Shewanella: Sneaky Sulfur Cyclers?

Katherine Mateos, Carleton College

Like Shewanella, I too thrive at cold temperatures!

Life can survive almost anywhere! From hot pools on volcanoes to beneath ice-cold glaciers, pretty much all of the inhabitants in these hostile environments are so small that you cannot see them with your bare eye. These extremophiles, as they are often called, include tiny single-celled microbes—bacteria and archaea. By studying tiny microbes we can answer big questions: How did life begin on Earth?  How can we find life on other planets? How will our planet respond to its changing climate?

Outflow of Blood Falls on the Taylor Glacier. Image credit: Dr. Jill Mikucki.

This summer, I am working with one of these extremophiles, a type of bacteria separated out from a sample from Blood Falls, Antarctica. This lake is a pool of brine (very salty water) covered by more than 150 feet of ice from the Taylor Glacier. Blood Falls gets its name from the bright red stain that the brine leaves on the Taylor glacier as it leaks out from beneath the glacier. As you would expect, this location is cold and dark, but the chemicals in the brine are what truly make this ecosystem extreme. For one, Blood Falls is super salty, over twice as salty as the ocean. Most water has oxygen trapped within it, but Blood Falls has very little. Two important chemicals are also found in unusually high quantities: iron and sulfur.

Electron Microscope image of Shewanella BF02. Image credit: Bruce Boles.

The bacteria that I am studying makes good use of the iron in this environment. Like a battery produces energy from a variety of chemical reactions, Shewanella (strain BF02) gets most of its energy by harnessing the energy that is released when one chemical form of iron changes to another. However, there might be another source of energy Shewanella can live off of—perhaps a chemical that contains sulfur. Sulfur is one of the most common elements on earth, found in pesticides, foods, and in humans. Sulfur can form compounds with other common elements including hydrogen, carbon, and oxygen. Some of these chemicals, known as volatile organic sulfur compounds (VOSCs), easily evaporate into our atmosphere and affect our environment. We want to know if the Shewanella are creating these VOSCs, and if they do, what chemicals the Shewanella turn into VOSCs.

The strain of Shewanella that I am studying is from an extreme ecosystem but similar Shewanella are found throughout many ocean ecosystems. We can treat Blood Falls as a model to learn about the way that our oceans will affect our environment.  Even though Shewanella are too small to see with your bare eyes, figuring out what compounds they break down can help us understand the future of the environment around the world.

Thank you to my mentor, Dr. Peter A. Lee, and our collaborators, Dr. Jill Mikucki and Abigail Jarratt, for their guidance in the research process. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

References

Mikucki, J. A. et al. A contemporary microbially maintained subglacial ferrous ‘ocean’. Science 324, 397–400 (2009).

Sievert, S. M., Kiene, R. P. & Schulz-Vogt, H. N. The sulfur cycle. Oceanography 20, 117–123 (2007).

Haloarchaea: Getting molecular

Ben Farmer, University of Kentucky

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The Approach: In my last post, I introduced the concept of extremophiles. My study organisms this summer, the haloarchaea, are extremophiles adapted to thrive in the most hypersaline environments on Earth. My goal this summer in Dr. Rhodes’ lab is to examine what makes these adaptations possible.

We know that the surface of proteins found in haloarchaea are not only acidic, but they are also negatively charged. Haloarchaea proteins likely use this negative charge to better compete with salt ions for water molecules and keep from unfolding in hypersaline water. Changes in the type of amino acids that make up haloarchaea proteins explains why their proteins are able to become abnormally acidic. To add another layer of molecular complexity, there are a set of molecules at play here called tRNA: tRNA mediate the transition from genetic code to proteins in the cell. We are investigating whether expression of certain tRNA corresponds with the change in overall acidity of haloarchaea proteins.

To test this hypothesis, first we needed to obtain haloarchaea species. The particular species I am analyzing is Haloferax sulfurifontis (or just Haloferax), which was isolated from a sulfur-rich spring in Oklahoma, USA (Elshahed et al. 2004). We created hypersaline solutions (called media) to grow the cells in, which in this case was tailored to support Haloferax. The most important part was providing enough salt to make the media hypersaline, which meant nearly 10x more concentrated than seawater. When the media was ready, we introduced cells of Haloferax in small quantities in a process called inoculation. Now that we had cells growing, we next determined which tRNA were present in their genomes with the help of the Genomic tRNA database (Lowe and Eddy 1996).

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Falcon tubes containing cultures of several archaea species. Growing archaea cells turn the media orange/red as a result of carotenoid pigments.

To begin tRNA analysis, we filled the wells of a plastic plate with an oligonucleotide (oligo, or short DNA sequence) for each tRNA gene we found in Haloferax. Readying the well plate involved suspending the dry oligos in water and then making sure that the concentration of DNA was the same for each mixture. A microarray was created by randomly placing 8 samples of each of the 40+ oligos on a larger plate, and then printing the results. Using 8 random samples ensured higher accuracy. The tRNA was then “labelled,” by incorporating a radioactive phosphate (32P) into the living Haloferax cells. Each tRNA oligo produced signals corresponding to how much they reacted to the radioactivity. These signals showed up as spots on the array, and the spots were directly proportional to abundance (expression) of tRNA. Through image analysis of the spot intensity, we determined the expression of each type of tRNA.

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Machine used to produce microarray results, courtesy of Dr. Renaud Geslain’s lab

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An example of the spots produced by radioactivity. Each black spot indicates a specific tRNA, and the intensity of the spot tells us the abundance of that tRNA in Haloferax. I aligned the yellow circles to the spots using imageJ software, which allowed me to quantify tRNA abundance.

Organisms require physiological adaptations to cope with environmental disturbances, and this often is apparent on the level of proteins. Amino acids are the building blocks of proteins, and mRNA provides the code for these building blocks. We are assuming that mRNA expression is changing in Haloferax to cope with extreme salinity, and it would follow that tRNA expression is changing in tandem. So, analyzing tRNA in haloarchaea provides us with a better of how organisms like extremophiles manage to adapt to the outer limits of environmental conditions on Earth.


Acknowledgements

Many thanks to my mentor, Dr. Matthew Rhodes, who has introduced me to everything from cell culturing to python. This project is funded through the National Science Foundation and supported by the Fort Johnson REU Program, NSF DBI- 1757899.



References

Elshahed MS, Savage KN, Oren A, Gutierrez MC, Ventosa A, Krumholz LR (2004) Haloferax sulfurifontis sp. nov., a halophilic archaeon isolated from a sulfide- and sulfur-rich spring. Int J Syst Evol Microbiol 54:2275–2279

Grelet S, McShane A, Hok E, Tomberlin J, Howe PH, Geslain R (2017) SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels. PLoS One 1–17

Lowe TM, Eddy SR (1996) TRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964

Reed CJ, Lewis H, Trejo E, Winston V, Evilia C (2013) Protein Adaptations in Archael Extremophiles. Archaea 2013:1–14