Wednesday, August 31, 2011

Musical Confusion


All I know is that somewhere, there is a fiddler in the straw.

Sunday, August 28, 2011

You can take a geneticist out of the lab...

...but it's harder to take the lab out of the genetecist.

The morning routine here involves me making a cappucino for the Real Doctor. The standard recipe for this drink is one third espresso, one third steamed milk, and one third milk foam, which is exactly how the Real Doctor likes it: 10 ml espresso, 10 ml milk, and 10 fluid ounces of foam.

This last week, we had a bottle of milk that just would not produce satisfactory foam. This morning I opened up a fresh bottle of milk, and whoosh! excellent foam. Groggy though I was, my first thought was "hmmm...is there a foam inhibitor present in the old bottle, or is it lacking a necessary reagent for foam, one that's present in the new bottle?"

So, more or less on genetics autopilot, I made a heterozygous diploid by combining the two milks, and tried to steam it up. It would not foam at all. So, it looks like there's a foam inhibitor in the old bottle.

My next thought as a geneticist was "My work here is done. Give it to a biochemist to identify the inhibitor." The thought after that was about my own cocoa.

Disclaimer--of course there are other options, but this is 6:30 AM genetics autopilot.

Friday, August 26, 2011

Friday Flora, somewhat risque edition


Amaryllis belladonna, "naked lady." It's somewhat awkward to be a six year old and have your mom enthusiastically urging you to look out the car window at all the naked ladies.

As a Californian, I'm used to these blooming in March. I guess I need to get used to an August bloom.

Thursday, August 25, 2011

Cultural references

I suppose that my first thought on reading yesterday's article about blinking bacteria should have been the Doctor Who episode "Blink and you're dead." However, what actually sprang to mind was a cartoon I think I saw--or maybe dreamed up--years ago. I could not find it, so here's my version of it:

Wednesday, August 24, 2011

;-) ;-) (wink, wink)



No living thing is in equilibrium with its environment. I mean this in a physical-chemical sense; ecologists will discuss how ecosystems are at equilibrium, meaning that populations stay constant. However, to a biochemist, equilibrium is the same thing as death. A system at equilibrium has zero energy, and since life by any definition requires energy, equilibrium is death. It follows that if you can measure how far out of equilibrium a living thing is, you can get a clue as to how much energy it has at its disposal.


The way almost all living things get energy is sort of like the way energy is stored in a battery:

In a battery, there’s too many electrons over at one end, and not enough at the other. This potential difference is a form of energy. We can let the battery get to equilibrium by letting the charges become uniformly distributed, and we can use that movement to do some work for us:

If it achieves equilibrium—an equal concentration of charges on either end—no more work can be done.

A living cell functions almost exactly the same way. The only changes are that the charge difference is between inside and outside, and the charged particles are (positively-charged) protons (H+) instead of (negatively charged) electrons.

And, just like a battery, this potential difference stores energy. If we let the cell get to equilibrium by letting the charges become uniformly distributed, the cell can use that movement to do some work. Instead of powering light bulbs, cells have enzymes in their membranes that use that energy to make a chemical form of energy, ATP, or wiggle their flagella around so they can move, or pack food into the cell and expel wastes.

Also like a battery, equilibrium means no more work can be done.


Establishing the disequilibrium in a battery takes work, and making the proton gradient across a cell membrane also takes work. Respiring cells use the oxidation of food to move protons out of the cell. Cells that can use light for energy have evolved a couple of mechanisms to move protons out of the cell. The simplest of these is called proteorhodopsin. It’s an enzyme that straddles the cell membrane, like so:

When it’s hit by light...

it can use that energy to change its chape, grab a proton from inside the cell and force it out of the cell (and away from equilibrium). Like so:

To understand the physiology of a cell, it’s nice to know the charge difference, or membrane potential, of the cell. Keeping track of membrane potential is pretty straightforward in cells like ours. Our cells are large enough that we can make tools that can investigate to our cell membranes the way that a stethoscope can check in on a guinea pig. However, bacterial cells are much too small for this approach, and their membranes are covered up by cell walls. So until recently, we haven’t been able to probe the membrane potential of individual bacterial cells, despite knowing that membrane potential is essential for the well-being of the cell.


A bunch of researchers at Harvard, led by Adam Cohen, have devised (and are in the process of patenting) and exceedingly clever way to look at membrane potential in single bacterial cells. Remember proteorhodopsin, which absorbs light energy and uses it to pull a proton out of the cell? The reason it’s able to absorb light energy is that it has a color. Grabbing onto a proton changes the energy state of the molecule, so it changes its color a little bit. So, Cohen and his gang figured out a way to modify proteorhodopsin, essentially reversing its normal behavior: if a proton were forced onto this modified enzyme, it would not only change color, it would fluoresce. How can a proton be forced onto this modified proteorhodopsin? Simply have a higher-than-normal concentration of protons inside the cell. So, here's a normal cell, and the engineered proteorhodpsin is dark:

And here's a depolarized cell. The relatively high concentration of protons inside the cell forces one onto the engineered bacteriorhodopsin, and it fluoresces:

Bear in mind that what triggers the light is not the concentration of protons, just the difference in concentration across the cell membrane.

This designed membrane protein doesn’t help the cell out any—it can’t do what normal proteorhodopsin does—but it does serve as a sensitive monitor of the membrane potential of a single bacterial cell. It is, in effect, a very sensitive tool. Cohen’s group embedded single bacterial cells in a gel matrix, hooked one end of the gel matrix up to the positive end of a battery and the other end up to the negative end. The electrical field pushed all of the protons in the matrix towards one end of the cell, depolarizing the other end. Sure enough, that end of the cell lit up like a Christmas tree. Here it is in cartoon form...

and the actual microscope view, as they flipped the polarity from one end of the cell to the other:


So far, this is just a cute tool. Unlike our cells, which are so large that small patches of the membrane can depolarize, bacterial cells are so large that they essentially can’t have any regionalized depolarization under normal conditions. Also, this modified enzyme only lights up when there’s essentially no difference in charge across the cell’s membrane—which condition, if sustained, is the same thing as death. So, what’s the point?


Here’s where this gets interesting, and actually tells us something new and unexpected about how bacterial cells live. When Cohen looked at a bunch of these cells under the microscope, he saw them “blink” at him at irregular intervals. Whatever the reason, something about the cells’ membrane potential would periodically just give out. (This movie is sped up four times actual speed.)

video

The modified bacteriorhodopsin essentially senses the difference in proton concentration across the cell membrane. So, there’s a couple ways that a blink could happen—either too many protons inside the cell, or not enough protons outside the cell. “Concentration of protons” is simply a long way to say “pH,” so to test this possibility, Cohen loaded his cells with a pH indicator (like Litmus paper, only in convenient liquid form!). When a cell “blinked,” its internal pH stayed the same.


The cell uses this proton concentration difference to do stuff—make ATP, and rotate its flagella, among other things. It’s kind of a rude trick, but if you glue a cell’s flagellum down, the whole cell rotates…

video

…as long as there is a proton gradient. (By the way, that movie is actually slowed down three times!) When Cohen’s group examined a cell that was “blinking,” they found that the blink correlated with the cell’s rotation slowing or stopping. Since the concentration of protons inside the cell is remaining constant, the concentration of protons outside the cell must drop during a blink. (I’m not going to include a movie of a cell…not moving.)


So why would a cell blink? Why would it periodically deprive itself of energy? There’s one thing that it’s much easier for a cell to do if it no longer has a membrane potential, and that is push positively charged substances out of the cell. There are some toxins and metals that are harmful to the cell that are positively charged, and cells are known to spit these out of the cell under stressful conditions. So, perhaps this is happening during a blink: the cell deliberately depolarizes, and purges itself of toxins. Cohen tried feeding his cells a toxic dye, and sure enough, it could be observed flowing out of the cell only during a blink.


So, this Cohen’s cleverly modified protein ends up being more than just an example of clever engineering; it tells us something new about how cells work. Until very recently, the only way we could study the physiology of bacterial cells was by studying the average properties of billions of cells all at once. Very useful information can be gained that way (just ask the US Census Bureau), but vital information about how individuals work is lost. This modified proteorhodpsin is one of several neat tools that have emerged in the last decade that allow us to study single cells or even single proteins. Just as doing interviews with individuals would change your views of census data, these new tools are changing how we view the lives of bacteria.


Joel M. Kralj, Daniel R. Hochbaum, Adam D. Douglass, and Adam E. Cohen (2011). Electrical Spiking in Escherichia coli Probed with a Fluorescent Voltage-Indicating Protein. Science 333: 345-348.



Tuesday, August 23, 2011

When 3.4 billion years old you reach, look as good you will not

I finally got a chance to read the latest microbial news that is creating a buzz, the discovery of some tantalizing maybe-fossils of cells over 3.35 billion years old. (News reports here and here; a couple of blogs’ opinions here and here.) I’m not a geologist, so I can’t adequately judge everything about the paper, but as a microbiologist I can offer my two cents.


I’ll confess that I rolled my eyes a bit when I first heard the news, for several reasons. First, I’m not a fan of science by press release. Fortunately this is actually in the legit literature*. Second, the topic of ancient microfossils is one that leads to a lot of squinting at grainy pictures and seeing what one wishes to see. Third (and partly because of the very subjective nature of interpretation) this has been a field of heated, occasionally quite personal debate, and I am conflict-averse. Finally, I’m not sure it matters.


Here’s the problem. It would be nice to find a beautifully preserved, clearly cellular structure over 3 billion years old with unambiguous evidence of biological functions. It would also be nice if a unicorn came by and pooped golden bricks for me, and about as likely. First, it’s really hard to find nice sedimentary, fossil-bearing rocks that are that old—plate tectonics being what it is, any really old rocks are likely to have been recycled, squeezed and cooked so much that fossils wouldn’t exist. Second, any life present over 3 billion years ago would have been unicellular and very small. It’s hard to find small things. Finally, it’s hard to find something that old that is unambiguously biological. Non-biological processes can generate mineral patterns that look cell-ish, and even mimic some of the chemical processes of life. Remember the martian meteorite that was supposed to have evidence of life on Mars?


So, here’s this report from Martin Brasier and his associates at the University of Western Australia. It’s actually pretty good.


To solve the first problem, they found one of the few places on earth with really old, relatively un-metamorphosed rock—the boonies of Western Australia. These are sandstones, sedimentary rock that was laid down on top of some volcanic rock that’s about 3.5 billion years old, then buried by more volcanic rock 3.35 billion years ago. That this rock is sandwiched between these layers is nice—the volcanic stuff is relatively easy to date, and it helps to protect it from modern organisms (there are some bacteria that live in surprisingly deep hot basalts; this geological setting minimizes the chances of contamination by these organisms, but does not eliminate it).


To solve the problem of finding fossils, they looked in likely places—places where carbonaceous traces were present. Here’s some pictures of what they found:

These are, of course, not convincing on their own to the layman or the specialist. Which brings up the third problem, and the meat of the report. As the authors say with modest understatement, “determining the biogenicity of putative [3 billion year old] microfossils is notoriously difficult.”


The authors provide some pretty good structural arguments. The candidate microfossils are cell-shaped, cell-sized (unlike the supposed martian fossils), and (like modern cells, but unlike some geological artifacts) quite uniform in size. They are located in the sediments like normal cells would be in such an environment. Some pretty amazing work with spectrometry shows that the things that look like cell walls are actually where most of the carbon is found. So, the geology is good and the taphonomy (I love that word—it means the conditions of fossil formation) is pretty good.


Some more convincing evidence comes from the chemistry of the supposed fossils. The researchers determined not just that carbon and nitrogen and sulfur were present, but which isotopes of carbon and sulfur were present. (Isotopes are versions of atoms with different numbers of neutrons—the number of protons and electrons remains the same, but an atom may have more or fewer neutrons. So, most carbon has 6 neutrons per atom, but a very small amount of carbon has 7 neutrons per atom.)


Isotope fractionation is one of the more reliable fingerprints of life. When living things such as plants and microbes take carbon dioxide from the atmosphere and make it into part of a living thing, they preferentially take the certain isotopes of carbon. To “fix” carbon dioxide, a biological enzyme first has to catch a molecule of the gas. The enzyme is not all that great at this, so it tends to catch relatively more of the heavier, slower-moving molecules that have heavier carbon isotopes. So, it’s possible to examine a carbon sample’s isotope ratios and determine whether it’s of biological origin. (Not only that, but different carbon-fixing enzymes are better or worse at snatching carbon dioxide from the atmosphere, so with modern carbon samples it’s possible to determine what sort of organism actually did the job—we can even determine how much of you is made from corn-based carbon vs. carbon fixed by wheat and other crops!)


The carbon isotopes reported with these microfossils check out as biological, which is reassuring. There are some arcane ways to make slight isotope imbalances without using biological processes, but the carbon compounds that are made are not consistent with what was found.


The researchers also presented evidence of isotope fractionation with sulfur found in the samples. This is particularly suggestive about what may have occurred so long ago. One place you may think of sulfur in your own biology (especially after the posts from this last week!) is in flatulence—and in a very real way, what is being looked at here are 3.4 billion year old bacteria farts.


The way we get most of our energy is through what are called oxidation-reduction, or “redox” reactions. In these reactions, an electron goes from a high-energy situation to a low-energy situation. Electrons don’t really like to be attached to carbon, so that’s a high-energy situation; electrons love to be attached to oxygen, so that’s a low energy situation. So, we get energy by letting electrons move (accompanied by a proton, to make a hydrogen atom) from carbohydrates to oxygen. To a very rough degree, you can think of these reactions moving hydrogen atoms from carbon to oxygen:


C6H12O6 + O2 --> CO2 + H2O **


There’s plenty of evidence that 3 billion years ago, there was no oxygen in the atmosphere. However, something like this process probably went on, and goes on today in your intestines, where there is no oxygen available. Although electrons love to be on oxygen, sulfur is a pretty good second choice. So, the microbes in your gut can do this reaction:


C6H12O6 + S --> CO2 + H2S


The cells won’t get as much energy, but they’ll get enough to live—and your farts will smell like rotten eggs.


Surrounding the supposed fossils, the researchers found grains of pyrite. If this metabolism (or one very like it, using hydrogen instead of carbohydrate and sulfate instead of sulfur) occurred, the resulting H2S would react with iron in the environment and form pyrite. This reaction can happen without biological intervention, but if it’s done by life as we know it, you would again expect to see an enrichment of heavier isotopes of sulfur—and this is just what was found. The spatial relationship of the supposed fossils and the pyrite grains is also the same as what one sees with modern cells performing this type of metabolism.


So, Brasier and his colleagues present a very good (but I hesitate to say iron-clad) case for having discovered 3.4 billion year old evidence for life. Their geology and taphonomy are sound, and they provide a couple of lines of evidence consistent with life, especially life under the conditions of the young earth. How young was the earth when these maybe-cells maybe lived? There’s geological evidence that the young earth was pummeled by a massive barrage of meteorites. This “late heavy bombardment,” which would have made life impossible by vaporizing the oceans, ended about 3.8 billion years ago. So, as one news report has it, life may have emerged “very soon” after the late heavy bombardment—although I think of 400 million years as being a pretty long time.


This is some impressive work, and it may well be the “oldest evidence for cellular life.” However, as I mentioned, I’m not sure it matters beyond who gets bragging rights. Although it’s old, it doesn’t change anyone’s estimate about when life started. It doesn’t change our views of ancient biochemistry, as it is consistent with what has been predicted. It would be nice to know if the purported cells were Bacteria or Archaea, but that information can’t be recovered. Finally, unambiguous proof of cells that old (or for that matter, if we every find anything on Mars or Europa) is just about impossible.


On the bright side, I suppose it is nice not only to know that you had a great-great grandmother, but also to have a picture of her. It makes for a better lecture to show a slide of these pictures rather than a slide of isotope data.


*David Wacey, Matt R. Kilburn, Martin Saunders, John Cliff and Martin D. Brasier (2011). Microfossils of sulphur-metabolizing cells in 3.4-billion-year-old rocks of Western Australia. Nature Geoscience (Published online 21 August 2011 | DOI: 10.1038.


**Yes, I'm not balancing the equation; I'm just interested in the chemicals, not their amounts. If you're concerned, I encourage you to do the stoichiometry.

Monday, August 22, 2011

Nature vs. Nurture in the gut microbiota III: what's in a name?

(Part I, Part II)

The population of microbes in our gut are a personal trait, like height or weight. The precise make-up of that population, its balance of different types of microbes, is significant as it can have major beneficial or harmful effects on our health. Like many other traits, the composition of that population is partly due to nature—our genes create an environment welcoming to certain types of microbes—and nurture, the food we eat.


Given the importance of the composition of the microbial community to our health, it’s not surprising that there is a lot of interest in trying to figure out just how significant are the contributions of nature and nurture. Comparing genetically identical mice with a drastically simplified community of microbes in their gut, researchers found that changes in diet led to predictable changes in community structure. However, comparing genetically distinct strains of laboratory mice, a different group of researchers found that the subtle genetic differences between two inbred lines of mice led to measurable differences in the gut microbial community (see part I).


The roles of nature and nurture can be probed further by comparing the microbial community of different, but related, types of animals. What if two animals are closely related, but have different diets? What if they are only distant relatives, but have similar diets? An extreme example of the latter case is a comparison of the intestinal microbes of the tammar wallaby and the cow (see part II). While the comparison is informative, these two mammals are about as distantly related as possible, so a closer comparison is useful.


In a recent study from Washington University at Saint Louis, a group led by Jeffrey Gordon attempted to compare a family tree of mammals, a family tree of the microbes that lived in their guts, the diets of those mammals, and the genetic abilities of their microbes. Any four-way comparison gets complicated pretty quickly, but there are some clear facts that emerge from the fog of statistics. These facts emphasize some basic biology of microbes, as well as telling us something about how mammals and their microbes co-evolve.


A family tree of the mammals comes out pretty much the same, regardless of whether you use genetic data or anatomical differences:

Looking at this family tree, there is little convergence between relatedness and diet. For instance, elephants, capybaras, gorillas, horses, and rabbits are all “hindgut-fermenting herbivores,” with a specific method of digesting cellulose—and yet, are found on different branches of the tree. Sheep, hyraxes, kangaroos, colobus monkeys, and pigs are all “foregut-fermenting herbivores,” with an anatomically distinct way of digesting plant matter—but again, they are only distant relatives. One can find groups, such as the primates, comprising foregut-fermenters, hindgut-fermenters, carnivores, and omnivores. So, genetics on a deep level does not determine diet.


If genetic heritage doesn’t determine diet, does it at least determine something about a mammal’s intestinal microbial community? To approach this question, you can imagine a goodly branch of that mammalian family tree. A diverse bunch of modern mammals evolved from a common ancestor:

We obviously don’t know anything of the DNA sequence of their common ancestor, but we can see the genetic similarities of the modern animals, and work backwards to predict what their ancestor was like, as well as which modern animals are most closely related. If the ancestor had a certain genetic composition—symbolized by the color blue—then its descendants would have evolved variations on that genetic composition—symboized by variations in the hue in the modern beasts.


Now, if genetic heritage determines an animal’s gut microbes, then the genetic character of that community should show a pattern of evolution sort of like the mammalian family tree:

This is a trickier tree than the previous one. Rather than looking at a lineage of organisms, we are looking at the composition of a community of thousands of species. We can make an analogy with the white pages of a phone book: in this tree, we are looking at how the content of a community’s phone book changes over time. (Also, it’s important to remind ourselves that we don’t know what the original community was like—we only have the present one, and we try to work backwards.)


So, what did Gordon’s group find? If mammals and their intestinal microbes co-evolved, then we could examine a diversity of modern mammals and the microbial communities in their poop—which is just what they did. We’d expect related mammals to have related communities. Lining up the tips of the family trees (that is, just currently living animals and the microbes in their poop), we’d see something like this:

There was good reason to expect this result, as similar patterns of co-evolution are seen with insects and their symbiotic bacteria. Co-evolution is common. But here’s what they found:

What to make of this? Obviously, genetic heritage has very little to do with the community structure of an animal’s intestinal microbes. However, Gordon’s group looked at the same data again. This time, instead of looking at mammals by their ancestry, they considered their diet:

Aaaaah, much better! So, it seems like diet—nurture, in our nature/nurture dichotomy—is a much better predictor of a mammal’s microbial community. (This is a much simplified version of the data presented; my apologies to Gordon et al, who studied 33 mammal species and hundreds of microbial species. It’s also useful to note that in several instances they looked at the communities of different individuals of the same species, and found little variation between them.)


Interestingly, despite the differences in community structure between mammals, all mammals have pretty much the same “core” community of microbes. To return to the metaphor of a phone book, any big city in this country will have roughly the same collection of names, it’s just that one city will have a lot more Schmidts and Webers and Mullers and another will have many more Vasquezes and Diazes and Gonzaleses.


Although there was a decent correlation between mammalian diet and community composition in Gordon’s data, his group found an even more significant relationship. The best correlation was not between mammalian diet and community composition. It was between mammalian diet and what genes were present in the microbial community.


Let’s consider that phone book analogy again. If I wanted to find somebody to turn some wheat into flour, I probably wouldn’t turn to the white pages and call up somebody named Muller or Miller. (Heck, I wouldn’t say I’m a good horticulturalist because I’m named Appleman.) Trades are not inherited with family names, and this is true in the microbial world as well. So, if you’re a vegetarian mammal looking for a microbe to turn your b-glucosides into monosaccharides, you can’t assume that you’re looking for Brevibacter—all you care about is whether the organism has the genes for doing the job.


Microbial genomes—the collection of genes a given species has—are incredibly fluid. Genes can disappear from one genome in the space of a few generations, and can establish themselves in a new genome in an equally short time. Genes can travel with relative ease between bacterial species that are barely related to each other. (There are many mechanisms for doing this, and it makes firmly describing a microbial species next to impossible.) So, when a hyrax feeds its intestinal microbes a blend of grass and insects, it is (unwittingly) selecting a specific assemblage of genes, not a specific community of microbes.


These results raise some interesting questions about evolution. The community of microbes that lives in a mammal’s gut—its microbiome—can easily be viewed as an organ like the lungs or kidneys. However, its inheritance and evolution are quite distinct from that of the rest of the animal. As Gordon’s group suggests, it would be fun to compare a lot of different carnivores, and try to extrapolate just when differences in their microbiomes evolved. Do such evolutionary jumps correlate with events such as continental drift or other significant milestones? Are the changes gradual or rapid? Can one community be dislocated by another? Just how fluid are the communities in the gut?


These questions bring us back to the tammar wallaby and its oddly non-methanogenic digestion. Interestingly, in this study, the microbiome of a kangaroo clearly grouped with other foregut-fermenting mammals, such as sheep and gazelles. It was an outlier in that group, but still clearly part of it and not some other group. Well, the tammar wallaby was found to have a small number of methanogens and acetogens in its gut, giving it at least some similarity to the sheep. The big difference in digestion (and flatus) may be traceable to the very small community difference of one organism with one interesting metabolic pathway, a factor that simply can’t be revealed by this sort of analysis. The situation is reminiscent of human societies, which can be hugely affected by a single individual—and knowing the name of that individual won’t tell you anything about how they affect society.


Brian D. Muegge, Justin Kucyznski, Dan Knigts, Jose C. Clemente, Antonio Gonzalez, Luigi Fontana, Bernard Henrissat, Rob Knight, and Jeffrey Gordon (2011). Diet Drives Convergence in Gut Microbiome Functions Across Mammalian Phylogeny and Within Humans. Science 332, 970-974.

Friday, August 19, 2011

Ferocious Friday Flora


Ferocactus from my Mom's garden. So soft and fuzzy!

Suddenly, nose!

(Douglas County Fair)

Wednesday, August 17, 2011

Nature vs Nurture in the gut microbiota, part II: the music of wallaby farts


I recently wrote about a study showing the impact of host genetics on gut microbes. This was done by comparing different strains of mice, eating about the same diet. These animals are closely related, having had common ancestors only a hundred years ago. What if we looked at animals eating about the same diet, but whose most recent common ancestor lived a hundred million years ago?


Consider the tammar wallaby: This cute little kangaroo-let has recently found interest as a laboratory animal. As a marsupial, the last ancestor it shares with a cow lived over a hundred million years ago. It eats essentially the same diet as a cow. However, while cow farts are rich in methane, wallaby farts are not. This suggests that, despite identical diets, these animals have very different gut microbes. Until very recently, the nature of these differences was quite puzzling.


It’s useful to have a little background on what goes on in the gut of a cow eating grass. Most of what the animal eats is cellulose and other polysaccharides. These are compounds that are made by stringing together hundreds of sugar molecules, and they are tough—the table that I’m writing on and the cotton I’m wearing are made of polysaccharides. Breaking these polysaccharides down to release their constituent sugars is chemically very difficult, and breaking down the cellulose found in forage is almost impossible. Surprisingly, cows can not do this. It’s a job done by microbes living in the guts of these animals. Of course, the microbes that release the sugars from cellulose don’t do it out of charity to their hosts—as soon as the sugar is freed from the cellulose, they eat it. How does this help the cow?


You probably know fermentation as what yeasts do, taking sugar and making ethanol and CO2 as wastes. However, there are other ways that microbes get energy by fermenting, and most of the microbes in a cow’s gut take sugar and make acetic acid, CO2, and hydrogen gas as wastes. So, after the microbes have broken down the cellulose and fermented the sugars, the cow thanks the microbes nicely, and absorbs the acetic acid as a nutrient. But the CO2 and hydrogen pose a real problem.


Picture a worker on an assembly line: he takes two widgets, screws them together, and passes them on to the next guy. He can keep working as long as the widgets keep coming down the assembly line and the next guy takes his finished product away. If the next guy falls asleep, the product will build up, and our worker will be unable to do his job. This is what could happen to the fermentation in a cow: CO2 and hydrogen are the products of fermentation, and if they build up, the fermenting microbes will stop (and the cow will starve). Fortunately for the cow and the microbes, there are other microbes in its gut, called methanogens, that get energy by “burning” the hydrogen—they oxidize it, using CO2 instead of oxygen, and produce methane. So, the production of methane in cow farts is absolutely necessary if the cow is to continue breaking down cellulose.


However, as was noted, wallaby farts don’t have so much methane, even though they eat the same amount of cellulose. How can wallabies and their microbes digest cellulose?


One possibility that seemed likely was that there were some other microbes in the wallaby gut that were able to eat CO2 and hydrogen. It was already known that termites (who also eat cellulose) house bacteria called “acetogens” that combine hydrogen and CO2 to make acetic acid rather than methane. So, it was thought that if an animal ate cellulose, it had to have either methanogens or acetogens in its gut to help the fermentation reactions along (and in fact, we have some of each in our guts—as evidenced by the flammability of farts—just not as many, since we don’t eat as many polysaccharides). When I first heard that wallaby farts were low in methane, I assumed that wallabies must be full of acetogens.


However, it’s always good to check your assumptions. An analysis of the wallaby gut community showed few methanogens, and somewhat more acetogens—but not enough to make fermentation (as it was understood) work. This was a real puzzle—without microbes to consume the CO2 and hydrogen from fermentation, how could the wallaby eat cellulose? The answer, according to an international group of researchers, may be that methanogens and acetogens are unnecessary for the fermentation that happens in a wallaby gut—their microbes use a unique fermentation reaction that doesn’t produce hydrogen, and consumes CO2.


The overall theme of fermentation by the cow’s microbes is to break sugars into smaller and smaller pieces—start with a molecule of six carbons, then go to two molecules of three carbons, then two molecules of two carbons and two molecules of CO2 and some hydrogen atoms.


The researchers observed that a significant component of the microbial population of a wallaby gut was similar to a previously identified bacterium called Succinovibrio, named after its unusual metabolism. Sugar fermentation in Succinovibrio starts the same way as in the cow; however, in the final stages, the fragments of sugar molecules are partially reassembled to make a four-carbon compound, succinate. This process (which also consumes CO2) provides the bacterium energy to live—and what’s more, the succinate gives the wallaby more energy than a similar amount of acetate gives to a cow.


The researchers showed very nicely that this sort of fermentation can go on in the wallaby’s gut, and that this could explain the way that wallabies are able to eat the same diet as a cow yet not produce so much methane. So, why do wallabies play host to Succinovibrio while cows play host to methanogens? It’s obviously not diet—the original studies showing differences in methane production were done with wallabies and cows on the same feed, and wallabies do host a few methanogens. There may be structural issues; cow anatomy is quite different from that of the macropodes. But most likely, as we have learned from studies on mice and humans, is that the genetic background of the host sets up a molecular environment that is particularly hospitable to specific microbes. The genomes of both the cow and the wallaby have been sequenced, so the information is there. We just have to do the hard work of understanding it.


There is a practical aspect to this question, beyond the flammability of wallaby farts. Methane is an extremely powerful greenhouse gas; it is over twenty times more efficient at retaining solar heat than CO2. The human fondness for cattle grazing has increased methane production, contributing to global climate change. There are researchers who entertain the pipedream of converting cattle to a wallaby metabolism—producing more meat per munch of hay, and producing less pollution to boot. A noble cause, but I’ll probably only be able to eat a burger from such an animal when I pull up to the drive-through in my flying car.


Paul N. Evans, Lyn A. Hinds, Lindsay I. Sly, Christopher S. McSweeney, Mark Morrison, and Andr ́e-Denis G. Wright (2010). Community Composition and Density of Methanogens in the Foregut of the Tammar Wallaby (Macropus eugenii). Applied and Environmental Microbiology 75: 2598-2602.


Emma J. Gagen, Stuart E. Denman, Jagadish Padmanabha, Someshwar Zadbuke, Rafat Al Jassim, Mark Morrison, and Christopher S. McSweeney (2010). Functional Gene Analysis Suggests Different Acetogen Populations in the Bovine Rumen and Tammar Wallaby Forestomach. Applied and Environmental Microbiology 76: 7785–7795.


P. B. Pope, S. E. Denman, M. Jones, S. G. Tringe, K. Barry, S. A. Malfatti, A. C. McHardy, J.-F. Cheng, P. Hugenholtz, C. S. McSweeney, and M. Morrison (2010). Adaptation to herbivory by the Tammar wallaby includes bacterial and glycoside hydrolase profiles different from other herbivores. Proceedings Natl Acad Sci USA 107:14793-14798.


P. B. Pope, W. Smith, S. E. Denman, S. G. Tringe, K. Barry, P. Hugenholtz, C. S. McSweeney, A. C. McHardy, M. Morrison (2011). Isolation of Succinivibrionaceae Implicated in Low Methane Emissions from Tammar Wallabies. Science 333: 646-648.

Monday, August 15, 2011

Nature vs. Nurture in the gut microbiota

It’s fun to watch how scientific truth changes with time. It shows that we can learn. But it’s amusing for me to compare what I told my first students with what I told my last students. When I started teaching, we didn’t know much about the microbes that lived in our gut—there were a lot of them, but they were generally regarded as being neither particularly harmful nor helpful, but just along for the ride. In the last decade, we have learned that these cells (and there are about ten of them for every one of our cells) are essential for providing nutrients, regulating metabolism, regulating the immune system, regulating the development and maintenance of the gut, and much more.


Although everyone has more or less similar general types of organisms in their gut, the balance of types varies considerably. Comparing the intestinal microbiota of two individuals is similar to comparing the yellow pages of Los Angeles and Fairbanks—they’ll both have the same classifications, but one will have a lot more acting coaches and the other will have more hunting guides. While the origin of the differences in yellow pages is largely due to environment, it’s more difficult to pin down the origin of the differences in our gut microbiota. Does diet have an effect on community composition? Does your genetics determine what microbes live in your gut?


A group from Washington University in St. Louis looked at an extremely simplified system to examine the influence of diet on gut microbes. Rather than studying humans, they used “gnotobiotic” mice—these are animals delivered by C-section, and raised in an absolutely sterile environment. These animals are pretty sickly (since the microbes that normally aid in development and digestion are absent), but researchers can deliberately add specific, known species of microbes to their food. This way, they know exactly what microbes are in there (gnotobiotic = “known life”), and by comparison with germ-free animals, they can know the effect of specific types of microbes.


To simplify their experiments, the researchers infected these mice with a community of 10 different types of microbes, chosen to represent the most general characteristics of the microbes in the human gut: one good at digesting starches, one good at fermenting amino acids, etc. They then gave these mice extremely simplified diets: mostly corn oil, mostly casein, mostly sucrose, or mostly cornstarch. After two weeks, the researchers collected the mouse poop, and analyzed the composition of the microbial community therein. This was repeated, until each mouse had enjoyed each diet.


After they had amassed a lot of mouse poop, they were able to develop a pretty good model of how the structure of the microbial community in the mouse gut was shaped by the mouse’s food intake. The model is actually relatively simple, and quite predictive: such a percent of protein in the diet leads to such a percent of Clostridium cells, and so on. The researchers were able to test their predictions by feeding the mice slightly more complex food (baby food, actually—pureed turkey, pureed carrots, etc). Sure enough, by knowing what went into the mouse, they were able to roughly predict the microbial community structure.


So, are we what we eat? Sorta. This was an extremely simplified system; thankfully our diets are more than corn oil, corn starch, casein, sucrose, and mashed peas, and we have thousands of different microbes in our guts. However, despite its simplicity, this study suggests that we can alter our gut microbiota at will. This can be medically important. For instance, the amino acid fermenting bacteria associated with the high protein diet are also associated with a variety of inflammatory syndromes; it would be nice to reduce their relative numbers.


However, another study reminds us that our family background is also a significant factor in determining our personal microbial community structure, and our health. Using mice, a group of researchers from Nebraska found a correlation between certain regions of DNA and certain types of bacteria in the gut.


Genetecists use the term “Quantitative Trait” to discuss something that can be measured (quantified) and inherited (trait). So, height is a quantitative trait. Once you have a quantitative trait, you can look for genes that influence it. In the case of height, there are dozens of such genes, each of which makes a small contribution to overall height. Each gene occurs in a specific region, or locus, in the DNA, so a geneticist could refer to a "Quantitative Trait Locus" (QTL) associated with height. It's useful to note that a QTL is not a gene; it's a region of DNA, potentially carrying many genes, associated with a particular trait.


The researchers decided to treat the composition of the gut microbiota as a quantitative trait. They did this by making a mathematical model that combined the relative abundances of 64 different microbes into one measurable variable; you could measure the relative numbers of these 64 microbes in the poop of any mouse, and compare it with any other mouse. Given the existence of so many different laboratory strains of mice, this made for a lot of data to analyze.


The big question, then, was whether there were any genetic loci that corresponded with variation in this quantitative trait. There were. At least 13 different places in the mouse genome corresponded with definite variations in their gut microbiota. If two mice had different versions of the same gene in one of these places, then they would likely have different ratios of different microbes in their guts.


So what genes determine what microbes we have? The answers are still a bit vague; we know that certain regions of the DNA are associated with these traits, but each region has several genes. Nonetheless, there are some cases where there are candidate genes that really stand out. For instance, there’s one QTL associated with increased numbers of two specific types of bacteria; this QTL contains genes specifically involved in controlling immune responses in the gut.


This study is highly suggestive, but not conclusive. It establishes a correlation, but the results were all retrospective; no predictions were tested. For example, it would be great to see a cross of two different strains of mice predicted to favor certain different bacteria. Would it be possible to accurately predict what bacteria the offspring would favor? Even without this bit of proof, this study represents a buttload of work, and a very promising entrĂ©e into a challenging question. Like the study with gnotobiotic mice, this study also has relevance to health and medicine, as it may explain the observed linkage between certain QTLs and diseases such as Crohn’s disease.


This research also raises a big question in the symbiosis between mammals and our gut microbiota. The authors of this study use the link between certain immune system genes and certain microbes to support the argument that the immune system evolved for the purpose to maintaining favorable microbes in the gut. However, I’m a microbiologist, and I look through the other end of the telescope. To me, this is further evidence that certain microbes have been exerting selection pressure upon us, in order to make a better environment for themselves.


Either way, your doctor’s advice for good health and longevity remains much the same: eat right, and choose good parents.


Andrew K. Benson, Scott A. Kelly, Ryan Legge, Fangrui Ma, Soo Jen Low, Jaehyoung Kim, Min Zhang, Phaik Lyn Oh, Derrick Nehrenberg, Kunjie Hua, Stephen D. Kachman, Etsuko N. Moriyama, Jens Walter, Daniel A. Peterson, and Daniel Pomp (2010). Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. Proceedings Natl. Acad. Sci. USA 107: 18933-18938.



Jeremiah Faith, Nathan P. Mc Nulty, Federico E. Rey, Jeffrey I. Gordon (2011). Predicting a Human Gut Microbiota’s Response to Diet in Gnotobiotic Mice. Science 333: 101-104.

Sunday, August 14, 2011

Gluing up

Sheesh--I still haven't finished posting all the work I did at the violin workshop, and I still haven't been able to do any violin work here in Roseburg. Things are starting to settle somewhat, so hopefully I can fix both of those issues.

Let's see, I left off with a fingerboard that had been planed flat on one side and curved appropriately on the other side. That meant it was time to glue it onto the neck, and here's one of the places where the Book said to do it one way and Michael said to do it another. Being as how Michael was there and the authors of the book were not, I went with Michael.

When you attach the neck to the body, it's really useful to have the fingerboard in place. Otherwise, it's very easy to get the neck slightly skewed along one of several axes. On the other hand, if you want to do a perfect job of varnishing the body, it's annoying to have the fingerboard in the way. Also, if you want to have your fiddle catch some sun while the varnish cures, the fingerboard will absorb a lot of heat and warp. So, the Book recommends just using a dab of glue to attach the fingerboard. Once everything is all assembled and ready to varnish, you pop the fingerboard off; then, varnishing completed, you reattach the fingerboard with a full dose of glue.

Bosh, sez Michael. (OK, he didn't say "Bosh," but words to that effect.) What did the old guys do? They glued the fingerboard on, then they varnished the fiddle. You can see it when you look at their work. It's quicker and easier, and the people who check the varnish under the fingerboard are the ones who look for dust behind your oven.

So, there it is, setting with a bunch of spring clamps.

While that was in progress, it was also time to glue the top onto the body. That was heaps of no fun. Hide glue sets very quickly, and I did not have the sang-froid to do it all at once. So, a dab here, some clamps, pry open a little bit, a dab more, re-clamp, and so on all the way around. It turned out OK, as far as I can tell--no visible gaps or buzzes. Yet.

Saturday, August 13, 2011

Comparing apples to apples

Sometimes it can really be hard to make meaningful comparisons between apples and apples…

But a recent review article makes a valiant effort at making such a comparison. Which is more efficient—this solar panel:

Or this one?

(The answer is coming…but make your guess now!)


It’s useful to note that this really is an apples-to-apples comparison. We’re used to thinking of life as being powered by chemical energy—you know, breaking down ATP or burning glucose, or photosynthesis making glucose. It may come as a shock that the energy underlying all these chemical processes is electrical energy—the movement of electrons from high-energy states to low energy states.


A surface view of what goes on in a photosynthesizing leaf is that energy from sunlight is used to combine carbon dioxide and water to make glucose. However, a deeper view is that this is an electrical process. Energy from sunlight is used to take a low-voltage electron, one slumming around on a molecule of water, and exalt it to an amazingly high potential. Once energized, the electron can be put onto a carbon atom*. This trick is managed by a handful of pigments, including chlorophyll, and a whole mess of protein enzymes.


The point of chlorophyll is to do the first part of photosynthesis: use light energy to give an electron a kick in the pants. Chlorophyll absorbs only certain colors of light. It loves blue and red, can use a little green and infrared, but essentially can’t use any of the other UV or other light energy that hits the earth. Different colors of light have different energies, which is why you will get a nasty burn from UV, but not red light. When chlorophyll absorbs blue light, it wastes a bunch of the energy stepping the light down in energy until it’s essentially the same energy as red light. Only then will it energize an electron, and the remaining energy is wasted as heat.


So here’s one powerful strike against photosynthesis—it only uses a fraction of the solar energy that hits the earth, and it makes inefficient use of most of that fraction. Compare that with a silicon solar cell: in principle, it can make use of any photon from UV through the visible spectrum to far infra-red. Here’s a chart (very loosely adapted from Blankenship et al) showing how many photons of different colors hit the earth:

So, lots of different colors besides the visible ROY G BIV hit the earth. In fact, since a UV photon packs more energy than a visible photon, most of the energy hitting the earth is invisible. How many of these photons—how much of the sun’s energy—can photosynthesis use?

Not so much—it can’t make much use of yellow or green, or any of the UV. How does this compare with a silicon solar cell?

Woah!


The second part of photosynthesis is the synthesis: using a hot-to-trot electron to make glucose. From a casual inspection, this is amazingly efficient—nearly 100% efficient, in that every electron that gets energized finds its way to glucose, without any losses. However, this estimate has to be tempered by biological reality. Unlike solar cells, whose raison d’etre is to make voltage for our use, the point of a plant—a point shaped by billions of years of evolution—is to make another plant. So, this photosynthetic system is not just making glucose for us to burn, it’s making membranes and proteins and pigments and DNA and so on. If we measure efficiency in terms of how much of the original sunlight gets converted into energy we can use, 100% gets whittled down to slightly over 1%.

How does this compare with a silicon solar cell? The best of these converts photon energy into voltage with an efficiency of about 18%. If we want to make an apples-to-apples comparison with a leaf, then we can use our solar cell to electrolyse water and make hydrogen gas. This process has some efficiency losses, so it brings the efficiency of a silicon solar cell down to about 14%.


OK—did you guess right about which was more efficient? I sure didn’t. But, as the authors say, “the efficiency advantage clearly goes to photovoltaic systems.”


So, is silicon really greener than a leaf? Well, yes and no. photosynthesis is an evolved, not a designed system. So, many key elements of photosynthesis were jury-rigged from other parts. And, if you start with a jury-rigged system, there’s going to be severe limits on how much it can be improved. (The authors of this review article use a wonderful euphemism, “legacy biochemistry,” to describe this historical baggage that all living things carry around.) Also, there’s the pesky fact that organisms are interested in making more organisms, not helping us.


However, we now know enough about biology to do a little bio-engineering. We have reached a point where we can contemplate taking an inefficient, evolved system and subjecting it to some intelligent re-design. We can make the components more efficient, and make the system’s main purpose energy production rather than reproduction.


Chlorophyll is a good start. It’s thought to have evolved on earth at a time when other organisms had already figured out a way to use green wavelengths of light for making energy. (These organisms are still around—they give salt ponds their spectacular purple hue. If you take the spectrum of visible light and absorb all the green and a little yellow-orange, as these guys do, you are left with purple.) Therefore, chlorophyll evolved to make use of the leftovers, blue and red. UV and infrared were eschewed because they’re just too dangerous for living things to deal with. Some researchers have been tinkering with modifications to chlorophyll, and have succeeded in making it absorb new wavelengths of light.


The synthesis part of photosynthesis is also subject to tinkering: the enzyme that starts the process of making glucose is notoriously inefficient, since it first evolved on earth when there was a much higher concentration of CO2 in the atmosphere, and virtually no oxygen. In this light, it is unsurprising that this enzyme is really inefficient in the presence of oxygen. Certain plants and bacteria have developed work-arounds for protecting this enzyme from oxygen and locally increasing the concentration of CO2, but it’s easy for us to simply grow algae in a bioreactor that’s kept nearly free of oxygen, and pump in lots of CO2 from burning biomass.


There are even more radical proposals for bio-engineering photosynthesis. These are pretty far in the realm of science fiction, but who knows—they may be used to power your oft-promised flying car. The authors of this review suggest a re-engineered algae, something that could only grow in a bioreactor, a slave to our demands for energy. It would have a short life span, because its engineered chlorophylls would absorb all wavelengths of light. It would not grow especially well, because most of the energy it absorbed would be used for making fuel, rather than making more cells. And, since glucose isn’t the best fuel to power your flying car, it would energize electrons from water and use them to make hydrogen gas. Such a system may not achieve the same efficiency of a silicon cell, but the peripherals (processing, hazardous waste produced, etc) may well make it much greener.


There’s no doubt that, sometime in the next century, big oil will be replaced by something else, and that it will probably be solar. The question is, will it be big silicon or big algae?


Robert E. Blankenship et al (2011). Comparing Photosynthetic and Photovoltaic Efficiencies and Recognizing the Potential for Improvement. Science 322, 805-809.


*a proton also goes along for the ride, and an electron and a proton together make a hydrogen atom—so chemically, it looks like hydrogen is being added to CO2.