Fish Guts and Cancer – Giant Bacteria, part 2

The gut of a fish is a strange place to go looking for bacteria. It’s an even stranger place to find the second largest bacterium on Earth.

Epulopiscium fishelsoni (E. fishelsoni) hangs out in the intestinal tract of the brown surgeonfish, commonly called the lavender tang. While it seems logical that E. fishelsoni would be named for the site where it was found – inside a fish – it was actually named for its discoverer, Lev Fishelson of Tel Aviv University.  

Epulopiscium fishelsoni is shown in the left image. The white line is approximately 100 µm. On the right is the Lavender Tang.  E. fishelsoni lives in this fish’s gut, and only in this fish’s gut.

Before T. namibiensis (last week’s post) was discovered, E. fishelsoni was the biggest kid on the block, having been first seen in 1985. It can be seen with the naked eye, reaching a maximum length of 0.7 mm, but it also has large size variations. In fact, this is one of the keys to its success.

E. fishelsoni’s changing size is a daily routine. In the early morning, E. fishelsoni is only about 10 µm long, only 2-5 times bigger than typical bacteria. As the surgeonfish starts to feed, more food is available to the bacteria in its gut. With this signal, E. fishelsoni starts to grow. By late afternoon into evening, the maximum size has been reached and they can be seen with the unaided eye (if you happened to be in the fish’s gut to see it – I wouldn’t recommend it as a holiday destination).

However, after the night passes, you would find just the small cells again in the morning. You would also see that the number of bacteria has increased.  The large cells have divided into daughter cells, splitting their cellular contents between their two or three new partners. Then, as a new day passes and food becomes available in the gut, these cells grow large and divide overnight. 

Could you imagine having your baby grow 75x bigger in one day?

Think of it this way: you bring home your 22-inch long newborn baby in the morning and place it in its crib.  That night, you find that you have a baby that is 140 ft. tall. You start to build the world’s largest crib, but by morning, the giant is gone and you find two 22-inch babies in the crib. It would continue like this everyday. Parenting is difficult.

E. fishelsoni’s shape is also different than that of T. namibiensis. E. fishelsoni is shaped like a long grain of rice, as opposed to the spherical T. namibinesis. This can help meet diffusion needs (see this post), since the distance to travel is much shorter for molecules brought in on its long sides. The elongated shape is enough to make the new daughter cells viable. But as the cells grow during the day, merely being longer than they are wide isn’t enough to overcome diffusion rate, mixing rate, and traffic time limits. E. fishelsoni must know another trick in order to survive at is maximum size.

In the majority of molecular interactions, it is a cellular protein that partners with a molecule that has diffused into the cell. What might E. fishelsoni do to increase the chance that an enzyme will find its substrate (the molecule an enzyme acts on and changes in some way) quickly?

Remember in the “It’s all in the Numbers” post, we saw that one way to reduce traffic time was to increase the number of one or the other interacting molecule. It is impossible for the bacterium to raise the concentration of nutrients, but it can raise the number of proteins made by the bacterium.

The central dogma of molecular

biology.

We need a bit of background to help explain E. fishelsoni’s trick to producing more copies of its proteins. There is a central dogma (core belief) to cell molecular biology: DNA goes to RNA goes to protein. This means that DNA is transcribed to a message (mRNA), which is then translated into a protein. However, if you want to make more protein, you can’t just transcribe more RNA from the DNA in the cell. This process is highly regulated and can only be manipulated to a certain degree. The other problem with this solution is that the proteins would be produced near the site of the DNA, so these extra proteins would have to travel a long distance to mix through the entire cell – this wouldn’t solve the mixing time (diffusion) problem.

What if the cell made more copies of its DNA and spread them out through the cell? Then the cell could produce much more RNA and hence much more protein. Having the DNA spread throughout the entire bacterium would solve the mixing time problem.

Fold number of chromosomes is a cell’s ploidy. 

N= haploid number of chromosomes, N in humans = 23, 

but we are diploid, so the total number of 

chromosomes is 2 x 23 = 46.

How would a bacterium make more copies of its entire DNA (its genome)? Isn’t the number of copies of DNA determined and unchangeable? In general, bacteria are haploid, meaning that they have one copy of each (usually just one) chromosome. Human cells (except for sex cells) are diploid, meaning they have two copies of each chromosome (one from Ma and one from Pa). Some plants exhibit triploidy, especially the seedless varieties of fruit, like bananas and watermelons. Finally, while polyploid cells (poly = many and ploid = fold) can occur naturally in lower animals and some plants, in humans it is often associated with cancer cells. The more copies of the genome there are in a cancer cell, the worse the prognosis (predictable outcome) for the patient.

E. fishelsoni has found a way to make being polyploid work for it. The early morning version of the bacterium (the small cell) is haploid, but as the cell volume increases hour by hour, the amount of bacterial DNA also increases through the circadian cycle (the daily sequence of physiological events).

Green color in inset shows the huge amount of DNA dispersed throughout  

E. fishelsoni. Courtesy of: Ward, R.J., Clements, K.D., Choat, J,.H. 

and Angert, E.R..  2009.  Cytology of terminally differentiated  

Epulopiscium mother cells.  DNA and Cell Biology 28:  57-64.

By evening, the mega-E. fishelsoni has 85,000 copies of its genome! Scientists don’t have a -ploidy name for a number that big; just plain polyploid. This is a huge amount of DNA for a prokaryotic cell, and is 25% more DNA than contained in a human cell.  The new DNA copies are spread throughout the cytoplasm to provide thousands of local protein factories. Wherever there is a diffused nutrient, the proper protein it needs to interact with won’t be too far away. Therefore, E. fishelsoni can disregard the usual size limitations placed on it by diffusion.

This bacterium still has much to teach us; for instance, I wondered about all that extra DNA. If there are 85,000 copies in the parent cell, but the two or three daughters that result from it are haploid (1 copy/daughter cell), what happened to the other 84,997 or 84,998 copies of the genome? I asked Dr. Fishelson about this, and he said, “there are several questions concerning this enigmatic bacterium, one of which is what you are asking about - what is the fate of the ‘surplus DNA’ as the daughter cells are produced?” If we figure out how E. fishelsoni gets rid of its extra DNA, we could take advantage of the process. Wouldn’t it be something if we learned how to beat cancer by studying a bacterium in the gut of a fish?




Bresler V, Montgomery WL, Fishelson L, Pollak PE. (1998). Gigantism in a bacterium, Epulopiscium fishelsoni, correlates with complex patterns in arrangement, quantity, and segregation of DNA J Bacteriol., 180 (21), 5061-5611 : 9791108


  For more information and activities on ploidy, central dogma, see below:

Ploidy –

http://www.bedfordorchids.com/ploidy.htm

www.cytopathology.org/website/navdispatch.asp?navitemid=1034

http://www.prostate-cancer.com/prostate-cancer-treatment-overview/overview-dna.html

http://wps.prenhall.com/esm_freeman_biosci_1/7/1951/499632.cw/index.html

http://www.biology.iastate.edu/Courses/211L/Pteroph/FernIndx

www.hartwick.edu/Prebuilt/BiolJBR3_Giusto.pdf

Central dogma –

http://homepage.mac.com/hjconklin/ConkWare/CD_index.html

http://employees.csbsju.edu/hjakubowski/classes/chem%20and%20society/cent_dogma/olcentdogma.html

http://www.accessexcellence.org/RC/VL/GG/central.php

http://cnx.org/content/m11415/latest/

http://www.youtube.com/watch?v=-ygpqVr7_xs

http://www.nature.com/news/2011/110519/full/news.2011.304.html

http://www.cbs.dtu.dk/staff/dave/DNA_CenDog.html

http://www.indiana.edu/~ensiweb/connections/genetics/dna.les.html

http://mvhigh.net/jnevis/central_dogma_activity.htm

It’s All in the Numbers - Sizes in Nature

If all the animal species are broken up into groups, the light 

blue section includes insects, and the rest of the 

circle colors represent every other animal on Earth!

Comparisons help to make very big or very small numbers meaningful, and biology is chock full of big and small numbers. For instance, there are more insects in the world than there are humans. By more, I mean ALOT MORE, something like 1.5 x 10¹⁸ insects. But what does that number mean? Consider looking at it this way; the world population hit 7 billion last year and that's a big number, but even if we were to double our population again in the next ten minutes, there would still be 100 million insects for every human on earth. This certainly makes an impression, but it seems small when comparing the most numerous organisms, bacteria, to humans.

Bacteria outnumber us by orders of magnitude more than insects do; they live everywhere, in every environment. They have been found in 0.5 million year-old permafrost as well as 40 miles up in the atmosphere. There are approximately 100 million to 1 billion bacteria in every teaspoon of dirt, so in total there are currently 5 x10³⁰ bacteria carrying out their daily routines. That means there are about 5 x 10¹⁹ living bacteria (that is 50,000,000,000,000,000,000) for every person who has EVER LIVED. Another way of visualizing this might be to imagine that each bacterium is a penny being stacked. The column would be a trillion light years high. That’s about five times the diameter of the observable universe.

Nanobacteria are still controversial, the 0.2 µm diameter is 

close to the smallest size that could still hold DNA. 

For comparison, the white line in panel A is 1 µm long, 

and in Panel C the line is just 0.1 µm.

While the redwoods might be slightly taller than the sequoias, 

the mass of the sequoias is much greater because the 

trunks have such a large diameter.

Even using comparisons and analogies, these numbers are almost too big to comprehend. It isn’t much easier when talking about sizes. The scale of life is amazing, from the smallest bacteria (called nanobacteria), just 0.2 µm in size (1/5,000,000 of a meter), to the biggest living thing on Earth, a Giant Sequoia called General Sherman. This behemoth of a tree is more than 83 meters (272 ft.) in height and 1,225,000 kilograms (2,701,000 lb.) in mass. This means that from smallest to largest, life spans more than eight orders of magnitude. In terms of biomass, the difference between the smallest bacterium and General Sherman is even greater, about 1 x 10²³, about the same as difference in mass as one human compared to seven Earths.

On a smaller scale, the difference in size between bacteria and nucleated cells (eukaryotic cells) is still pretty stunning. A single macrophage cell of your immune system can ingest more than 100 bacteria without flinching, and macrophages are nowhere near the biggest eukaryotic cells. These different sizes demand some distinctions in how cells conduct their business; for example, how they move molecules into and within themselves.

A macrophage reaching out and ingesting bacteria.

The bacteria are the small, connected rods.

Eukaryotic cells, unlike prokaryotic cells (bacteria and Archaea), have specialized systems, like actin filaments, cytoskeleton, and microtubules. These apparatus are designed to act like conveyor belts; they carry different molecules through the cell to their needed destinations. Eukaryotes also have specific receptors for bringing in specific molecules. These are fast systems of uptake and movement, and can work against a concentration gradient.

The cytoskeleton of the eukaryotic cell stretch out like fibers.

They help it move, can convey molecules from place to place,

and holds the cells shape.

Unfortunately, bacteria only have diffusion to move molecules around their insides. This makes things doubly hard on them because bacteria have limited access to resources; most often they meet up with few molecules that are important to them (being a small cell in a big environment). Therefore, they need to get as many of these resources into their cell as possible and move throughout their entire volume quickly.

Diffusion is the movement of molecules from places where there a lot of them toward places here there are fewer of them (from high concentration to low concentration). Think of a crowd pouring out onto the football field after a big win. You start with many people in the stands and very few on the field, but end up with about an equal number of people in all parts of the stadium. Bacteria count on consuming their nutrients this way. Important molecules diffuse into the cell, and then get metabolized for energy or other building blocks. This breaking down and reassembly of molecules helps ensure that the concentration of important molecules is always lower inside the cell, so diffusion into the cell can continue. Importantly, as the width or length of a cell doubles, the volume increases by a factor of eight; therefore, prokaryotic cells remain small so that they can get molecules everywhere they need them quickly. It is the only way for diffusion to remain profitable for them.

Diffusion is the movement of from where there are 

many to where there are few. If it is water 

molecules that are moving, then call it osmosis.

Diffusion is not quite as simple as people pouring out the stands. There are several aspects of this process that are important to bacteria. The first of these is the diffusion rate, which is based on a diffusion coefficient for each different molecule, and the liquid it is moving through. For oxygen moving through water, the diffusion rate is about 1 mm/hr. This means that for an average sized bacteria it only takes 1 millisecond (1/1000th of a second) for an oxygen molecule to travel across the entire cell.

There is also the mixing rate; this refers to the time it takes for a molecule that enters the cell to have an equal probability of being found in any part of the cell. A 1µm (1/1,000,000 of a meter) bacterium has a mixing time of roughly 1 millisecond. But since the volume increases by a factor of eight as the size doubles, it would not take much growth for the mixing time to become problematic if a cell was to rely on diffusion alone.

Finally, there is the issue of traffic time. Every reaction that takes place in a cell involves two or more molecules finding one another and then interacting. In both prokaryotic and eukaryotic cells there are some systems designed to help bring molecules together, but in the end, it is basically luck – they have to run into one another. The number of molecules can affect this time; say you want molecule A to meet molecule B. If the cell contained only one of each molecule, this could take a while, but if there are 1000A’s and 1000B’s, then the traffic time will be decreased considerably. For average sized bacteria, traffic times exist in the range of 1 second, but again, if they are much bigger, the chances of molecules meeting their partners goes down dramatically.

If the bacterium grows too big, the diffusion rate, mixing time, and traffic time can become too long to permit survival. Therefore, size limitations seem to be set for bacteria. However, some bacteria just have to be rule breakers. There are two excellent examples of bacteria that have evolved ways to overcome the diffusion problems associated with increased size, and we'll start to look at them next week.



Schulz, H., & Jørgensen, B. (2001). Big Bacteria Annual Review of Microbiology, 55 (1), 105-137 DOI: 10.1146/annurev.micro.55.1.105



For more information on numbers in nature, diffusion, and cytoskeleton, as well as web-based activities and experiments, go to:

Cell size and volume:
http://staff.jccc.net/pdecell/cells/cellsize.html
faculty.massasoit.mass.edu/whanna/121_assets/15-week_2_prelab.pdf
http://www.youtube.com/watch?v=qdvKM1m0jnE
http://www.cellsalive.com/howbig.htm
www.nsa.gov/academia/_files/collected_learning/high.../surface_area.pdf
www.smccd.net/accounts/bucher/modules/DuzSizeMatter.pdf
http://www.accessexcellence.org/AE/AEC/AEF/1996/deaver_cell.php


scaling in nature:
http://www.nature.com/scitable/content/the-sizes-of-organisms-span-21-orders-15321100
http://learn.genetics.utah.edu/content/begin/cells/scale/
http://www.dnatube.com/video/596/Size-Analogies-of-Bacteria-and-Viruses
http://www.smithsonianeducation.org/educators/lesson_plans/size_shapes_animals/index.html


diffusion:
http://www.biologycorner.com/bio1/diffusion.html
http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__how_diffusion_works.html
http://staff.jccc.net/pdecell/cells/diffusion.html
http://hyperphysics.phy-astr.gsu.edu/hbase/kinetic/diffus.html
http://www.wisc-online.com/objects/ViewObject.aspx?ID=ap1903
http://www.biologycorner.com/2009/09/16/diffusion-lab/
http://chem.lapeer.org/Bio1Docs/Diffusion.html
http://www.biologyjunction.com/osmosis__diffusion_in_egg_lab.htm
http://phet.colorado.edu/en/contributions/view/3415


cytoskeleton:
http://www.cellsalive.com/cells/cytoskel.htm
http://www.youtube.com/watch?v=5rqbmLiSkpk
http://www.biochemweb.org/cytoskeleton.shtml
http://www.biology.arizona.edu/cell_bio/tutorials/cytoskeleton/page1.html
http://www.biology.arizona.edu/cell_bio/tutorials/cytoskeleton/main.html
http://www.youtube.com/watch?v=zlYyoi5vpE8