps Hoc enhances adherence to host cells in the natural environment. Indeed it might, but Barr and colleagues found that Hoc can play another role.

Phage T4 Adheres to Mucus

Comparing wildtype Hoc+ T4 with a Hoc– mutant, they found that the Hoc+ T4 and only the Hoc+ T4 bound selectively to mucin-coated agar plates. Would this binding be sufficient to retard phage movement through the mucus layer? To address this, they used multiple particle tracking to measure the diffusivity of fluorescently labeled T4 phage. They found that Hoc– T4 phage diffused through a 1% (w/v) mucin suspension at the same rate as through buffer, while the mucin decreased the diffusion rate for Hoc+ T4 eight-fold.

This is consistent with their hypothesis, but additional evidence was needed to support the view that the phages bind specifically to the mucin glycans. For this, they assayed the binding of Hoc+ and Hoc– T4 phage to each of 610 glycan targets in printed mammalian glycan microarrays. Only the Hoc+ T4 phages bound to any of those glycans. They bound weakly to a large number of diverse glycans, especially to O-linked glycan residues found in mucin glycoproteins.

While this is interesting, from our metazoan point of view the number one question is whether these adherent phages actually protect us from invasion by bacterial pathogens. Our mucosal surfaces are our most vulnerable zones, the main point of entry for infectious agents. To explore this possibility, Barr and colleagues set up an in vitro model system with various tissue culture cells, some mucus-producing and some not. Initial experiments determined that Hoc+ T4, but not Hoc– T4, adhered to the two types of mucus-producing tissue culture (TC) cells tested (T84, a human colon epithelial cell line, and A549, a human lung epithelial cell line). Even Hoc+ T4 did not adhere to these TC cells if the mucus was first removed with a mucolytic agent (N-acetyl-L-cysteine). Furthermore, Hoc+ T4 did not adhere to non-mucus-producing mutant cells derived from the A549 cell line, again indicating that the phage binds to the mucus, rather than to some other cell surface component.

Figure 3. Two TC cell lines were used in these experiments: mucus-producing A549 lung epithelial cells and a non-mucus-producing “mucus knockdown” line (MUC^–)derived from A549. Confluent cell monolayers were incubated overnight and cell death determined by propidium iodide staining and subsequent flow cytometry. (Left) Untreated controls. (Center) Overnight incubation with E. coli. (Right) Pretreatment with Hoc⁺ T4 for 30 min, phage removal by repeated washings, followed by overnight incubation with E. coli. (ns = not significant). Phage pretreatment completely protected mucus-producing A549 cells from bacterial challenge (n=12, ****P<0.0001, Tukey’s oneway ANOVA); protection of MUC^– cells was 3.1-fold less (n=12, *P=0.0181). Source.

Adherent Phage Protect Mucus-Producing Cells

Next these researchers used their in vitro model to demonstrate that adherent phages reduced bacterial colonization of mucus-producing TC cells. For these experiments, monolayers of TC cells, both mucus-producing and non-mucus-producing, were incubated with Hoc+ T4 phages for 30 min, washed to remove the phages and then incubated for 4 h with E. coli. The cells were scraped from the plates, fluorescently stained, and the attached phages and bacteria counted (see figure 2). The phage pretreatment significantly protected the mucus-producing cells from E. coli colonization. This protection mattered, as similar experiments showed that pretreatment with phages significantly decreased cell death.

Those experiments measured how well the phages protected the TC cells during a 4 h incubation with E. coli. Under their conditions, 4 h was long enough for the T4 phages to complete multiple cycles of replication, thereby increasing in number by several orders of magnitude. Repeating the experiments with Hoc+ T4 phages that could not replicate due to amber mutations in essential genes demonstrated that this replication was essential for effective protection of the TC cells.

A Phage-Mediated Immunity

The observations reported here could be the tip of an unexplored immunological iceberg. Future experiments are needed to determine if phages indeed protect mucosal surfaces in vivo, and if so, how significant this protection might be. Of particular interest is the potential for rapid adaptation of this non-host-derived immunity. Ig-like domains, such as those in Hoc, can accommodate large sequence variation (potentially >1013 variants), and a mechanism for rapidly generating such variation is known to be present in phages in the human gut. That mechanism employs the diversity generating retroelements (DGRs) first recognized in Bordetella phage (see our 2008 blog post). Moreover, continual sloughing of the mucus layer means that even adherent phage populations turnover quickly, thus facilitating rapid phage response to new bacterial invaders.

Figure 4. The Bacteriophage Adherence to Mucus (BAM) model.1) Mucus is produced and secreted by the underlying epithelium. 2) Phage bind variable glycan residues displayed on mucin glycoproteins via variable capsid proteins (e.g., Ig-like domains). 3) Phage adherence creates an antimicrobial layer that reduces bacterial attachment to and colonization of the mucus, which in turn lessens epithelium cell death. 4) Mucus-adherent phage are more likely to encounter bacterial hosts, thus are under positive selection for capsid proteins that enable them to remain in the mucus layer. 5) Continual sloughing of the outer mucus provides a dynamic mucosal environment. Source.

A Phage-Metazoan Symbiosis

As noted in Elio’s recent review of a most pertinent paper, the word is getting out: symbioses with microbes are a hallmark of successful metazoans. But we are just beginning to learn how we go about selecting our microbial associates, how we distinguish symbionts from pathogens, how we destroy the latter while welcoming the former. Mucosal surfaces are key sites for this interplay. In the eyes of Barr and colleagues, a major participant in this coalition has been overlooked until now—a partner on our side. They see their research as the first demonstration of a phage-metazoan symbiosis. With this development, the story of microbial-metazoan symbioses just became a whole lot more complicated.

Merry is a free-lance microbiology editor and writer living on the Big Island of Hawaii. After working side-by-side on STC with Elio for five years, she retired to become STC’s first Blogger Emeritus. As such, she continues to participate, but in a more relaxed capacity.

Barr JJ, Auro R, Furlan M, Whiteson KL, Erb ML, Pogliano J, Stotland A, Wolkowicz R, Cutting AS, Doran KS, Salamon P, Youle M, & Rohwer F (2013). Bacteriophage adhering to mucus provide a non-host-derived immunity. Proceedings of the National Academy of Sciences of the United States of America PMID: 23690590

A Whiff of Taxonomy – The Phylum Elusimicrobia

2013-05-23T04:00:00-07:00

by Elio

If you happen to look, you’ll find that new bacterial phyla spring up with amazing frequency, and that taxonomic names and facts accumulate at a staggering rate. As a public service, we’ll try from time to time to nibble away at this huge salami, slicing off and serving up one unfamiliar phylum at a time. Today it’s the turn of the Elusimicrobia. I admit that I chose them on account of their name. What could be more enticing than an elusive living being? (BTW, the name is derived from the Latin elusus, escaped from capture.)

An unrooted maximum-likelihood tree of 280 bacterial genomes, including the two sequenced representatives of the phylum Elusimicrobia, representing the regions of the bacterial domain currently mapped by genome sequences. Source.

This phylum used to be referred to as Termite Group 1, a monophyletic group of bacteria found as endosymbionts of flagellates in the hindguts of termites and other insects. Actually, Elusimicrobia are far less constrained than that, inhabiting also the ocean, soils, and sewage sludge. Since they form a deep branch of the bacterial tree, they are thought of as being quite old. The first one to be cultivated was given the apt name Elusimicrobium minutum. It is minute, an “ultramicrobacterium” only 0.17- 0.3 μm wide and variable in length. For an organism that can be grown in the lab, its genome is a puny 1.64 Mbp. It is a strict anaerobe but can grow on a variety of organic substrates. No pili have been seen on its surface although it is equipped with many of the genes needed for constructing pili. It presents the typical Gram-negative two-membrane system at its surface, but, perhaps due to its extreme thinness, it displays an interesting partitioning of its nucleoid (see the electron micrograph).

The story of the first cultivation of E. minutum bears telling. The authors had prepared culture tubes with what they assumed were sterile filtrates from the larval gut of a scarab, presumably sterilized by passage through 0.2 μm filters. The tubes were stored for future use, but, to their surprise, showed evidence of microbial growth after 3-6 months. Subculturing in “deep agar” yielded small, lens-shaped colonies.

TEM image of longitudinal and radial sections of E. minutum. Note the partition along the major axis between the nucleoid (n) and the ribosome-rich cytoplasm (r). The authors point out that this unusual partition may be due to the cells being limited in thickness. Source.

This phylum is split into two classes, the Elusimicrobiales (to which E. minutum belongs) and the Endomicrobiales. The latter also have small, spindle-shaped cells (0.6 μm in length and 0.3 μm in diameter). They, too, are abundant within the flagellates, which are in turn so plentiful that they can make up 1/3 the body weight of the termites. These protists are essential for degrading the wood cellulose on which the insects feed, but apparently the bacteria are not. Termites treated with antibiotics continue eating wood, unperturbed. So, what else might these elusive bacterial endosymbionts contribute to the partnership? Might their story, once further elucidated, provide yet another absorbing tale of cooperation between insects and their nested endosymbionts?

Herlemann, D., Geissinger, O., & Brune, A. (2007). The Termite Group I Phylum Is Highly Diverse and Widespread in the Environment Applied and Environmental Microbiology, 73 (20), 6682-6685 DOI: 10.1128/AEM.00712-07

Tit-for-Tat: A Bacterial Counterattack System

2013-05-20T04:00:00-07:00

by Spencer Scott & John De Friel

Schematic diagram of a type VI secretion system by Y. M. Cully, C. Cambillau, and E. Cascales. The lower bilayer membrane is the attacker’s, the upper one the host’s. Notice the complex structure of the Type VI secretory apparatus. Source.

Microbial ecology may be a young field but it is well understood already that there is a broad spectrum of interactions between bacterial species, ranging from cooperative to competitive. In a recent paper researchers from John Mekalanos’ lab further characterized a recently discovered mechanism for inter-cell communication. This system, called the Type VI secretion system (T6SS), is a multi-protein complex native to many bacterial strains and structurally and functionally similar to a bacteriophage tail. The T6SS system is unique in that it is used as a weapon for injecting toxic proteins into the cytoplasm not only of animal host cells but also of neighboring bacterial cells by propelling its components through the neighbors’ membrane. The toxic effector proteins, Tse1 and Tse3, are peptidoglycan-degrading enzymes that can cause cell lysis in the absence of antitoxin proteins. For reviews, click here and here.

The Type VI Secretion System Differs in Two Species

To elucidate how and when a cell decides to inject a neighboring cell with its T6SS, these workers studied its behavior in Vibrio cholerae and Pseudomonas aeruginosa. In V. cholerae, the T6SS seems to shoot off at random, constantly showing up in different areas on the cell, which endows it with high pathogenicity and with the ability to kill off many other species of cells. In this light, V. cholerae can be seen as a sort of Yosemite Sam, a character renowned for his excessive and poorly aimed shooting. The analogy to this character and to Batman (see below) was thought up by Robert Cooper and appeared in his fine blog at Science 2.0.

On the other hand, in P. aeruginosa, T6SS often shows up in a cell only in one place exactly corresponding to the T6SS activity of an adjacent cell, something the group dubbed as “dueling activity.” Instead of the haphazard random firing of V. cholerae, P. aeruginosa seems to duel with other nearby cells, so that when one cell fires its T6SS, the other cell would fire right back in the same spot. Thus, P. aeruginosa has a more sophisticated T6SS response system that will fire only in a spatially-targeted fashion and only in response to being attacked first. Because of this more refined offensive strategy (at least with respect to Vibrio), P. aeruginosa can be likened to Batman, a figure that classically attacks aggressors only. The paper unknowingly continues this analogy by showing that P. aeruginosa will happily coexist with non-aggressive cells even though it has the capability to destroy them, just like our hero Batman.

Who Attacks Whom?

Yosemite Sam. Source.

The study began by characterizing the “Yosemite Sam” firing of the wild-type Vibrio cholerae. To appreciate the chaotic firing nature of the aggressive V. cholerae, we suggest watching the paper’s video abstract. The group then focused on P. aeruginosa alone. They noticed that one sister cell would fire its T6SS, the neighboring cell would fire back after a short delay. However, the P. aeruginosa makes an antitoxin to the injected toxic protein, so it can survive attacks from sister cells.

When V. cholerae and P. aeruginosa were co-cultured, the overly aggressive V. cholerae were killed effectively by the P. aeruginosa whereas the P. aeruginosa proved invulnerable to the Vibrio’s attacks. The authors speculate that P. aeruginosa’s immunity to V. cholerae may be due to the impermeability of its outer-membrane since it can survive attacks from V. cholerae even if its own T6SS system is knocked out and has no way of offensively defending itself. The toxin protein P. aeruginosa secretes is called Tse, for which Vibrio doesn’t have an antitoxin. However, even without these proteins (using tse1-3 null mutants) P. aeruginosa could still kill the vibrios. This is likely due to the mere puncturing of the cell, which ruins the structural integrity of its outer membrane.

Is Pseudomonas Like Batman?

Batman. Source.

To test if Pseudomonas is simply a cold-blooded killer, these researchers mixed and grew it together with a non-aggressive E. coli (-T6SS) strain. Through microscopy, they showed that E. coli is killed very slowly, if at all, by the righteous P. aeruginosa. In the same vein, they created a non-aggressive V. cholerae strain by knocking out the T6SS, and mixing it with P. aeruginosa. This knockout proved to be very beneficial in that these mutants were killed at rates ~100 times slower than the wild type vibrios. Its T6SS system is so specific that even when P. aeruginosa was mixed with both the passive vibrio mutants and the Yosemite Sam wild-type vibrios, the aggressive strain is killed but the cooperative strain is spared. Pseudomonas is clearly Batman-like, showing mercy to those who do it no harm.

Lastly, the group made sure that this system was not specific to V. cholerae by growing T6SS+ Acinetobacter baylyi cells together with P. aeruginosa. Once again the P. aeruginosa effectively and efficiently killed the aggressive A. baylyi, proving that it is capable of responding to a variety of T6SS+ organisms. In short, P. aeruginosa can carry out coordinated counter-attacks to a spectrum of aggressive T6SS+ cells. The attack is spatially localized and used as a response to aggression. It is also parsimonious, in that P. aeruginosa saves its ammo solely for counterattacks, as if using its powers only to subdue aggressors.

An Offensive Weapon Can Be Defensive

There are several important conclusions to draw from these different uses of the T6SS system. Essentially, as the authors state “V. cholerae uses the [T6SS] as an offensive weapon, whereas P. aeruginosa uses the organelle as a defensive weapon.” This has a strong evolutionary implication since, as the paper states, biofilms composed of diverse but cooperative bacterial species likely have more growth potential than biofilms composed of a single bacterial species. See here and here. Thus, P. aeruginosa’s ability to discriminate between friend and foe may evolutionarily reflect the adage “don’t bite the hand that feeds you.” This suggests that P. aeruginosa has developed a way to coexist with other bacterial species as long as they are not aggressive. On the other hand, its ability to counterattack an aggressive bacterial species demonstrates a “tit-for-tat” evolutionary strategy. If you are a non-aggressive bacterium, P. aeruginosa is a great choice to create a symbiotic relationship with. It would be easy to create a biofilm with the P. aeruginosa and provide it with nutrients in exchange for protection from predatory bacteria like V. cholerae. And you thought that bacteria live a simple life.

Spencer and John are Bioengineering graduate students participating in the 2013 UCSD/SDSU Joint Doctoral Integrative Microbiology graduate course.

Basler M, Ho BT, & Mekalanos JJ (2013). Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions. Cell, 152 (4), 884-94 PMID: 23415234

Pictures Considered #4. Koch’s Development of Early InstaGram Positive Photography

2013-05-16T04:00:00-07:00

by Daniel P. Haeusser

Figure 1A. Koch’s photograph of B. anthracis, one of several photomicrographs in his 1877 paper, the earliest published bacteria photos. Source.

Robert Koch is one of the key figures in early bacteriology, helping develop culture techniques (e.g. solid media), critical reasoning (e.g. Koch’s postulates), and disease etiology (e.g. cholera and tuberculosis). He also published the first photomicrographs of bacteria (Figure 1A) in his 1877 paper Verfahren zur Untersuchung, zum Conservieren und Photographiren der Bakterien.

Discontent with communicating microscopic observations with hand-drawn illustrations, Koch pioneered the photography of bacteria. On suitable days, Koch would set up to shoot outdoors. In his 1877 publication Koch explains how to take photographs outside through a basic microscope:

“Clean all the lenses, screw them in completely, and place the illuminating mirror on the sunny side of the microscope. With a dark cloth over your head, look through the ground glass and adjust the light and focus the specimen… Once the image is in focus… go inside to prepare the photographic plates. In the darkroom…remove a clean glass plate with forceps and pour over its surface the iodized collodion solution, making sure the film spreads evenly and completely. Once the collodion film is ready, close the darkroom door and carefully lower the plate into the silver bath… Allow it to drain and put it in the cassette… Go back outdoors to the photomicrographic apparatus. Remove the black cloth…and check to be certain that the proper image is still in focus… Then carefully place the cassette, being careful not to move anything. After the exposure… push the slide back in the cassette, remove the cassette from the microscope, and cover the microscope again with the black cloth. This whole procedure must be done quickly! Run back to the darkroom with the closed cassette, develop the plate, and fix the negative. If the photographic image is not completely sharp, or if there are imperfections in the emulsion… it is necessary to repeat the whole process, since nothing is more disheartening in the photographic technique than to try to make prints from unsatisfactory negatives.¹

Quite a process! Following this protocol, Koch reports it took at least three hours to obtain four to six good pictures. Note that this time does not include setting up the equipment, preparing the bacterial sample, or freshly mixing the photographic chemicals. Yet with skill, patience, and perseverance during his early studies on anthrax, Koch obtained photos of the Gram-positive B. anthracis in a quality that rivals images taken today. Indeed, as an exercise of curiosity, members of my department² took an Instagram picture of B. anthracis using a cell phone camera. The image took seconds to capture with modern technology, yet doesn’t come close to rivaling Koch’s.

Figure 1B. A 1982 stamp from Zimbabwe celebrating the centennial of Koch’s discovery of M. tuberculosis (DPH personal collection).

Koch’s realization of bacterial photography was not merely a pretty picture. The communication of accurate images of bacterial shape was vital to the understanding that multiple species of bacteria existed, and Koch’s practice in imaging relatively easy-to-cultivate organisms like B. anthracis allowed his eventual rapid success in discovering the extremely fastidious M. tuberculosis in diseased tissue. It is this attention to detail and scientific resolve that led Koch to the discoveries that have immortalized him in scientific, medical, and indeed general human history (Figure 1B).

Notes:

¹Brock, Thomas D. (1999) Robert Koch: A Life in Medicine and Bacteriology. ASM Press, p. 61.
²Thanks are given to Katie McCallum for her Instagram account and to Lori Horton, Malik Raynor, and Michelle Swick of the Kohler Lab for the microscopy equipment and B. anthracis sample.

Fecal Transplants in the “Good Old Days”

2013-05-13T08:00:00-07:00

by Stanley Falkow

I had a conversation with some colleagues last week about “personalized medicine,” which has been transformed now into the term “precision medicine.” The conversation revolved around what to do about the perceived effects of antibiotic treatment on the microbiota of individuals. How does one treat a patient without disrupting their microbiota? Do we create new classes of antimicrobials that target only a precise pathogen? I opined that I thought the day was coming when all individuals might have the microbiota from each anatomic site preserved so that it could be reconstituted after some catastrophic disruption caused by antimicrobial therapy for an infection, transplantation, surgery etc. The topic of fecal transplantation and how successful it has been for the treatment of intractable Clostridium difficle infection then came up. Would fecal reconstitution really work?

I answered truthfully that I did not know, but my experience many years ago led me to believe it would. One of the people in this conversation, John Mekalanos, no stranger to stools, asked when I participated in a fecal transplantation study. It occurs to me that my experience in this study it might be of interest, or at least titillate, those who read this blog.

I was a 23-year-old medical technologist (MT, ASCP) in 1957 working as a journeyman bacteriologist in several clinical laboratories in Rhode Island and Massachusetts. It was a time when the Staphylococcus aureus 80/81 phage type was raising a specter of uncontrolled hospital infections. It was a time when large doses of antibiotics were administered to patients pre-operatively and continued until they were discharged some days later. Many of these patients reported to their physicians that they suffered from diarrhea, flatulence, indigestion and generally felt terrible after their surgery, though the operation was deemed a success. This was before anyone knew about C. difficle, of course, but antibiotic-associated diarrhea was known even in those days. One of the internists I knew well came to talk with me. I should point out that in the late 1950s many (most) of the physicians were frequent visitors to the bacteriology laboratory because they wanted to look at the Gram stains and the cultures obtained from their patients. The physician in question, who I will simply call Dr. S, thought after examining and talking to patients who had not “felt right” after their surgery had suffered from the aftereffects of the antibiotics that had been given them to sterilize their bowel flora before surgery. The feces of many of these patients would yield no growth on blood agar plates and MacConkey agar for days after their surgery. (We didn’t do anaerobic cultures in those days though). The stools were even odorless. Few stools can make that claim. S thought that their normal flora had been disrupted by the antibiotics. ‘Healthy bowels, and regularity made a happy patient”, he said.

Empty gelatin capsules, ready for filling. Source.

Dr S thought it would be prudent to ask patients to bring in a stool specimen when they came to the hospital for their surgery. He said to me, “Now, Stan, how do we get it back into them?” We decided that gelatin capsules from the pharmacy might do the trick. The pharmacies at that time still made a good deal of their own formulary. We set up a protocol. Stools were obtained from the patients immediately after admission. I would transfer the stool as quickly as possible into 12 large gelatin capsules,. This was a messy and not a precise or enthusiastic process on my part. I would wash the capsules in water and rinse in a dilute solution of mercuric chloride to disinfect the outer surface of the capsule, and then the capsules were rinsed again and put in a small ice cream carton and put in the refrigerator labeled only with the patient’s initials.

Upon discharge, Dr. S and one other physician who became a convert to this “protocol” would obtain the capsules from me. They would tell the patient to keep them refrigerated, to take 2 twice a day until they were all consumed. At least Dr. S told them, “Eat lots of salad.” This uncontrolled trial continued for some months, and, according to the anecdotal reports of Dr. S and his colleague Dr. B was quite successful in comparison to the patients of other physicians who did not have the benefit of the autogenous fecal sandwich. I don’t recall that we ever thought about the ethics of this. It was a time before informed consent. I’m pretty sure, however, that the esthetics of this practice was understood and that the patients in question never knew the contents of the capsules they ingested.

The chief hospital administrator discovered what was up. He confronted me and exclaimed, “Falkow, is it true you’ve been feeding the patients s**t!” He used the Anglo-Saxon phrase for feces. I responded: Yes I had been a participant in a clinical study that involved the patients ingesting their own feces. You’re fired! was the reply, although Dr. S came to my rescue. I was rehired two days later. Thus, the “experiment” came to an abrupt end. I left in June, 1958 to study for my PhD with C. A. Stuart and Seymour Lederberg at Brown University.

Now I am not going to claim that I knew that feeding patients their own feces after intense antibiotic therapy would be beneficial. Dr. S was sure it was the case based on his years of clinical experience. I understood the point that the indigenous flora was important. I had examined hundreds of stool specimens from sick and well people for too long. I routinely Gram-stained fecal samples and examined them in a wet mount. I didn’t have deep sequencing but I could discern differences in the flora of individuals. One fecal flora did not reflect all. I understood this even better after I met Rene Dubos and Russell Schaedler at the Armed Forces Epidemiology Board in the early 1960s and even more when I read the wonderful book by Theodor Rosebury, Life on Man.

My experience presaged the current excitement and exciting information that has deluged us in the past few years about the wonders of the human microbiota. The understanding and the appreciation for the sanctity of the “normal flora,” however, is not a new thing. Fecal transplants, of a sort, were practiced some 50 years ago because of the recognized untoward effects of antibiotic therapy. Mekalanos after hearing this story said: It once was "Eat s**t and die!" Maybe now it will be "Eat s**t and live!”

This experience also reminds me of something I have learned over the years. Experiences that occur during experiments or facts learned in a seminar or read in a paper have a way of reappearing, often decades later, with new meaning.

Stanley Falkow is the Robert W. and Vivian K. Cahill Professor Emeritus of Microbiology and Immunology and Medicine in the Department of Microbiology and Immunology at Stanford University.