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

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