Thursday, December 14, 2017

It’s A Plant World, We’re Just Living In It

Biology concepts – cell walls, chloroplasts, myco-heterotrophs, holoparasites,

Life on Earth is easy. It can be boiled down to three sentences. “The mitochondria and the chloroplasts are, in a fundamental sense, the most important things on Earth. Between them, they produce oxygen and arrange for its use. In effect, they run the place.” Lewis Thomas wrote this in his award winning book, The Lives Of The Cell: Notes Of A Biology Watcher, in 1975.

Nature’s carbon recycling center. The sun’s energy is used to 
polymerize carbon (CO2) into carbohydrates (CHO) and releases 
oxygen (O2). Then the mitochondria use the O2 to break down 
the CHO, resulting in chemical energy (ATP) and carbons (CO2
ready to be polymerized again.
He was so right - for the organisms that use them. I guess he didn't consider the exceptions. These two organelles mesh seamlessly in their functions. One produces carbohydrate and oxygen, while consuming carbon dioxide. The other consumes carbohydrate and oxygen and produces carbon dioxide. The ultimate recyclers.

If these two organelles are the most important things for life, then doesn’t that make plants the kings of life on Earth, since they have both chloroplasts and mitochondria? Makes you feel a bit more humble now about your place in world, doesn't it.

However, this brings up an essential question – and the main focus of today’s topic and exceptions. What makes a plant cell a plant cell? Green algae have chloroplasts and mitochondria, but they aren’t plants, they belong to the kingdom Protista. We have discussed the sea slug, E. chlorotica, and its ability to photosynthesize – it is certainly not a plant. So what makes a cell a plant cell?

Leaving the chloroplast out of the equation for a minute, you could argue that a plant cell is one with a cell wall and cell membrane. That surely separates them from animal cells, since animal cells only have the cell membrane. But many bacteria, archaea, fungi, and algae have cell walls. If the argument is refined to define a plant as having a certain kind of cell wall, then we must look a little closer. Many cell walls are made of sugars, but are plant cell walls unique in their constituents?

True bacteria have two large groupings, Gram+ and Gram -,
based on their cell wall structures. The gram stain sticks to
the peptidoglycan layer, so the thick layer on G+ bacteria make
them stain deeply. The lipopolysaccharide (LPS) layer of the G-
species keeps them from staining, and is highly toxic.
Endotoxin (LPS) and causes about 70% of septic shock cases.
Bacteria cell walls are made of peptioglycan (peptido = amino acid containing, and glycan = polymer of two sugars). One of the two components is always N-acetylmuramic acid, and the other is often poly-N-acetylglucosamine, but other things can be included as well. The exception is the Mycoplasma, a group of small bacteria that don’t have a cell wall at all. Since many antibiotics function by disabling the bacterial cell wall or preventing its formation, they don’t work against mycoplasma infections like M. genitalium, which a 2011 study linked to pelvic inflammatory disease in women.

Fungal cell walls are also made of a polysaccharide (poly = many, and saccharide = sugar), in a polymer called chitin. Chitin is also the rigid polymer that makes so many insects crunch when you step on them. Chitin cell walls are defining for fungi, as many cellulose containing cell wall fungi have been moved out of the kingdom of Fungi. But this still doesn’t tell us what is unique to plant cell walls.

Plant cell walls contain cellulose, and is complex. Plant cell walls can contain up to three layers, with different sugars involved, including cellulose, hemicellulose, and pectin, and lignin. Lignin is a more rigid polysaccharide that gives strength. It is what makes bark hard, protective, and water resistant.

If the hydrogens (H) bound to the #1 and #4 carbons
up on the same side, the polymer is starch. If they
are on different sides, the polymer is cellulose.
We can digest starch: we can’t digest cellulose.
Plants make both – the part we can’t digest we call
dietary fiber.
Cellulose is made of a chain of glucoses, yet we can’t digest it. The number one carbon in glucose has an –H that is sticking up or down. If the –H sticks “down”, then it is an alpha glucose. If it sticks “up”, then it is a beta-glucose. Cellulose is linked chains of beta-glucose. Starch is linked chains and branches of alpha-glucose. Just that difference in –H position determines if it is food for us or not. Herbivores have the enzymes (and bacteria) to digest cellulose, but not us.

So is it the inclusion of cellulose that makes a plant cell wall unique? Well, no. Algal cells also use cellulose in their cell walls. You might try to argue that algae are plants, since many of them also have chloroplasts and are primary producers – but you would be wrong. Algae can be unicellular (although they can also be multicellular) while plants are all multicellular. Algae don’t have specialized reproductive cells or parts like plants do; algae reproduce by spore or from broken parts of themselves. Finally, DNA analysis shows that while plants and algae are monophyletic (one ancestor), they diverged from one another long ago.

Then there is the issue that not every plant cell has a cell wall. In angiosperms (angio = chest or vessel, and sperm = seed; plants with enclosed seeds and flowers), the gamete (sex) cells of the male in the pollen and the gamete cells of the female in the ovary do not have cell walls, at least not on all sides. The ovary contains the ovules (latin for small egg), and the pollen contains the sperm cells and the tube cell, that forms the pollen tube and delivers the sperms cells to the ovules.

After the ovules are fertilized by the sperm cells of the pollen, the ovules form the seeds, and the ovary forms the fruit. From here on in, all the daughter cells will have cell walls. For fertilization, it would make sense that the involved cells would not have a cell wall that would just get in the way of love.

The Sago Palm isn’t a palm, but is one of the most
primitive plants that reproduces with seeds. It
presents a problem to pet owners because every part
is toxic to pets, but it tastes good to them. They don’t
know not to eat it; then they bleed to death.
And even weirder, not all plants use just this strategy. Cycads (like the sago palm, which isn’t really a palm at all), and gingko biloba plants have sperm cells with flagella, long projections that whip and move them along, hopefully toward an egg cell. They don’t use a tube cell or pollen tube; these plant cells without cell walls swim. Plant cells that move, now there is an exception worth noting! Some more primitive bryophyte plants (liverworts, mosses) also have motile sperm, but the cycads and gingko are the only examples of seed plants with motile cells.

So cell walls aren’t a defining characteristic of plant cells either. Maybe it is the chloroplast that defines a plant cell --- maybe not.

As you can guess, there are exceptions going both ways. There are organisms that have chloroplasts that aren’t plants, namely the algae. But a more interesting exception are many of the protozoan Euglenids. Euglena gracilis is a prototypical euglenid that can produce carbohydrate by photosynthesis. However, most euglenids can also eat things, which makes them both autotrophic and heterotrophic.

As for the other direction, there are many plants that don’t have chloroplasts. Of the roughly 350,000 different species of plants on earth, almost 3000 of them are non-photosynthetic. Therefore, the most common characteristic that people use to tell a plant from a non-plant (photosynthesis by chlorolplasts) isn’t true for almost 1% of the species on Earth. That is a pretty big exception. That would be like saying 1% of people on earth don’t have a brain! O.K., maybe that's a bad example.

Indian Pipe is Monotropa Uniflora. Monotropa means
one turn, and uniflora means one flower. The plant is
called the ghost plant – obvious, or the corpse plant –
because it turns black as it matures. This naming thing
is easy!
Indian pipe (Montropa uniflora, or ghost plant) is one such plant. Related to the blueberry of all things, the ghost plant has gone its own way and become parasitic. It garners its nutrients and energy from the tissue of another plant. The roots of the Indian pipe penetrate the rhizoids (root-like projections) of certain types of fungi and sponge off their hard work. In fact, the fungi themselves are symbiotic, having invaded the roots of certain pine tree species.

The fungus and tree live together in a mutualistic relationship, making the fungus a mycorrhizal (myco = fungus and rrhizal = root) variety. The tree supplies the fungus with carbohydrate, and fungus supplies the tree with mineral nutrients. However, Indian pipe does not respect this mutualism and is a parasite of the fungus, taking some of the carbohydrate supplied by the tree. This makes the Indian pipe a myco-heterotrophic parasite.

Other plants without chloroplasts are holoparasitic (gain nutrients only by parasitism).  These would include the rafflesia species of the Indonesian rainforests. These plants are know for having the largest single flowers in the world, some the size of car tires! The plant doesn’t have a stem or root or leaf, it is a vine that grows inside another type of vine. Only when it is ready to flower does it bud out from the bark of the host. The flower takes nine moths to develop, and then smells like rotting flesh in order to attract fly pollinators.

Rafflesia is also known as the corpse flower, as opposed
to the corpse plant (Indian pipe). This is because it
smells like a corpse in order to attract the flies that
pollinate it. This young man is either holding his breath,
has no sense of smell, or is just really odd.
In addition to holoparasitic plants, plant cells without chloroplasts would include those same gamete cells we discussed above as not having cell walls. And neither to do most root cells. However, there are exceptions, like many of the orchids. The ghost orchid has photosynthetic roots, which is a good idea, since they grow directly on other plants; their roots are not buried in the dirt.

Maybe it is not a single characteristic that makes a plant cell a plant cell, or a plant a plant. Maybe it is the combination of cells with cell walls, central vacuoles and in most cases, chloroplasts that make it a plant. I guess it is like beauty; you can’t define it, but you know it when you see it.

Next week we will take another shot at finding a defining characteristic of plant cells, namely the plastid, the mother of all chloroplasts – might there be an exception?

Mizukami I, & Gall J (1966). Centriole replication. II. Sperm formation in the fern, Marsilea, and the cycad, Zamia. The Journal of cell biology, 29 (1), 97-111 PMID: 5950730

Nikolov LA, Tomlinson PB, Manickam S, Endress PK, Kramer EM, & Davis CC (2014). Holoparasitic Rafflesiaceae possess the most reduced endophytes and yet give rise to the world's largest flowers. Annals of botany, 114 (2), 233-42 PMID: 24942001

For more information and classroom activities on cell walls or parasitic plants, see:

Cell walls –

Parasitic plants - 

Thursday, December 7, 2017

Many Paths To The Top Of The Mountain

Biology concepts – hydrogenosome, FeS cluster protein, loricifera, erythrocyte

More than one way to skin a cat seems to
be a newer version of the old British saying,
“there are more ways to kill a cat than by
choking it with cream.” Mark Twain was one
of the first to use the cat skinning version, in his
classic A Connecticut Yankee in King Arthur’s
The old Chinese proverb says, “There are many paths to the top of the mountain, but the view is always the same.” Put somewhat less delicately, “There’s more than one way to skin a cat.” Who wants to skin a cat? I think there is something to be said for the wisdom gained in 4000 years of culture, to say nothing of the ability to say it better.

In biology, this is particularly relevant; organisms have found different ways to do the same things, and different ways to do different things, but the end goal is always the same – live long enough to reproduce and the more offspring the better.

Last week we talked about how some organisms have degraded their mitochondria into mitosomes, and how they get along fine just using glycolysis and fermentation for energy (and maybe some arginine dihydrolase action). But there is another mitochondrial remnant in some other species of anaerobic eukaryotes called the hydrogenosome, and it works more like a mitochondrion than does the mitosome.

Here is the T. vaginalis protist. The blue probe
binds to DNA (just one nucleus for this guy) and
the yellow probe binds to a hydrogenosome
protein. The strands at the top are the flagella it
uses to move, not its hair.

Trichonomas vaginalis is a eukaryotic amitochondriate, and therefore is an anaerobic (without oxygen) protozoan. Unlike many protozoans, T. vaginalis does not have an environmentally resistant form (something that can live outside the host for a prolonged time – often called a cyst). It is transmitted directly from host to host, in this case sexually. Trichomoniasis is the most common curable sexually transmitted disease, but 70% of cases have no symptoms (asymptomatic). This is unfortunate because T. vaginalis infection can predispose to HIV infection and even cervical cancer. Having symptoms initially might prevent some of the later tragedies.

Unlike the mitosome containing protists, T. vaginalis does use its mitochondrial remnant (hydrogenosome) to make ATP. The hydrogenosome was discovered much earlier than the mitosome, although they have the same origin and general morphology. Because of this difference in timing, amitochondrial organisms with hydrogenosomes are called type II amitochondriates. Type I’s were the organisms that presumably didn’t have any mitochondrial-like organelle (and were seen first), like the Giardia and E. histolytica that we now know have mitosomes.

Pyruvate generated by glycolysis enters the hydrogenosomes just like it does in mitochondria. The Krebs cycle would be next for aerobic organisms, but in the hydrogenosome, iron-containing enzymes convert the pyruvate into an intermediate that has CoA (coenzyme A) bound to it. When this CoA is removed, energy is released, and this energy is used to convert ADP to ATP.

Because ATP production occurs at the level of substrate (a molecule being chemically changed, in this case by an enzyme), it is called substrate level phosphorylation. This is in contrast to the use of oxygen and the electron transport chain of proteins to produce ATP through the proton gradient (oxidative phosphorylation). One of the byproducts of the pathway is hydrogen, hence the name of the organelle.

In terms of energy production, the pyruvate:ferredoxin oxido-reductase (the iron/sulfate-containing enzyme in hydrogenosomes, often abbreviated as FeS cluster enzymes) pathway is about as efficient as the arginine dihydrolase pathway (ADH) in some mitosome-containing organisms. However, T. vaginalis also contains the ADH pathway, so it comes out ahead of Giardia in terms of energy production.

While the hydrogenosome has some activity in energy production via the FeS-protein mediated metabolism of pyruvate with production of ATP, the mitosome seems to be limited to the assembly of the FeS clusters only. A study of the proteins of the mitosome show the parts are there to make the FeS clusters, but that there are not the enzymes needed to break down pyruvate and produce ATP.

A study trying to quantify the amount of methane
gas produced by cows was carried out recently
in Argentina. The method involved a big backpack
and a delicately placed rubber hose. At some point,
scientist A approached scientist B and said, I’ve
got a great idea….”
Other hydrogenosome-containing organisms include the anaerobic unicellular fungus, Neocallimastix frontalis (it lives in the guts of rumen animals like cows). N. frontalis byproducts are used by gut methanogens (methane-producing bacteria) and therefore contributes to the generous amount of gas produced by cows. Many estimates name dairy and beef cattle flatulence as a bigger source of greenhouse gases than automobiles!

Another hydrogenosome-containing protozoan is Nyctotherus ovalis. It lives in the GI tract of cockroaches, and efficiently works with an archaeal bacterium that uses the hydrogen that the hydrogenosomes release. Just one more reason that cockroaches will outlast us all. The fact that some fungi and some protozoans have hydrogenosomes indicates that this organelle has evolved independently from mitochondria at least three different times in history – they must be a good idea.

Even with the exception of anaerobic protists and fungi, it was believed until just recently that at least all multicellular eukaryotic (metazoan) organisms depended aerobic respiration for energy production. However, there are even metazoan exceptions. A 2010 study of the bottom of the Mediterranean Sea found three different animals that survive without using oxygen and therefore don’t have mitochondria.

The deepest basin of the Med, near Greece, is nearly anoxic (an environment without oxygen).  In the muds of this basin were found three loriciferan (lorici = corsette and fera = bearing, so organisms with a sort of girdle) species that live in this area all the time. Other animals can survive in an anoxic environment for a while, but they don’t call it home.

Loriciferans weren’t even discovered until 1983.
Now we have some that live as anaerobes. Most
species of this phylum live in the deep waters,
but only a few are obligate anaerobes, meaning
they can only perform anaerobic respiration.
Oxygen can be damaging, it likes to scavenge
electrons, I wonder if it is toxic to the loriciferans.
These new loriciferans have hydrogenosomes instead of mitochondria, and produce ATP in the same ways as T. vaginalis and the other anaerobic eukaryotes. This is a completely new door being opened in biology, because the multicellular animals evolved after Earth turned from an anoxic environment to a place where oxygen was plentiful. It seems that even some of the more advanced organisms don’t have a problem reverting to more ancient systems if they find themselves in a place where they need it.

Would you believe that some of your cells might not have mitochondria? Well, about 26 trillion of your cells (if you’re an adult male) are amitochondrial – your red blood cells. That’s right; the erythrocytes that deliver oxygen to your cells in order so they can make ATP in their mitochondria don’t have any mitochondria of their own! In an attempt to carry as much oxygen as possible (bound to a big molecule called hemoglobin) your red blood cells have evicted their mitochondria.

This is probably a good idea, since making energy in the erythrocytes would use up the oxygen they are supposed to deliver to other cells. Instead, they act more like prokaryotes, and carry out glycolysis and lactic acid fermentation in their cytoplasm for the energy they need. To gain more room for hemoglobin, the RBCs have also done away with their nucleus.  They have no way to produce more proteins or repair themselves, so they work as long as they can and then they are replaced.

Old erythrocytes are phagocytosed (eaten) by macrophages in the spleen and liver and are destroyed. New RBCs (about 2 million per second) are produced in your bone marrow. The spleen also acts as a reservoir for blood cells, a ready supply for when you need them, but you can get along without it, you are just more susceptible to infections, since the spleen houses many white blood cells just waiting to recognize a pathogen that needs to be taught a lesson.

Human red blood cells (left) are round and biconcave,
but the camel RBCs are oval. You can see why so many
people believe they have a nucleus, but what you are seeing
is their biconcave side staining darker. The large cell in the
middle is an immune cell.
Anucleate (a = without, and nucleate = pertaining to a nucleus) erythrocytes are the norm for mammals. Many people think that camels are the exception, that they have nucleated RBCs, but this is not so. But they do have ovoid RBCs. When they run low on water, camels can remove water from their blood and use it in their cells. This leaves their blood thicker and harder to push through the small capillaries. Round RBCs would be impossible to squeeze through when the blood is viscous, so the camel has evolved RBCs that are longer in one direction and smaller in the other, to help blood flow in times of dehydration.

On the other hand, almost all non-mammalian vertebrates do have erythrocytes that do have nuclei. The only exceptions are a few salamander species that have some anucleate erythrocytes. For example, 95% of the Batrachoseps attenuatus salamander’s RBCs are anucleate. There is also the pearlside fish which is known to have non-nucleated red blood cells.

However, the crocodile icefish is even a bigger exception; it is the only vertebrate animal that has gotten rid of its RBCs altogether. This species lives in cold, highly oxygenated waters. The oxygen it needs just travels in the blood as a dissolved gas and is carried to every cell. These fish have even lost the DNA for making hemoglobin – now that is efficiency!

Given our apparent complexity, it is amazing
just how few genes humans have; the grape
has almost 30% more. The chicken doesn’t have
many fewer than us, and we don’t have to worry
about laying eggs. What is more amazing is that nine
years after the completion of the human genome
project, we still aren't exactly sure how many
genes we have.
Or is it? We have recently discovered that the majority of proteins have more than one function. Scientists gave this idea more thought when the results of the human genome project started to role in and we discovered far fewer genes than we expected. It is now accepted that humans have about 22,000 genes, not even as many as the grape, which has 31,000. Even the lowly fruit fly has 15,000 genes! How do we get so many functions out of so few gene products? Multitasking!

Take hemoglobin for example, it doesn’t just carry oxygen in the blood. It also acts as an antioxidant in several types of immune cells, and in certain neurons. It is a regulator of iron uptake and metabolism, since it carries iron at its core. It destroys nitric oxide, which is one reason why the little blue pill doesn’t work forever. You have to wonder what else the crocodile icefish has lost by giving up its hemoglobin and how it has made up for these losses. One change probably requires many more to be made as well.

We have seen how some organisms get along without mitochondria. What about the other end of the energy equation? Plants can make their own carbohydrate in the chloroplast – but is that what makes it a plant? Let’s look at this next time.

Roberto Danovaro, Antonio Dell'Anno1, Antonio Pusceddu, Cristina Gambi1, Iben Heiner and Reinhardt Møbjerg, & Kristensen (2010). The first metazoa living in permanently anoxic conditions. BMC Biology DOI: 10.1186/1741-7007-8-30

For more information or classroom activities on hydrogenosome, FeS cluster protein, loricifera, erythrocyte, see:

Hydrogenosome –

FeS cluster protein –

Loricifera –

Erythrocytes –

Thursday, November 30, 2017

A Biological Energy Crisis

Biology concepts –  mitochondria, aerobic respiration, anaerobic respiration, glycolysis, fermentation, mitosome

The bee hummingbird is the smallest bird in the world. 
Living on the 2 largest islands of Cuba, this little 
guy is only 5 cm (1.9 in) long and weighs just a bit more 
than a paperclip. The males and females live in separate
nests and never see each other again after mating.
Birds in flight use an astounding amount of energy, and the smallest birds use the most energy. Hovering hummingbirds must flap their wings 50-80 times a second, which requires a lot of energy. To meet this demand, they use 10x the amount of oxygen that a person uses (per gram of body weight)! To move this much oxygen in their blood when flying, their hearts must beat over 1200 times per minute. At that rate, a red blood cell can traverse the bird’s entire circulatory system in less than one second!

It is a vicious circle; the hummingbird must eat constantly in order to have the energy to hover, and it must hover in order to eat constantly. Hummingbirds convert their carbohydrate intake into cellular energy (ATP) on the fly, using the sugars ingested only a few minutes earlier to support up to 90% of their need. Contrast that to humans; elite athletes can draw only about 15% of their needed energy from the sugars they ate recently. 

So how is all this energy made? Since we have been talking about the mitochondria on and off for several weeks, you would be right to guess that this organelle is involved, but it doesn’t start there.

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This is an extremely simple cartoon of glycolysis.
If you want more detail, like which step calls for
glyceraldehyde phosphate dehydrogenase, then
look here.
Dietary glucose ends up in the cytoplasm after it is eaten and transported through the blood to each and every cell in the body. In the cytoplasm, the sugar is broken down in a process called glycolysis (gly = glucose and lysis = splitting). This process takes the carbohydrate from a six-carbon sugar down to two three-carbon sugars (pyruvate). In the process, there is a net gain of two ATP molecules (four are actually made per glucose but you have to invest 2 ATPs to get the process rolling). That isn’t much of a payoff. There must be something more, and this is where the mitochondria figure in the process.

The pyruvates are taken into the mitochondria and a second process begins to consume them. First there is a carbon and two oxygens removed from each pyruvate to form acetyl-CoA in what is called a linking reaction, since it links glycolysis to the next step - the citric acid cycle (Kreb’s cycle).   In this cycle, a series of reactions takes place to sequentially remove carbons from the sugar, leaving a four-carbon molecule (oxaloacetate) that then joins to the acetyl-CoA produced from another pyruvate. The series of reactions results in 2 ATPs and 6 NADH’s formed. This latter molecule (long name = nicotinomide adenine dinucleotide + hydrogen) will become important in the final step.

Remember that the mitochondrion has two membranes, and the inner membrane is folded into many cristae, in order to increase its surface area. The NADH’s produced during the Krebs cycle work with a series of proteins embedded in the inner mitochondrial membrane (called the electron transport chain) to create a proton gradient.

This is a Goldilocks version of the electron transport chain; the level 
of detail is juust right. Keep an eye out for the NADH, the water, and 
the protons moving in and out. They are important, as is the flow of 
the electron, hence the name; the electron transport chain.
When the NADH is broken down, a hydrogen ion (the same thing as a proton) is pushed into the inner membrane space. This is against its gradient and creates a high-energy situation, since it wants to move back into the matrix (the space inside the inner membrane). The ATP synthase allows the proton to move back in, but uses the energy of the gradient to convert ADP into ATP. One ATP is made for every proton that is pushed out and then allowed back into the matrix by oxidative phosphorylation.

The driving force behind NADH’s release of an electron and a proton (hydrogen atom) is that some atom must be waiting to scoop the extra electron, and this something is oxygen (this is why it is called oxidative phophorylation). This is why we have to breathe, the oxygen is a big magnet (metaphorically speaking) for the electron. The oxygen plus the electron plus two hydrogens bind together to form water. This is the metabolic water that is so important to many animals that don’t drink water

All told, the electron transport chain produces 36 ATP molecules per glucose, much more than the paltry 2 resulting from glycolysis (called substrate level phosphorylation as opposed to oxidative phosphorylation). It is a good thing that hummingbirds have mitochondria to wring so much energy out of their food (not so bad for us either).

And herein lies the exception, some eukaryotes have decided to try to live without mitochondria. It isn’t as though they just never underwent endosymbiosis; recent evidence is showing us that all eukaryotes had mitochondria at some point in their evolution. These exceptional organisms just worked out another way to produce energy, and allowed their mitochondria to disappear or change over time.

The human gut pathogen Giardia intestinalis (or lamblia) is a good example. Look as long as you like, but you won’t find a mitochondrion in this protozoan. Until 2003, scientists hypothesized that the lack of mitochondria in G. lamblia meant that it was a very early eukaryote, diverging from other eukaryotes before the endosymbiotic event that created mitochondria. But, then we discovered it was an even bigger exception.

Meet Giardia intestinalis; he looks happy to see you.
The blue probe binds to DNA, those are the two nuclei.
The green probe binds to the mitosomes. Just like the
duck in A Christmas Story – “it’s smiling at us!”
Instead of mitochondria, Giardia has 2-50 cryptons, also called mitosomes. These are mitochondrial remnant organelles (crypton = cryptic mitochondrion), with no genome of their own. They are completely reduced; all of their DNA has been transferred to the nucleus or lost, so mitosomes do not replicate on their own.

In Giardia, the mitosomes line up and down the sides of the organism’s two nuclei, with some between the nuclei. Yes, you're right -  Giardia doesn’t have any mitcohondria, but it has two nuclei – go figure. This specific and repeated arrangement suggests a specific function for these organellar remants. We aren’t sure what the functions might be, but it is not energy production. G. intestinalis produces its energy by glycolysis and by fermentation – the same process that yeast use to produce alcohol.

In alcohol fermentation of yeast, the 3-carbon pyruvates from glycolysis are converted to 2-carbon ethanol and some NADH is converted back to NAD+. This prevents a critical shortage of NAD+ in the cell. The amount of NAD+ in the cell is limited, so if glycolysis is to continue there must be NAD+ must be recycled from NADH.  The conversion of NADH back to NAD+ is the main purpose behind fermentation; it doesn’t produce any more energy than glycolysis alone.

Notice how fermentation doesn’t make more
ATP than glycolyis alone. In both lactic acid
fermentation and alcohol fermentation change
NADH to NAD+. This is the purpose behind
fermentation. Lots of energy is left on the
table -you can power a car engine on ethanol.
By the way - you ferment too. Yes, you. When oxygen is scarce, mammals will resort to fermentation, we just don’t produce alcohol. Instead, our waste product is lactic acid. In 1929, Nobel laureate Archibald Hill stated that it was the buildup of lactic acid in the muscles that caused muscle soreness after exercise, but his experiment was flawed. It wasn’t until just a few years ago that we discovered that lactic acid is crucial in keeping the muscles working (and brain) working when they are taxed. Lactic acid isn’t the problem, it is part of the solution.

But back to Giardia. Unlike yeast, G. lamblia doesn’t have a choice, it undergoes alcohol fermentation all the time. Make that almost all the time. Without oxygen (even though it doesn’t use it to make ATP) most of the pyruvate is converted to alanine, an amino acid, during fermentation. With even a little bit of oxygen, this switches over to alcohol production.  But there is another way Giardia can make some energy.

A mechanism called the arginine dihydrolase pathway has been seen only in prokaryotes and two eukaryotic anaerobes (Giardia and Trichomonas vaginalis). This speaks to the primitive nature of Giardia; no wonder scientists thought that it didn’t ever have mitochondria, like prokaryotes. In the arginine dihydrolase pathway, a whole bunch of steps lead to a little bit of ATP formation. It must make a difference for the organism’s survival, otherwise they wouldn't invest the energy in maintaining the pathway.

Giardia isn’t the only eukaryote to choose mitosomes over mitochondria. Entamoeba histolytica also causes diarrhea when it takes up residence in your gastrointestinal tract. I think this suggests that we are providing them with all the carbohydrates they need so that glycolysis and fermentation pay off. Was there less diarrhea before twinkies and french fires? Could be – there is probably grant money available for that study.

Entamoeba histolytica and Giardia intestinalis
are not closely related, they are very different
types of protozoa. For instance, Giardia is a
flagellete (moves by flagella), but E. histolytica
is an amoeboid (moves by body movement).
But they both cause diarrhea, and Giardia has
two nuclei and E. histolytica has four!
E. histolytica was also thought to be an ancient eukaryote that never had a mitochondrion, but mitosomes were discovered in this pathogen way back in 1999; the good old days. Another pathogen, Cryptosporidium parvum is also a mitosome-containing amitochondriate. Again, this is an intestinal parasite that causes diarrhea. I think that living in the gut must have turned these organisms into mutants, like the 1950’s animals exposed to radiation in great old movies like Them! and Godzilla.

C. parvum is closely related to the organism that causes malaria (Plasmodium falciparum), but they make ATP in different ways. P. falciparum  has mitochondria and can carry out oxidative phosphorylation via the electron transport chain. So how can they be related?

Here’s how: P. falciparum might have mitochondria, but they look like they are on their way out. They only have a few genes, and at least one principal enzyme is completely missing. In one stage of the infection, Plasmodium survives only by fermentation (although it goes to lactate, not alcohol), so maybe these two parasites are not so different after all. They have another similarity, but we will talk about that in a couple of weeks when we discuss plants without chloroplasts.

Fermentation is one way eukaryotic organisms get along without mitochondria, but there are many paths to the top of the mountain. Next time we will look at organisms that found another path.

Makiuchi T, & Nozaki T (2014). Highly divergent mitochondrion-related organelles in anaerobic parasitic protozoa. Biochimie, 100, 3-17 PMID: 24316280

Raj D, Ghosh E, Mukherjee AK, Nozaki T, & Ganguly S (2014). Differential gene expression in Giardia lamblia under oxidative stress: significance in eukaryotic evolution. Gene, 535 (2), 131-9 PMID: 24321693

For more information or classroom activities on glycolysis, oxidative phosphorylation, or fermentation, see:

Glycolysis –

oxidative phosphorylation –

fermentation –

Thursday, November 23, 2017

Life Outside The Chromosome

Biology concepts – plasmid, linear organelle genomes, extrachromosomal circular DNAs, conjugation,

Planet of the Apes (1968) – a good movie, but not a great movie.
Every ape was a ventriloquist; you never saw their lips move.
But it did have the first reciprocal interspecies kiss. The pan and
scan version loses the, see no evil, hear no evil, speak no evil joke;
you only see what is in the red box.
I love older movies, but only if shown in full aspect (wide screen or letterbox format). So much of old cinema had interesting things going on outside the field of focus.  Take Charlton Heston testifying before the panel of apes in Planet of the Apes. In the pan and scan version, you see one ape covering his ears when he doesn’t like what Heston is saying, but you miss the other two apes – one is covering his eyes and one is covering his mouth! You only get the joke in wide screen.

Biology can be the same. So much emphasis is placed on chromosomal DNA that we sometimes miss interesting things going on elsewhere, or we start to investigate years later than we might have if we would just look at the whole picture.

Last week we focused on the big DNA in prokaryotes, the chromosome(s). But this doesn’t mean prokaryotes don’t have other DNA. Most prokaryotes have extrachromosomal DNA in the form of plasmids (plasma = shape, and id = belonging to). These are smaller loops of DNA that have fewer genes than a chromosome, and the genes are not essential for survival.

However, "smaller than chromosomes" doesn't mean they have to be small. The "megaplasmids" are over 100,000 nucleotides, and can be more than 2 million nucleotides in length, but even these are smaller than the chromosome. The exception might be in bacteria that have multiple chromosomes. Often one chromosome is much smaller; a megaplasmid could be larger than the secondary chromosome.

Plasmids replicate on their own, so sometimes they are called autonomously replicating elements. As such, they do not depend on the chromosome for their existence. Plasmids have internal control features that keep the number of a certain plasmid within limits in any one bacterium. Some plasmids have other controls that keep certain plasmid types from surviving in cells that have other types of plasmids. But this doesn’t mean that a cell may have only one type of plasmid. Our lyme disease-causing example of last week, B. burgdorferi, has 21 different plasmids. What is more, some are linear and some are circular. It just can’t help but be an exception in all things molecular.

The plasmid is different from the chromosome. It is
smaller and is not tethered to the cell membrane.
New data is showing that eukaryotes also possess
plasmids, especially yeast. They are being used to
produce complicated proteins in a system more
like our own cell
Even though plasmids do not carry genes essential for survival, they can still have an influence on the life of the cell. For instance, most antibacterial resistance genes are carried on plasmids. These extrachromosomal elements can be transferred from bacterium to bacterium, and can be passed on to the daughter cells, producing populations of bacteria that can laugh at our puny efforts to kill them.

Plasmids may also transfer metabolic genes, allowing the recipient cell to degrade other sources of food, or virulence genes, allowing them to colonize different portions of the body. This is sometimes what happens with E. coli.  Species that live in the large bowel pick up a plasmid that codes for a system that lets them cling to the wall of the small intestine, higher in the gastrointestinal tract. Having them live here can cause diarrhea in several different ways, but it all depends on the presence or absence of  that plasmid.

One type of plasmid, called the F plasmid, has a role in bacterial sex determination. O.K., it isn’t like the sexes we think usually think of; bacteria with the F plasmid are considered F+ or “male” and those without are considered F- or “female.” The F plasmid codes for proteins that will create a tube (pilus) that can link one bacterium to another and permit the replicated F plasmid to be transferred to the F- cell, thereby turning a female in to a male. Tada – sex change the easy way.

The F plasmid contains tra genes that build the pilus
and control the integration of the DNA into the
chromosome. Helicase, the enzyme that unwinds
DNA for replication or insertion, was first identified
in the F plasmid.
Most of the time this is not such a big deal, but sometimes the F plasmid sequences can integrate into the chromosome of the bacterium, and when it cuts itself back out and becomes circular again, it may bring piece of the chromosome as well. This is now a F’ plasmid. When the F’ gets transferred to a F- cell, it takes those chromosomal sequences with it. This is one important source of genetic diversity in bacteria, called conjugation.

Plasmids are an integral part of the prokaryotic genome, so I have never considered them exceptions. What is more, you and I both know that there are circular DNAs in eukaryotic cells. Remember that the mitochondrion and chloroplast have their own chromosomes, although significantly reduced from what they had when captured by our ancestor cells underwent endosymbiosis.

Since the organelles were derived from prokaryotes, it would follow that their DNA is kept in a single, circular chromosome. In most cases this is true, but there are those organisms that demonstrate linear organelle DNA or multiple chromosomes in their organelles.

For example, the human blood sucking louse Pediculus humanus doesn’t have a single mitochondrial chromosome. Its 34 remaining mitochondrial genes are housed on 18 separate minichromosomes. Why ? – IDK (with a nod to my texting children). Even stranger, the fungus Candida parapsilosis has a linear mitochondrial genome, while its very close relative, the human pathogen C. albicans, has a conventional mitochondrial genome geometry.

The moon jellyfish is a cnidarian. Cnidarins are named
for cnidocytes, the stingers that allow them to defend
themselves or catch food. However, the sea turtle is
immune to the toxin of the moon jelly, so they are
happy with jellyfish sandwiches, like on SpongeBob.
Many other examples of linear organelle chromosomes exist, especially in the cnidarians (animals like corals and jellyfish). The relationships between these groups, phylogenetically speaking, have been hard to work out. The evidence that the hydrozoans (like the fire coral and the Portugese man-o-war) and scyphozoans (like moon jellyfish) have linear mitochondrial genomes indicate that they are probably closely related to each other and are younger than the other groups of cnidarians, like anthozoans (most corals and sea anemones).

Finally, corn (maize, species name Zea mays) cells have been show to have linear, complex, and circular forms of the chloroplast genome. In seedlings, the areas of high cellular division seem to be more active in the linear copies of the chloroplast chromosome. This may indicate that while the circular form is still present, it is the linear form that is functional in the Z. mays cells. Maybe we are catching a peak at evolution in action.

Most prokaryotes have circular chromosomes, and most eukaryotic species have organelles with circular chromosomes. It would follow that the instances of linearization of mitochondrial or chloroplasts sequences occurred after endosymbiosis was established, but why? What is their advantage? What would the text abbreviation be for “nobody knows?”

The above examples indicate that extrachromosomal DNA in eukaryotes can be more dynamic than previously surmised. But we haven’t touched on the interesting part. Eukaryotic linear chromosomes can sometimes give rise to circular pieces of DNA that then replicate on their own and stick around for varying lengths of time, just like plasmids.

Probably for reasons of "species prejudice" we don’t use the term plasmid for circular DNA in higher organisms; it makes us sound too similar to our prokaryotic ancestors. Circular DNA in plants and animals is called extrachromosomal circular DNA (eccDNA) or small poly-dispersed circular DNA (spcDNA) – and the scientists are right, these sound much more advanced: a plasmid that a eukaryote can be proud of.

The sources of these eccDNA sequences are several. They can be formed from non-coding DNA (sequences that don’t lead to the production of a particular RNA or protein), or they can be derived from tandem repeat (two copies of the same gene) DNA that are plentiful in the eukaryotic genome. A June, 2012 study identified a new type of eccDNA in mice and humans that actually has coding sequences that are non-repetitive.

eccDNA has been found in every species in which it has been looked for, so its presence is not unusual. What is unusual is that eccDNA can come and go, and can be formed from normal intrachromosomal recombination (the crossing over of sequences within one chromosome) or by the looping out of sequences from a chromosome and then being cut out. As of now, we don’t know what controls their occurrence or why they form.

Importantly, they do seem to have a function. Small numbers are seen in normal cells, but the number is increased in cancer cells or normal cells that have been exposed to cancer-causing or DNA-damaging agents. This was first demonstrated using a cancer cell line called HeLa, named for the mother from whom they were isolated, Henrietta Lacks. I highly recommend the biography of her tumor cells called, The Immortal Life of Henrietta Lacks, authored by Rebecca Skloot.

Xenopus laevis is a good model organism for
Studying development. Notice how the tadpole
Only takes 3 days to develop into a tadpole, and
every stage can be visualized. Plus, they can lay
up to 2500 eggs at a time.
The function of eccDNA in normal tissues is suggested by a study in Xenopus laevis, the African clawed frog. This animal is a much used model for studies of development because the eggs and embryos are big, the frogs can be induced to mate year round, and the embryos develop outside the body.

During development of the embryo, different levels of eccDNA are seen. Some sequences are seen early, while different sequences are seen later, and most of the eccDNA is gone by the time the embryos mature to tadpoles. This suggests specific functions for eccDNA in normal development. We wish we knew what the specific functions are – again, your opportunity for a Nobel Prize. 

The type of eccDNA in X. laevis is called a t-loop circle. The “t” stands for telomeres, like we mentioned last week. Telomeres have many units of a repeated sequence and are used to help replicate the ends of linear chromosomes. We have talked about how each replication of the chromosome leads to a slightly shorter telomere and how some scientists hypothesize that telomere shortening has something to do with aging defects.

Early in development, embryonic cells are dividing rapidly; in the 4-week human embryo, new cells are produced at a rate of 1 million/second! All this cell division requires replication, and replication shortens the telomeres. Could it be that the t-loop circle eccDNA has a function in preserving telomere length?

The telomere has many copies of a repeat sequence. Each repeat 
is recognized by an enzyme that helps to replicate that end of 
the chromosome. The enzyme called telomerase contains 
an RNA primer that can’t be converted to DNA, so the last
repeat is always lost. The telomere gets shorter with every 
replication. Sooner or later, this is going to cause a problem.

A study in 2002 suggested just that, these eccDNA telomere sequences might serve as a reserve of long telomeric sequences. These repeats could later be added back on to the telomeres through recombination events, thus preserving telomere length despite high levels of chromosome replication.

One the other hand, eccDNA is more plentiful in ageing cells and damaged cells. This might be an attempt to save the cell from the defects induced by telomere shortening or by damaging agents, or it may have a completely different function, perhaps even to induce cell suicide (apoptosis), so as to prevent damage to other cells. Once again, the small DNAs that are so easy to ignore may very well be the ones that allow us to live.

We have talked directly and indirectly about the mitochondria for the past few weeks; a crucial structure for energy production. Next time lets talk about the organisms that think they can do without this organelle.

Shibata, Y., Kumar, P., Layer, R., Willcox, S., Gagan, J., Griffith, J., & Dutta, A. (2012). Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues Science, 336 (6077), 82-86 DOI: 10.1126/science.1213307

For additional information or classroom activities about plasmids, extrachromosomal DNA, or telomeres, see:

Plasmids –

Extrachromosomal DNA –

Telomeres -