Breaking the Size Barrier – Giant Bacteria, part 1

If you double the size of a cell in each direction, the volume 

increases eight fold. This makes take eight times longer 

for a molecule to diffuse through the whole cell.

In the last post we talked about how the reactions that must take place inside cells often limit the maximum size of bacteria. Because important molecules can reach every part of the bacterial cell only by diffusion, the organism can’t have too large a volume. At the same time, the bacterium needs as much surface area as possible for important molecules to diffuse into the cell. This means that they need a high surface area: volume ratio. We showed last time that if you double (2x) the length of a bacterium in three directions, then the volume is increased eight fold (8x). This would result in cubing (2³=8) the mixing rate and traffic time as well. If the size of a bacterium was increased from a typical size of 1 µm to a theoretical 100 µm bacteria, it could take almost a day for two molecules to find one another (traffic time). It wouldn’t seem plausible that bacteria this size could remain alive.

HOWEVER, I want to show you two bacteria that have found ways around this size limitation. Even more impressive (and a sign of how inventive nature can be), each of these organisms has found a different way to beat the system. Our two examples are the two largest prokaryotes known, and can be seen by the naked eye. This is really something considering that we can’t see our own cells without a microscope.

Our first size offender is called Thiomargarita namibiensis (T. namibiensis). The thio- part of the name means that this is a sulfur oxidizing bacterium, while the last part of its name records that it was first found on the ocean floor just off the coast of the African country, Namibia. Sulfur bacteria change elemental sulfur (S₀) into sulfur oxides (SO_2-4). These reactions release enough energy to make ATP (the chemical energy of the cell). In order to carry out these oxidation reactions, some sulfur bacteria use nitrate as an electron acceptor during ATP production. This works out just fine when there is a lot of nitrogen present in the immediate environment, but at the bottom of the ocean this is not always the case. Most of the nitrogen comes within reach of the bacterium only after a storm disturbs the ocean floor.

Our “sulfur pearl of Namibia” bacterium (arrow) is as big 

as the head of the fruit fly. To compare, each 

eye of the fruit fly contains over 16,000 cells!

Therefore, T. namibiensis must scavenge as much nitrogen as possible and store it within a large central vacuole (a membrane bound sac) for the lean times. It also stores sulfur in smaller granules, leading to a speckled pearl-like appearance over the clear nitrogen vacuole (which explains the middle part of name, margarita = pearl. Often, these bacteria stick together in a line and look like a string of pearls).

T. namibiensis is a spherical bacterium. Round cells are least well equipped for good mixing and traffic times; the center is far from any cell surface. But if the cell was flattened out or narrow in one dimension the traffic times could be reduced, even if the organism was larger. For this reason, many bacteria are not round, but perhaps rod-shaped or flattened rhomboids. Here we see that T. namibiensis is huge (up to 750 µm) while still spherical. That size makes it just about the size of the period at the end of this sentence; not much compared to a beach ball, but 3 million times the volume of a typical spherical bacterium.

T. namibiensis usually occurs in chains of ten or so bacteria, with pearlescent sulfur granules as shown in the left image. In cross-section on the left, you can see both the thin band of cytoplasm and the large nitrogen-containing vacuole.

The first key to Thiomargarita’s size is that large central vacuole of nitrogen. As shown in the righthand photomicrograph (courtesy Woods Hole Oceanographic Institute), there is only a thin layer of cytoplasm (the essential, viscous, water-based medium that fills the cell) between the vacuole and the cell membrane. The vacuole itself consumes almost 98% of the total cell volume. This small layer of cytoplasm means that all the important molecules are close to the surface through which they diffuse; therefore, the large size of the cell does not violate any limitations placed on its mixing rates or traffic times. While the size of the bacterium is huge, the distance any one molecule has to travel is still small. In fact, the amount of cytoplasm in T. namibiensis is just about the same as in a normal sized bacterium.

The large diameter of T. namibiensis also helps it survive in two ways that are less evident. One advantage has to do with the diffusive boundary layer. Because of the natural friction between all molecules, there is always an area next to any surface where the flow of liquid is reduced to near zero. Reduced flow means reduced numbers of important molecules can be picked and carried; therefore, the concentration of important molecules is reduced, a bad thing for bacteria trying to survive. However, because of the huge size of T. namibiensis, much of the cell sticks up above the sea floor’s diffusive boundary layer, into the area where diffusion can be more productive.

The second survival advantage is slightly more straightforward. T. namibienisis and other megabacteria are just too big to be bothered by predators. T. namibiensis doesn’t have to worry about being eaten, because no bacterial predator is big enough to “swallow” it. This is similar to the ancient sauropod species, like Brachiosuarus or Diplodicus, which had no predators once they grew to adult

Just like a T. Rex couldn’t bring down or swallow

a brachiosaur, a normal bacterium (the white dot

in the top right hand corner) can’t eat T. namibiensis.

size – a healthy sense of self-preservation would keep any T. Rex from trying to eat an adult brachiosaur.

We have seen that limitations on bacterial size imposed by diffusion can be overcome if natural selection results in some advantageous characteristic and if there is a reproductive advantage to be being big. The development of a central vacuole permitted T. namibiensis to become bigger, and being bigger provided an advantage for survival on the sea floor. It seemed designed to end up just so, but remember that evolution is not purposeful. It is merely a series of random changes and random environmental changes that render some characteristic advantageous, disadvantageous, or moot.

Next time we will look at another giant bacterium. This second rule-breaker has a completely different solution to the diffusion/size limitation. Just as we highlighted with the nylon metabolizing bacteria a few weeks ago, nature can find an infinite number of ways to overcome a single problem. It just takes random mutation (a change), environmental pressure (a need for the change) and time (for the reproductive advantage afforded by the change to have an effect on the population).

For more information on surface area: volume, sulfur bacteria, and T. namibiensis, please see below:


Cell surface:volume laboratories:
http://www.oocities.org/capecanaveral/Hall/1410/lab-B-24.html
www.nnin.org/doc/SurfaceVolumeRatioB_TG.pdf
http://illuminations.nctm.org/LessonDetail.aspx?id=L609
http://www.neiljohan.com/projects/biology/sa-vol.htm


sulfur bacteria:
http://www.moldbacteria.com/bacteria_testing.html
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Eubacteria.html
http://filebox.vt.edu/users/chagedor/biol_4684/Microbes/greensul.html
http://bmb-it-services.bmb.psu.edu/bryant/lab/Project/GSB/index.html
http://m.biotecharticles.com/Biology-Article/Green-and-Purple-Sulfur-Bacteria-705.html
http://filebox.vt.edu/users/chagedor/biol_4684/Microbes/purprnb.html


Thiomargarita:
http://web.mst.edu/~microbio/BIO221_2005/T_namibiensis.htm
http://www.sciencenews.org/sn_arc99/4_17_99/fob5.htm

When Evolution Goes Sideways – Sea slug hybrids, part 3

This is the third and final installment in our story of photosynthesis by an animal. E. chlorotica, a simple sea slug, has proven to be not so simple. We have seen that it is an exception to the "animals as heterotrophs" rule, as well as an exception to the "horizontal gene transfer is an activity of unicellular organisms" rule. Can we learn more from this mollusk? There must be more, otherwise the post would end right here.

Horizontal gene transfer has occurred between E. chlorotica and its algae food, V. litorea, with gain of function for photosynthesis by E. chlorotica. This is an exception to the current idea of how evolution works. More than 50 years before Darwin’s theory of natural selection, there was another natural philosopher (the term “scientist” didn’t gain popularity until the 1850’s) who had his own ideas about inheritance.

According to Lamarck, giraffes acquired their longer neck, then passed it on.

Jean Baptiste Lamarck was a retired French soldier who became a famous zoologist and botanist; he coined the terms "biologist" and "invertebrate" among other accomplishments. In the late 1700’s and early 1800’s, Lamarck suggested inheritance followed strict natural rules. His idea was called "soft inheritance" and was the first comprehensive theory of evolution. Lamarckism, or inheritance of acquired characteristics proposes that new or changed features that an organism acquired during its life would be passed on to its offspring. His classic example was the giraffe. The food was high in trees, so the giraffe strained its neck to reach the leaves, and its neck got longer. Therefore, its offspring were born with longer necks. There are two main features to Lamarckism: 1- mature organisms can change their characteristics permanently; and 2- these acquired changes are passed on to progeny.

Then came Charles Darwin. His voyage on the Beagle took place from 1831-1836 and he formulated his theory soon after that, but he found it hard to reconcile his religious beliefs with his scientific findings and theories. Therefore, he did not publish his work until 1859, when he learned that another scientist, Alfred Russell Wallace, had come to the same conclusion and was about to publish. After years of public debate and testing the theory, Darwin’s and Wallace’s theory of natural selection as the instrument of evolution won out over Lamarckism, and is now widely accepted.

The principle behind natural selection is that changes in organism characteristics are always occurring, but when changes in the environment result in a reproductive advantage for the organism that has a inherited a certain new characteristic, then that characteristic will be passed on to the next generation at a higher rate (the organism with that change is more likely to survive long enough to mate and produce offspring – if you don’t survive to mate, your genes aren’t passed on – duh!).

A prototypical example of natural selection and evolution is nylon eating bacteria. Nylon wasn’t invented until the 1940’s, so the ability to feed on it must be new as well. In the 1970’s, a strain of Flavobacterium was found producing enzymes to digest nylon. Even more amazing were the results of multiple experiments carried out with a non-nylon digesting bacterium, Pseudomonas aeruginosa. When this bug was placed in an environment where nylon was its only possible food source, it took only a few generations before every bacterium on the plate contained enzymes to metabolize nylon, and these were not the same enzymes as found in the Flavobacterium. It happened in trial after trial, showing that life can adapt to whatever the environment offers. There are many other concrete examples of natural selection in the scientific literature – it is one of the most solidly supported theories in all of science.

In an ironic twist of fate, scientists are now helping bacteria evolve the ability to process garbage into adipic acid, one of the two major chemicals in nylon. Man invented nylon, nylon-eating bacteria taught us about evolution, we used this knowledge to evolve bacteria to make nylon!






We know that natural selection takes place, causing species to diverge, converge, and move along ever changing paths. We have seen it take place and can follow the clues it has left behind. However, horizontal gene transfer has made following those paths more difficult. Using molecular methods, scientists have ways of tracking the rate of change in DNA over time. With this data they can put together family trees and charts (cladograms) to show the points at which different species diverged from one another. But if a certain organism can pick up one or more new traits in one horizontal transfer, it makes patterns for small changes harder to use in developing the evolutionary cladograms. Is a new trait the result of mutation and natural selection or horizontal gene transfer?

A simple cladogram for apes and hominids is on the left. It shows evolutionary relationships between species. The right cladogram shows things can be confusing when horizontal gene transfer (crossing arrows) takes place.




Other features have entered the debate on the nature of Darwinian evolution as well. Does natural selection proceed slowly, with very small changes over time adding up to a measurable change in some characteristic or species (called gradualism), or does a species remain stagnant for a long time, until a mutation brings some rapid, even instantaneous, change (called punctuated equilibrium)?

Gradualism has been the prevailing theory for decades now, but examples of horizontal gene transfer would argue for punctuated equilibrium, especially if it is occurring in higher eukaryotes. Instead of many small changes adding up to a measurable difference over time, horizontally transferred genes bring the potential for big changes from the time of transfer down through all subsequent generations.

Is the retention and function of chloroplasts in E. chlorotica an example of punctuated equilibrium? Definitely not - the instantaneous gain of photosynthetic ability is a sudden change, but remember, the chloroplasts themselves are not passed on to the next generation – this is not an example of horizontal gene transfer in itself. But the fact that photosynthesis-specific genes are found in the nucleus of the slug is evidence that horizontal gene transfer has taken place and that they are passed on vertically. This would argue for punctuated equilibrium over gradualism. So, could this be an exception to the commonly held idea of evolution?

What is more, the combination of heritable plant genes AND acquisition of photosynthetic ability in an already living individual smells a lot like evolution by acquired characteristic – Lamarckism! There is a change in the individual (kleptoplasty of chloroplasts) and this acquired ability is passed on to future generations (via germ line inheritance of the photosynthetic genes). That seems like a more complex, two-step version of Lamarck’s giraffes.

If true, a blow will be dealt to neo-Darwinian evolutionary theory - but not a fatal blow. Darwin himself acknowledged that a mechanism of acquired characteristic inheritance or loss of disused characteristics might exist. He called it pangenesis. His idea was deeply flawed in its proposed mechanism; Darwin thought that cells would throw off small particles (pangenes) that would carry the essence of the change throughout the body and pass the characteristic to the next generation. It sounds silly to us now, but remember that changes in environment can initiate communication between cells that are far apart, and can affect DNA function. This is called epigenetics, and we may have an opportunity to talk about it in the future. However, in the case at hand, it might turn out that Darwin was right to hedge his bet against Lamarck.

It would be nice if the story were this simple – O.K., it’s already not simple – but there has been a catch. Even though the genes for some photosynthesis elements are present in the slug’s nucleus, scientists had not been able to show that they were doing anything. Logic says they must be, since the chloroplasts are functional for so long. One could track this is based on the fact that DNA genes are converted to RNA messages before being translated into protein. If the nuclear genes are making proteins, you should be able to see their RNA transcripts - but scientists went a long time without seeing the transcripts. They surmised that the transcripts were short-lived or present in low amounts that couldn't be detected.

All this speculation has been put to rest by recent evidence from Case Western Reserve University. Investigators in late 2011 studied the transcriptosome of E. chlorotica. Whereas the genome is the sum of all genes present in an organism, a transcriptosome represents all the mRNAs present in an cell or organism, but only at a specific point in time or under a specific set of conditions.

This is real difference between genomes and transcriptosomes; genomes are basically the same in all somatic cells under all conditions. But different cell types need different gene products, and reacting to different conditions will call for changes in which genes are transcribed to mRNAs and then translated into proteins. Therefore, the transcriptosome will be different for different cells and at different times

The scientists in the Case Western study isolated mRNA from whole E. chlorotica organisms afaer they had been starved for 2 months and then exposed to sunlight for two hours. Using starved animals ensured that the dose of sunlight would stimulate expression of as many photosynthesis genes as possible.

In all 111 chloroplast transcripts from 52 different genes were identified, many encoded by the stolen cholorplasts, but many others that represent nuclear genes - once found only in the nucleus of the E. chlorotica's algal food, but now found in the nucleus of the sea slug.

This is the first direct evidence of functional lateral gene transfer in a kleptoplastic organism - and think, they only had to study a mere 98,238,204 separate sequences and 8.9 billion nucleotides of code in order to make this discover. This suggests that the copy number and the transcription rate of laterally transferred genes are low, but who cares. We now have proof of horizontal gene transfer  and the production of a true plant/animal hybrid!



Pierce, S., Fang, X., Schwartz, J., Jiang, X., Zhao, W., Curtis, N., Kocot, K., Yang, B., & Wang, J. (2011). Transcriptomic Evidence for the Expression of Horizontally Transferred Algal Nuclear Genes in the Photosynthetic Sea Slug, Elysia chlorotica Molecular Biology and Evolution, 29 (6), 1545-1556 DOI: 10.1093/molbev/msr316


For more information on Jean Baptiste Lamarck, Charles Darwin, gradualism and punctuated equilibrium or pangenesis, as well as web-based activities and experiments, go to:

Jean Baptiste Lamarck –

www.victorianweb.org/science/lamarck1.html
www.ucmp.berkeley.edu/history/lamarck.html
www.lamarck.cnrs.fr/?lang=en
http://necsi.edu/projects/evolution/lamarck/intro./lamarck_intro.html
www.britannica.com/blogs/2009/02/the-rebirth-of-lamarckism-the-rise-of-epigenetics/
http://videosift.com/video/Darwinism-vs-Lamarckism-cute-short-about-evolution
www.indiana.edu/~ensiweb/lessons/lam.darw.html
www.sciencenetlinks.com/lessons.php?DocID=387
www.liftminds.com/lesson/60/Mechanisms_of_evolution_lamarckism_principles
www.clas.ufl.edu/users/ufhatch/NSF-PLANS/4-1_ORIGIN.htm
http://thinkwell.mindbites.com/lesson/3915-biology-contrasting-lamarck-and-darwin
www.imarksweb.net/book/lamarck+vs++darwin+worksheet/
www.iteachbio.com/Life%20Science/Evolution/Earlyevoltheory.doc

Charles Darwin –
www.aboutdarwin.com/
www.blupete.com/Literature/Biographies/Science/Darwin.htm
www.darwinday.org/
www.pbs.org/wgbh/evolution/educators/lessons/lesson2/act1.html
http://teacher.scholastic.com/activities/explorations/adaptation/
www.tes.co.uk/teaching-resource/Charles-Darwin-Cartoon-Strip-activity-6077894/
http://peer.tamu.edu/curriculum_modules/ecosystems/module_1/activity.htm
www.biologyinmotion.com/evol/index.html
http://serendip.brynmawr.edu/sci_edu/waldron/
http://science.pppst.com/evolution.html
http://ats.doit.wisc.edu/biology/ev/ns/ns.htm
www.accessexcellence.org/AE/AEC/AEF/1995/wartski_natural.php
www.mhhe.com/biosci/genbio/virtual_labs/BL_12/BL_12.html

Gradualism and punctuated equilibrium –
http://necsi.edu/projects/evolution/evolution/grad+punct/evolution_grad+punct.html
www.blackwellpublishing.com/ridley/a-z/Phyletic_gradualism.asp
www.talkorigins.org/faqs/punc-eq.html
www.indiana.edu/~ensiweb/lessons/peek.html
www.youtube.com/watch?v=Islx7y62I6M
www.zartistry.net/pgvspe/index.html
www.lessonplanet.com/lesson-plans/punctuated-equilibrium
www.truthinscience.org.uk/tis2/custom/Fossils%20Lesson%20Plan.pdf
http://evolution.berkeley.edu/evosite/evo101/VIIA1bPunctuated.shtml

Pangenesis -
www.answersingenesis.org/home/area/cfol/ch2-pangenesis.asp
www.neoteny.org/2009/12/04/gemmules/
www.chemistrydaily.com/chemistry/Pangenesis
www.softchalk.com/lessonchallenge09/lesson/genetics/an_introduction_to_Mendelian_genetics_print.html