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 -

Thursday, November 16, 2017

On Geometry And Genomes

Biology concepts – linear chromosomes, circular chromosomes, taxonomy, replication, telomere

Organization is helpful in learning and work,
and apparently in crafts. But there is a fine
line between organization and obsessive
compulsive disorder.
Everyone (teenagers excepted) knows that getting organized helps you to learn and work. When you group tasks, items, or facts, it helps in remembering or working with them. In biology, grouping organisms has a history as old as language.

In the older grouping systems, the name of an organism was a phrase that described some characteristic of the organism. When a new relative was identified, the name phrase had to be lengthened to separate this new organism from those similar to it. As you can imagine, the names got very long very fast.

In the 1750’s, Carolus Linnaeus developed a much easier system of naming. In his “trivial system,” each organism had two descriptors in its name; a binary naming system. Linnaeus’ system (and others) of taxonomy (taxis is Greek for “arrangement”) is based on shared characteristics.

Carolus Linnaeus (he let me call him Carl) had many
names. His knighthood name was Carl von Linne, his
born name was Carl Nilsson Linnaeus. In his naming
system Linne came up with the name mammal, so I guess
he named himself again.
At first, it was the characteristics people could see that were used to group organisms. Then it was the characteristics on the macroscopic and the microscopic levels. Now it is based on molecular characteristics, forming both a taxonomic classification and an evolutionary tree; this is now called the science of phylogenetics.

Molecular characteristics usually mean DNA. Differences in DNA sequence and in the number of mutations that have occurred provide a relationship between organisms. Using these factors, a time line for their divergence can be estimated. We changed the ways we determine similarity, and that changed the rules. With new rules come new exceptions.

Many of the DNA rules start with chromosomes (chromo = color and soma = body, this comes from the dark and light banding pattern of stained DNA). Cellular DNA is very long and very thin, perhaps only 12-22 nanometers wide (about 1/5000 the width of a human hair). In this form, it can only be seen with an electron microscope.

In eukaryotes, this DNA becomes complexed with many proteins during cell division so that all the DNA can be packed up and moved more easily to the daughter cells.  Called chromosomal packaging, the DNA is wound around proteins called histones, then folded many times over, so that the finished chromosome is packed 10,000 times more compact than the original DNA helix. This is the packed DNA that we see as dark and light bands and gives it its name.

DNA packaging with proteins is a eukaryotic characteristic, unless 
I find an exception! The DNA wraps around the histones, then the 
histones line up into a coil, then the coils fold up into the
chromatid. Total packing – about 10,000 fold; it takes a piece of 
DNA 1.5 cm long and makes it 0.0000002 cm long!
By definition, a chromosome is a piece of DNA that contains genes that are essential for the survival and function of the organism. This implies that there may be other pieces of DNA that contain genes that are not necessary for survival.

The molecular rules of biology state that prokaryotes have one chromosome, a single piece of double stranded DNA that contains all the genes that the prokaryote (archaea or bacteria) needs. This is efficient for the organism; it is one stop shopping for replication of all its instructions and only two chromosomes (after replication) need to be segregated to the two daughter cells that are being made.

And here begins our exceptions. There are several prokaryotic organisms that have more than one chromosome. That is to say, their essential genes are located on more than one piece of DNA.

The first identified example of multiple chromosomes in a prokaryote was Rhodobacter sphaeroides, a photosynthetic species of true bacteria that can also break down carbohydrates it takes up. This bacterium was found to have two chromosomes, although one was more than three times the size of the other.

Genes encoding essential products for making proteins and carrying out day-to-day functions are located on each of the two R. sphaeroides circular chromosomes. There are other genes that exist on both of the chromosomes, but appear to be turned on and off via different signals. This implies that the same gene may serve its function at different times in the organism's life, or under different environmental conditions.

R. sphaeroides is by no means the only prokaryote that possesses multiple chromosomes. More than a dozen different groups of bacteria have at least some members with more than one chromosome. This includes Vibrio cholerae, the causative organism of the disease cholera. V. cholerae is responsible for a diarrheal infection that affects more than 3-5 million people per year and causes 130,000 deaths each year.

This is a crown gall in a birch tree caused by R. radiobacter.
Like in cancer tumor in animal tissues, a gallis unregulated 
growth. In grape vines, it has been responsible for the ruin 
of entire Kentucky vineyards. Kentucky makes wine?
In addition to these organisms there is Agrobacterium tumefaciens, whose name was recently changed to Rhizobium radiobacter. This is a very interesting two chromosome bacterium. It usually is a pathogen of plants, forming galls (tumors) on several cash crops, such as nut trees and grape vines. This is an important tool in the molecular biologist’s toolbox, since it has been found that R. radiobacter easily transfers DNA between itself and the plants it infects, via lateral gene transfer (a subject we have discussed in depth, When Amazing Isn’t Enough and Evolution of Cooperation). But R. radiobacter goes further, it can also cause disease in humans who have poorly functioning immune systems. For folks battling cancers, HIV, or other diseases that wreak havoc with their ability to fight off infections, R. radiobacter can cause bacteremia (bacteria colonizing the blood) or endopthalmitis (infection of the two hollow cavities of the eye).

The second molecular rule of biology is that prokaryotic chromosomes take the shape of a circle; the DNA forms a single loop. This shape is helpful in terms of replicating the prokaryotic chromosome prior to cell division. Start anywhere, and you can keep going to replicate the entire thing.  In point of fact, they don’t start just anywhere, but one start point (called an origin of replication) leads to complete replication.

There are advantages to having a circular chromosome. Prokaryotic chromosomes do not complex with proteins to become more densely packed, so it remains as a thin, long molecule. This means that fewer proteins are needed to maintain a circular, prokaryotic chromosome. In addition, since replication requires the doubling of just one piece of DNA from one origin of replication, this takes less time and fewer proteins to accomplish. Together, these features of a circular chromosome result in a more efficient and simpler process, with fewer chances for mistakes to be made.

Borrelia burgdorferi, a spirochete (spiral) bacterium was
Named for the researcher who discovered, it in 1982, Willy
Burgdorfer. It is one of the few pathogens that can function
without iron; it uses manganese instead. The ways this bug
gets around the rules is astounding.
However, there are exceptions in which prokaryotes have linear chromosomes. The Borrelia burgdorferi bacterium has a single chromosome, but it has the geometry of eukaryotic chromosomes, a line segment with two ends. This was the first prokaryote found to have a linear genome, way back in 1989. This lyme disease pathogen has one major linear chromosome and other pieces of smaller DNA that are circular or linear (which we will discuss in the next post); you just can’t trust a pathogen to follow the rules. Other prokaryotes that have linear chromosomes include our friend R. radiobacter. Even more interesting, while this pathogen has two chromosomes; one is circular and one is linear. How does that happen?

The previous discussions do not mean that all prokaryotes with multiple chromosomes or linear chromosomes are disease-causing agents, just the interesting ones. Since they cause pathology in animals or crops, they hit us in the wallet. It makes sense that we have studied them in more detail and have discovered their hidden exceptions. There are probably thousands of innocuous prokaryotes that have more than one chromosome or have linear chromosomes, we just don’t have a reason to look at them in that much detail.

There may be more than one way that prokaryotes end up with linear chromosomes. In some cases, the linear chromosomes still have bacterial origins of replication, indicating that they may have evolved from circular chromosomes. There is also evidence that some linear chromosomes might have developed from other linear DNAs in the cell, something we will talk about next time.

The rules of defining prokaryotes and eukaryotes also state that eukaryotes have linear chromosomes. The essential genes are stored on more than one piece of DNA, and these pieces have two ends apiece, like a line segment in geometry.

Linear chromosomes are a disadvantage because it is hard to replicate the ends. Because of the way that DNA replicates, the ends of the chromosomes, called telomeres, end up being shortened every time the DNA is replicated. Over time, this leads to shorter chromosomes that might lose DNA sequences that the cell needs in order to function.

Some lines of evidence suggest that telomere shortening is a direct cause of ageing. The loss of important sequences at the ends of chromosomes cases cells to perform at less than optimal levels, and mistakes and toxic products then build up and lead to larger dysfunctions of cells, organs, and systems, ie. getting old.

This is a very simple cartoon depicting recombination. When
sequences are exchanged, it isn’t necessarily a 1:1 exchange.
Sometimes parts of genes are sent one way but not the other,
So new genetic sequences can result. Some help, some hurt, and
some have no effect until the environmental conditions show
them for what they are. Most exchanges do not increase diversity
to any great degree, but the fact that some do has helped move
evolution along.
On the other hand, linear chromosomes may promote genetic diversity. In eukaryotes, the division of the cell requires each chromosome to be replicated, then the matching chromosomes of a pair (one from mom and one from dad) line up together. This is a prime opportunity for the chromosome to exchange some sequences in a process called homologous recombination; a mixing of genes beyond just getting one from each parent.

However, a study published in 2010 indicates that the geometry of the chromosome doesn’t matter when it comes to recombination rates. Scientists took a circular chromosome organism and linearized its genome (they cut it so it had ends). They also did the reverse experiment, taking a linear chromosome organism and circularizing its DNA.

In both cases, there was no change in the rate that its DNA recombined and produced slightly different offspring (the two circular chromosomes after replication can swap some pieces). So geometry does not appear to affect genetic diversity – so why did each type evolve? Good question – that can be your Nobel Prize project.

Next week we will continue the discussion of exceptions in DNA structures, including DNA that isn’t part of a chromosome, and mitochondrial and chloroplast genomes that don’t look like they should.

Casjens SR, Mongodin EF, Qiu WG, Luft BJ, Schutzer SE, Gilcrease EB, Huang WM, Vujadinovic M, Aron JK, Vargas LC, Freeman S, Radune D, Weidman JF, Dimitrov GI, Khouri HM, Sosa JE, Halpin RA, Dunn JJ, & Fraser CM (2012). Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids. PloS one, 7 (3) PMID: 22432010

Ramírez-Bahena MH, Vial L, Lassalle F, Diel B, Chapulliot D, Daubin V, Nesme X, & Muller D (2014). Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid. Molecular phylogenetics and evolution, 73, 202-7 PMID: 24440816

Suwanto A, & Kaplan S (1989). Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. Journal of bacteriology, 171 (11), 5850-9 PMID: 2808300

For more information or classroom activities on prokaryotic chromosomes or eukaryotic chromosomes, see:

Prokaryotic chromosomes –

Eukaryotic chromosomes –

Thursday, November 9, 2017

The Evolution Of Cooperation

Biology concepts – biological timeline, serial endosymbiosis, endocystosis, evolution

Taxonomy, the placing of species in different
groups based on their characteristics, changes
everyday – literally everyday – organisms are
placed in different groups and groups are created
and eliminated. That better be a temporary tattoo!
If we look at the 3.5 billion year history of life on Earth, we see that out planet was lifeless for almost a quarter of its span, and animals have been around just a short blip of time, a mere 760 million years. Often, it seems that the big numbers to get in the way of understanding the time line as a whole.

If we treat the entire history of earth as one year, we might get a clearer picture. Earth coalesces from space dust on January 1st, but it isn’t until March 22nd that we find the first evidence of life. These most primitive fossils are of the prokaryotes called Archaea (Greek for “ancient”). Not long after this, maybe a week or so, the eubacteria and Archaea separate from one another.

Then we have to wait until August 7th to find a big change; the first eukaryotic organisms are seen. These represent a fundamental change in the organisms, having nuclei and membrane bound organelles. It's amazing that we must travel 3/4 through our one year time line before we see a cell that looks somewhat like ours!

Here is one of the Namibia sponge fossils recently
discovered in Africa. It represents the oldest animal
in the fossil record. Just how that was recognized as a
fossil is beyond me – I think I have six of those in my
Later in the year, around October 30th at noon, we see the first animals. Fossils of Namibia sponges in Africa were first reported in February of 2012. This fossils are 100 million years older than the previously oldest animal remains, so our new data means that animals have been around for an additional week in our time line of a year.

Insects appear about Nov. 26th, while mammals first show up around Dec. 8th. The dinosaurs became extinct sometime in the afternoon of Dec. 26th, so they had very little time to play with their Christmas presents. Homo sapiens (us) didn’t appear on the doorstep looking for holiday cheer until 11:40 pm on New Years Eve, Dec. 31st!

Our time line analogy shows us that prokaryotes are the wise old ancestors; we aren’t even old enough to be rebellious teenagers, although we still think we know everything. The key question is: how did we progress to analogy-makers from single celled Archaea? If we put together several of the topics we have been discussing in the past three weeks, we may come up with an interesting step in the process. Our clues include:

1) Microcompartments exist in bacteria, like organelles, and they also exist in eukaryotic cells, especially in nucleus' function. This links eukaryotes to prokaryotes.

2) Sometimes cells will engulf objects, parts of other cells, or other cells. Depending on the size of the particle or cell, we may call this endocytosis or phagocytosis, and is similar to how we saw keratinocytes take up melanosomes.

3) Three eukaryotic organelles, the nucleus, the mitochondria, and the chloroplast have double membranes, and they each have their own DNA.

4) There are two different types of prokaryotes, archaea and bacteria.

Bacterial microcompartments give prokaryotes some compartmentalization in order to carry out necessary chemical reactions. Eukaryotes also have some prokaryotic microcompartment remnants, like the nuclear vault complex. This shows crossover between prokaryotes and eukaryotes, and gives us clues about eukaryotic origins. In fact, the currently accepted theory about the evolution of organelles - the very thing that makes cells eukaryotic - has to do with both types of prokaryotes - archaea and bacteria.

There are three types of endocytosis (with exceptions).
Endocystosis of large objects and cells is called phagocytosis.
Internalization of very small molecules and fluid is called
pinocytosis. Other molecules of various sizes have specific
receptors that recognize them on the cell surface. They are
brought in by receptor-mediated endocytosis. Notice that no
matter what method is used, the internalized particle ends up
surrounded by part of the cell membrane.
The key to their interrelationship has to do with endocytosis (endo = into, cyto = cell). Most prokaryotic and eukaryotic cells eat other cells; they do it all the time – it is how heterotrophic organisms (those that can't make their own carbohydrates, ie. non-plants) gain their nutrients. We do it too, just on a larger scale; we eat millions of cells at a time; often these millions of cells can take the shape of a steak or a carrot.

When a cell, protein, other molecule is engulfed by another cell, it is wrapped in a portion of the aggressor cell’s membrane. The naked molecule is now contained in a vesicle, a membrane bound sac, like the melanosome. If the endocytosed material is an entire cell, something that has its own membrane, then it ends up with two membranes, just like the mitochondrion, chloroplast, and nucleus.

Most often, when one prokaryote phagocytoses another, the story is over….gulp, yum, digest. But scientists believe that long ago (sometime in the first week of August in our time line) an endocytosed cell did not go gentle into that good night. Instead, it took up residence in the cell that ate it. In this rare case, it turned out that both cells gained from the situation.

The endocytosed cell was protected from other predators and had a ready supply of nutrients from the parent cell. The captured cell made lots of ATP, but it didn’t need much because it was being supplied with everything it needed; it didn't need to make energy to move or hunt or escape. Most of its ATP production went unused. Perhaps it moved this excess ATP out into the parent cell. So the parent cell gained a source of ATP production. This was mutualism, a type of symbiosis in which both parties benefit.

Clownfish clean the sea anemone and keep it
parasite free. The poisonous anemone provides
a safe environment for the clown fish; no
unwanted house guests! This is a good example of
mutualistic symbiosis. Bet you didn’t know you
learned things from Finding Nemo.
Imagine if the same thing happened with a cyanobacterium, a cell that could perform photosynthesis. The same sort of symbiosis might be set up, with the endocystosed cell providing carbohydrates and the parent cell providing protection.

Now imagine that these captured cells, the photosynthesizer and the ATP maker, replicated themselves inside their parent cells just as they would if they were outside, living on their own. They could easily do this since they still retained their own DNA and cell division mechanisms.

This is in fact what scientists believe happened. The endocytosed cells that produced extra ATP evolved into our mitochondria. Endocytosed cells that could do photosynthesis became the chloroplasts of plants. Not all cells are plants because not all cells with an ancestral mitochondria also ate a cyanobacterium. The fact that plants cells have mitochondria as well as chloroplasts tells us that plant cells developed AFTER cells with mitochondrial ancestors.

But the nucleus may be a tougher nut to crack. It may be that an endocytosed cell good at keeping DNA safe and producing ribosomes became the nucleus, by endocytosis. The data suggests that our DNA is closer to archaeal DNA than bacterial DNA, so it would have been a eubacteria endocytosing an archaea. Or perhaps the archaea invaded the bacterium rather than being endocytosed. The nucleus does have a double membrane and uses some prokaryotic microcompartments to this day, so this could make sense.

But other theories also exist, including one that says an intermediate eukaryotic cell, theoretically called a chronocyte, had developed some organelles on its own or by endocytosis, including a cytoskeleton. This internal structure allowed the cell become bigger, and engulf a cell large enough to evolve into the nucleus.

Another theory uses an evolutionary exception as its basis. Some aquatic bacteria, called planctomycetes (planktos = drifting and mycete = fungus-like), have an organized interior, with something that looks like a nucleus with pores, called a nucleoid. In fact, when they were first discovered, planctomycetes were mistaken for small fungal cells. However, we know they are prokaryotes by DNA sequencing. I thought prokaryotes didn’t have nuclei! Remember that in biology, there is almost always an exception. The planctomycete nucleoid structure suggests that the nucleus may have evolved on its own, without endocytosis.

The planctomycete species, Pirellula (latin for small pear),
is an exceptional bacterium. It has a primitive nucleus
and a stalk that makes it look like a eukaryotic
fungal cell. It was misidentified for a long time, and is
a prime example of why the tattoo above was a bad
Finally, another theory posits that the nucleus originated from a virus infecting a primitive prokaryote, and this internalized virus forming a nucleus or causing the cell to be predated by another cell. Even though there are different theories for the nucleus, we can see that the three organelles that have double membranes look like they could have been endocytosed cells, that then evolved into the organelles we see today. Endocytosis resulted in symbiosis, so the theory of organelle development is called endosymbiosis.

Endosymbiosis is a cool idea and has lots of support. Besides the double membrane evidence, lets look at how dividing cells get more mitochondria and chloroplasts. These organelles replicate on their own by binary fission, just like bacteria. They can replicate on their own because they have their own DNA. Mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) are smaller pieces of DNA than nuclear chromosomes, mtDNA and chDNA look much like the small genomes of bacteria. They are also circular pieces of DNA, not linear like our nuclear chromosomes.

By replicating through binary fission, they can be portioned in the dividing cell so that each daughter gets some of these crucial organelles. But it isn’t as if mitochondria and chloroplasts of today look just like the engulfed ancestors. Mitochondrial and chloroplast genomes are greatly reduced from what they used to be.

Serial endocytosis is also called secondary (2˚) endocytosis.
This refers to the movement of DNA from internalized
cells to the nucleus of the endocytosing cell by lateral
gene transfer. This strengthens the symbiotic relationship
between the two organisms until they can be considered
one total organism.
The mitochondria only codes for about thirteen proteins, just enough for it to replicate on its own. The DNA that codes for the rest of the 1500 or so proteins needed for mitochondrial function have been transferred to the nucleus over time. For a discussion of the chloroplast and its horizontal gene transfer to the nucleus, see the posts on C. litorea, the photosynthetic sea slug.

We know that these gene transfers were actual events based on the structure and nucleotide ordering of the mitochondrial and photosynthetic sequences in the eukaryotic chromosomes; they are structured and coded in ways that are typically bacterial. Because of this slow transfer of DNA to the nucleus, endosymbiosis has evolved over time, changing again and again until we got today’s organelles. Therefore, our idea of organelle development is sometimes called serial endosymbiosis theory (SET), because it must have had several different changes through evolution.

Now that we have laid out the evidence and sense for the serial endosymbiosis theory, next week we can talk about some exceptions that show us that that some organisms just can't stick with something that seems to work. Some life just has to take the road less traveled.

Okie JG, Smith VH, & Martin-Cereceda M (2016). Major evolutionary transitions of life, metabolic scaling and the number and size of mitochondria and chloroplasts. Proceedings. Biological sciences / The Royal Society, 283 (1831) PMID: 27194700

Kostygov AY, Dobáková E, Grybchuk-Ieremenko A, Váhala D, Maslov DA, Votýpka J, Lukeš J, & Yurchenko V (2016). Novel Trypanosomatid-Bacterium Association: Evolution of Endosymbiosis in Action. mBio, 7 (2) PMID: 26980834

Erbilgin O, McDonald KL, & Kerfeld CA (2014). Characterization of a planctomycetal organelle: a novel bacterial microcompartment for the aerobic degradation of plant saccharides. Applied and environmental microbiology, 80 (7), 2193-205 PMID: 24487526

For more information or classroom activities on history of life time lines, endocytosis,  serial endosymbiosis theory, evolution of eukaryotes, or planctomycetes, see:

History of life on Earth timelines -

Endocytosis –

Serial endosymbiosis theory –

Evolution of eukaryotes –

Planctomycetes –