Breaking down the wall: Illustration of the cellulosome as an impressive bacterial machine to degrade plants…think Legos

Components of a major cellulosome of Clostridium thermocellum

I have heard of the cellulosome for quite some time. It discribes a extracellular ‘factory’ of enzymes some bacteria (or fungi) are equipped with to degrade the components of the plant cell wall. These enzymes are held by a scaffold structure projecting out of the cell. Several bacterial species to date are known to encode some sort of cellulosome. Here, I will focus on a model species, Clostridium thermocellum.  I never really thought twice about cellulosomes until recently when researching for an upcoming project. Now, however, I have a great appreciation and respect for this massive, impressive apparatus.

The backbone of sorts for the cellulosome is the scaffoldin CipA. CipA is a monstrous protein with many domains, most of which necessary to attach the enzymes needed to break down plant cell walls. CipA contains 9 cohesins, domains used to securely allow different proteins to interact. The enzymes ( I will describe soon) contain dockerin domains that interact with cohesins. CipA also contains a carbohydrate binding module (CBM) which allows it to directly interact with cell walls.

Many of the cohesins are used to attach carbohydrate degrading enzymes, usually of two classes: endoglucanases and exoglucanases.  These work in concert to breakdown cellulose and other carbohydrate polymers. Apart from the catalytic portions of these proteins are dockerin, needed to bind to CipA, and other domains like the Ig domain or X domain.

These enzymes ‘fit’ onto the scaffold protein like Legos. This makes them very modular. Now consider other scaffold proteins have a different type of cohesin (cohesin II) that can be used to attach other scaffold proteins thus making polycellulosomes. For example, Cthe_0736 is a scaffold protein with 7 type II cohesins. This means Cthe_0736 can have 6 other scaffold proteins attached to it meaning this polycellulosome could contain up to 63 individual enzymes which is potentially common considering isolated cellulosomes vary in molecular weight considerably.

Illustration of a polycellulosome attached to cell (blue) on substrate (brown)

Closer look at the polycellulosome

In a later post, I will go into a little detail on how these cellulosomal enzymes actually are able to degrade anhydrous polymers of carbohydrate.

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Artistic take on ethanol-producing yeast

Write text here…

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Living at the Boiling Point: What we can learn from extreme heat-loving microbes

Thanks to the decreasing costs of sequencing genomic DNA, finding novel microorganisms that add to our understanding of metabolism in myriad environments is becoming common place. Not only are we learning about the diversity of life in extreme environments, like heat, cold, pressure, and altitude, but we are also learning what life on other planets may be like. With each additional genome added into ‘the cloud’, our synthetic biology toolbox gets a little bit bigger and our ability to manipulate tiny organisms to produce novel compounds is possible. Enter the “rushing fireball”.

Pyrococcus furiosus is an archeal species that thrives near deep-sea thermal vents where temperatures are between 90 and 100 degrees Celsius (or 194 to 212 degrees F). P. furiosus can grow at temperatures as low as 70 degrees C (158 deg F). To live in such conditions, this organism’s proteins must be tolerant to what we would consider harsh conditions. This organism’s ambient conditions makes wild-type proteins well-suited for industrial processes where temperatures are near boiling.

So far, P. furiosus has been utilized to produce 3-hydroxypropionic acid, a common industrial chemical used to make various products including acrylics. The kicker is that these cells were wired to make this chemical from atmospheric carbon dioxide. It is not crazy to think of what other useful products can be produced by P. furiosus with small modifications within the genome; products like ethanol or butanol as biofuels.

My illustration of Pyrococcus furiosus

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Reference

Keller, M., Schut, G. J., Lipscomb, G. L., Menon, A., Iwuchukwu, I., Leuko, T., Thorgersen, M. P., Nixon, W. J., Hawkins, A., Kelly, R. M. and Adams, M. W. W. (2013) “Exploiting microbial hyperthermophilicity to produce an industrial chemical using hydrogen and carbon dioxide”Proc. Natl. Acad. Sci. U.S.A. (in press).

Combing the Earth One Genome at a Time: In Pursuit of “The Next Big Thing” in Sustainability

Illustration of Clostridium thermocellum cells (orange) on the surface of a cellulose fibril (multicolor)

There is one thing that can be said about scientists: they’re never satisfied…thankfully. Observation and curiosity leave them on a never-ending quest to understand Mother Nature and improve humanity. One great example of this is the field of alternative energy science. Through the efforts of the Bioenergy Research Centers (BRCs) and Joint Genome Institute within the U.S. Department of Energy‘s Office of Science, there is a perpetual search for Nature’s best metabolic machinery. This search requires thinking outside the box and sometimes outside your comfort zone. For example, last year researchers from the Joint BioEnergy Institute published findings that originated in the El Yunque National Forest in Puerto Rico, a rain forest and home to Enterobacter lignolyticus, a bacterium that is tolerant to ionic liquids (liquids with salts that are not crystaline, but are liquid). This discovery began with the observation that soil microbes at El Yunque have a high rate of organic decomposition and tolerance to osmotic pressure.

Another example are bacteria from the genus Caldicellulosiruptor that are able to degrade biomass, however, they live in extremely thermophilic environments like hot springs from New Zealand to Russia to Yellowstone. Researchers at the BioEnergy Science Center were able to isolate these microbes and start characterizing the enzymes responsible for degrading woody biomass into simple sugars.

Or what about  researchers at the Great Lakes Bioenergy Research Center essentially dissecting a leaf-cutter ant colony in Panama to examine its ecology; from the fungus the ants use as food, to the bacteria that help degrade the leaves. Or what about isolating microbes from termite guts or wasp guts?

Then there is the champion for raising scientific curiosities, Clostridium thermocellum which holsters woody biomass degradation factories attached to the outside of its cell membrane. These factories are known as cellulosomes.

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From vinegar, a potential cheap energy alternative: Bacterial nanowires Part 2 with three animations

Acetate entering the cell

I know you are wondering how vinegar fits into all of this. Acetate, or acetic acid, is what constitutes vinegar. It also is the basic two carbon currency in organic metabolism. It is the basis of acetyl-CoA, a molecule utilized in carbohydrate metabolism as well as fatty acid synthesis and degradation. It can be a major source of the precious hydrogen atoms to drive energy production in Geobacter.

Once entering the cytoplasm, acetate is converted to acetyl-phosphate via acetate kinase (black, right) and ATP. Then acetyl-phosphate is converted to acetyl-CoA via phosphate acetyltransferase (black, left) and coenzyme A.

Once acetate enters into the cell, it undergoes phosphorylation by acetate kinase (ACK) to form acetyl-phosphate. Phosphorylation is an important way to activate molecules so they can be utilized in metabolic pathways. Once formed, acetyl-phosphate is a substrate for phosphate acetyltransferase (PTA) which converts acetyl-phosphate to acetyl-CoA. Once formed, acetyl-CoA can enter to many different metabolic pathways.

Research studies have shown biostimulation (addition of some nutrient to stimulate growth of organisms) of uranium-contaminated sites with acetate can increase the reduction of uranium from a soluble form to an insoluble form that is no longer a threat for entrance into the water shed. Increased uranium reduction is the equivalent to increased electricity generation just by swapping the terminal electron acceptor from the electron transport system from uranium to electrode of a microbial fuel cell.

So there you have it, adding vinegar (acetate) can stimulate the generation of bioelectricity rather cheaply.

Electrons (glowing ball) are transferred from MacA (maroon) via cellular respiration to PpcA (bright red) in the periplasm to the awaiting geopilus (green) for extracellular electron transfer to a waiting electron acceptor.

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